INTERLEUKIN-7

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13 INTERLEUKIN-7 Peter K.E. Trinder ~ a n d M a r k u s L M a e u r e r 2 1Nemod Immuntherapie AG, Berlin, Germany 2University of Mainz, Mainz, Germany W h a t is n o t fully u n d e r s t o o d is n o t p o s s e s s e d J o h a n n Wolfgang v o n Goethe

INTRODUCTION Interleukin-7 (IL-7) is an exceptional cytokine, as it mediates lymphopoiesis in mice in a non-redundant fashion. In contrast, targeted gene deletion of other cytokines, including IL-2, IL-4 or IL-10 (Schorle et al., 1991; Kuhn et al., 1991, 1993), revealed that these cytokines are not essential for development and proper function of either B or T lymphocytes. IL-7 is secreted by both immune and non-immune cells and appears not only to be involved in the development of an effective immune system, but also in the generation and maintenance of strong and effective cellular immune responses. IL-7 serves as the major growth and differentiation factor for both thymic and extrathymic development of 7~+ T lyrnphocytes. IL-7 promotes immune effector functions in T lymphocytes, natural killer (NIO cells and monocytesmacrophages, and modulates the quantity and quality of immune responses in vitro and in vivo. The availability of IL-7-targeted gene-deleted mice or IL-7

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze [SBN 0-12-689663-1, London

transgenic animals has allowed a more detailed study of the physiology and pathophysiology of paracrine and systemic effects of IL-7 which represents a key regulator of T-cell homeostasis. The implementation of IL-7 in the treatment of different diseases, including immunodeficiency disorders and malignancy, suggests that IL-7 may facilitate a number of therapeutic endeavors including bone marrow and organ transplantation, cancer immunotherapy, the treatment of infectious diseases and, in general, 'immunereconstitution'. Despite the substantial work on IL-7 in development and differentiation of B and T cells, novel co-factors have been identified: the pre-pro B cell growth-stimulating factor (PPBSF), the variant beta chain of the hepatocyte growth factor, which is covalently bound to IL-7. In addition, an 'IL-7-1ike' factor has been debunked as thymic stromal lymphopoietin (TSLP) which interacts with the respective TSLP-receptor, but also with the IL-7 receptor alpha chain. The identification of IL-7-1ike molecules and IL-7-hybrid cytokines, as well as soluble IL-7

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receptors suggests that IL-7-mediated effects are finely tuned and tightly controlled. To date, IL-7 represents the single non-redundant cytokine responsible for shaping the humoral as well the cellular immune system.

CLONING, PURIFICATION AND STRUCTURE Following the development of culture techniques for studying in vitro bone marrow cultures it became apparent that B-cell maturation occurred in the presence ofbone marrow stromal cells, suggesting the existence of a growth and/or maturation enhancing cytokine (Hunt etal., 1987). Namen and coworkers subsequently demonstrated that conditioned medium from stromal cell cultures stimulated the growth of B-cell precursors. They immortalized a stromal cell line by transfecting it with the plasmid pSV3neo (large and small T antigens of SV40) and isolated a clone (I • N/A6) which produced a factor initiaUy called lymphopoietin1 (LP-1) that stimulated the growth of B-cell precursors. Conditioned medium from the growth of this clone was then purified. High-performance liquid chromatography (HPLC) column fractions containing LP- 1 bioactivity were isolated. A single unit of LP-1 activity is that causing half- maximal 3HTdR incorporation in a culture of precursor B cells (LP-1 bioassay). At this stage of purification it was clear that several proteins were present in the fraction that could account for the biologic activity. Additional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis under non-reducing conditions associated bioactivity with a protein of 25 • 103 Da, which was substantiated by 125I-labeled LP-l-binding experiments. The purified protein exhibits a specific activity of approximately 4 X 10 6 U/mg ofprotein and is active at a half-maximal concentration of 10 - 13M(Namen et al., 1988). The same murine stromal cell clone provided a cDNA library which was screened for LP- 1 activity following expression in COS-7 cells. A clone (1046) was identified that was associated with high biological activity. The sequence contains a 548 bp 5' non-coding region which may be involved in expression regulation. The sequence includes a 462 bp open reading flame and a 579 bp 3' non-coding region containing a consensus polyadenylation signal and terminating in 15 adenine

residues. Purified protein was subjected to N-terminal analysis, which suggested that the nucleotide sequence from clone 1046 codes for the same protein, the protein was designated IL- 7. The mature protein has a 25 amino acid leader sequence followed by 129 amino acids with two N-linked glycosylation sites and six cysteine residues leading to intramolecular disulfide bond formation. The importance of disulfide bond formation is suggested by loss of activity following treatment with 2-mercaptoethanol breaking disulfide-bond formation. The disparity between calculated molecular weight and that predicted by migration of the native protein may be accounted for by glycosylation (Cosenza et al., 1997; Namen et al., 1988). Two such N-linked glycosylation site in murine IL-7 are located at amino acid residues 69 and 90 (Namen etal., 1988). IL-7 mRNA has been detected in murine thymus spleen, kidney and liver by Northern blot analysis. Interestingly, although message was present in thymus and spleen, no biological activity could be detected in these tissues. Goodwin and colleagues characterized human IL-7 by nucleic acid hybridization of cDNA prepared from a hepatocarcinoma cell line (SK-HEP-1, ATCC HTB 52) with the murine IL-7 probe. There is considerable homology between the two IL-7 nucleotide sequences (81% in the coding region) and up to 60% amino acid homology with all six cysteine residues being conserved (Goodwin et al., 1989). The human IL-7 gene contains 6 exons over 33 kb (Lupton et al., 1990). The human IL-7 cDNA is composed of 534 nucleotides encoding a protein of 177 amino acid residues (17.518 Da) with a signal sequence of 25 amino acid residues and three potential N-linked glycosylation sites (Goodwin et al., 1989). There is a 19 amino acid insert for human IL-7 (coded for by exon 5 in the human genome) which does not exist in murine IL-7 (Plate 13.1) and appears not be essential for biological IL-7 activity using a proliferation assay of progenitor B cells (Goodwin et al., 1989). Additionally, an apparently alternatively spliced human IL-7 mRNA lacking the entire exon 4 (44 amino acid residues) results in loss of ability to stimulate proliferation of murine progenitor B cells isolated from the SK-HEP-1 line. Alternatively spliced IL-7 isoforms have also been identified in different tissues, or human cell lines. Human small intestinal epithelial cells, which secrete IL-7, express two alternatively spliced mRNA tran-

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scripts (Madrigal-Estebas et al., 1997). Alternatively spliced IL-7 mRNA has also been identified in h u m a n malignant hematopoietic cells obtained from children with acute lymphoblastic leukemia (ALL). Three IL-7 splice variants have been identified which lack either exon 3, exon 5 (like the original cDNA clone identified by Goodwin and coworkers), or both exons 3 and 5 in combination with exon 4 (Korte et al., 1999). In addition to these in-flame IL-7 variants, several out-of-flame variants have also been identified. The role of these isoforms is still enigmatic. Differentially spliced IL-7 may act as a natural antagonist, a situation which has already been observed for IL-4 (Atamas et al., 1996). In addition, the readout system, e.g. proliferation of progenitor B cells, may not be able to detect alternate functions by such IL-7 variants. Human recombinant IL-7 is active on murine and h u m a n B-cell progenitors. In contrast, murine IL-7 acts only on murine, but not on h u m a n cells. To date, there is only a limited source of biological assays available to detect IL-7 bioactivity and the readout is based on proliferation of a murine precursor B-cell line. However, these assays detect only a single facet out of a magnitude of IL-7 activities, e.g. induction of V(D)J recombination, proliferation of mature T cells, maintenance ofT-cell survival or activation of monocytes. Thus, different biological readout systems may be advisable if either mutant IL-7 forms or differentially spliced IL-7 isoforms are scrutinized for biological activity. To date, most studies have used the cell line I x N / 2 b (Park et al., 1990) or the pre-B cell line 2E8, which was initially maintained on stromal cells and then in IL-7-conditioned medium (Pietrangeli et al., 1988) to define IL-7 bioactivity. Recently, a limited number of biological assay systems has been added to the panel of IL-7-responsive cells, e.g. the acute myeloid leukemia (AML)-derived cell line MUTZ-3 proliferates in response to IL-7 and to thymic stromal lymphopoietin (TSLP) (Quentmeier et al., 2001). Alternatively, the murine pre-B-cell line PB-1 is dependent on the presence of IL-7 and can be used to detect IL-7 in both plasma and serum samples with a sensitivity in the range of 50 pg/ml IL-7 (Mire-Sluis et al., 2000). This bioassay appears to be more sensitive for detecting IL-7 as compared with the IL-7dependent 2E8 cell line and it shows no response to cytokines (e.g. TGF[~ and IL-13) which may interfere with 2E8 proliferation (Mire-Sluis et al., 2000). A stable

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pre-pro B-cell line (YS-PPB), derived from AA4.1 + yolk sac cells from day 10 mouse embryos may also be implemented to study the role of IL-7 in B-cell differentiation and expansion (Lu and Auerbach, 1998). Human IL-7 is predicted to conform to the 'small hematopoietin' subclass members, forming a fourhelix bundle structure. Human or murine IL-7 has not yet been crystallized. However, using a combination of theoretical sequence structure recognition prediction and disulfide bond assignments (human IL-7 exhibits six cysteine residues, see Plate 13.1), three-dimensional models of IL-7 have been constructed (Cosenza et al., 2000) by superimposing IL-7 helices into the IL-4 template (Walter et al., 1992a, 1992b) which has been defined by X-ray structure crystallized IL-4 (Bajorath et al., 1993), incorporating the disulphide bond assignments (Cys3-Cys142, Cys35-Cys130, Gys48-Cys93) into the model. The disulphide bridges have been determined by matrixassisted laser desorption/ionisation (MALDI) mass spectroscopy and by cysteine to serine mutational analysis (Cosenza et al., 1997). No biological activity was retained, using a murine pre-B-cell precursor assay, if all six cysteine residues were substituted with serine (Cosenza et al., 1997), which is in keeping with earlier results that [3-mercaptoethanol treatment abolishes bioactivity (Henney, 1989). Cysteine residues were reintroduced into the mutant IL-7 proteins and assayed for bioactivity. Apparently, only a single disulfide-bond forming IL-7 mutant protein is able to form a tertiary structure capable of stimulating precursor B-cell proliferation. These data were used to define the potential IL-7 structures involved in receptor binding. This is not only of interest in the context of structural analysis, but also pertaining to the development of 'designer' cytokine molecules which either act as antagonists or as 'superagonists' as compared with the wild-type cytokine protein. Circular dichroism analysis suggested that h u m a n IL-7 is primarily approximately 35% a helical, 31% random coil, 23% [3 sheet and 11% [3 turn. The threedimensional IL-7 structure has been postulated based on data obtained from IL-4 and GM-CSF: IL-7 belongs to the group of short chain four a helical bundle cytokine molecules defined by four (lefthanded) ~ helices in an up-up-down-down position. These helices are designed with A to D (Bazan, 1990). In general, amino acid residues of helices A to C bind

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the individual cytokine (i.e. IL-7Ra) receptor and residues located on helix D bind the to common 7 chain (7c) shared with different cytokines of the same family (see Plate 13.1). Of practical interest, these structural analyses provided the means to identify potential IL-7 molecules endowed with antagonistic effects, at least in regard to precursor B-cell proliferation. Two approaches have been reported. First, mutational analysis results in the construction of IL-7 variant proteins with amino acid substitutions at different positions (i.e. K121-A, L136-A, K140-A, W143-A), which ablated proliferation of murine precursor B cells. It is worth noting that the mutant hIL-7 (W143-A) is able to replace the wild-type IL-7 from the receptor and acts as an antagonistic molecule (Cosenza et al., 2000). A second approach has been reported by Kroemer and coworkers. Three-dimensional IL-7 models were altered based on the observation of alternatively spliced IL-7 isoforms and analyzed for IL-7 receptor binding by computer modelling (Kroemer et al., 1998). IL-7 isoforms with altered disulfide bonding patterns may show a different three-dimensional structure and also act as antagonists, at least in the murine precursor B-cell assay. However, other readout systems may help in better defining the biological impact of such IL-7 protein variants. The human IL-7 gene appears to represent a single copy gene located on the chromosome 8q12-13 in proximity to the p53/p56 ~yngene, a member of the Src tyrosine kinases and the HYRC gene which is potentially involved in VDJ recombination located at 8ql 1 (Corey and Shapiro, 1994; Seckinger and Fougereau, 1994). The murine IL-7 gene lacks transcription regulatory elements which have been commonly identified in eukaryotic promoters, e.g. the TATAbox, CAAT sequences, SP1 or GC-rich regions. Only one SP1 binding site has been identified in the human IL-7 gene. Additionally, a potential binding site for El2 is conserved in both murine and human IL-7 genes, as well as other sequences confirming to 'helix-loop-helix' class of DNA-binding proteins (Lupton et al., 1990). Of note, several IL-7 mRNA species of 1.5, 1.7, 2.6 and 2.9 kb have been identified in murine tissues. However, all four transcripts appeared to be present in the thymus. In contrast, only the 2.6- and the 2.9-kb IL-7 mRNA transcripts could be detected in kidney

(Namen et al., 1988). Regulation of murine IL-7 mRNA expression has been addressed by examining IL-7 RNA resolution in murine PAM 212 keratinocytes (Ariizumi et al., 1995). Treatment with IFN7 yields preferential expression of the 1.5- and 2.6-kb mRNA species in addition to the constitutively expressed 2.9- and 1.7-kb mRNAs by the use of alternative transcription initiation sites. The 1.5- and 2.6-kb mRNAs are transcribed within 250 bp from the coding sequence. In contrast, the 1.7- and 2.9-kb mRNA species contained >400 bp in the 5' untranslated region. IFN7 promotes conversion to 1.5- and 2.5-kb mRNA expression through the IFN-stimulated response element (ISRE) located 270 bp upstream from the coding sequence (GAAACTGAAAGT). This ISRE is immediately followed by a non-TAT-type transcription 'initiator' element (CTTACTCTTG). It appears that IFNT-induced transcription of IL-7 may be controlled through the ISRE-control complex, whereas other 'initiator sequences' may be responsible for the base-line IL-7 transcriptional activity in certain cell types (Ariizumi et al., 1995). It has been suggested that the IFNT-inducible IL-7 transcripts may be more translationally active as compared with the conventional 'base-line' 1.7- and 2.9-kb transcripts, a concept which may impact on the molecular definition of the cellular interaction of keratinocytes, a source of IL-7 secretion, and IFNT-producing cells in skin (see below). IL-7 has also been cloned from different species, for instance from swine (Ueha et al., 2001), rat (Visse et al., 1999) or bovine origin. IL-7 cDNA from a bovine leukemia virus-induced B-cell lymphosarcoma was characterized constituting a protein of 176 amino acids and showing 75% homology with human IL-7 and 65% homology with murine IL-7 (Cludts et al., 1992; Barcham et al., 1995) (Table 13.1).

Cytokines similar to IL-7 and IL-7-associated cytokines A number of studies have addressed the nature of stromal-derived growth factors capable of regulating pro-B and pre-B cell development (for review see (Baird et al., 1999).Only a limited number of studies address the factors that are involved in pre-pro B-cell differentiation. Using a long-term bone marrow culture system, a 30 kDa pre-pro B-cell factor (PPBSF)

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TABLE 13.1 IL-7 expression ( m R N A or protein) in cell lines a n d tissues Tissue / Cell Type

Detection of mRNA

Detection of protein

Reference

Bone marrow stromal cells

+ (h,m)

+ (h, m)

Spleen

+ (h, m)

n.d.

Kidney Kidney allograft Renal cell cancer (RCC) tissue sections, or RCC-cell lines Fetal and adult thymus

+ (h,m) + (h) + (h) a

n.d. n.d. + (h)

N a m e n et al., 1988; Sudo et al., 1989; Witte et al., 1993 N a m e n et al., 1988; Goodwin et al., 1989 N a m e n et al., 1988 Strehlau et al., 1997 Trinder et al., 1999

+ (h, m)

+ (h, m)

Thymic stromal cells

+(m)

+

Hassall's corpuscles Keratinocytes

+(h) + (h.m.)

n.d. + (h, m)

Intestinal epithelial cells, epithelial goblet cells Colorectal cancer cells Cervical cancer cells Uterus Brain Head and neck squamous cancer Adult liver Hepatocarcinoma Activated/mature dendritic cells (CD80 § CD83 +, CD86 § CD40 § EBV § B-cell lines Burkitt's l y m p h o m a cells Chronic B-lymphocytic leukemia cells

+(h) a

+(h)

+(h) a +(h) a +(m) +(h) n.d. +(r) +(h) a +(h)

+(h) +(h) n.d. n.d. +(h) n.d. n.d. +(h)

+(h) +(h) +(h) a

+(h) +(h) +(h)

+ (h) + (h)

n.d. n.d.

+ (h) a + (m) + (h) + (h) + (h)

+ (h) + (h) + (h) n.d.

+ (h) b

n.d.

+(h) n.d.

+(h) +(h)

Bladder cancer Inflammatory malignant fibrous histiocytoma Follicular dendritic cells Fibroblasts Oral m u c o s a vascular endothelial cells Hodgkin's cell line, nodularsclerosing type Sezary l y m p h o m a cells Lesions from tuberculoid lepra Lymph nodes from HIV+ patients

N a m e n et al., 1988; Goodwin et al., 1989; Montgomery and Dallman, 1991; Sakata et al., 1990; Wiles et al., 1992 Sakata et al., 1990; Gutierrez and Palacios, 1991 He et al., 1995 Heufler et al., 1993; Matsue et al., 1993a, 1993b Watanabe et al., 1995; Madrigal-Estebas et al., 1997 Maeurer et al., 1997 our unpublished observations Appasamy, 1997 Appasamy, 1997; Paleri et al., 2001 Appasamy, 1997 Goodwin et al., 1989 Sorg et al., 1998 Benjamin et al., 1994 Benjamin et al., 1994 Frishman et al., 1993; Long et al., 1995 Kaashoek et al., 1991 Melhem et al., 1993 Kroncke et al., 1996 Aiba et al., 1994 Kroncke et al., 1996 Kroncke et al., 1996 Bargou et al., 1993 Foss et al., 1994; Asadullah et al., 1996) b Sieling et al., 1995 Napolitano et al., 2001

acloning and sequence analysis of mRNA exhibits alternatively spliced forms(s) of IL-7. bIL-7 mRNA did not appear to be overexpressed as determined by semiquantitative analysis in skin biopsies from patients with mycosis fungoides, or with pleomorphic T-cell lymphoma when compared with biopsies obtained from normal skin, psoriatic lesions or atopic dermatitis. n.d., not determined, h, human; m, mouse, r, rat.

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has been identified which formed a heterodimer with IL-7 (Lai et al., 1998; McKenna et al., 1998). The PPBSF cofactor (PPBSF-coF) is produced in stromal cells obtained from IL-7 -/- mice (McKenna and Goldschneider, 1993; McKenna et al., 1998). PPBSFcoF alone (i.e. without IL-7) maintains pre-pro B-cell viability (Lai et al., 1998). More recently, the PPBSFcoF has been identified as the free mitogenic 13chain of the hepatocyte growth factor (HGF)/scatter factor (Lai and Goldschneider, 2001), which represents a heparin-binding, stromal-derived cytokine closely related to plasminogen (van der Voort et al., 2000). Interestingly, the 13 chain of HGF had not been reported to be independently produced of the HGF chain. IL-7/HGFI3 represents a new member of 'hybrid' cytokines which are composed of the bioactive forms of cytokines which are independently produced. IL-7 complexes with HGFI3 in the presence of low molecule weight heparin-sulfate to the heterodimer (PPBSF) which may enable IL-7 to participate in stromal interactions and to trigger effectively the IL-7 receptor. IL-7 appears to be present almost exclusively as the PPBSF (IL-7/HGFI3) heterodimer in pre-B cell culture systems (Lai et al., 1998). In contrast to the generation of IL-7 heterodimeric molecules, 'IL-7-1ike' cytokine(s) have also been described, the thymic stromal lymphopoietin (TSLP), which was originally identified in conditioned medium of a thymic stromal cell line that supported the development of IgM § B cells from fetal liver hematopoietic progenitor cells. Both murine (Sims et al., 2000) and human TSLP (Quentmeier et al., 2001) have been cloned and characterized (Sims et al., 2000). The TLSP activities partially overlap IL-7-mediated functions. Both cytokines facilitate B lymphopoiesis in fetal liver cultures, bone marrow lymphocyte precursors and both cytokines are able to stimulate thymocytes and mature T cells (Friend et al., 1994). The TLSP receptor has recently been identified and shares high homology with the 7c chain molecule (Pandey et al., 2000; Park et al., 2000). Binding of TSLP to the respective TSLP receptor results in a low-affinity interaction. Binding of TSLP to the TSLP-R is markedly increased in the presence of the IL-7Ra chain, which explains why IL-7Ra gene-deleted mice show a different phenotype as compared with IL-7 -/- animals, as TSLP is still able to interact with the IL-7Ra (Pandey et al., 2000; Park et al., 2000). Thus, TLSP binds to its indi-

vidual receptor with low affinity and does not bind to the 7c receptor molecule. Both the IL-7RR and the TLSP receptor are required for high-affinity binding of TSLP (see Plate 13.2b). Thus, IL-7 is able to form heterodimeric molecules and is 'mimicked' by other proteins, e.g. TSLP. However, IL-7 -/- mice clearly demonstrated the non-redundant nature of the IL-7 protein. Loss of IL-7 results in a severe reduction of a13§ T cells and the entire loss of 75 § T cells.

THE IL- 7 RECEPTOR A cell line absolutely dependent on IL-7 for growth (I• (Park et al., 1990) was used to characterize the IL-7 receptor (IL-7R) designated as CD127 (Schlossman et al., 1994; Kishimoto et al., 1997). The IL-7R complex, exhibiting both high-affinity (-Kd 100 pM) and low-affinity (-Kd lnM) binding sites (Page et al., 1993) is composed of at least two subunits: the IL-7Ra chain, identified by a direct expression cloning strategy (Goodwin et al., 1990), maps to the human chromosome 5p13 (Lynch et al., 1992) and the common 7 chain (7c) shared with receptors for IL-2, IL-4, IL-9 and IL-15 (Noguchi et al., 1993; Kondo et al., 1993, 1994; Russell et al., 1993, 1994; Giri et al., 1994). The 7c chain appears to be constitutively expressed (Taniguchi and Minami, 1993). The IL-7Ra receptor subunit forms a heterodimer with the 7c chain which is required for the high-affinity IL-7 binding (Noguchi et al., 1993; Kondo et al., 1994). It has been suggested that IL-7 receptors expressed on some cells may contain an as yet poorly defined subunit, since IL-7 binds (albeit at low affinity) to COS cells in the absence of transfected IL-7R~ and 7c chains (Goodwin et al., 1990; Noguchi et al., 1993; Kondo et al., 1994). Alternatively, since COS cells represent primate kidney cells, endogenously expressed IL-7R may provide IL-7 binding sites. Six extracellular and four intracellular cysteine residues are present in human as well as in the murine IL-7Ra coding for a 439 amino acid protein with a calculated molecular weight of 49.5 kDa (Plate 13.2a). The IL-7Ra domain exhibits the characteristic features of the cytokine receptor superfamily: the cytoplasmic IL-7Ra domain, composed of 195 amino acid residues, does not exhibit consensus protein kinase sequences (reviewed in Sugamura et al., 1996). A

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differential splicing event results in mRNA encoding for the secreted form of the IL-7 receptor capable of binding IL-7 in solution (Plate 13.2a), a form that may be important for binding circulating IL-7 (Goodwin et al., 1990; Park et al., 1990; Mehrotra et al., 1995). IL-7Rcz has been detected on pre-B cells, thymocytes, some T-lineage cells (Park et al., 1990; Rich et al., 1993), on human intestinal cells (Reinecker and Podolsky, 1995), colorectal cancer cells, renal cell cancer cells (our unpublished observations), and on a bone marrow-derived macrophage but not on mature B cells (Foxwell et al., 1992), on cutaneous T-cell lymphomas (Bagot et al., 1996), and thymic NKI.1 + T cells (Miyaji et al., 1996). The human IL-7Ra (CD127) is expressed on both naive or activated memory CD4 § or CD8 § T cells (Plate 13.3). Similarly to the alternatively spliced IL-7 transcripts, the IL-7Ra not only codes for the 'canonical' IL-7Ra or the soluble form thereof (see above), but also for differentially spliced IL-7Ra isoforms (Korte et al., 2000). These transcripts have been identified in human acute lymphoblastic leukemia. These isoforms apparently lack a part of the cytoplasmic domain, but are still capable of binding IL-7 protein, similar to the intracellularly truncated erythropoietin receptor that fails to mediate proliferation and is capable of inhibiting the functions of the wild-type receptor in a dominant negative fashion (Nakamura and Nakauchi, 1994). The total number of IL-7 binding sites on individual cells may range from 1 • 104 up to 5 • 10S/cell (Armitage et al., 1992b). Interestingly, IL-2 and IL-7 reciprocally induce IL-2Ra and IL-7Ra receptor expression on 75 + T lymphocytes, which may be important for proliferation and T-cell response to locally produced cytokines of intraepithelial lymphocytes (iIEL). However, there are two different subsets of such iIEL: 7~ + T cell receptor (TCR) dim cells (7~+ TCR dim) are responsive to IL-2 (presumably provided in situ by ~ + T cells) and IL-7 (presumably provided in situ by epithelial cells or macrophages). In contrast, 7~+ TCR bright cells did not respond by up-regulation of their IL-2 and IL-7 receptors (Fujihashi et al., 1996). Additionally, IL-7 binding to the IL-7R leads to expression of the transferrin receptor and the 4F2 antigen. Sequence analysis of the 5' flanking region of the murine IL-7Ra revealed that it contains CAATT and TATA sequences, potential glucocorticoid receptor binding sites, as well as a potential ISRE element.

31 1

Using DNA microarray technology in order to analyze gene expression in peripheral blood cells obtained from healthy individuals upon exposure to glucocorticoids, IL-7Rczhas been identified to be induced as the prominent gene by glucocorticoids both on the mRNA as well as on the protein level (Franchimont et al., 2002). Conversely, TCR signaling decreases IL-7Rcz expression and the fine balance between IL-7Ra down-regulation and induction by glucocorticosteroid determines the final IL-7Ra level expression on T cells. Enhanced IL-7 expression has been shown to be associated with enhanced IL-7-mediated signaling and immune effector functions in responder cells (Franchimont et al., 2002). Interestingly, activation of PBMCs with anti-CD3 results in a four-fold downregulation of IL-7 receptors (high and low affinity) (Foxwell et al., 1992). These findings have recently been substantiated by the observation that IL7Ra- 7c chain complexes are detectable in activated, but not in resting T cells, independent of total cell surface 7c chain expression. Thus, stimulation of T cells may lead to assembly of IL7Ra-TC chain complexes which correlates with JAK3 expression. However, the work by Franchimont and coworkers suggests that expression of the IL-7Ra on T cells not only reflects T-cell activation status, but also the response to 'stress' mediated by glucocortocoids at high levels, either by exogenous application or by endogenous production. Thus, triggering the TCR may lead to activation-induced cell death (apoptosis) and the same may be true for apoptosis induced by stress hormones (Boumpas et al., 1993). Triggering the TCR and glucocorticoids may be able to prevent apoptosis and promote T-cell survival. Since IL-2 or IL-4 gene-deleted mice do not exhibit severe defects in T-cell differentiation, such as those observed in either IL-7 or IL-7Ra gene-deleted mice, IL-7 may account for most of the immunological defects observed in murine models of the X-SCID defect associated with defects of the common 7c chain receptor unit (Takeshita et al., 1992; Noguchi et al., 1993; Leonard et al., 1994; DiSanto et al., 1995). The X-SCID defects can also be observed in humans (Lai et al., 1997). In human unstimulated T cells, the IL-7Ra is constitutively associated with the Src kinase enzymes p59~ and p56 ~ck(Plate 13.2b) (Page et al., 1993). IL-7 binding the IL-7R leads to both increased p59 fynand p56 ~cklevels in stimulated and unstimulated T cells (Page et al.,

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1993). Signaling via the IL-7R also leads to increased activity of the Src kinases in stimulated and unstimulated T cells, suggesting that activation of p59~ and p56 ~ck is not exclusively responsible for IL-7-driven T-cell proliferation and that other signaling events (e.g. mediated through the 7c chain) are required (Page et al., 1993). However, targeted gene deletion for p59~ in mice did not show a major impact on lymphopoiesis (Stein et al., 1992; Grabstein et al., 1993; Sudo et al., 1993). In contrast, in p56 ~ckgene-deleted mice, a thymocyte maturation block at the double negative state could be observed (Molina et al., 1992). However, similar effects could not be detected in CD4, CD8 or IL-2 gene-deficient deleted mice. These observations suggest that p56 ~ckis also involved in the signaling pathways (Fung-Leung et al., 1991; Schorle et al., 1991). Thus, the observed effects of p56 ~ck on lymphopoiesis may be attributed to the lack of IL-7-driven p56~ck-mediated cellular responses. IL-7mediated phosphatidylinositol-3 (PI-3) kinase activation induced by tyrosine phosphorylation of the PI-3 kinase p85 subunit appears to be essential to the IL-7 proliferative signal (Sharfe et al., 1995). A different protein tyrosine kinase, termed pim-1, may also be involved in IL-7-mediated signaling, as IL-7mediated pre-B-cell expansion is decreased in pim-1deficient mice (Domen et al., 1993). IL-7 activates members of the Janus (JAK) family of non-receptor tyrosine kinase, JAK1 and JAK3 (Russell etal., 1994; Zeng etal., 1994; Musso etal., 1995), which are both activated by 7c chain-sharing cytokines including IL-2, IL-4 and IL-9. These kinases may serve as the signal transduction pathway to the nucleus by phosphorylation and activation of signal transducers and activators of transcription (STATs) (Plate 13.2b). IL-7 has been shown to activate STAT-1, -3 and -5 (Zeng et al., 1994; Lin et al., 1995; van der Plas et al., 1996; Perumal et al., 1997) by interacting with an area spanning the tyrosine residue 409 at the C-terminal end of the IL-7 receptor (Lin et al., 1995). Thus, at least several alternate signal transduction pathways (e.g. p56 ~ck,p59~, JAKs, STATs) may be operational in IL-7responsive cells (e.g. T cells or epithelial cells). It is possible that IL-7 may exert its functions in a cell- or tissue specific manner dependent on differential activation of the IL-7R signaling transduction pathway(s). For instance, the IL-7 receptor complex delivers signals of different quality to lymphoid progenitor cells

during rearrangement of the antigen receptors (reviewed in Candeias et al., 1997b). IL-7-mediated effects through the IL-7 receptor are also dependent on separate IL-7 receptor domains. These IL-7Ra regions have been mapped by transgenic expression of the mutant IL-7 receptor genes in the thymus of IL-7 Ra -/- mice (Porter et al., 2001). The IL-7 receptor controls different individual properties during thymic development. The IL-7Ra tyrosine-containing carboxy-terminal T domain is crucial for restoring thymic cellularity in IL-7 Ra -/- animals, important in pro/pre T-ceU progression and T-cell survival. The functional differentiation of TCR a~ + T cells and 75 + T cells are partially independent of this IL-7 receptor region. Both PI-3 kinases and STAT-5 have been demonstrated to signal through the T-domain (Lin et al., 1995; Corcoran et al., 1996). Gene-deleted mice for either IL-7 (von Freeden-Jeffry et al., 1995), IL-7RR (Peschon et al., 1994), 7c (DiSanto et al., 1995), ]AK1 (Rodig et al., 1998) or JAK3 (Nosaka et al., 1995; Park et al., 1995; Thomis et al., 1995) exhibited a similar block in the early stage of T-cell development. Of interest in the context of IL-7-induced T-cell proliferation, JAK3 is intimately associated with the T-cell receptor (TCR) signalling complex: ]AK3 is phosphorylated upon TCR triggering. This event is independent of the 7c receptor, but dependent on the TCR-associated signaling molecules Lck and ZAP-70 and physically associated with the TCR ~ chain, one of the key components of mediating downstream TCRmediated signaling events (Tomita et al., 2001). To date, the steps associated with these signaling molecules have not yet been clearly defined. Although IL-7 stimulation does activate the PI-3 kinase, the src kinases and the transcription factor STATS, genedeleted mice with loss of each of these molecules have not indicated a critical role for these components in the IL-7 downstream effects (Appleby et al., 1995; Yasue et al., 1997; Teglund et al., 1998; Terauchi et al., 1999). In contrast, the Pyk2 (Avraham et al., 1995; Lev et al., 1995; Sasaki et al., 1995; Yu et al., 1996), a protein-tyrosine kinase related to focal adhesion kinase is critically involved in IL-7-mediated effects (Benbernou et al., 2000). Pyk2 appears to be physically associated with ]AK1 prior to IL-7 stimulation and critically involved in cell survival, since antisense Pyk2 resulted in accelerated cell death. In addition, Pyk2 can be activated independently of IL-7

THE CYTOKINES AND CHEMOKINES

THE I L - 7 RECEPTOR

through muscarinic and nicotinic receptors: either carbochol or ionomycin/phorbol myristate-activated Pyk2 promotes cell survival in the absence of IL-7. Thus, Pyk2 appears to be one of the essential cofactor(s) associated with the trophic effects mediated by IL-7. Of note, Pyk2 has not only been shown to be involved in T-cell survival, but also in mediating IL-7 effects in neurons, e.g. by affecting the potassium channel KV1.2 (Lev et al., 1995). Thus, the IL-7Ra mediates a 'trophic' or a 'maintenance' effect regarding cell viability during gene rearrangement. Earlier studies showed that immature thymocytes undergo apoptosis when separated from the thymus. IL-7 is capable of sustaining these cells without inducing significant cell proliferation (Watson et al., 1989). These anti-apoptotic effects delivered by the IL-7Ra can also be observed in mature lymphoid cells (Komschlies et al., 1994) and may be attributed to the induction of Bcl-2 members (Hernandez-Caselles et al., 1995; Lee et al., 1996; Vella et al., 1997). However, other Bcl-2-related proteins, inducing Bcl-xL or Bcl-w, or other anti-apoptotic factors, may also be involved, since bcl-2 knock-out ( - / - ) mice exhibit a different picture concerning T-cell development as compared with alterations identified in I L - 7 R a - / - mice (Veis et al., 1993; Matsuzaki et al., 1997). The IL-7Ra may also deliver 'mechanistic' signals required for gene rearrangement. IL-7Ra -/- mice exhibit impaired 7 gene rearrangement (Maki et al., 1996; Candelas et al., 1997b). The same was found to be true for IL-7Ra-mediated signals, required for immunoglobulin (Ig) heavy chain and TCR~-chain rearrangement (Corcoran et al., 1996; Crompton et al., 1997). The TCR5 rearrangement may not be exclusively IL-7 dependent, as IL-7Ra -z- mice exhibit 5-chain rearrangements in vivo (Peschon et al., 1994; Corcoran et al., 1996; Oosterwegel et al., 1997; Candelas et al., 1997b). Such effects may derive from several factors. First, IL-7 induces RAG1 and RAG2 expression (Muegge et al., 1993). IL-7Ra -z- mice exhibit decreased RAG expression in double-negative, but not in double-positive, cells (Crompton et al., 1997). Therefore, decreased recombinase activity may affect recombinational events in distinct thymic cells. Second, IL-7Ra-mediated signals may be required to prevent untimely apoptosis in thymocytes. It has been suggested that IL-7Ra-mediated signals may

313

unmask genes associated with proliferation and antiapoptotic properties (Peschon et al., 1998). This is substantiated by the observation that peripheral T cells in IL-7Ra -/- mice undergo apoptosis upon stimulation (Maraskovsky et al., 1996). Recent studies addressed these points in detail (Schlissel et al., 2000; Carvalho et al., 2001; Huang et al., 2001; Huang and Muegge, 2001; Ye et al., 2001), particularly the dissection of IL-7-mediated effects on lymphocyte progenitors, i.e. survival, proliferation and 'recombination'. IL-7Ra-mediated effects (Ye et al., 2001), i.e. proliferation of lymphocyte precursors, are transmitted through the PI-3 (Corcoran et al., 1996; Pallard et al., 1999). Different signaling molecules are crucial for recombination of the T-cell receptor and immunoglobulin genes. In general, different 'conserved' recombination signals as well the RAG1/RAG2 recombinases are crucial for T- and B-cell development. It has been postulated that specific mechanisms should exist which make the appropriate gene locus accessible to recombinases according to the developmental stage of B-or T-cell differentiation (see Figure 13.4). IL-7 transmits an 'early' signal in lymphocyte development when V(D)J recombination takes place. The 'recombination' signal promoting variable (V), diversity (D), joining (J), recombination in the IgH and TCR7 locus is also transmitted through the IL-7Rcz. IL-7R-induced signaling leads to germline transcription and DNA rearrangement in D-distal variable segments in pro-B cells (see Figure 13.4) (Corcoran et al., 1996); the variable-joining recombination and germline transcription of TCR7 genes is severely impaired in IL-7Rcz-deficient animals (Durum et al., 1998; Ye et al., 1999). It is evident that the IL-7 receptor is required for TCR7 accessibility (Schlissel et al., 2000). The STAT5 proteins (signal transducers and activators or transcription), activated through the IL-7R (see Plate 13.2b), interact with consensus motifs in the 5' regions of J7 segments leading to germline transcription (Ye et al., 1999). Thus, STAT5 is able to induce germline TCR transcription, V-J recombination of the TCR7 genes and is also able to 'rescue' 73T-cell development in IL-7Rcz-/- mice. These STAT5-mediated actions are transmitted through recruitment of transcriptional coactivators to the TCR J7 germline promotor and by controlling accessibility of the TCR7 locus via histone acetylation (Ye et al., 2001).

THE CYTOKINES AND CHEMOKINES

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INTERLEUKIN-7

~,,.~CR-13,y,8 rearrangement//

TCR-o~ rearrangement

I D25+ CD44+

HPC

Pro-T1

CD44+ CD25+

TCRp+ Pre-To~-

Pro-T2

TCRI3+ Pre-To~-~

pre-pro B pro B:early Fraction-

A

CD43+

B

Pro-T3

Pro-T4

late

pre-B

C

D

--4

::3" TCRo~I3+ ,<

CD8+

DP

3

CD8+

r

TCRo~I3+

O~

SP

Immature Mature B E

F

CD43+

CD43+

HSA+

B220'o'/

B220intJ

HSA+

HSA+

SLC/p+

B 2 2 0 int

B220hig h

SLC+/-

SLC/p+

BP-I+

IgM+

IgM+

BP-I+ D-JR

CD4+

CD4+ TCRo~p-

CLP

J

O3

O

3 -,,1

O

IgD+

V-DJH VK-J K rearrangement

FIGURE 13.4 B- and T-cell differentiation. Cell surface marker expression, V(D)J rearrangement, and IL-7 responsiveness according to the following (Hardyet al., 1991; Hardy and Hayakawa, 1991; ICltamuraetal., 1991, 1992; Peschon et al., 1994; von Freeden-Jeffry et al., 1995; Li et al., 1996). All blood cells are derived from hematopoietic stem cells (HSC). Antigen-receptor rearrangements as well as phenotypic cell surface analysis help in segregating the differentiation status of cells of the B, T or NK lineage. Six developmental subsets of B cells have been identified. T cells can be segregated based on receptor expression (CD3- CD4- CD8-, triple negative (TN); CD4 + CD8 +, double positive (DP); and CD4+/CD8 +, single positive (SP)). TN cells are divided into four subsets based on CD44/CD25 expression (for review see Baird etal., 1999; Schatz and Malissen, 2002). HPC, hematopoietic progenitor cell; CLP, common lymphoid progenitor; SLC, surrogate light chain.

IL-7 and B lymphocytes All lymphocytes derive from hematopoietic stem cells (HSC). Apparently, the IL-7 receptor mediates nonredundant signals for T as well as for B cells originating from HSC. Not only animal models with target gene deletion, but also examination of the IL-7associated signaling pathways in the thymus as well as in bone marrow indicated that the up-regulation of the IL-7 receptor starts at the stage of the clonogenic

common lymphoid progenitor, which is capable of developing into T, B or NK cells (reviewed in Akashi et al., 1998; Killeen et al., 1998). Detailed analysis of the fate of either B or T cells underlined the central role for IL-7 in reconstituting an functional immune repertoire which is not only crucial for a better understanding of immune cell development and differentiation, but also in the context of designing novel strategies for immune reconstitution (see Figure 13.4). In addition, novel data suggest that central par-

THE C Y T O K I N E S A N D C H E M O K I N E S

THE IL-7 RECEPTOR adigms, e.g. T-cell lineage models, may have to be altered: T-cell development/differentiation may not be as 'static' as previously postulated. For instance, one of the central paradigms in T-cell development is that CD4§ § (double-positive, DP) thymocytes develop into single (CD4 § or CD8+)-positive T cells by terminating transcription of the respective coreceptor molecule. In contrast to this model (see for overview Figure 13.4), DP thymocytes initially terminate CD8 transcription when differentiated into CD8§ -) T cells (Brugnera et al., 2000). CD8 § thymocytes terminate CD8 transcription, even if differentiation into CD8 § T cells has already been started. IL-7 is capable of mediating to 're-start' CD8 transcription by silencing CD4 transcription in thymocytes which selectively terminate CD8 transcription, an observation coined as 'coreceptor reversal' which sheds new light on IL-7 in modulating the ultimate fate of CD4/CD8 T lymphocytes (Brugnera et al., 2000). A different example indicating that IL-7 substantially shapes the immune cell repertoire is that IL-7 also may act on the neonatal T-cell pool. Earlier models postulated that IL-7 and the IL-7Rcz are critical for delivering signals during the early phases of thymic development (i.e. at the stage of CD4-CD8- precursor evolution) but not necessarily in intrathymic expansion of positively selected lymphocytes. However, recent data show that intrathymic positive selection is associated with up-regulation of the IL-7Rcz and that the MHC class II § epithelial cells, driving postselection proliferation, express IL-7 mRNA. In addition, detailed examination of thymic tissue from IL-7Ra -~- mice also showed that postselection expansion is reduced if signaling through the IL-7Rcz does not occur (Hare et al., 2000). Thus, IL-7 may also be critical for driving the antigen-independent proliferation of either CD4 § or CD8 § T cells after positive selection, which impacts on the peripheral Tcell pool and immune receptor repertoire. This is a crucial observation helpful for designing new ways for effective immune reconstitution either in patients with HIV infection or in bone marrow transplant recipients. Most of the data pertaining to the role of IL-7-mediated effects on T- or B-cell development and differentiation is derived from animal models; data from humans are scant. 'Experiments of nature' reflecting gene defects in humans are examples of the importance of I1-7-derived signals. Severe combined immunodeficiency (SCID) can be derived from a

3 15

number of different defects (Leonard, 1996; Buckley et al., 1997; Fischer et al., 1997). The most common form of SCID is the lack of the cytokine receptor 7 chain shared among IL-2, IL-4, IL-7, IL-9 and IL-15 receptors (Noguchi et al., 1993). The defective IL-7Rcz chain leads to a T-B*NK*SCID phenotype in humans (Puel et al., 1998), a phenotype which is likely to be expected in patients who lack functional IL-7 protein. The most compelling evidence that IL-7 represents an important lymphopoietin and possibly one with clinical importance comes from a number of in uiuo investigations. IL-7 administration to normal mice (5 mg) twice daily for 4-7 days results in a two- to fivefold increase in the number of peripheral and splenic white cells without significant change in bone marrow cellularity. Analysis of the bone marrow showed an increase in B-cell precursors (B220 § sIg-) with a concurrent decease in 8C5 and MAC-1 cells (myelomonocytic markers) (Damia et al., 1992). In addition, injection of murine or h u m a n IL-7 into mice resulted in an increase of B-cell precursor (pro-B and pre-B cell) production (Valenzona et al., 1998). A general scheme for B-cell maturation is outlined in Figure 13.4. For the purpose of clarity, we have adapted the nomenclature of Hardy and coworkers defining early stages of differentiation of murine B cells. These cells can be identified in liver or in bone marrow and are divided into distinct classes (A-F) based on marker cell surface expression (Hardy et al., 1991; Li et al., 1993). Adult stem cells develop into 'conventional' B2 cells. Fetal liver stem cells are capable of differentiating into B1 cells, which persist in adult animals, reside primarily in the peritoneal cavity and stain positive for the CD5 antigen. The role of B1 and B2 cells in the context of IL-7 is further discussed below in the section titled 'IL-7 and microbial immune responses'. The early stages of B-cell development will occur in the bone marrow in response to stromal cell contact and cytokines. Hematopoietic stem cells (HSC) of the adult bone marrow have been characterized by cell surface marker analysis. HSC can be derived from murine BM using the CD34 (sialomucin) antibody, other cell surface markers include the antigens CD4, MHC class I, ER-MP12, and AA4.1 (Katz et al., 1985; Berenson et al., 1988; Szilvassy et al., 1989; Wineman et al., 1992; Orlic et al., 1993; Slieker et al., 1993; Szilvassy and Cory, 1993). Additionally, B-cell differentiation may be defined by D-J,

THE CYTOKINES AND CHEMOKINES

316

INTERLEIJKIN-7

or V-D-] rearrangement (Hardy et al., 1991). The antigen receptor of B (and T cells) are encoded in the germline by individual DNA segments, termed V, D and J, which are joined during lymphocyte differentiation. This process (VDJ recombination ) is initiated by the RAG1 and RAG2 proteins which act together at the junctions between the coding segments and the recombination signal sequence in order to produce two types of DNA ends: a signal end, terminating in a blunt double-strand break, and a coding end, which terminates in a DNA hairpin. The involvement of double-strand DNA cleavage has suggested that this process is linked to the cell cycle: several lines of evidence indicate that the initiation of VDJ recombination takes place at the G0/G1 phase (Oettinger et al., 1990; Lewis, 1994). IL-7 appears to sustain expression of the RAG1 a n d RAG2 genes (Muegge et al., 1993). IL-7 does not alter the RAG mRNA levels, but rather affects posttranscriptional regulatory mechanisms. In order to discriminate progenitor cells from cells which are already committed to the B-cell lineage, Hardy and coworkers have recently investigated bone marrow stromal cells for expression of the B-cell lineage marker B220 and HSA in combination with the CD4 and AA4.1 markers (Li et al., 1996). The latter marker is expressed on HSC, B-cell/myeloid progenitors, and early B-cell lineage cells (McKearn et al., 1985; Loken et al., 1988; Cumano and Paige, 1992). About 50% of the B220 § CD43 § and HSA cells (formerly termed A) stained positive for AA4.1 expression (Li et al., 1996). This cell population was capable of proliferating on a stromal cell layer, indicating that it may indeed represent B-cell lineage precursors. Thus, the earlier designation of fraction A B cells had to be revised. Two AA4.1 fractions (A1and A2) appear to represent the earliest stages of B-cell lineage development. The B220-, AA4.1+, CD4 ~~ fraction has been designated as A0 cells and appears to represent yet uncommitted progenitor cells. However, these 'earliest' stages identified in B-cell development will have to be characterized for activity of B-cell differentiation factors such as IL-7, kit-ligand (Flanagan and Leder, 1990; Williams et al., 1990), and ilk2/ilk3 (Rosnet et al., 1991). IL-7 does not support in vitro growth of cells of the granulocytic-monocytic or erythroid lineage but does stimulate eosinophil colony formation. This activity can be abolished by anti-IL-5 antibody treatment which suggests that IL-7 acts by stimulating

release of IL-5 or that potentially IL-5 represents an obligate cofactor (Vellenga et al., 1992). More recent data pertaining to transcription factor expression at different B-cell differentiation stages (Nutt et al., 1999; Rolink et al., 1999) also help in better understanding the role of IL-7 in concert with different bone marrow-derived growth factors in B-cell development (Cory, 1999); for instance, the commitment to B-cell production as associated with the paired box transcription factor 5 (Pax5) which is identical to the B-cell-specific activator protein (Adams et al., 1992). Pax-deficient B-lymphoid cells stop at the pre-B1 stage (Figure 13.4). In general, B cells can be cultured in the presence of stromal cells and IL-7. If IL-7 is withdrawn, the Ig receptor genes are rearranged. Not surprisingly, pre-B1 cells which are Pax-5 deficient are not able to differentiate into B cells in the absence of IL-7 unless IL-7 is restored. Surprising was the fact that IL-7-starved Pax-5 -~- cells exhibit a wide range of developmental potential, e.g. giving rise to T cells or antigen-presenting cells, depending on the environmental factors including stromal cells or cytokines (Nutt et al., 1999; Rolink et al., 1999). The growth factor combination of IL-11 and MGF (mast cell growth factor) supports bipotential progenitor cells to commit either to the B or to the macrophage lineage (Kee and Paige, 1996). Single cell cloning assays suggested that IL-7 does not act directly on the decision of whether cells commit to the B cell or macrophage lineage. However, bipotential cells responded to IL-7 by an increase in number and IL-7 added to the IL-11/MGF mixture promoted expression of mRNA transcripts coding for B-cellspecific genes (Kee and Paige, 1996). Furthermore, the growth factor combination of IL-11/flt3/IL-7 appears to maintain the potential of bipotential precursors (Ray et al., 1996). However, in a different report, uncommitted Lin-SCA-1 § bone marrow progenitor cells have been demonstrated to differentiate into B220 +, CD43 +, HSA + B cells (without expressing cytoplasmic ~ heavy chain or sIgM) using a combination of Flt3 and IL-7, which proved to be superior in regard to driving B-cell differentiation as compared with the combination of stem cell factor and IL-7; the latter combination leading to production of mature granulocytes (Veiby et al., 1996a, 1996b). Concerning already committed B cells, early pro-B cells require a combination of IL-7 and factors provided by stromal

THE CYTOKINES AND CHEMOKINES

THE I L - 7 RECEPTOR

cell layers; late pro-B cells are capable of proliferating in IL-7 without stromal cell support. The same was found to be true for early pre-B cells, but probably not for late-pre-B cells (Hardy et al., 1991). IL-7-mediated effects in B-cell differentiation may in part be mediated by regulation of the G1/S transition of the cell cycle (Yasunaga et al., 1995). Rearrangement of ~clight chains and sIgM expression correlates with IL-7RR down-regulation and therefore IL-7 unresponsiveness (Cumano et al., 1990; Park et al., 1990; Era et al., 1991; Henderson et al., 1992). However, the most precise data concerning the role of IL-7 in B-cell development are provided by IL-7 -zor IL-7Ra -z- mice. Evaluation of B lymphopoiesis in bone marrow appeared to be blocked at the transition to pre-B cells (see Figure 13.4). IL-7 -z- mice were blocked in the transition between the pro-B (fractions B/C B220+/IgM-/S7+/HSA § to the pre-B-cell population (fraction D, B220 +, IgM-, $7-, HSA+). Thus, differentiation and maturation of B/C fraction B cells to fraction D appears to be IL-7 dependent (von Freeden-Jeffry et al., 1995, Moore et al., 1996; Peschon et al., 1998). IL-7Ra gene-deleted mice showed a block in B-cell development at the transition of pre-proB cells (formerly fraction A) to pro-B cells (fraction B) (Peschon et al., 1994). This may be due to the action of other growth factors, potentially the thymic stromalderived lymphopoietin (TSLP) (Friend et al., 1994; Peschon et al., 1994) or fit3 ligand (Namikawa et al., 1996). Indeed, recent studies addressing TSLP and its receptor showed that these differences in B-cell development are linked to the TSLP receptor: TSLP has been demonstrated to impact on B-cell development in vitro (Levin et al., 1999). Ultimately, the study of TSLP -z- or TSLP receptor -z- mice will provide the answers pertaining to signaling pathways of TSLP and the involvement in lymphocyte development. In addition, more recent data suggest an IL-7independent pathway in B-lymphocyte differentiation. In IL-7 -z- mice, B-lymphocyte production takes place exclusively during fetal and perinatal life and stops 7 weeks after birth (Carvalho et al., 2001). However, the B-cell pool in the periphery of these mice appears to be stable during life and is exclusively constituted of B 1§ cells and cells which belong to the marginal zone (MZ) compartments. These 'MZ cells' are characterized by high IgM and CD21 and low IgD and CD23 cell surface expression. So-called 'multi-

317

reactive' B cells with 'sticky' antigen receptors are recruited into MZ and respond to LPS or CD40 ligation. As discussed above, B 1 cells are enriched in the peritoneal cavity and derived from early B-cell precursors (Hayakawa et al., 1985; Hayakawa and Hardy, 2000). These B1 cells show less N region diversity as compared with conventional B2 cells and are thought to be involved in autoantibody production (Gu et al., 1990; Lalor and Morahan, 1990; Tornberg and Holmberg, 1995). Of interest, serum immunoglobulins are increased up to five-fold in these mice. Thus, there appears to exist an IL-7-independent pathway of B1 and MZ B-cell generation which is active in early life: TSLP -/- mice will reveal whether TLSP is able to drive development of this distinct B-cell subpopulation. Application of IL-7 neutralizing monoclonal antibodies of mice resulted in a similar B-cell maturation blockade compared with those observed in IL-7RR knockout animals, but not to the B-cell maturation blockade observed in the IL-7 gene-deleted animals (Grabstein et al., 1993; Peschon et al., 1994). One potential explanation is that other cytokines (e.g. TSLP) may utilize the IL-7 receptor as well. Other cytokines, e.g. TLSP, stem cell factor (SCF)/c-kit, or flk2/flk3 may synergize with IL-7 to regulate B-cell development (Veiby et al., 1996b). The stem cell factor SCF/kit ligand which represents a growth factor for myeloid and erythroid progenitor cells synergizes with IL-7 in stimulating B-cell precursor cells (McNiece et al., 1991, Billips et al., 1992; Funk et al., 1993). However, some cytokines appear to counteract the IL-7-mediated effects. For instance, IL-1 (Suda et al., 1989), IFN7 (Garvy and Riley, 1994) and TGF~ (Lee et al., 1989) are able to inhibit IL-7-mediated B-cell precursor growth. Additionally, a number of genes involved in B-cell development may be up-regulated by IL-7, including n-rnyc, c - m y c (Morrow et al., 1992), CD19 (Wolf et al., 1993), the precursor lymphocyte-specific regulatory light chain (PLRLC) (Oltz et al., 1992) and the aminopeptidase BP-1/6C3. Incubation with IL-7 is associated with an increase in 6C3Ag expression by pre-B cells, but not mature B cells. The BP-1/6C3 molecule is expressed by early B-lineage cells and some stromal cells and represents a type II integral membrane glycoprotein that belongs to the zinc family of metallopeptidases (Sherwood and Weissman, 1990).

THE CYTOKINES AND CHEMOKINES

318

INTERLEUKIN-7

In humans, IL-7 did not stimulate proliferation of B-ceU lineage cells expressing CD24. Human pro-B cells but not pre-B cells respond to IL-7 (Ryan et al., 1994; Dubinett et al., 1995); this is in contrast to the data for murine cells which suggests that speciesspecific differences in mode of action exist between humans and mice (Tushinski et al., 1991). This human-rodent dichotomy exists for other cytokinesperhaps most notably IL-4. In general, human HSC commitment and differentiation has not been extensively characterized as compared with that of the murine system. However, recent data suggest that certain stages of human B-cell development may not necessarily depend on the presence of IL-7. Using a human bone marrow stromal cell culture system, human HSC CD34 cells underwent commitment, differentiation and expansion into the B-cell lineage as defined by loss of CD34, increased CD19 cell surface expression and appearance of Ig receptor-expressing immature B cells. This was not significantly influenced either by exogenously added IL-7 or by addition of anti-IL-7 neutralizing antibody (Prieyl and LeBien, 1996). The implementation of the fit3 ligand in combination with IL-7 or IL-3 using human fetal bone marrow-derived CD34 CD19 + pro-B cells in a stromal cell-independent and serum-deprived culture system revealed that fit3, like IL-3, synergizes with IL-7 in promoting B-cell growth and differentiation of the majority of cells into CD43-, CD19 +, cIgM § sIgM- pre-B cells; a minority of pro-B cells matured into sIgM § B cells (Namikawa et al., 1996). After the productive rearrangement of Ig heavy chains, pre-B cells undergo a number of divisions in response to IL-7 (Smart and Venkitaraman, 2000). This stage of B-cell expansion is constrained by an inhibitory signal initiated by receptor assembly (Smart and Venkitaraman, 2000). In mice, immature B cells divide in response to IL-7 according to their developmental program. Pro-B cells, which have not yet completed IgH chain rearrangement, require stromal contact and IL-7 (Hayashi etal., 1990; Hardy etal., 1991; Ray et al., 1998). After productive IgH rearrangement, pre-B cells need only IL-7 for proliferation (Namen et al., 1988; Lee et al., 1989; Sudo et al., 1989). These B cells undergo limited proliferation until they finally develop into IL-7-unresponsive mature B cells (Suda et al., 1989; Decker et al., 1991). The loss of IL-7 responsiveness appears to be mediated through an as

yet ill-defined molecule which interacts with the tyrosine residue (tyr 410) in the cytoplasmic tail of the IL-7 receptor (Smart and Venkitaraman, 2000). The future characterization of this interaction will be essential in order to define mechanisms which are involved in gauging peripheral B-cell numbers.

IL-7 and T lymphocytes IL-7 added to murine fetal thymic organ cultures (day 13) causes a preferential expansion of immature cells exhibiting the CD4-, CD8-CD3-, CD2-, SCA-1+ phenotype. Cells expressing 75- TCR are increased and the number of a[3+ TCR are decreased. Neutralizing anti-IL-7 antibody inhibits growth of fetal thymocytes (Leclercq et al., 1992; Plum et al., 1993). In vitro culture of human fetal thyrnocytes in rIL-7 results in the proliferation of CD4 § and CD8 § thyrnocytes and partial differentiation of thyrnocytes with preferential expansion of the CD4 + CD8- population (Uckun et aL, 1991). IL-7 promotes the growth of pre-T cells from fetal liver at day 14 and promotes the expression of TCR ~, a and 13genes. Culture of fetal liver cells in IL-7 is associated with the appearance ofThy-1 and Pgp-1 Ag phenotypes as occurs in day 14 fetal thymus (Appasamy, 1992). IL-7 mRNA can be detected in the fetal thymus as early as day 12 and peaks at day 15 (Wiles et al., 1992). IL-7 stimulates the generation of CD3 § cells from human bone marrow cultures with the production of both CD4 + and CD8 + populations (Tushinski et al., 1991). These results suggest that IL-7 may be produced locally in the thymic and bone marrow microenvironments and that IL-7 plays a role in the proliferation and potentially differentiation of immature T cells (Watanabe et al., 1992). Similar studies indicated that IL-7 induces proliferation and maintenance ofT-lymphocyte numbers, but not T-cell differentiation. However, with the advent of IL-7, or IL-7Ra gene-deleted mice, several central questions concerning the role of IL-7 in lymphopoiesis could be addressed in more detail. The macroscopic examination of IL-7 -/- mice indicated apparently normal development of both fertile sexes. The lymphatic organs or tissues, including thymus and spleen, were dramatically reduced in size and the peripheral lymph nodes and immune cells within the Peyer's patches were not detectable (von Freeden-Jeffry et al., 1995). Accordingly, the reduced

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white blood count in IL-7 gene-deleted mice was caused by an absolute reduction in lymphocytes, the normal ratio as well as the absolute numbers of granulocytes and monocytes were decreased. Overall, the massive lymphocytic reduction in these animals was due to decreased B- and T-cell numbers (von Freeden-Jeffry et al., 1995; Moore et al., 1996) reflecting the inefficient thymic development of IL-7deficient mice. Only 5% of normal thymocyte numbers and only 15% of splenic cell numbers could be detected in IL-7 gene-deleted mice (von FreedenJeffry et al., 1995; Moore et al., 1996). However, the immune responses of the remaining cells appeared to be similar to those observed in normal mice with regard to their function as defined by testing B cells in response to LPS, splenic T cells to Con A, or proliferation of thymocytes to a mixture of Con A + IL-2 (von Freeden-Jeffry et al., 1995). Similar T-cell abnormalities to those observed in IL-7-gene deleted mice have been identified in IL-2R7 receptor chain knockout mice (Takeshita et al., 1992; Noguchi et al., 1993; DiSanto et al., 1995). As discussed above, the common 7c chain is shared by several other cytokines, including IL-2, IL-4, IL-9 and IL-15 (Takeshita et al., 1992; Kondo et al., 1993; Girl et al., 1994). Since IL-2 or IL-4 gene-deleted mice do not exhibit defects in T-cell development, IL-7, but not other cytokines, appears to account for most of the lymphocyte defects observed in murine models of X-SCID associated with abnormalities of the 7c chain receptor (Takeshita et al., 1992; Kondo et al., 1993; Noguchi et al., 1993; DiSanto et al., 1995). Thymic T-cell development has been segregated into sequential stages based upon expression of distinct cell surface markers (see Figure 13.4). Thymic IL-7 is produced primarily during fetal development (Chantry et al., 1989; Conlon et al., 1989; Okazaki et al., 1989). CD4-CD8- fetal and adult immature thymocytes proliferate well in response to IL-7. In contrast, CD4*CD8 § thymocytes respond rather poorly. The capability to respond to IL-7 correlates with expression of the IL-7 receptor cz chain (IL-7Rcz) expressed by CD4-CD8-, CD4§ - and CD4-CD8 § but not by CD4+CD8 § thymocytes (Chantry et al., 1989; Conlon et al., 1989; Okazaki et al., 1989; Everson et al., 1990; Suda and Zlotnik, 1991). Earlier studies indicated that IL-7 mediates effects on TCR rearrangement. T-cell precursors from thymus or fetal

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liver cultured in IL-7 express rearranged [3 or 7-chain transcripts (Appasamy, 1992; Appasamy et al., 1993; Muegge et al., 1993). IL-7, sustaining expression of the RAG genes (Muegge et al., 1993) induces rearrangement of V72 and V74, but not V73 or V75, TCR chains in mice (Appasamy et al., 1993). Further evaluation of IL-7 gene-deleted mice showed reduced numbers of total T lymphocytes with preservation of the normal CD4/CD8 ratio and increased percentage of a[3§ T cells compared with 76 § T cells (von Freeden-Jeffry et al., 1995; Moore et al., 1996). However, more recent data indicate that IL-7 may also be involved in T-cell differentiation, since the IL-7Ra controls chromatin accessibility (see above, section IL-7 receptor) due to histone acetylation (Huang and Muegge, 2001). Again, the IL-7-mediated effects on T cells are two-fold: trophic (survival signal) and 'mechanistic' (reviewed in Candeias et al., 1997a; Hofmeister et al., 1999). Thus, cellular immune deficiencies observed in IL-7Ra -/- mice can, in part, be restored by reintroducing bcl2 as a transgene (restoring the trophic function), or alternatively, a rearranged TCR (Crompton et al., 1997). Most of the signaling molecules associated with IL-7Ra triggering in thymic development have been identified; however, recent studies showed that the Piml proto-oncogene may be critical for the IL-7 signaling pathway, since Piml has been demonstrated to reconstitute cellularity in IL-7 -/- and (7c -/mice. Of interest, Pim-1 transgenic, but Rag-deficient mice are able to expand CD4§ + thymocytes: Pim-1 appears to bypass the pre-TCR controlled checkpoint in T-cell development (Jacobs et al., 1999). Immature thymocytes have been divided into four distinct phenotypes based on differential expression of the cell surface markers CD25, CD44 and CDll7 (c-kit). CD4 ~~ (CD44 +, CD25-, CDll7 +, CD3-CD8-) and pro-T cells (CD44 +, CD25 +, CDll7+), representing the early stages of thymic differentiation, are present in IL7 -/- mice. In contrast, transition of pro-T cells to pre-T cells (CD44-CD25 +, CDll7-) and post-preT cells (CD44-, CD25-, CD 117-) could not be detected in IL-7 gene-deleted mice (Moore and Zlotnik, 1995; Moore et al., 1996;). In the absence of signals mediated by the IL-7R~, T-cell precursor cells are not able to initiate cleavage at the TCR7 locus, as the chromatin structure is not accessible for RAG-mediated cleavage (Tsuda et al., 1996; Oosterwegel et al., 1997). Interestingly, IL-7R~-mediated signals can be

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replaced by Trichostatin A, which is a specific inhibitor of histone deacetylases, which suggested a role for histone acetylation in chromatin opening associated with IL-7. However, these actions were global and not specific for the TCR7 locus: IL-7 specifically modulates chromatin accessibility by targeting the histone acetylation at the TCR7 locus (Huang et al., 2001). The failure of IL-7 -z- mice to rearrange the TCR7 has been shown to represent a failure to initiate cleavage, but as a failure to relegate broken DNA ends (Schlissel et al., 2000). The detailed examination of IL-7R~ -/- mice in the context of V(D)J recombination also resulted in novel insights pertaining to T-cell development: V(D)J recombination was thought to start at the pro-T2 stage (see Figure 13.4) after the arrest of IL-7Ra -/- thymocytes at the pro-T1 stage. However, novel studies showed (Schlissel et al., 2000) that both TCRI3 and 7 recombination takes place in normal T1 cells. Exclusively TCRI3 recombination intermediates were detected in IL-TR-/- mice underlining the role for IL-7Rcz-mediated signals in TCR7 recombination. More recent studies have scrutinized the role of IL-7 in the development of 75 + T lymphocytes. IL-7 -/showed a profound reduction of CD4-CD8- 78 + T cells to approximately 1% of normal levels (yon Freeden-Jeffry et al., 1995; Moore et al., 1996). A substantial body of evidence supports the notion that IL-7 preferentially promotes development of 78 TCR § thymocytes as compared with el3+ thymocytes, due to differential IL-7Ra expression on 78 + thymocytes compared with ~13+ TCR thymocytes. This notion is supported by the fact that 78 + T cells are absent in thymus, gut, liver and spleen in IL-7Ra -/- mice. 78 + T cells are not only strictly dependent on IL-7, but also on the source of IL-7. Development of intrathymic 78 T cells is dependent on intrathymic IL-7, while 'peripheral' IL-7 is sufficient to drive extrathymic 78 + T cell development (Laky et al., 1998). In contrast, czl3 TCR + lymphocytes, and NK cells appeared to be reduced in number, but to develop normally (He and Malek, 1996; Maki et al., 1996). However, NK1 § T cells can be detected in thymus, liver and spleen of IL-7Ra -/- mice. Recent data have suggested that differentiation of these NK1 § cells is dependent on signaling via the 7c chain and expansion on IL-7Rcz-mediated signals (Boesteanu et al., 1997). These results provide reasonable evidence that

signal transduction mediated by the IL-7 receptor is a prerequisite for 78 T-cell development in both thymic and extrathymic pathways. Expression of the RAG-1 and RAG-2 genes is also significantly reduced in the thymus of IL-7Ra -/- mice, but restored in doublepositive thymocytes observed in TCR transgenic IL7Rcz-/- mice (Crompton et al., 1997). Thus, signaling through the IL-7Rcz appears be necessary for RAG expression and initiation of VD] rearrangement, as described forVD] recombinatorial events in B-cell differentiation (see above). VDJ rearrangement may impact on organ-specific immunity. For instance, pulmonary cells with the canonical fetal-type V76 chain are missing in nude mice owing to a preferred thymic pathway of TCR gene rearrangement, but not to thymic selection. These cells can be restored in vitro and in vivo by administration of IL-7 (Hayes et al., 1996). In murine fetal development, T-cell production can be detected at day 15 of gestation. T cells at this stage express the invariant TCR complex composed of V73 and VS1 chains. Maturation of thymocytes is accompanied with differential expression of the CD24 (heatstable antigen) expression. First, immature V73 cells exhibit a TCR V73~~ and CD24 § phenotype and progress to m a t u r e V~3 high and CD24- cells. These 75 § T cells may populate the epidermis, or potentially other epithelial sites, and represent the dendritic epidermal T cells (DTEC). Alternatively, 75 § T cells may also mature extrathymically. Interestingly, IL-7 -/mice characteristically exhibit a block of maturation of V73 ~~ CD24 + T cells to V73 high CD24 ~~ T cells (Moore et al. 1996). This observation provides another piece of evidence that IL-7 does not serve exclusively as a 'maintenance' factor for thymocytes, but may also be involved in T-cell maturation and differentiation. In recent years, characterization of T lymphocytes residing primarily in the intestine (intestinal intraepithelial lymphocytes; iIEL) has revealed a distinct phenotype as well as a different functional activity of such immune cells compared with 'conventional' ~13T cells in the periphery (Van Kerckhove et al., 1992; Boismenu and Havran, 1994; Guy-Grand et al., 1994; Havran and Boismenu, 1994; Rocha et al., 1994). Of note, thyrnic and intestinal epithelial cells share the same embryologic origin as they are both derived from entoderm and may both be capable of secreting IL-7 in situ (Namen et al., 1988; Heufler et al., 1993; Matsue et al., 1993a, 1993b; Ariizumi et al., 1995;

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Watanabe et al., 1995; Maeurer et al., 1997). Thus, given that fact that IL-7 -/- mice (Moore and Zlotnik, 1995; von Freeden-Jeffry etal., 1995; Moore etal., 1996), 7c chain knockout mice (Takeshita et al., 1992; Kondo et al., 1993; Noguchi et al., 1993; DiSanto et al., 1995) as well as JAK3-deficient mice (Nosaka et al., 1995; Park etal., 1995) lack 76+ T cells, IL-7 appears to represent the major growth/differentiation factor required for thymic and extrathymic development of 76 T cells. Of note, ~13+ TCR ilEL are detectable in IL-7 -/- mice, but not in 7c or in JAK3-deficient mice, suggesting that other cytokines may be critical for generation of ~13 TCR + ilEL, but not necessarily for 76 + TCR ilEL (Takeshita et al., 1992; Noguchi et al., 1993; DiSanto et al., 1995; Moore and Zlotnik; 1995; Park et al., 1995; von Freeden-Jeffry et al., 1995; Moore et al., 1996). To date, there is strong experimental evidence that TCR + ilEL may develop in situ. Such immune cells are present in both congenitally athymic nude mice, as well as in athymic radiation chimeras (for review see Poussier and Julius, 1994; Klein, 1996). Much more controversy surrounds the origin of the various subsets of iIEL which show a limited TCR repertoire (Van Kerckhove et al., 1992; Guy-Grand et al., 1994; Poussier and Julius, 1994). IL-7 gene-deleted mice may help to define the impact of IL-7 in the generation and TCR composition of a[3+ TCR lymphocytes at different anatomic sites, preferentially in the intestine. Recently, clusters of lymphocytes located in crypt lamina propria (designated cryptopatches) have been characterized within the murine small and large intestinal mucosa. Such lymphoid cells are characterized by cell surface expression of CDll7 + (c-kit), IL-7Ra +, Thyl + and the absence of markers for CD3, ~13TCR, 78 TCR, slgM and B220. It has been proposed that immune cell population, first detected on days 14-17 after birth, may represent the lymphohematopoietic progenitors for T and B cells in the intestine. The prominent role of IL-7 in lymphopoietic development is further underscored by the observation that such cryptopatch-associated lymphoid cells are virtually absent in IL-7Ra-deficient mice. Earlier studies showed that intestinal epithelial cells express stem cell factor (Puddington et al., 1994; Laky et al., 1997) and IL-7 (Fujihashi et al., 1996; Murray et al., 1998; Watanabe et al., 1998), thereby creating an environment that facilitates immune cell development. In order to define the role of I1-7 expressed by intestinal

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epithelial cells, IL-7 expression was restored in situ using the tissue-specific intestinal fatty acid binding protein (iFABP) promotor (Laky et al., 2000). T-cell development in bone marrow as well as in the thymus was similar to that in IL-7 -/- mice, but 11-7 elaborated by enterocytes was sufficient for extrathymic 78 + T-cell devlopment and crucial for development of mucosal lymphoid tissue (Laky et al., 2000). IL-7Ra + CD3- cells have been postulated to act as direct induces of Peyer's patches (Yoshida et al., 1999).

Tissue-specific i m m u n e responses and IL-7 The fact that IL-7 has been found to be synthesized in such a large variety of tissues is confirmation of the cytokine's role in promoting local immune responses. IL-7 is produced by human and murine keratinocytes (Heufler et al., 1993; Matsue et al., 1993a, 1993b; Ariizumi et al., 1995) and is a major growth factor for dendritic epidermal T cells (DTEC) which express the 76 TCR (Matsue et al., 1993a, 1993b). The mouse epidermis harbors a T-cell population characterized by expression of CD3, asialo-GM1, CD2, Thy-1, Ly48, and E-cadherin, but not CD4 or CD8 phenotypic markers (Steiner et al., 1988). These DETC express the 78 TCR composed of the V73 and V61 chains without junctional diversity (Matsue et al., 1993a, 1993b; Moore et al., 1996; Steiner et al., 1988) and may play a role in monitoring stressed keratinocytes, or recognize class Ib antigens, such as TIa, or Qa (for review see Haday, 1995). Keratinocytes constitutively express IL-7 mRNA and secrete in vitro biologically meaningful amounts of IL-7 protein. In addition to being the principle growth factor for DETC, IL-7 also prevents apoptosis in DETC exposed to ultraviolet B radiation, or corticosteroid treatment (Takashima et al., 1995). This agrees with earlier observations that IL-7 is better than IL-2 at maintaining viability and responsiveness in antigen-specific T-cell lines. Interestingly it is IFNT, secreted by 73 T cells, that modulates the growth of murine keratinocytes (Takashima and Bergstresser, 1996). IL-7 augments LFA-1 and VIA-4 expression in human PMA and Ionomycin-ca stimulated PBL, thus enhancing the capacity of these cells to adhere to parenchymal cell monolayers (Fratazzi and Carini, 1996). IL-7 also induces cell surface expression of the costimulatory molecule B7 as well as ICAM-1 (CD54)

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on pre-B cells, this being biologically relevant ifB cells act as antigen-presenting cells (Dennig and O'Reilly, 1994). The involvement of IL-7 in 75 T-cell homing to the epidermis is fundamental to the evolution of contact sensitivity to trinitrochlorbenzene, which can be abrogated by administering mAbs to 75 T cells in viuo. 75 T cells invading the site of antigen challenge typically exhibit a CD8~ +, CD813-, V73 + phenotype, and proliferate in response to IL-7, but not to IL-2 or IL-4. Furthermore, in vivo application of IL-7 neutralizing antibodies inhibits accumulation ofV73 + T cells in the skin, as well as in the regional lymph nodes adjacent to the site of application (Dieli et al., 1997). Other cell types such as fibroblasts within the epidermis may also provide biologically meaningful quantities of IL-7 in vivo (Mba et al., 1994). Some of the strongest evidence supporting IL-7's role as the major growth factor for intra-epithelial lymphocytes is provided by Williams and Kupper (Williams et al., 1997), who showed that epidermal densities of DETC increase substantially in keratin 14 promotor-driven IL-7 transgenic mice, in which ectopic IL-7 is produced exclusively by keratinocytes (personal communication, Takashima and Bergstresser, 1996). Overexpression of IL-7 in transgenic mouse keratinocytes results in a lymphoproliferative skin disease in which mice develop dermal and epidermal T cell infiltrates associated with alopecia (Williams etal., 1997). Elevated IL-7 levels have also been observed in sera from patients with psoriasis, although no correlation with disease intensity was observed (Szepietowski et al., 2000). The role of IL-7 in skin immune reactions is supported by the observation that IL-7 mRNA is upregulated in mite allergen patch test reactions in patients with atopic dermatitis (Yamada et al., 1996). The site of positive patch reaction is also a site of eosinophilic infiltration, and IL-7 also up-regulates the low-affinity receptor for IgE (CD23) in activated PBL (Fratazzi and Carini, 1996). Studies have also indicated that IL-7 is involved in bullous pemphigoid, and IL-7 levels both in the blister fluid and in serum appear to correlate with disease intensity (D'Auria etal., 1999). In addition to creating an interactive environment between keratinocytes and 76 T cells, IL-7 may also be involved in the germinal center reaction (Kroncke et al., 1996a). Both IL-7 mRNA and protein have been detected in human follicular dendritic cells (FDCs) obtained from tonsils. However, mature peripheral

IgM § B cells appear to be unresponsive to IL-7, whereas antistimulated tonsilar B cells proliferate in response to IL-7 without secreting immunoglobulins, indicating that IL-7 may well regulate B-cell responses in the periphery. In addition to skin and tonsils, IL-7 mRNA and protein have also been detected in human intestinal cells (Reinecker and Podolsky, 1995; Watanabe et al., 1995) and overexpression of IL-7 is believed to play a role in chronic colitis. IL-7 transgenic mice developed a colitis histopathologically similar to human ulcerative colitis. This, together with the observation that IL-7 eliminates activated lymphocytes in the inflamed mucosa of ulcerative colitis in humans while stimulating the proliferation of inactivated mucosal lymphocytes, indicates that disregulation of IL-7 expression in epithelial cells results in chronic inflammation of the colonic mucosa (Watanabe et al., 1999). Additionally, IL-7 mRNA and protein have also been detected in human colorectal tumor cells (Maeurer et al., 1997), in normal kidney (Goodwin et al., 1989) and in human renal cell cancer cell lines (Trinder et al., 1999). These cells produce significant amounts of IL-7 protein in vitro. Of interest, elevated IL-7 mRNA expression appears to represent one of the most sensitive markers of graft rejection in patients after kidney transplantion (Strehlau et al., 1997). In a recent study it was shown that administration of IL-7 following allogeneic bone marrow transplantation enhanced lymphoid reconstitution but failed to aggravate graft-versus-host-disease while maintaining graft-versus-leukemia activity. This appears to be related to the only minimal expression of IL-7R in activated and memory alloreactive donorderived T cells from recipients of allogeneic bone marrow transplants (Mpdogan et al., 2001). IL-7 promotes the growth of lamina propria lymphocytes but inhibits their CD3-dependent proliferation (Watanabe et al., 1995). Like IL-2, IL-7 promotes the preferential expansion of (short-term, day 14) cultured tumor-infiltrating lymphocytes obtained from patients with colorectal cancer (Maeurer et al., 1997). Long-term in uitro culture of human iIEL harvested from patients with colorectal cancer with IL-7 results in preferential outgrowth of VS1 + T cells (Maeurer et al., 1995, 1996) which recognize colorectal cancer cells, renal cell cancer and pancreatic cancer cell lines (Maeurer et al., 1996). Such immune effector cells release significant amounts of IFNT. Human intestinal

THE CYTOKINES AND CHEMOKINES

THE IL-7 RECEPTOR cells also express IL-7Ra, and stimulation of such cells with IL-7 leads to rapid tyrosine phosphorylation of proteins (Reinecker and Podolsky, 1995). The physiologic role of these IL-7-responsive epithelial cell lines is unclear, although IL-7 may represent a m e m b e r of a family of epithelial growth factors that promote homing, maturation and maintenance of IL-7-responsive immune cells. Thus, IL-7 may be actively involved in creating an interactive environment of epithelial cells and lymphocytes. Murine 76 T cells secrete keratinocyte growth factor (KGF), which promotes proliferation of epithelial cells (Boismenu and Havran, 1994). Conversely, h u m a n epidermal growth factor (EGF) (Reinecker and Podolsky, 1995) increases IL-7Ra mRNA expression in h u m a n colorectal cancer cell lines. Human keratinocyte growth factor stimulates IL-7 mRNA expression and IL-7 protein secretion by h u m a n intestinal cells (our unpublished observation). Therefore, EGF and/or KGF and IL-7 may represent cytokines involved in the homeostasis of epithelial and immune cells in vivo. The conditioning role of intestinal cells is further supported by the observation that bone marrow cells develop into phenotypically mature T cells when co-cultured with the intestine epithelial cell line MODE-K (Vidal et al., 1993; Maric et al., 1996). To what extent IL-7 is involved in mediating these effects is not known. The effects of IL-7 on IL-7Ra § immune cells are well understood. Less is known about IL-7-mediated effects on non-hematopoietic cells. Recent data underlined the 'trophic' effects of IL-7 on gastrointestinal cells. Intestinal stem cells show increased radiosensitivity within intestinal crypts in IL-7Rt~-/mice and IL-7 is able to confer protection of radiation-induced apoptosis in intestinal stem cells (Welniak et al., 2001). Several recent studies have addressed the roles of IL-7 and IL-7Ra mRNA expression in developing tissues. The observation that IL-7 stimulates maturation of embryonic hippocampal progenitor cells in culture indicates a role for IL-7 in the proliferation and differentiation of immature cells of non-hematopoietic origin (Mehler et al., 1993). IL-7 has also been observed to both enhance survival of hippocampal neurons in culture and increase numbers of astroglia and microglia (Araujo and Cotman, 1993). IL7 and IL-7R mRNA expression can also be observed in the developing brain, and treatment of culture of embryonic

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brain with exogenous IL-7 leads to increased neuronal survival and greater numbers of cells exhibiting neurite outgrowth. Changes in gap junction properties have been observed in hippocampal multipotent progenitor cells undergoing differentiation under the influence of IL-7 (Rozental et al., 1998). Such effects on neuronal differentiation may be the result of the restriction and differential modulation of glial and neuronal signaling compartments. IL-7 may also be involved in local immune reactions affecting the eye. The neuroectodermis derived retinal pigment epithelium (RPE) contributes to the blood-retina barrier that regulates infiltration of immune cells in retinal diseases. Activation of RPE cells leads to the expression of MHC class II antigens as well as adhesion molecules. IL-7 also induces monocyte chemotactic protein-1 and IL-8 in RPE cells (Elner et al., 1996). Further studies are needed, however, to address whether IL-7 can be detected in retina-associated diseases in vivo.

IL-7 and cancer IL-7 plays different roles in cancer-bearing hosts, depending both on the tumor and status of the immune system. IL-7 mRNA, IL-7 protein and IL-7R~ have all been identified in some hematologic malignancies, indicating that IL-7 functions as a growth factor in an autocrine fashion. Some tumor cells exhibit expression of IL-7R~ without IL-7 expression and these may be responsive to IL-7 provided by different cell types. IL-7 may be used as a treatment for cancer since IL-7 increases immune effector cell functions byT lymphocytes, NK cells and macrophages. IL-7 may be applied systemically, or it may be secreted by genetically engineered tumor cells in order to induce a strong and long-lived immune response. IL-7 may be one of several growth factors suitable for use in recovery from bone marrow transplantation both in the setting of treatment of hematological malignancy and of bone marrow rescue following high-dose chemotherapy treatment of solid tumors (e.g. breast cancer).

Expression of IL-7 and IL-7Ra in cancer Several solid tumors, including colorectal and renal cell cancers, have been found to be positive for IL-7

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mRNA and also to express IL-7 protein (Watanabe et al., 1995; Maeurer et al., 1997; Trinder et al., 1999). Cells from both tumor types express the IL-7Ra receptor as well as the common ~/c chain. Immunohistochemical staining of IL-7 has been observed in head and neck squamous cell cancer (Paleri et al., 2001) (see Plate 13.5) and IL-7 mRNA has been detected in tumor cells of nodular sclerosing and mixed cellularity type of Hodgkin's disease (Bargou et al., 1993; Foss et al., 1995). The prominent immune cell infiltrate observed in most cases of Hodgkin's disease may be attributed to local delivery of IL-7 in vitro. Increased IL-7 serum levels have been detected in patients with Hodgkin's disease (Trumper et al., 1994; Gorschluter et al., 1995). Similarly, S6zary's lymphoma cells express IL-7Ra and proliferate in response to IL-7. However, some of these lymphoma cells obtained from different patients (3/5) were also found to express IL-7 mRNA (Foss et al., 1994). It is believed that keratinocyte-secreted IL-7 serves as growth factor for cutaneous T-cell lymphomas. This hypothesis is substantiated by examination of IL-7 transgenic mice in which the IL-7 gene is expressed under the control of the mouse MHC class II (Ea) promotor (Mertsching et al., 1995). These mice develop a lymphoproliferative syndrome characterized by early polyclonal expansion of T lymphocytes followed by development of pro-pre-B and bipotential myeloid/ B-cell tumors, which can be observed in about 25% of C57B1/6 and in up to 100% of Balb/c mice (Mertsching et al., 1995, 1996). If the IL-7 gene is controlled by of the Sra promotor, which is constitutively expressed in many tissues, development of cutaneous (?~+ TCR) lymphomas is observed (Uehira et al., 1993). A number of leukemia and lymphoma cells isolated from patients have been screened for their growth responses and/or dependence on IL-7: many but not all proliferate when exposed to IL-7 and the cell types include B- and T-cell malignancies (Eder et al., 1990; Touw et al., 1990; Makrynikola et al., 1991; Shand and Betlach, 1991; Skjonsberg et al., 1991; Lu et al., 1992; Yoshioka et al., 1992). Evidence of lymphoid maturation of the tumor cells in response to IL-7 incubation was not observed (Eder et al., 1990). In a separate study, pre-B cells transformed by a variety of oncogenes were tested for IL-7 production. None produced any IL-7 bioactivity. IL-7 overexpression achieved by removing portions of the 5' flanking

region was not associated with dramatic colony formation in agar and most clones were not tumorigenic in vivo (syngeneic mice) (Young et al., 1991). It seems therefore that production of IL-7 does not represent a final common step in the malignant transformation of lymphoid cells, but that in selected malignancies it may represent a target for therapeutic intervention. For instance, IL-7 may represent an 'antiapoptotic' factor for some hematopoietic malignancies: in murine T-cell lymphoma cells (CS-21) IL-7 induces expression of the Bcl2 protein and suppression of the CPP32-1ike protease (Lee et al., 1996). The observation that IL-7 up-regulates ICAM expression by melanoma cells, a phenotype correlated with metastatic behavior, indicates an additional role for IL-7 in malignant progression (for review see Moller et al., 1996). IL-7Ra expression in several types of cutaneous and nodal lymphomas has been studied in detail. IL-Rct is not expressed in cutaneous B-cell lymphomas, benign cutaneous lymphoid infiltrates or in reactive lymph nodes, but is expressed in over 50% of all histological types of cutaneous T-cell lymphomas (Bagot et al., 1996). IL-7 mRNA and protein are also readily detectable in chronic B-cell chronic lymphocytic leukemia (B-CLL). The coincidence of IL-7 mRNA down-regulation and apoptosis in B-CLL suggests that IL-7 gene expression may be required for B-CLL viability in vitro. Interestingly, IL-7 down-regulation and apoptosis could be prevented by co-culture of B-CLL cells with human umbilical cord endothelial hybrid cells (EA.hy926). Cell-cell contact appears essential as cell culture supernatant was unable to reconstitute the effect, suggesting that poorly defined integrins expressed on B-CLL cells may affect IL-7 gene expression and apoptosis (Long et al., 1995). Furthermore, IL-7 mRNA and protein elaborated by B-CLL cells may account for some of the clinical symptoms: some patients with CLL experience suppression of immune responses and also autoimmune symptoms (Frishman et al., 1993). Interestingly, cloning of the IL-7 gene product from B-CLL cells revealed that at least one different, alternatively spliced, IL-7 mRNA is expressed in tumor cells. The alternatively spliced form appears to be identical to an original IL-7 cDNA clone obtained by screening a (hepatocarcinoma) cDNA library for the human IL-7 gene (Goodwin et al., 1989). The alternative transcript

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THE IL-7 RECEPTOR lacks the entire exon 4 (132 bp) coding for 44 amino acid residues, as depicted in Plate 13.1 (Goodwin etal., 1989; Frishman et al., 1993). We have also observed that IL-7 mRNA expressing cells derived from renal or colorectal cancer cells contain the 'canonical' IL-7 full-length IL-7 mRNA and additionally differentially spliced IL-7 mRNA (our unpublished observations). The biology of these IL-7 mRNA species remains unclear. Thus, it appears that IL-7 exerts several differential effects on tumor cells. IL-7 may exert growthpromoting, but potentially also growth-arresting, activities. For instance, proliferation of some pre-B acute lymphoblastic leukemia (B-ALL) cells can be specifically inhibited by exogenous IL-7. This effect can be abrogated by blocking of the IL-7 receptor (Pandrau-Garcia et al., 1994). In contrast, other acute lymphoblastic leukemia cells appear to be IL-7 responsive (Greil et al., 1994). Furthermore, targeting of IL-7R-positive cells using a recombinant fusion toxin (DAB389-IL-7) has been suggested as a treatment for lymphomas (Sweeney et al., 1995). Thus, IL-7induced effects mediated by IL7Rcz may also be dependent on the actual cell type, as proliferation of early pre-B cells can be augmented by IL-7.

IL-7 in cancer therapy Immunotherapy is in the process of becoming a feasible treatment option for some cancer patients. Theoretically, the approach assumes the existence of antigenic differences between malignant and normal cells, and that these differences can be manipulated in a manner beneficial to the host. In addition, it must be assumed that the tumor-bearing host is functionally immunodeficient in that the tumor somehow blocks or inactivates the patient's own antitumor response. For instance, alterations in expression and function of signal transduction molecules associated with the TCR are responsible for inefficient immune responsiveness in T lymphocytes in several human malignancies, such as renal cell cancer, colorectal cancel ovarian cancer and melanoma. Decreased CD3 expression and inefficient CD3mediated signaling in tumor infiltrating lymphocytes (TIL) and in PBL have been observed in tumor-bearing mice (Mizoguchi et al., 1992; Salvadori et al., 1994; Levey and Srivastava, 1995), and also recently in

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cancer-bearing patients (Finke et al., 1993; Nakagomi et al., 1993; Matsuda et al., 1995; Tartour et al., 1995; Zea etal., 1995; Lai etal., 1996; Rabinowich etal., 1996). One of several mechanisms of CD3 down-regulation and inefficient signaling involves reduced expression of the ~ chain of the TCR, presumably related to hydrogen peroxide secretion elaborated by tumor-derived macrophages (Kono et al., 1996). This defect can be reversed in vitro and in vivo using exogenous IL-2 or IL-2 transfected into tumor cells and used as a vaccine (Salvadori et al., 1994; Rabinowich et aL, 1996). IL-7 is, however, also able to up-regulate the TCR (Ono et al., 1996) and can enhance protein expression of molecules associated with TCR expression and signaling functions (e.g. ZAP-70, ~ chain, p561ck and p59~, our unpublished data). Additionally, suppressive factors released by tumors may impair antitumor-directed immune responses, such as TGFI3. Macrophagederived TGF[3 mRNA can be down-regulated by IL-7 (Dubinett etal., 1993). The same effect of IL-7 has been found to be true for TGF[~ down-regulation in a murine fibrosarcoma (Dubinett et al., 1995). In contrast, TGF[3 is able to reduce stromal IL-7 mRNA expression and protein secretion using a human in vitro lymphoid progenitor cell culture system (Tang et al., 1997). Biologic therapy approaches including immunotherapy seek to reverse this apparent state ofanergy and to augment antitumor-directed immune responses. Clinical trials utilizing IL-2 as a cytokine-based immunotherapy have demonstrated that this approach is successful in treating some patients. The challenge for both clinicians and researchers is to increase the efficacy and decrease the non-specific effects of the therapy. IL-7 appears to have a number of'IL-2-1ike' properties and preclinical testing is supportive of the use of IL-7 in clinical trials. The IL-7-mediated effects can be segregated into those due to non-specific, MHC non-restricted, lysis of tumor cell targets (e.g. due to lymphoid-activated killer (I_AK) cells), MHC class Ior class II-specific recognition of tumor cells by alp T lymphocytes and tumor-restricted and presumably classical MHC non-restricted recognition by 75 § T-cell effectors. The LAK phenomenon was first reported in 1980 by Yron and associates (Yron et al., 1980) and describes the in vitro lysis of labeled flesh tumor targets by lymphoid cells that have been preincubated in IL-2 or other lymphokines. The effect is not MHC restricted

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and is relatively non-specific, in that a variety of different fresh tumors are lysed yet most normal cells are spared. IL-7 is able to generate LAK activity from thymocytes and peripheral blood mononuclear cells. Compared with IL-2, IL-7 is a relatively weak LAK inducer. IL-2 stimulates five-fold more LAK precursors than IL-7 (Alderson et al., 1990). Thymocytes from cultures grown in IL-2 are highly cytolytic whereas those grown in IL-7 exhibit minimal cytolytic activity, however, cultures grown in IL-7 and then switched to IL-2 become cytolytic. The addition of IL-4 does not induce cytolytic activity of the cells grown in IL-7 but rather down-regulates IL-2-induced proliferation and cytoytic activity (Widmer et al., 1990). IL-7 can generate h u m a n LAK activity in the absence of IL-2 and induces or up-regulates expression of CD25, CD54 and CD69. LAK generation is negatively influenced by TGF[3 and IL-4. Anti-IL-4 antibody and anti-IL-4 antisense enhances IL-7induced LAK activity (Stotter et al., 1991). IL-7 promotes secretion of TNF but not of IFN~/. The nature of the LAK cell precursor for IL-7induced LAK is not totally clear. One study showed that LAK cell activity (comparable to that obtained using IL-2) could be generated from a population of NK cells (CD56 +) whereas no LAK activity was generated in PBMCs (Naume and Espevik, 1991). Another study using murine cells compared IL-7-induced LAK with IL-2 LAK. IL-7 LAK peaked on days 6-8. IL-7 was more effective at maintaining cytotoxic activity over longer periods of time than IL-2. IL-7 LAK were induced from secondary lymphoid tissue (spleen and nodes) but not from primary lymphoid tissue (thymus and bone marrow). I2KK activity was abrogated by anti-CD8 or anti-Thy-1 +C and unaffected by antiCD4, anti-asialo GM1 or anti-NKl.l+C suggesting that IL-7 LAK activity is probably mediated not by NK cells, but by T lymphocytes (Lynch and Miller, 1990). When compared with IL-2 and IL-12, IL-7 stimulates CD56 § NK cells to secrete significantly lower amounts of soluble TNF receptor as well as lower levels of GM-CSF, but significantly higher GM-CSF levels (Naume et al., 1993). Assuming that IL-7-induced LAK activity resides within the T-cell population, it might then be possible to create a LAK immunotherapy treatment regimen that lacks some of the deleterious effects of IL-2 treatment which have been blamed on the NK cell popula-

tion. Circulating h u m a n T cells also proliferate when incubated in IL-7. Both CD4 + and CD8 § subsets respond to a similar degree; although, when T cells are separated on the basis of reactivity with an antibody (anti-CD45) that reacts with 220 kDa isoform (CD45RA) of the c o m m o n leukocyte antigen, m e m o r y T cells (CD45RO) appear to respond more readily than naive T cells (CD45RA) (Welch et al., 1989). In addition, a variety of effects of IL-7 on monocytes has been reported. Activation of monocytes with IL-7 can result in the development of a tumor lytic phenotype using melanoma cells as targets (Alderson et al., 1991). Induction of mRNA for both IL-8 and macrophage inflammatory protein-1 13 gene is induced in monocytes by IL-7 (Ziegler et al., 1991; Standiford et al., 1992), and monocytes incubated with IL-7 secrete large quantities of IL-6 as well as IL-1R, IL-1[3 and TNFR. This response can be abrogated by addition of IL-4. T cell-based immunotherapy has the advantage of increased specificity compared with LAK cell therapy. T-cell tumor lysis is MHC restricted and highly specific. Various cytokines including IL-2 and IL-4 are active in promoting the clonal expansion in vitro of T cells while maintaining their tumor lytic activity. IL-7 appears to have similar properties. IL-7 alone generates modest CTL activity which is augmented by IL-2, IL-6 or IL-4 (Bertagnolli and Herrmann, 1990; Hickman et al., 1990). Removal of CD8 § cells results in decreased killing whereas removal of CD4 § cells enhances the CTL response. IL-7 has been found to enhance cell proliferation and duration of growth more than IL-2. Addition of antiIL-4, anti-IL-2 or anti-IL-6 decreases the proliferation of CTL in culture (Bertagnolli and Herrmann, 1990; Jicha et al., 1992). CTLs harvested from draining nodes of tumor-bearing animals and incubated in IL-7 were four-fold more effective than CTL grown in media alone in adoptive transfer experiments (Lynch et al., 1991). CTLs incubated in IL-7 and adoptively transferred to mice bearing 3-day pulmonary metastases (MCA tumor) were effective in mediating tumor regression (Jicha et al., 1991). IL-7 stimulates proliferation of h u m a n TILs derived from renal cell carcinoma, but only if the TILs are first incubated in either IL-2 alone or in IL-2 + IL-7. IL-7 stimulated proliferation of CD4 + or CD8 + TIL lines specific for renal cell carcinoma. IL-7 synergized with

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anti-CD3 in the induction of IFN7 from short-term TIL cultures (Sica et al., 1993). Human T cells harvested from peripheral blood and incubated in IL-7 when restimulated with phorbol ester and ionomycin secrete IL-2, IL-4, IL-6 and IFNT. This effect was not seen as readily in cultures initiated with either IL-2 or IL-4. Both CD4 § and CD8 § subsets responded by cytokine secretion. Almost all the potential to secrete IL-4 and IL-6 in response to IL-7 pre-incubation resides within the memory subset as opposed to the naive population (Armitage et al., 1992a). The aforementioned observations suggest that IL-7, either alone or in conjunction with IL-2, acts to stimulate proliferation and tumor lytic activity in sensitized T cells and therefore may be clinically useful in the immunotherapy of malignancy. Some of the most promising data come from a study demonstrating that antitumor-specific T lymphocytes can be grown and expanded in vitro without restimulation for extended periods (up to 22 months) compared with T lyrnphocytes grown in IL-2 (Lynch and Miller, 1994). IL-7 alone (Maeurer et al., 1997), admixture of IL-7 to IL-2 and INF7 also appears to preferentially expand and maintain tumor-specific and MHC class IIrestricted CD4 + T lymphocytes (Cohen et al., 1993) from tumor-bearing patients. Of note, some of these tumor-reactive and MHC class II-restricted T lymphocytes preferentially secrete IFN7 in response to autologous tumor cells (Maeurer et al., 1997). This observation substantiates an earlier report demonstrating IL-7-mediated effects in adoptive immunotherapy in human colorectal cancer xenografts in SCID mice. Exclusively the combination of IL-7 treatment and passive transfer of human autologous T cells resulted in enhanced survival of mice engrafted with the respective tumors; treatment with IL-7 alone showed no effect. The antitumor effects correlate with IFN7 secretion by the passively transferred T cells and not by their cytolytic capacity (Murphy et al., 1993). The ability of IL-7 to generate antitumor-directed immune reactivity may also depend on the tumor type and the availability of T cells capable of recognizing tumor-associated peptides presented either in the context of MHC class I, or MHC class II molecules. For instance, application of IL-7 resulted in an up to 75% reduction in pulmonary metastases of the murine renal cell cancer line Renca (Komschlies et al., 1994). However, the pharmacoki-

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netics of IL-7 administered to humans have not yet been evaluated in detail. Some toxic side-effects have been observed in mice treated with IL-7 systemically (Komschlies et al., 1994). IL-7 may also be used to reconstitute the immune sytem in primary or secondary immunodeficiencies (e.g. induced by viral infections, by inherited abnormalities, such as Di George syndrome) or after BMT. For instance, the successful outcome of autologous BMT is limited by susceptibility to infections. Since the effective reconstitution of the immune system requires more than just the quantitative replacement of immune cells (usually achieved within 3-4 months after transplantation), the quality of the immune system is often impaired. Since IL-7 has not only growthpromoting, but also differentiation-promoting effects on both B- and T-cell lymphopoiesis, it may represent an attractive cytokine, potentially in combination with flt2/flt3, to reconstitute a competent immune system. Several studies have addressed this issue. For instance, IL-7 treatment of Balb/c mice after syngeneic BMT leads to increased thymic cellularity, increased RAG-1 expression, and to promotion of V[~8(D)J gene rearrangement of TCRs. The increased 'quality' of IL-7-treated mice is reflected in better mitogenic responses of thymic cells and in enhanced cytokine production provoked by influenza virus challenge (Abdul-Hai et al., 1996). Additionally, IL-7 accelerates PBL recovery of mice after cyclophosphamide, 5-fluorouracil treatment (Damia et al., 1992) or radiation (Faltynek et al., 1992). Using a metastatic breast cancer model in mice, IL-7 and BMT could significantly prolong survival, presumably due to enhanced immune cell reconstitution after splitdose chemotherapy using cyclophosphamide, cisplatin and nitrosourea (Talmadge et al., 1993). In another study IL-7 was shown to mobilize longterm reconstituting peripheral CD34 § stem cells (Grzegorzewski et al., 1994). Such cells may be useful for stem cell transplantation, or for therapies using CD34 § cells either for gene transduction or for maturation in vitro in order to generate potent antigen-presenting cells capable of initiating potent antitumor-directed cellular immune responses. Additionally, development of tumors in older individuals may reflect not only accumulative genetic alternations, but also a decreased capacity of the humoral and cellular immune system to identify and eradicate

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transformed cells efficiently. Several studies have demonstrated age-related alteration in T and B lymphocytes. In murine models, the ability of pro-B-cells to proliferate in response to stroma cells decreases with age (Stephan et al., 1996). This functional alteration is due to an impaired response of pro-B cells to IL-7, but not to other stromal-associated cytokines including stem cell factor or insulin-like growth (Stephan et al., 1997). The reduced IL-7 responsiveness appears not to be induced by inefficient IL-7R expression, but rather by as yet poorly defined intracellular signalling events mediated through the IL-7 receptor complex (Stephan et al., 1997). Thus, IL-7 may not only be implemented for primary or secondary immunodeficiency disorders: the functional impairment of the immune system in older individuals may in part be mediated by reduced IL-7responsive immune cells. Future studies may devise therapeutic strategies to overcome age-related immunodeficiencies which may play a role in decreased immune-surveillance. The effects of locally secreted IL-7 elaborated by genetically engineered tumor cells may overlap with some of the effects observed by systemic application. Transfection of cytokine genes into tumor cell lines has been developed as a theoretical strategy to increase the local regional response to the tumor in the hope that a heightened in situ response might translate to an enhanced systemic response, not only to the transfected tumor but also to the nontransfected or wild-type tumor. IL-7 transfection experiments have yielded some provocative results. Transfection of IL-7 into the murine tumor line (J5581) leads to tumor rejection in uiuo. CD8 § cells have been shown to be required for longterm tumor eradication although short-term regression has been noted in the absence of CD8 § cells. While tumor transfected with IL-2, IL-4, TNF or IFN7 regresses when placed in nude or SCID mice, IL-7transfected tumor requires the presence of CD4 § cells for regression and no regression was observed in nude mice bearing tumor transfected with IL-7. In most of the murine studies, tumors were eventually rejected by the animals, while the in vitro growth was not affected by IL-7 (Aoki et al., 1992; Hock et al., 1991, 1993; McBride et al., 1992; Miller et al., 1993; Allione et al., 1994; Tepper and Mule, 1994). It appears that CD8 § T cells play a major role in mediating tumor rejection

(Hock etal., 1991, 1993; Aoki etal., 1992; McBride etal., 1992; Miller et al., 1993) and that antigen-specific T cells are elicited upon immunization with IL-7secreting tumor cell lines (Aoki et al., 1992). However, other immune cells may also contribute to antitumor responses, since not only T lymphocytes, but also macrophages, eosinophils and basophils are present at the site of tumor rejection (Hock et al., 1991; McBride et al., 1992). In another study, the effects of locally secreted IL-7 and induction of tumor-specific cellular immune responses were examined (Cayeux et al., 1995). In B7-transfected m a m m a r y adenocarcinoma cells TS/A, T cells showed predominantly CD28 § and CD25- marker expression and in IL-7 transduced tumor cells CD28 § and CD25 § marker expression, while in B7 +/IL7 + tumor cells, the T-cell infiltrate typically showed CD28+/CD25 + expression. The doubletransfected tumor elicited enhanced immunity as compared with tumor cells expressing IL-7, or B7 alone, or non-transfected tumor cells admixed with C o r y n e b a c t e r i u m p a r v u m (Cayeux et al., 1995). Human non-small-cell lung cancer cell lines infected with a retroviral construct containing the h u m a n IL-7 cDNA lead to alterations of cell surface expression of molecules (e.g. MHC class I, LFA-3) on co-cultured PBL favoring antitumor-directed immune responses (Sharma et al., 1996). In a rat model of glioma, IL-7transfected glioma cell clones, immunized into the hind leg, were found to inhibit growth of their intracerebral parental tumors, thereby enhancing recipient survival (Visse et al., 1999). Thus, despite the unfavorable location of intracerebral tumors, therapeutic immunization with IL-7-transfected tumor cells at a site distant to the brain, appears to be a viable therapy option. IL-7-transfected tumors cells may therefore represent a reasonable vaccine for eliciting strong antitumor-directed immunity in a variety of cancer types (Moller et al., 1996). Vaccination with IL-7 genemodified autologous melanoma cells was found to enhance antimelanoma cytolytic activity in patients participating in a phase I study (Moller et al., 1998). In three of six patients, antimelanoma cytolytic precursor cell frequencies increased by between 2.6- and 28fold, and two of these patients exhibited a minor clinical response. In a further clinical study, patients received autologous tumor cells which had been simultaneously transfected with IL-7 and GM-CSF using the MIDGE system (Minimalistic, Immunologically-

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Defined Gene Expression). All ten patients had progressive disease at trial onset. Cytotoxicity of patients' PBLs increased significantly during treatment and five patients showed responses ranging from complete (1 / 10) response to stable disease (3 / 10). The remaining five patients progressed (Wittig et al., 2001).

IL-7 and the i m m u n e response to infection The role played by cytokines in regulating host immune responses to intra- and extracellular pathogens is becoming clearer. It is the THl-type response, involving secretion of IFN~,, IL-2 and IL-12, which represents the dominant reaction to obligate intracellular pathogens in mouse and man. Interestingly, a number of studies have recently suggested that IL-7 plays a central role in infections involving intracellular bacteria or parasites. IL-7 has been found to have both positive and negative effects depending on the time of IL-7 application and also on the infection model studied. IL-7 has been found to be beneficial in murine models of mycobacterial infection, or in infections with the parasite Toxoplasma gondii. Female A/] mice treated with IL-7 from the time of infection (2 mg daily for 2 weeks) with Toxoplasma gondii survived, while mice treated following infection died, as did untreated infected mice. Antibody-based depletion experiments revealed that both CD8 + T cells and asialo-GM1 + NK cells are required for protection against this intracellular parasite in vivo. It appears that these IL-7mediated effects are predominantly mediated by IFN~, secretion, since in vivo depletion of IFN~/abolished any IL-7 protective effects (Kasper et al., 1995). In another mouse model, combination of IL-7 with IL- 113 augments anti-Listeria monocytogenes-directed i m m u n e responses. There is a predominance of peritoneal ~,~ T lymphocytes which specifically react to heat-killed Listeria preparations in the presence of macrophages as accessory cells in an MHCindependent manner. The responsiveness of 7~ T cells to IL-7 was found to be enhanced in the presence of accessory cells. This effect could be replaced by exogenous IL-1 (Skeen and Ziegler, 1993). IL-7 also appears to be involved in the successful immune response to infection with mycobacteria. In Mycobacterium leprae infection the cellular immune

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response has been found to correlate both qualitatively and quantitatively with clinical manifestations. Increased IL-7 mRNA and IL-7-receptor mRNA expression correlates strongly with the tuberculoid form of the disease, in which the infection is limited. In contrast, no significant IL-7 mRNA expression is observed in the progressing lepromatous form of the disease (Sieling et al., 1995). Furthermore, IL-7 has been found to inhibit the intracellular growth of M. avium complex (MAC) in h u m a n macrophages in vitro (Tantawichien et al., 1996). MAC is a common opportunistic pathogen often found in patients with HIV infection. The major reservoir for MAC in the susceptible host appears to be the mononuclear phagocyte and consequently such infections are often resistant to standard treatment protocols. Additional treatment modalities may therefore be required in order to control MAC infections. Treatment of h u m a n macrophages with TNFcz or GM-CSE for example, leads to mycobacteriostatic or mycobactericidal activity (Denis, 1991). Furthermore, MAC infection of h u m a n macrophages leads to generation of TGFI3 which inhibits the capacity of infected cells to control bacterial growth (Bermudez, 1993). Despite this, treatment of h u m a n macrophages with IL-7 brings about a dose-dependent reduction in the number of intracellular bacteria. Addition of IL-7 to cultured macrophages prior to infection, results in diminished anti-MAC activity as compared with that obtained when IL-7 was added to cells post MAC infection (Tantawichien et al., 1996). We have obtained very similar results using virulent M. tuberculosis bacilli (Maeurer et al., 2000). IL-7 treatment of Balb/c mice preinfected with M. tuberculosis resulted in up to 100% increased survival when compared with untreated mice, or mice treated with IL-2 or IL-4. Such IL-7-mediated survival can be passively transferred to animals preinfected with mycobacteria, using spleen cells derived from IL-7treated and M. tuberculosis-infected animals. In contrast, transfer of cells from mice treated with IL-7 alone failed to increase survival as compared with control animals, indicating that priming with M. tuberculosis is required to elicit anti-microbial immune responses facilitated by IL-7 treatment (Maeurer et al., 2000). In other studies, IL-7-mediated effects were abolished by anti-human TNFa antibody. In contrast, IL-7 failed to decrease TGF[3 secretion by

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macrophages upon infection, an observation found to be true for IL-7-mediated down-regulation of IL-2 as well as LPS-induced TGF[3 mRNA expression in murine macrophages (Dubinett et al., 1993). Thus, IL-7 may exert some of its effects by inducing or potentiating proinflammatory cytokines, such as IL- 1~, IL- 113, IL-6 and TNF~ (Alderson et al., 1991). Additionally, some of the antibactericidal effects of macrophages involve nitric oxide and superoxide radicals, both of which are induced by IL-7 (Alderson et al., 1991; Gessner et al., 1993). Unfortunately, IL-7 can also negatively infuence 'clinical outcome' in animals with intracellular infections. Previous studies have shown mediation by IL-7 of antimicrobial activity against the intracellular parasite Leishmania major in murine macrophages in vitro (Gessner et al., 1993). However, treatment of susceptible Balb/c mice with IL-7 at the onset of L. major infection leads to enhanced lesion development and accelerates death of treated animals correlating with up to 40-fold increased parasite burden in spleens and lymph nodes when compared with untreated animals. Analysis of cellular immune responses of such animals showed that lymphocytes from IL-7-treated mice produced comparable amounts of the TH2 cytokines IL-4 and IL- 10, but significantly less IFN? in response to antigen (Gessner et al., 1995). In agreement with this observation, decreased IFN? production resulting from increased levels of IL-7, leads to aggravation of disease in Schistosoma mansoniinfected mice (Wolowczuk et al., 1997). These observations suggest that a number of other factors may be involved in the complex interactions of cytokines. IL-7 has, for instance, been shown to up-regulate anti-CD3, or anti-CD3/anti-CD28-induced IFN? and IL-4 mRNA expression in human T lymphocytes (Borger et al., 1996). An increase in total cell numbers in the B-cell compartment has been observed in IL-7-treated mice. In order to elucidate the nature of any potentially deleterious B-cell responses, mice with the XSCID immunodeficiency were evaluated. Typically, these mice lack B 1 cells and have reduced numbers of B2 cells which are only partially functional. B1 cells (formerly referred to as Ly-1 B or CD5 § B cells) represent a small subpopulation with a distinct phenotype, and developmental and functional properties. B1 cells express a unique array of cell surface molecules, in addition to expression of the CD5 marker; they are

preferentially generated from fetal or neonatal sources of progenitors, and the antibodies derived from B1 cells are predominantly of the IgM class, show minimal hypermutation and a high frequency of low-affinity, poly- or self-reactive specificities (for review see Stall and Wells, 1996). The absence of these cells leads to reduced susceptibility against infections with intracellular parasites (e.g. Leishmania species). Treatment of X-SCID mice with a single IL-7 dose concomitant with Leishmania infection resulted in a clinical course resembling that of susceptible Balb/c mice with up to 100-fold enhanced parasite load in treated animals. Again, examination of CD4 § Leishmaniaspecific T lymphocytes revealed that IFN? secretion is reduced in IL-7-treated X-SCID mice compared with control animals, and that the population of B2 (B220 § sIgM § MHC class II +) cells appeared to be significantly enhanced. However, the mechanisms of the disease-aggravating effects of IL-7 remain unclear. One potential mechanism may be antigen presentation by B cells, an event which may lead to preferential activation and expansion OfTHz lymphocytes. As with parasitic infection, a similar dichotomy of IL-7 has emerged in infection with the human immunodeficiency virus (HW). Previous studies indicated that exogenous rhIL-7 can augment the generation of antiviral CTL responses (Carini and Essex, 1994). Examination of HW-infected individuals testing negative for anti-HW-l-specific CTL reactivity revealed that CD8 + and CD4 § T cells lack cell surface expression of IL-7R, which may be attributed to production of insufficient numbers of IL-7R upon retroviral infection, or alternatively, to increased shedding of IL-7R (Carini and Essex, 1994; Carini et al., 1994). In the case of CD8 § T cells, IL-7R expression is partially restored in patients undergoing effective antiretroviral therapy (MacPherson et al., 2001). Because HW infection is associated with loss of cytotoxic CD8 § T-cell activity, and also with reduced numbers in the CD4 § cell compartment, several cytokines capable of modulating the immune system have been considered for treatment of HIV-positive individuals. IL-7 represents one of these candidates (e.g. IL-2, IL-12, or IL- 15) as it not only enhances anti-HW-directed CD8 § T-cell responses, but also augments both CD4 § T helper cell-dependent humoral immune responses and CD8 § cytotoxic %cell reactivity in mice immunized with the HIV envelope protein (Bui et al., 1994).

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Caution must be exercised, however, prior to incorporation of cytokines including IL-7 into clinical protocols, since addition of exogenous IL-7 induced virus replication and increased proviral DNA levels in PBMC cultures, and increased the levels of doubly spliced HIV-1 tat RNA (Smithgall et al., 1996). These effects are not inhibited by neutralizing IL-I[3, IL-2, IL-6 or TNFa activity. Although CD8 § T cells inhibited the increase in viral replication induced by IL-7 stimulation, they failed to prevent virus replication following CD3 ligation in the presence of IL-7, an event that can also be mimicked by adding IL-7 to anti-CD3 antibody-stimulated HIV § PBMC cultures, resulting in enhanced HIV production (Moran et al., 1993). The results obtained from such studies have addressed the role of exogenous added IL-7 in HIV replication in vitro, but not the role of endogenous IL-7 on viral load or viremia. Serum levels of IL-7 are known to be elevated in HIV-1-infected subjects, and patients undergoing highly active antiretroviral therapy (HAART) continue to have elevated IL-7 serum levels despite successful treatment, thus bringing into question the impact of IL-7 immunotherapy on the process of immune reconstitution (Darcissac et al., 2001). This has, however, been studied. McCune and coworkers observed that not only were increased IL-7 serum levels accompanied by HIV-1-mediated T-cell depletion, but that they were also associated with increased viral load (Napolitano et al., 2001). The authors suggest that increased IL-7 production results from the existence of a compensatory feedback loop that leads to enhanced T-cell differentiation, and forms part of a homeostatic response to T-cell depletion. Raised IL-7 levels have also been observed in severe combined immunodeficiency syndrome, acute lymphocytic leukemia and various other lymphopenic conditions (Mackall, personal communication in Bui et al., 1994; Napolitano et al., 2001). The enhanced IL-7 levels associated with HIV-1 infection appear to be the result of increased synthesis by dendritic-like cells within peripheral lymph nodes. IL-7 is not just associated with enhanced viral load, it also accelerates HIV1 infection in vivo (mouse model, in Napolitano et al., 2001). Thus, although IL-7 has been touted as a possible treatment for HIE, its use may actually be detrimental, enhancing replication and therefore viral load and leading to accelerated disease progression. There are several other clinical conditions, in which elevated

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IL-7 levels have been determined. For instance, plasma and synovial fluid levels of IL-7 were significantly elevated in patients with systemic juvenile rheumatoid arthritis, but not in individuals with poly, or pauciarticular JRA, or in patients with other rheumatic diseases (De Benedetti et al., 1995). Activated T cells have been associated with increased osteoclast formation and bone resorption linked with inflammation. T cells stimulate osteoclast formation by producing osteoclastogenic cytokines. IL-7 stimulation of T cells leads to increased production of such osteoclastogenic factors from T cells and is therefore indirectly involved with osteoclast formation leading to bone loss (Weitzmann et al., 2000). In addition, IL-7 was found to be increased in patients with untreated Hodgkin's lymphoma (Trumper et al., 1994) and in some patients with colorectal, or renal cell cancer (Trinder et al., 1999). However, the precise source, and the functional consequence of such elevated IL-7 serum levels has yet to be determined. IL-7 mRNA has been detected in a number of different infections, and exogenously added IL-7 either provided by the recombinant protein or by retroviral infections, has been shown to augment specific cellular immune responses. For instance, IL-7 mRNA has been detected in Helicobacter pylori-positive gastritis, but not in H. pylori-negative controls (Yamaoka et al., 1995). Studies with an IL-7R neutralizing antibody have indicated a key role for IL-7R in the development of H. felis-induced gastritis in mice (Ohana et al., 2001), and IL-7R has also been shown to be up-regulated in epithelial cells following their infection with enteropathogenic bacteria in culture (Yamada et al., 1997). Salmonella t y p h i m u r i u m , enteropathogenic Escherichia coli and enteroinvasive E. coli all induced IL-7R expression in the colonic epithelial cell line T84, suggesting that communication between the epithelium and mucosal lymphocytes may be involved in the modulation of infection-induced mucosal inflammation. Others have shown that IL-7 assists in induction of antiviral specific T-cell responses using synthetic peptides and IL-7 as an adjuvant (Kos and Mullbacher, 1992) and that IL-7 overcomes anergy in parasite-specific cellular immune responses (Sartono etal., 1995), and facilitates expansion oftetanus toxoid (Kim et al., 1994), or dengue-virus-specific cytotoxic CD4 § T-cell clones (Berrios et al., 1996).

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T-cell h o m e o s t a s i s and ' i m m u n e reconstitution' Recent evidence has implicated IL-7 as being a key cytokine in at least two important clinical situations (Plate 13.6). First, IL-7 serves as a central cytokine in %cell homeostasis and second, IL-7 appears to be enhanced, potentially as a feedback mechanism, in response to low lymphocyte counts (for review see Fry and Mackall, 2001). In general, lymphoid homeostasis is required to ensure immune responsiveness to a wide variety of antigens and to prevent immunodeficiency. The immune system has to maintain naive T cells as well as memory T cells which are able to react effectively to the target antigen upon reencounter. Naive and memory, as well as effector T-cell subsets are defined by different phenotypic markers, exhibit different homing characteristics (Jourdan et al., 2000), and may also show different requirements for cytokines in order to ensure survival in the absence of antigenic stimulation (Lantz et al., 2000). Moreover, (CD4 § memory T cells are comprised of at least two populations, 'central memory' T cells, which express the chemokine receptor CCR7 and CD62 ligand (L) and home to secondary lymphoid organs, and 'effector memory' T cells, which are CCR7 negative and are capable of migrating into non-lymphoid tissues (Manjunath et al., 1999; Sallusto et al., 1999; Iezzi et al., 2001; Masopust et al., 2001; Reinhardt et al., 2001). IL-7 (as well as IL-15) is able to expand effector memory T cells effectively; central memory T cells do not respond well and naive T cells fail to respond to IL-7 (Geginat et al., 2001), which suggests that IL-7 may help in sustaining and expanding antigenexperienced T cells, a situation which is desirable in patients with chronic infectious disease or suffering from cancer. More interestingly, in T cell-depleted conditions, naive T cells undergo spontaneous proliferation in response to MHC-peptide complexes in a cytokine-dependent manner, a condition termed 'homeostatic proliferation'. Exclusively IL-7, but not IL-4 or IL-15, is able to induce 'homeostatic proliferation' (Tan et al., 2001), which makes IL-7 a key candidate to facilitate T-cell expansion in T cell-depleted hosts. The nature of this expansion could formally be two-fold: first, IL-7 may preferentially expand distinct T-cell subsets, alter the T-cell repertoire and induce

T-cell activation. Second, IL-7 may not directly impact on the T-cell pool, but rather lead to 'preservation' of the immune repertoire. Indeed, the latter possibility appears to be the case: IL-7, but not IL-2, IL-4 or IL-6, expands and maintains naive (CD45RA § T cells and directly induces telomerase activity which may help in preserving the life-span of naive T lymphocytes (Soares et al., 1998; Webb et al., 1999; Geiselhart et al., 2001; Hassan and Reen, 2001; Rathmell et al., 2001; Vakkila et al., 2001; Vivien et al., 2001). Most of the data pertaining to T-cell homeostasis have been generated by analyzing the CD4 § T-cell population. A similar central role emerged for IL-7 for CD8 § T cells: IL-7 appears to be crucial for survival and maintenance of CD4 § and CD8 § T cells if the host is lymphopenic. CD8 § (but not CD4 § T-cell survival in normal hosts also depends on IL-7. In contrast, an immediate T-cell response to infection does not require IL-7, but the generation of a strong and longlasting memory T-cell response is again associated with the presence of IL-7 (Schluns et al., 2000). A physiological 'immuno-deficiency' is represented by non-efficient functional activity of the cellular immune system in later life. Thymic atrophy has been postulated to contribute to the contraction of the T-cell repertoire and to a decline in immune effectiveness. Recent studies have shown that IL-7 alone is able to reverse the age-associated effects in thymocyte development (Andrew and Aspinall, 2001), leading to improved TCR-I3 rearrangement at the stage of the triple negative thymocytes (see Figure 13.4). In addition, IL-7 impacts on the development of thymic dendritic cells (Varas et al., 1998) and may interact at different levels within the thymic architecture with thymocytes: MHC class II § thymic epithelium and fibroblasts express fibronectin and heparan sulfate, extracellular matrix components which effectively bind IL-7 (Banwell et al., 2000). Recent studies linked age-associated thymic atrophy with a decline in IL-7 production (Andrew and Aspinall, 2002) and showed that a decline of thymic IL-7 is associated with the dose of (experimentally) applied radiation (Chung et al., 2001), which substantiates the notion that IL-7 may represent an interesting candidate to reverse an age-associated decline in immune responsiveness, particularly in the case of patients suffering from cancer at older ages which presumably requires a broad and effective immune repertoire. The second key

THE CYTOKINES AND CHEMOKINES

REFERENCES function m e d i a t e d by IL-7 represents the capacity to expand and establish a functional i m m u n e system in l y m p h o p e n i c hosts. As discussed above, I1-7 s e r u m levels show an inverse relationship to the n u m b e r of lymphocytes both in b o n e marrow recipients (Bolotin et al., 1999) as well as in patients with HIV infection (Llano et al., 2001; Napolitano et al., 2001). These data s u p p o r t the notion that IL-7 m a y be i m p l e m e n t e d as a therapeutic agent to restore i m m u n e competence. Indeed, IL-7 has b e e n shown to facilitate engraftment and to restore protective i m m u n i t y in athymic T celldepleted mice (Fry et al., 2001). Of note, IL-7 application after allogeneic b o n e marrow transplantation did improve i m m u n e reconstitution without the aggravating side-effects of graft-versus-host disease but with preservation of the beneficial effects of allogeneic transplantation. The graft-versus-leukemia effect r e m a i n e d intact, an observation which has b e e n associated with low IL-7R expression on activated and m e m o r y alloreactive donor-derived T cells (Mpdogan et al., 2001). Thus, IL-7 represents a key m e d i a t o r of i m m u n e c o m p e t e n c e and m a y hold great promise in clinical applications which require T-cell homeostatic expansion and m a i n t e n a n c e of a strong and effective i m m u n e response.

SUMMARY IL-7 is an i m p o r t a n t l y m p h o p o i e t i n and plays a critical role in both B- and T-cell development. IL-7 prom o t e s expansion of T lymphocytes exhibiting antigen-specific reactivity. IL-7 m a y be i m p l e m e n t e d to p r o m o t e strong and effective i m m u n e responses against t u m o r cells, or directed against microbial or viral infections. It m a y also be useful in reconstituting an effective and functional i m m u n e system after b o n e m a r r o w transplantation or helping to design novel strategies for i m m u n e reconstitution in patients with cancer or with HIV infection.

ACKNOWLEDGMENTS We thank Monika W i e d m a n n for expert secretarial assistance and editing. Dr Ingeborg Zehbe, Dept. of Med. Microbiology, contributed the picture of IL-7 in cervical cancer and Dr Torsten Reichert, the Dept. of

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Oral and Maxillofacial Surgery, University of Mainz, provided the picture of IL-7 protein expression in head and neck cancer. This work was s u p p o r t e d by grants DFG SFB 432/A9, SFB 490/C4 and the Stiftung ffir Innovation Rheinland-Pfalz (to M.M.).

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THE CYTOKINES AND CHEMOKINES

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INTERLEUKIN-7

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THE CYTOKINES AND CHEMOKINES

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THE CYTOKINES AND CHEMOKINES

IL-7 heterodimer: PPBSF (pre-pro B-cell growth-stimulating factor: IL-7+ hepatocyte g rowth(HGF)tScatte r-factor

IL-7 molecule:

Molecules with similar functions: TSLP (thymic stromal lymphopoietin)

25aa leader ~ 8 q l 2-1 3

,

A LP

l-11-24

h ,,e,ix

17518 Da

IL-7 R~ ~ ~

?c

20-27kDa

B ~t

Met_25_Ser1 Aspl_Leu24

~

177aa

1

D " i f

128-145 It

Asp25_Asn51 Lys52_Gin95 Va196_Leu113

Glu 114_His152

oonnet,no,oo,:, I ,ntron I c , teine residue

PLATE 13.1 IL-7 composition. The h u m a n IL-7 gene codes for a 177 amino acid molecule. A differentially spliced IL-7 mRNA was initially identified by probing a cDNA library derived from a h u m a n hepatocellular carcinoma cell line, with the murine IL-7 cDNA by nucleic acid hybridization (Goodwin et al., 1989; Lupton et al., 1990). The h u m a n alternative IL-7 transcript lacks exon 4 coding for 132 bp', thereby reducing the protein by 44 amino acids (aa). Both the entire IL-7 mRNA and the alternatively spliced IL-7 mtlNA have reproducibly been identified in chronic lymphatic B-cell leukemia (Frishman et al., 1993), in follicular dendritic cells (Kroncke et al., 1996a, 1996b), and in renal cell cancer (our unpublished observation). The murine IL-7 cDNA lacks a region which codes for 19 amino acids and would correspond to exon 5 of the h u m a n IL-7 gene (Namen et al., 1988). The lack of exon 5 apparently does not impair biological IL-7 functions in conventional assay systems. H u m a n and murine IL-7 exhibit up to 81% sequence homology with regard to the nucleotide sequence and up to 60% homology in amino acid residues. The leader peptide (25 aa) is shaded and coded in exons 1 and 2. The amino acids within each of the six exons are indicated. The contribution of the IL-7 components in forming the helices A-D are noted. Note that helices A-C may interact with the IL-7t/, the D domain interacts with the yc chain. In addition, the 'IL-7-1ike' thymic stromal lymphopoietin (TSLP) is indicated at the right (box) as well as the heterodimeric molecule composed of IL-7 and the (30 kDa) 13chain of hepatocyte growth factor (pre-pro B-cell growth-stimulating factor, PPBSF).

(a)

(b) IL-7 receptor (IL-7R~z) chain 439 aa

IL-7 R~z

TSLP-receptor system

soluble IL-7 receptor 242 aa

l TSLP-R

Survival and proliferation Bcl2

TSLP-R+IL-7R~x

V(D)J

recombinabon

IgH and TCI~ locus

cysteine residues

EX

219 aa Glut.AspS,9

215

_< -

aa

GluLSeP

WSXWS

i 16

motif 25

TM

CY

27

..

aa

aa

Jak3 ..

Gl~-Ile~ Pro~0.Try~

~-

"4

"

Ja~__~ Stat I

Box1

PI3 kinase

Box 2

195aa Lys~-G

/

.

-.

Stat 3

,.

lu4-,~

PLATE 13.2 IL-7 receptor and signal transduction. (a) Receptor composition. The IL-7Ra is composed of an extracellular (EX), transmembrane (TM) and cytoplasmic part (CM). The latter can be divided into three distinct parts. The membrane proximal part contains a proline-rich sequence (homology box 1) which is common in the cytokine receptor family. The less conserved box 2 is, in concert with box 1, essential for transmission of proliferative signals. The tyrosinase residues in the C-terminal receptor region are involved in phosphorylation in different downstream signaling pathways. The composition of the IL-7 soluble isoform is also indicated. (b). Signaling events associated with IL-7 receptor triggering. The phosphorylation of the Janus tyrosine kinases JAK1 and JAK3 initiates different downstream signaling events resulting in activation of the STAT1 and STAT5 proteins, or the phosphorylation of the insulin receptor substrate 1 and 2 (IRS-1/2) proteins. The middle part of the IL-7 receptor is involved with the src family tyrosine kinase p59 fyn and the membrane distal domain mediates 'differentiation signals'. The C-terminal receptor region exhibits three tyrosine residues (Y at positions 381,429 and 436) which play a role in phosphorylation of the SH2 homology protein tyrosine phosphatases (PTP), in the ras pathway. The interaction of the phosphytidylinositol 3-kinase (PI-3) takes place with the sequence Tyr (Y)-X-X-Met at position 429. Boxed: Note that the IL-7-1ike molecule TSLP binds to the TSLP receptor which shows a high degree of similarity to the 7c component of the IL-7 receptor. TSLP either binds alone to the TLSP receptor with low affinity, or alternatively, to a heterodimeric molecule, composed of the IL-7Ra chain and the TSLP chain with high affinity.

30%

"r D

18%

r-t, ...............

13%

;CD8

"i/ O

15%~

co-~---] --TL-......

"~

,..... : ......... i-----P i 4 , ...... I

, CD45RA CD4+CD45RA+

CD4+CD45RA-

("'~ - r . . . . . . . . . . . . . . . . . . . . . &.-.. ,' 80%

........... 76-~

Di

0 [__~.............. T

/35%

:.....................

-!

bx~Z:e ....... - CD45RA

CD8+CD45RA+ ~[ i

CDS+CD45RA-

27% I

,

SSC

PLATE 13.3 IL-7 receptor (CD127) expression. PBL were harvested from a patient with HIV infection and tested for IL-7Ra expression by flow cytometry. CD4 § and CD8 § T cells were either gated on CD45RA § (naive or terminally differentiated effector T cells) or CD45RA- (activated/memory T cells) and tested for IL-7Ra expression (side scatter versus CD 127 expression). Note that naive as well as m e m o r y T cells express the IL-7R~ receptor.

Head and neck cancer

(C[34+) T-cell depletion

Lymphopenia

BMT, HIV

Serum -

I

level omeostasis and Jrvival of naive T-cells

i ~ _ ~ ~ ~

~

~.~~

~1

~, ~~~i;~'~!~l

Cervical cancer

PLATE 13.5 IL-7 protein expression in cancer. IL-7 protein expression either in head and neck cancer (top) or cervical cancer (bottom) was determined by immunohistochemistry. The specifity of the polyclonal anti-IL-7 Ab has been demonstrated earlier (Kroncke et al., 1996a). Note that single tumor cells express abundant IL-7 protein. Elevated IL-7 levels in patients with head and neck cancer appear to be associated with better prognosis (Paleri et al., 2001). In contrast, IL-7 serum levels in patients suffering from cervical cancer appear to be associated with systemic disease and reduced survival (Chopra et al., 1998). These disparate results reflects different facets of IL-7. Enhanced serum levels may be associated with tumor load if the tumor cells produce IL-7. Not mutually exclusive, e n h a n c e d IL-7 levels may represent a counter-regulation to support lymphopoiesis in patients with cancer.

Lymphocytosis PLATE 13.6 IL-7 in T-cell depleted hosts and T-cell homeostasis. IL-7 levels are enhanced in HIV § individuals with a reduced peripheral CD4 § T-cell count. The same is true for lymphopenia: IL-7 levels are inversely correlated with age and absolute lymphocyte count in bone marrow transplant recipients. IL-7 levels in serum may have to be corrected by the age of the patient group. IL-7 serum levels were reported to be in the range of 2.82 ( 1.3 pg/ml in healthy adults (n = 30) and in the range of 4.32 ( 3.9 pg/ml in children (n = 21) (Bolotin et al., 1999). However, in a different study, IL-7 levels were reported to be in the range of 3.6 ( 3.05 pg/ml in healthy individuals (n = 49) and elevated in HIV + patients with 19.4 __ 5.7 pg/ml (n = 131) (Llano et al., 2001). In healthy individuals, IL-7 may represent a key cytokine in T-cell homeostasis, particularly in the maintenance and expansion of naive T-cell populations (for review see Fry and Mackall, 2001).

I

Signal Sequence

32aa

Potential N-glycosylation site

Extracellular Domain

173aa

(Sushi domain 65aa Linker Pro/Thr Rich Region)

I

Transmembrane Domain

21aa

Cytoplasmic Domain

37aa

PLATE 18.1 Regulation oflL-15 expression.