Recent progress in the discovery and invention of novel hematopoietic cytokines

Critical Reviews in Oncology/Hematology, 1992; 13:1-l 5 G 1992 Elsevier Science Publishers B.V. All rights reserved. 1040-8428/92/$15.00

ONCHEM 026

Recent progress in the discovery and invention of novel hematopoietic cytokines Frederick A. Fletcher and Douglas E. Williams Department of Experimental Hematology, Immunes R&D Corp., Seattle WA, USA

(Accepted 2 March 1992)

Contents I.

Introduction

II.

Regulators of the immune response A. IL-9/P40 . . . . . . . .._........_........_..._....._..._...._..._.,.... B. IL-lO/CSIF . C. IL-lZ/CLMF/NKSF

I

. . . . . . . . . . . . . . . . . . . .._..._...._..._..._....__...._..____

. . .._......._

I 2 5 6

.

_........ __..,_.., _.., .._

III.

Myeloid and erythroid cell regulators A. IL-11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .._........_..............._. B. MGFISCFIKLISL factor C. PIXY321.......................................................

7 7 8 11

IV.

Conclusion

12

References

...

.....

... .

... .. ...

.

..

.._..._......._..._............................._........._..

I. Introduction

Pluripotent hematopoietic stem cells in the bone marrow provide cells of all differentiated lymphoid and myelo-erythroid lineages. The production of differentiated hematopoietic progeny is controlled, at least in part, by exogenous cytokines that act at various stages of lineage committment. Prominent as suppliers of these cytokines are the stromal cells found in the hematopoietic organs, activated T cells, and tissue macrophages. It is clear that natural and recombinant cytokines have pleiotropic functions, and exert these functions in a poorly-defined cascase of interacting effects. The known effects of many well-characterized hematopoietic cytokines have recently been reviewed [1,2]. The purpose of this review is to consider more recently identified cytokines that affect the regulation of the immune Correspondence to: Dr. Douglas E. Williams, Immunex R & D Corp., 51 University St., Seattle, WA 98101, USA.

. . . .._........

I?

response, including interleukin-9, - 10 and - 12; and those that have effects on the production of myelo-erythroid cell lineages, including interleukin- 11, mast cell growth factor/stem cell factor/kit ligand, and a novel synthetic cytokine, PIXY 32 1. II. Regulators

of the immune response

The specificity of the immune response to antigen is defined primarily by T cells and B cells. T cells are broadly responsible for stimulating cell-mediated immunity and for coordinating functional activation of B cells. T cells can be divided into two functional classes, based on differential expression of the CD4 or CD8 antigens. CD4 or CD8 is expressed on T cells in association with an antigen-specific T cell receptor heterodimer, and together recognize a specific antigen presented in association with either class II- or class IMHC molecules, respectively. Activated CD8’ (cytotoxic) T cells can recognize and kill host cells that have

2 THI

TH2 f

I

IL-4

IL-2

IL-3

IFNy LT

GM-CSF

IL-5

TNFa

IL-6

J Cell-Mediated Immunity

IL-10

Humoral Immunity

Fig. 1. Differential cytokine secretion by T,l and T,2 cells. T,l cells provide help primarily for macrophage activation, thus enhancing cell-mediated immunity. T,2 cells, however, provide better help for B cell activation, leading to enhanced humoral immunity.

consumed, or that have been infected with, certain pathogens or tumor cells; while activated CD4’ (helper) T cells regulate the production and secretion of antibodies by B cells (humoral immune response). Helper T (TH) cells can be further subdivided into at least two classes, based on stable, differential expression of cytokine subsets (Fig. 1). T,l cells express interferon-y and IL-2, while T,2 cells express IL-4, IL-5 and IL-6. T,l cells are thought to preferentially induce macrophage activation [3] and the delayed-type hypersensitivity response [4], while T,2 cells provide superior help for B cell responses [5,6]. The recently identified cytokines IL-9, IL-lo, and IL12, directly interact in the proliferation and differentiation of cells involved in the immune response. One of these, IL-9, has effects that extend far beyond its ability to stimulate the proliferation of T cells and T cell precursors; while the others have known effects that are more restricted. II-A. IL-9/P40 P40 was originally described by Uyttenhove et al. [7] as a soluble activity derived from a cloned helper T (TH) cell line (TUCZ. 15) that supported the antigen presenting cell (APC)-independent proliferation of select T, clones in vitro, but not cytolytic (CD8’) T cell clones or primary T cells. The activity was not inhibited by neutralizing antibodies directed against IL-2 or IL-4, and co-purified with a novel 32-39 kDa glycoprotein. Subsequently, van Snick et al. [8] reported the cloning of a cDNA encoding murine P40 from a concanavalin A (ConA)-stimulated TH clone. A single cDNA was shown to express bioactive P40 in fibroblasts, indicating that the entire coding region was contained within this clone. The cDNA contained a 554 nucleotide insert,

with a single 432 nucleotide open-reading-frame encoding a predicted protein product of 144 amino acid residues. The predicted protein contained an amino-terminal string (18 residues) of hydrophobic amino acids, consistent with a signal sequence. The mature 126 residue protein had a calculated M, of 14 150 and contained four potential sites of N-linked glycosylation. The higher apparent M, of the native protein was due to variable glycosylation, as N-glycanase treatment of the native protein reduced the M, to approximately 15 kDa, as determined by SDS-PAGE. Yang et al. [9] were the first to report cloning the human homologue of P40; Renauld et al. [lo] also cloned the human equivalent. Yang et al. [9] identified an HTLV-I transformed T cell line [ll], that constitutively produced a soluble factor mitogenic for the megakaryoblastic leukemia cell line, M07e. An expression cloning strategy allowed identification of a single cDNA clone that encoded this activity. The protein produced by COS cells transfected with this clone was 20-30 kDa in size. The clone contained a 432 nucleotide open reading frame, encoding a predicted protein of 144 residues with a calculated M, of approximately 16 kDa. Sequence comparison between this cDNA and murine P40 revealed 56% identity at the nucleotide level and 65% identity at the protein level. More importantly, all 10 cysteine residues in the coding region were conserved between the two protein sequences, suggesting that this cDNA encoded the human homolog of P40. The cDNA cloning and production of recombinant murine and human P40 protein has allowed study of the biology in terms of both tissue distribution and function, and has led to re-naming the molecule IL-9. Renauld et al. [ 121 studied expression of IL-9 mRNA in fractionated and unfractionated human peripheral blood mononuclear cells (PBMC). The IL-9 message could not be detected in normal unfractionated PBMC, but stimulation with PMA and a calcium ionophore (A23187) transiently induced the 0.7 kb message. Stimulation of unfractionated PBMC with LPS or S. aureus did not induce the IL-9 message; suggesting that monocytes and B-cells do not normally produce IL-9, even after activation. Stimulation with the T cell mitogen PHA or anti-CD3 antibody, however, induced the IL-9 mRNA. Based on these findings, it was suggested that production of IL-9 mRNA is a physiological response of T cells to antigen stimulation. FACS separated CD4’ subsets of T cells preferentially expressed IL-9 mRNA (in contrast to CD8’ subsets), a finding consistent with in vitro observations [ 131 indicating that IL-9 is a product of CD4’ T cells. Functional characterization of both murine and human IL-9 has revealed effects that extend beyond the

3

original T cell stimulatory activity. Suda et al. [14] extended the initial observation that IL-9 was a stimulant for T cell lines, to include primary murine fetal thymocytes. They reported that murine IL-9 enhanced the IL-2-dependent proliferation of day 15 fetal murine thymocytes in vitro. This effect was not mediated by the secondary production of TNF-a, as neutralizing antiTNF-a serum did not inhibit the synergy between IL-9 and IL-2. IL-9 had little or no effect on the proliferation of fetal thymocytes induced by IL-4 or IL-7. Hueltner et al. [15] reported that murine IL-9 enhanced the response of certain mast cell lines to IL-3 (mast cell enhancing activity, MEA), thus being the first to identify

a novel factor combination (IL-9 + IL-3) active on myeloid lineages. Donahue et al. [16] reported that human IL-9 stimulated the formation of BFU-E from partially-purified human peripheral blood progenitors. The efficiency of this burst promoting activity (BPA) stimulated by IL-9 was approximately 50% of that induced by plateau levels of IL-3 or GM-CSF. The BPA of IL-9 was not affected by neutralizing antisera directed against IL-3 or GM-CSF. The IL-9 erythroid effect was suggested to be direct, based on low-cell-density-plating experiments using more fully purified progenitors (CD34’ immunoselection) from peripheral blood. A similar BPA of IL-9

TABLE 1 Molecular mass, expression distribution, and target cell specificity of recently identified hematopoietic cytokines Cytokine

Molecular mass (observed/predicted)

mRNA expression

Target cell(s)

IL-9

murine 32-39 kDa/l4 kDa human 20-30 kDail6 kDa

T, cell lines, inducible in CD4’ T cells

CD4’ T cells, primary fetal thymocytes, mast cell lines, BFU-E

IL-10

murine 17-21 kDa/18.7 kDa

T,2 cell lines, B cell lymphomas, mast cell lines

double-negative and single-positive thymocytes, CD4’ T,l cells, CD8’ T cells, and mast cells

IL-I 1

primate 20 kDa/ZO kDa

bone marrow stroma, trophoblast, placenta, fetal lung T cells

plasmacytoma, preadipocytes, murine B cells, primitive murine bone marrow progenitors, and BFU-MK

IL-12

human a-subunit 40 kDa/35 kDa B-subunit 35 kDa/22.5 kDa

human PBMC, lymphoblast cell lines

human CD8’ T cells, human lymphoblasts, LAK cells, NK cells

MGF/SCF/KL

murine 28-30 kDa/22 kDa rat 28-35 kDa/nd

bone marrow stroma, fibroblasts, embryonic brain, T cells, endothelial cells

melanocytes, primordial germ cells, multipotent and committed hematopoietic progenitors, mast cells, megakaryocytes

PIXY321

**35 kDa

**

myeloid cell lines, CFU-GEMM. CFU-GM, BFU-E, CFU-MK, BFU-MK

**Does not exist in a natural, non-recombinant nd, not determined or not reported.

state.

4

was reported on normal adult bone marrow and cord blood that had been prepared by density centrifugation through Ficoll and by adherance to plastic. IL-9 had no effect on the production of in vitro colony-forming cells (CFU-GM, -G, or -M) in these cultures. Williams et al. [ 171 reported that, unlike results presented by Uyttenhove et al. [7], murine IL-9 could support the proliferation of some IL-3-dependent myeloid cell lines in vitro. These lines included the mast cell lines MC-6 and H7, as well as the relatively undifferentiated line NFS-60. The effect of IL-9 was most pronounced on MC-6 cells, being equivalent to the stimulative effect of IL-3. IL-9 was further shown to synergise with suboptimal concentrations of murine IL-3 in stimulating the proliferation of these lines. Cultured primary mast cells or 5-fluorouracil (SFU)-treated bone marrow showed no detectable response to IL-9. Murine IL-9 was also shown to promote erythroid burst formation from murine bone marrow in vitro, a result consistent with the BPA activity of human IL-9 reported by Donahue et al. [16]. The mechanism of BFU-E stimulation by murine IL-9, however, was suggested to be indirect, and mediated by marrow accessory cells. Williams et al. noted strict cell concentration dependence of the murine IL-9 BPA. They further demonstrated that stringent removal of T cells and monocyte/macrophages from bone marrow prior to assay abolished the BPA of IL-9, while maintaining sensitivity of erythroid precursors to IL-3. Further characterization of the direct IL-9 target indicated that removal of MAC-l’ cells (monocytes/ macrophages) had no effect on the burst-promoting activity, but T-cell depletion abrogated this effect. Mock et al. [18] mapped the murine and human IL-9 genes to chromosomes 13 and 5, respectively. A panel of murine/hamster somatic cell hybrids was used to identify the chromosomal localization of murine IL-9. A panel of human/mouse hybrids was used to assign an unambiguous localization of human IL-9 to chromosome 5. Identification of a MspI restriction-fragmentlength polymorphism associated with the murine IL-9 gene allowed, through the study of interspecific backcross mice, its localization distal to tcrg and proximal to dhfr. Both of the IL-9 genes share similar sizes (approx. 4 kb), organizations, and encode five exons. Interestingly, the appearance of weakly-hybridizing bands other than those from the IL-9 gene on Southern blots of DNA from mouse/human hybrids suggested the possibility that a second IL-9-related gene, that did not co-segregate with chromosome 5, exists in the human genome. Renauld et al. [12] reported that the mouse and human IL-9 genes were also highly homologous in the 5’ and 3’ non-coding regions, including a conserved

consensus AP- 1 binding site (5’-TGACTCA-3’) upstream of the TATA box, consistent with reports that the mRNA was up-regulated in response to stimulation with phorbol esters. In addition, an interferon (IFN) regulatory factor-l binding site (5’-AAGTGA-3’) and an AP-2 binding site (5’-TCCCCAG-3’) were identified, but the functionality of these other regulatory sites is unknown. The presence of a weakly hybridizing band on Southern blots of murine genomic DNA probed with the IL-9 cDNA suggested the existence of an IL-9-related gene, but no similar band was detected by these investigators in the human genome. Kelleher et al. [19] mapped the human IL-9 gene to 5q3 l-32 by in situ hybridization of a IL-9 cDNA probe to human metaphase chromosomes. This region of human chromosome 5 contains many other known cytokine genes (IL-3, IL-4, IL-5, GM-CSF, a-FGF) and at least one cytokine receptor (PDGF-a) and is deleted in a number of hematologic disorders (5q-syndromes). In addition to the 5’ regulatory sequences identified by Renauld et al. [12], these authors identified other potential regulatory sites in the human gene, including recognition sites for other TPA-inducible transcriptional factors (AP-3, AP-5, NF-kB), a perfect octamer sequence (5’-ATTTGCAT-3’) and an SP-1 site. Druez et al. [20] reported the biochemical characterization of murine IL-9 receptors on a factor-dependent T, cell line (TSl), that responded to IL-9. Binding of iodinated IL-9 to TSl cells at 4°C reached a plateau at 1 nM, with half-maximal saturation occurring at approximately 100 pM. Scatchard analysis of this data suggested the existence of a single receptor class with a Kd of approximately 103 pM, and an approximate receptor density of 311 l/cell. Two percent receptor occupancy was sufficient to generate half-maximal proliferation of TSl cells. SDS-PAGE analysis of solubilized membranes from TSl cells that had been crosslinked to iodinated IL-9 revealed a single radio-labelled band of 86kDa, under reducing conditions, representing the crosslinked ligand-receptor complex. N-Glycosidase treatment revealed that the IL-9 receptor consisted of a 54 kDa protein with approximately 10 kDa of associated N-linked glycosylation. The presence of IL-9 receptor could not be detected on primary thymocytes, spleen cells, resting or Ag-stimulated lymph node cells, purified B- or T-lymphocytes, LPS- or ConA-stimulated lymphoblasts. Some T, cell lines, however, did express detectable IL-9 receptor, and the receptor level corresponded to the proliferative response generated with IL-9. In addition to the T cell lines that expressed IL-9 receptor, Druez et al. reported that two macrophage lines, P388D, and PU5-1.8, and two bone marrow-derived mast cell lines, L138.8A and

L138.C expressed significant levels of the IL-9 receptor. The reported cellular distribution of the IL-9 receptor corresponds to the known effects of IL-9, with the notable exception of primary thymocytes. It was suggested that fetal thymocytes, as opposed to adult thymocytes, might differentially express the IL-9 receptor, thus explaining the lack of detectable receptor on adult thymocytes in the present report.

Class

TCR/CD4

II llHC

Complex

t

Actlvatlon

II-B. IL-lO/CSIF

Humoral (Ab-mediated) or cell-mediated immunity can be induced in vivo in response to antigenic challenge. There is evidence that these two responses are mediated by different subsets of T, cells, the so-called Tnl and T,2 populations of T cells [21,22]. The defining feature of these two populations is the differential expression of cytokines: T,l cells differentially express IFN-y and IL-2, while Tn2 cells express IL-4, IL-5, and IL-6 [23]. T,l cells preferentially induce macrophage activation and delayed-type hypersensitivity (DTH) (cell-mediated immunity), while Tn2 cells provide superior help for B cell responses. While T,l cells can provide B cell help [24,25], at a high T,l/B cell ratio (> 1:1) T,l clones are lytic against autologous B cells pulsed with specific antigen [26]. A decrease in immunoglobulin production was correlated with the lytic activity of T,l clones, suggesting that this activity might be an important mechanism for the down-regulation of antibody responses in vivo. The Tnl product IFN-y inhibits proliferation of T,2 clones in vitro, perhaps explaining the in vivo dominance of DTH responses in some instances. The absence of a known cross-regulator of Tnl cells (produced by T,2 cells) led Fiorintino et al. [27] to search for such a molecule. Fiorintino et al. [27] discovered a protein (IL-lo), that they initially called cytokine synthesis inhibitory factor (CSIF). IL-10 was produced by murine T,2 clones and inhibited production of a limited subset of cytokines by Tnl clones, including IFN-)I and IL-2. The inhibition of IFN-y production by Tnl clones was most complete and is interesting regarding the activity of IFN-y in inhibiting the proliferation of T,2 clones. Although CSIF did not inhibit the proliferation of T,l clones, down-regulation of IFN-I/ production should inhibit their function (Fig. 2). The mechanism of T,l cytokine inhibition mediated by CSIF was suggested to be indirect and mediated by antigen presenting cells (APC). Although splenic and peritoneal B cells and macrophages, as well as B cell and macrophage cell lines, can function as APC to T, 1 clones in vitro, IL- 10 only acted on primary macrophages or macrophage cell lines to suppress IFN-y, and to a lesser extent IL-2 production

-IFNy

ILIO

-

Fig. 2. Interaction between T, cell subsets. Preferential activation of antigen-specific Tnl or Tn2 clones. dependent upon antigen presentation by different classes of APC. can result in a mixed or exclusive immune response. Cytokine cross-talk between T, cells, involving IFN-y and IL-IO. could lead to functional inactivation of one type of T, cell response, resulting in either a cell-mediated response or a humoral response.

by T,l clones [28]. The ability of splenic B cells to act as APC was unaffected by IL-10 treatment. Although it is possible that IL-10 induced production of a secondary secreted factor that inhibited cytokine production in Tnl cells, these authors suggested that it was more likely that IL-10 acted directly to inhibit an APC function in macrophages not related to class II MHC interaction with the T cell receptor. They suggested that down-regulation of an unknown macrophage co-stimulatory activity (such as a cell adhesion molecule), required for optimal T,l activation was responsible for the observed effect on cytokine secretion from Tnl cells. It has been reported that Tnl and T,2 cells have different co-stimulatory requirements [29], an observation that offers an explanation for the differential activity of CSIF-treated macrophages on T, 1 and T,2 clones. It is also formally possible that IL-10 acted to inhibit production of a secreted factor from the APC that was required for optimal activation of T, 1 cells. Differential activity on co-stimulatory molecules was also offered as an explanation for the lack of an effect of IL-10 on the APC ability of B cells. It is unlikely that B cells lack a functional IL-10 receptor, as IL-10 has been demonstrated to enhance the in vitro viability of splenic B cells,

6

and also to up-regulate class II MHC antigens on resting splenic B cells [30]. An additional finding made by Go et al. [30] was that IL-10 had no activity on small, dense B cells from the inbred immunodeficient mouse XID (X-linked Immunodeficiency). It is interesting to speculate that B cells from this strain might lack functional IL-10 receptors or other IL-lo-related signal transduction molecules. The cDNA sequence for murine IL-10 has been reported [31] from a ConA-stimulated T cell clone, DlO. The IL-10 clone was reported to encode a predicted protein of 178 amino acid residues. The first 18 residues constituted a cleaved signal sequence, resulting in a mature protein of 160 residues. The predicted amino acid sequence contained two potential N-linked glycosylation sites and was calculated to encode a protein of 18.7 kDa. Transfection of the IL-IO-encoding cDNA clone into COS7 cells resulted in the production of three protein species of 21, 19, and 17 kDa. These three species of recombinant IL-IO were identical in size, by SDSPAGE, to the native proteins produced by DlO cells, and were precipitated with monoclonal antibodies directed against three different epitopes of native IL-IO. Culture of DlO cells with the N-linked glycosylation inhibitor tunicamycin resulted in production of only the 17 kDa species; and N-glycanase treatment of the recombinant protein resulted in a single 17 kDa species. These results suggested that the size heterogeneity was the result of variable iv-linked glycosylation. RNA blot hybridization revealed mRNA species of 1.5 and 1.0 kb in ConA-stimulated T cell clones DlO and CDC35, but the message was undetectable in T,l clones. The IL-10 message was also detectable by blot hybridization in the CD4’ HT2 cell line and the Lyl’ B cell lymphoma CH12; and by PCR in the mast cell lines MC/9 and MM3. The IL-10 message was undetectable in fibroblast (NIH 3T3), early myeloid (NSF60), and macrophage (P388Dl) cell lines. Identification of IL-10 mRNA in the mast cell line MC/9 led Thompson-Snipes et al. [32] to investigate whether IL-10 was involved in growth regulation of mast cells. While IL-10 alone was unable to support the proliferation of MC/9 cells in vitro, it was suggested to extend their viability in the absence of exogenous IL-3 or IL-4. IL-10 was shown, however, to improve the IL-3- or IL-4-dependent proliferation of MC/9, even when IL-3 or IL-4 concentrations were sub-optimal. Enriched primary mast cells from parasitized mouse mesenteric lymph nodes were unable to respond, or responded poorly, to either IL-3, IL-4, or IL-IO alone, but two factor combinations of each of these cytokines resulted in a significant increase in mast cell colony formation in vitro. These results suggested that IL-IO,

in combination with IL-3 and IL-4, might play a significant role in certain immune response, such as parasiteinduced mastocytosis. IL-10 was also shown to improve the IL-2/IL-4-dependent proliferation of fetal and adult thymocytes [33], an activity previously ascribed to a novel B-lymphomaderived factor, B-TCGF [34]. Adult CD4-/CD8- (DN), CD4’/CD8-, and CD4-/CD8’, but not CD4’/CD8’ thymocytes responded to the co-stimulatory effects of IL-10 in this assay. Little or no change in the CD4/CD8 phenotype was noted in fetal or adult cells after costimulation with IL-lo, with the exception of adult DN cells, where almost 30% of cells were induced to express CD8 after 4 days of culture. Chen et al. [35] subsequently reported that IL-10 improved the IL-Zdependent proliferation of CDS’ T cells, and also augmented the IL-Zdependent cytolytic activity of effector CTL generated from ConA-activated CD8’ spleen cells in vitro. These results suggested that IL-10 acted as a cytotoxic T cell differentiation factor, a result in agreement with the results reported by MacNeil et al. [33]. Comparison of the IL-IO nucleotide and protein sequence to available databanks revealed high overall amino acid sequence homology (70%) with an uncharacterized open reading frame (orf> in the Epstein-Barr virus (EBV) genome, BCRFI (BamHl fragment C Rightward reading Frame No. 1) [31]. The suggestion was made that BCRFl represented a captured human gene (IL-IO) that facilitated survival of EBV in an infected host cell. Interestingly, IFN-y inhibits the generation and outgrowth of EBV-transformed B cells in vitro, suggesting that, if BCRFl is expressed in vivo, functional mimicry of an inhibitory host cytokine (IL10) might be exploited by EBV to evade host defense systems in vivo. Hsu et al. [36] subsequently demonstrated that BCRF 1 did encode an IL- lo-like activity. Recombinant protein from the BCRFl orf was shown to inhibit IFNy synthesis by cloned T,l cells in the presence of syngeneic APC. The BCRFl protein also inhibited IFN-7 synthesis by PHA- or IL-Zstimulated primary human PBMC in vitro. Since MHC-restricted cytotoxic T cells probably control persistent (latent) EBV infection in vivo [37], and since the BCRFl gene is transcribed in the late phase of the lytic life cycle of EBV [38], Hsu et al. suggested that the BCRFl protein exerted a protective effect (IFN-I/ inhibition) only during the lytic stage of infection. II-C. IL-l.&‘CLMFiNKSF

IL-12 was originally identified by Gately et al. [39,40]

as a soluble activity produced by PHA-stimulated human PBMC that synergized with IL-2 in the stimulation of allogeneic human CTL response in mixed-lymphocyte-tumor cultures. They called this activity cytotoxic lymphocyte maturation factor, CLMF. CLMF was subsequently purified to homogeneity by these same investigators [41], and demonstrated by SDSPAGE analysis to consist of a 75 kDa disulfide-linked heterodimer of 40 and 35 kDa subunits. N-terminal protein sequence obtained from each purified subunit revealed that they were not related to each other or any other previously-characterized cytokine. Purified CLMF was also shown to act alone and to synergize with IL-2 in stimulating PHA-stimulated human lymphoblasts; and to act in synergy with IL-2 in stimulating the induction of lymphokine-activated killer (LAK) cells [42]. Generation of partial protein sequence from CLMF demonstrated its identity with the natural killer cell stimulatory factor (NKSF) identified from the human B lymphoblastoid cell line RPM1 8866 by Kobayashi et al. [43]. This finding extended the known effects of CLMF to include novel activities of NKSF, i.e., induction of IFN-y from PBLs and augmentation of NK cell cytotoxicity [43,44]. cDNAs encoding the 35 and 40 kDa subunits of IL12 have been cloned [45] from the human lymphoblastoid cell line, NC-37. Partial protein sequence from each subunit allowed synthesis of mixed oligonucleotide primers that were used to amplify specific probes from NC-37 cDNA by use of the polymerase chain reaction (PCR), followed by hybridization screening of an NC37 cDNA library cloned into a ;1 phage vector. Both cDNAs were predicted to encode secreted products, with hybrophobic signal sequences followed by the Nterminus of the mature protein, as revealed by protein sequencing. The cDNA encoding the 40 kDa subunit consisted of 22 hydrophobic residues preceding the 306 residue mature protein. The protein encoded by this cDNA had a calculated molecular mass of 34 699 Da, contained 10 cysteine residues, and four potential Nlinked glycosylation sites. At least one of these sites (Asn-222) was known to be glycosylated, and another (Asn- 125) was reported to be not glycosylated. A cDNA encoding the 35 kDa subunit also contained a 22 residue signal peptide, followed by a 197 residue mature protein. The calculated molecular mass of the mature protein was 22 5 13 Da, and contained seven cysteine residues and three potential N-linked glycosylation sites. When analyzed by SDS-PAGE under reducing conditions, the 35 kDa subunit appeared heterogeneous in size, suggesting that it was variably glycosylated. Northern blot analysis revealed that the 40 kDa subunit of IL-12 is encoded by a 2.4 kb mRNA, and the 35

kDa subunit is encoded by a 1.4 kb mRNA. These two mRNAs were demonstrated to be co-ordinately upregulated upon induction of NC-37 cells with PMA and calcium ionophore. Co-expression of both cDNAs in transfected COS cells was required to recover secreted, biologically-active IL-12. The two constituent subunits apparently could not re-associate into biologically-active IL-12 after mixing in vitro. The activity of recombinant IL-12 was essentially identical to purified natural material in assays of ability to stimulate proliferation of pHA-treated PBL and generation of LAK in HC-containing cultures. III. Myeloid and erythroid cell regulators

The normal supply of differentiated hematopoietic cells occurs in the absence of any immunological stimulus. Bone-marrow stem cells provide a steady supply of committed hematopoietic progenitors to replenish expended cells. These developing hematopoietic cells are found in the marrow in association, with a complex network of stromal elements, including macrophages, fibroblasts, adipocytes, and endothelial cells. This stroma produces soluble and cell-associated factors that affect constitutive hematopoiesis by positively and negatively regulating the production of mature hematopoietic progeny cells. At least two naturally-occurring fdctors (IL-l 1, MGF), and one novel recombinant hybrid cytokine (PIXY321), that effect myelo-erythroid cell differentiation have been recently identified. These factors are described in the following sections. III-A.

IL-1 I

IL-I 1 was originally identified as a soluble product of the cloned non-human primate bone marrow stromal cell line PU-34 that was able to support proliferation of the IL-6-dependent murine plasmacytoma line, T1165 [46]. The proliferative activity on this line was resistant to neutralizing anti-IL-6 antibody, a result suggestive of a novel cytokine similar in function to IL-6. Kawashima et al. [47] reported independent discovery of human IL-l 1 as an activity from the cloned human stromal cell line, KM-102, able to inhibit progression of cultured pre-adipocytes to adipocytes. It is interesting to note that IL-6 and the leukemia inhibitory factor (LIF) also inhibit adipogenesis in vitro, further suggesting a functional similarity between IL-11 and IL-6. The cDNAs for human [47] and non-human primate [46] IL-l 1 have been cloned and expressed. Paul et al. [46] utilized an expression-cloning strategy in mammalian cells that ultimately yielded a 721 bp cDNA with a single long open reading frame of 597 nucleotides capa-

8

ble of encoding an 199 amino acid polypeptide. Surprisingly, the predicted protein contained no potential sites for asparagine-linked carbohydrate and encoded no cysteine residues. Expression of the cDNA in COS cells yielded a secreted product of approximately 20 kDa, a result consistent with the predicted size. Northern analysis of PU-34 and human cell lines derived from trophoblast (TPA30-l), placenta, fetal lung (MRCS), and T-cells (ClO-MJ2, C5-MJ2 and MO) indicated the presence of two transcripts (approximately 2.5 and 1.5 kb) encoding the same protein, differing only in the length of the 3’-noncoding regions. Both of these transcripts were detectable in PU-34 and MRC5 cells after stimulation with IL-l-a, while only the longer 2.5 kb fragment was detectable in TPA30-1 cells. Neither transcript was detectable in the other cell lines tested. Kawashima et al. [47] reported the cDNA sequence for human IL-l 1 isolated from the PMAA23 187-treated bone marrow stromal cell line, KM-102. Sequence comparison between the human cDNA isolated from the MRC5 line and the primate cDNA from PU-34 cells indicated approximately 97% identity at the nucleotide level. Two transcripts (2.6 and 1.3 kb) were constitutively expressed in KM-102 cells, but expression was significantly enhanced after stimulation with PMA and A23 187. Alternative poly-adenylation was suggested to be responsible for the different transcript sizes, but no explanation was offered as to the significance of this differential poly- adenylation. No IL-l 1 mRNA was detectable from primary human liver, heart or kidney. Assay of COS cell-derived primate IL-I 1 for activity in various hematologic and immunologic systems revealed that it enhanced the T cell-dependent formation of Ig-secreting murine B cells in a spleen cell plaqueforming assay and also enhanced the IL-3-dependent formation of megakaryocyte colonies (CFU-MK) from unfractionated murine bone marrow in vitro [46]. Bruno et al. [48], however, reported that human IL-l 1 did not synergize with IL-3 in stimulating colony formation by human CFU-MK in vitro, but did potentiate the positive effect of sub-optimal concentrations of IL-3 on formation of megakaryocyte colonies from the developmentally more primitive precursor BFU-MK. This discrepancy appears to be semantic, as the second result was obtained from highly-purified sub-populations of human bone marrow that allowed more precise definition of the target cell. The ability of IL-l 1 to stimulate proliferation of the IL-6-dependent plasmacytoma T1165 and inhibit adipogenesis in vitro highlights functional redundancy between IL-l 1 and the IL-6/G-CSF/LIF triad (reviewed recently by Metcalf [49]). Interestingly, Musashi et al. [50] reported that IL-l 1 shortens the G, phase of murine

bone marrow progenitors in vitro, resulting in enhanced proliferation of committed blood cell progenitors. Similar activities have been reported for both IL-6 [51], G-CSF and LIF [52], suggesting that IL-l 1 might be an additional member of the IL-6, G-CSF, LIF family of effecters. III-B. MGF/SCF/KL/SL factor

The discovery of MGF is an example of a directed approach to cloning a specific gene, somewhat akin to the reverse genetic approach, where the chromosomal localization of a mutant phenotype is first identified, followed by identification of the gene product responsible for the defect. Mice with mutations at the dominant white spotting (IV’) or Steel (SL) locus present with a similar phenotype, characterized by defects in hematopoiesis, gametogenesis and melanogenesis [53-551. Bone-marrow transplantation studies demonstrated that the hematopoietic defect at W was intrinsic to the pluripotent stem cell [53,56,57]; whereas the SL defect was stromal [53,58-601. The similar phenotypes and cross-complementatity of SI bone marrow transplanted into W recipients suggested that the products of Wand SL were interacting molecules, such as a receptor and ligand . Recent studies have demonstrated that, in fact, the W locus does encode a receptor, the c-kit proto-oncogene [61,62]. The product of c-kit is a receptor tyrosine kinase [63] closely related to c-fms [64]. This finding further strengthened the hypothesis that the product of SL was a ligand for the c-kit receptor, and resulted in intensive efforts to identify candidate ligands. A putative c-kit ligand was simultaneously identified by at least three different groups [65-671, and has been called mast cell growth factor (MGF), stem cell factor (SCF), and the kit ligand (KL). Nocka et al. [67] and Williams et al. [65] utilized a similar strategy to identify a candidate ligand, taking advantage of the Wand SL phenotype to identify a c-kit ligand. Recognizing that defective mast cell production was a primary defect in otherwise viable Wand SL animals, these investigators used mast cell proliferation assays to detect differences in factor production between wild type and SL stromal cell line. Nocka et al. [67] purified a 28-30 kDa protein, KL, from media conditioned by NIH-3T3 fibroblasts, that supported proliferation of normal mast cells, but not W/W” mast cells. Either type of mast cell proliferated in response to IL-3. suggesting that the novel protein interacted with the c-kit receptor. The purified protein was suggested to be monomeric, based on molecular mass studies under denaturing and non-denaturing con-

9

ditions, and of heterogeneous size due to variable glycosylation. Williams et al. [65] identified a putative kit ligand (MGF) from two previously reported [68] stromal cell lines derived from SL/SLd mice or normal littermates. The stromal line derived from normal mice was found to support the transient survival of at least two mast cell lines (MC6 and H7) in vitro, while the line derived from SL/SLd mice was unable to do so. A novel protein, MGF, was purified from media conditioned by the normal stromal line and further characterized. The mitogenie response of MC6 and H7 was shown to correlate with expression of the c-kit mRNA, and non-responders (FDC-P2, FDC-P2-lD, DA-l, 32D) did not express the c-kit mRNA. These results supported the hypotheses that MGF was a c-kit ligand and a product of the SL locus. Iodinated MGF was shown to specifically bind to native c-kit receptor on MC6 cells by receptor-ligand crosslinking and immunoprecipitation with anti-serum directed against the C-terminus of c-kit. MGF was further demonstrated to specifically bind and immunoprecipitate with anti-kit antiserum on COS cells transfected with a c-kit cDNA. Zsebo et al. [66] identified the rat homolog of MGF, a protein that they called the stem cell factor (SCF), as a soluble product of Buffalo rat liver cells (BRL-3A). SCF was identified based on its ability to stimulate hematopoietic colony (HPP-CFC) formation in vitro from 5-flourouracil (SFU)-treated murine bone marrow. Purification of this activity yielded a protein with a molecular mass of 28835 kDa on SDS-PAGE. The size heterogeneity was shown to be the result of both O-linked and N-linked glycosylation, with fully-deglycosylated protein migrating at approximately 18-l 9 kDa in SDS-PAGE. Purified SCF was reported to indirectly support the formation of hematopoietic colonies containing granulocytes, monocytes and megakaryocytes in vitro by unseparated or partially purified murine bone marrow progenitors. SCF was also shown to stimulate proliferation of the MCI9 mast cell line, a result consistent with that reported by Williams et al. Both Williams et al. [65] and Zsebo et al. [66] reported N-terminal sequence of their respective c-kit ligands, the reported sequences being similar. Zsebo et al. reported a blocked N-terminus on the rat protein, identified as pyroglutamate; while Williams et al. reported an unmodified lysine as the N-terminal residue of the secreted mouse protein. The sequence reported by Williams et al. was confirmed by Huang et al. [69]. Identification of mouse [69,70], rat and human [71] cDNAs encoding the c-kit ligand have been reported. Huang et al. [70] isolated a murine cDNA from NIH3T3 cells, corresponding to the deduced amino acid

sequence, using degenerate oligonucleotide primers and the polymerase chain reaction. A cloned PCR product was identified that encoded the correct polypeptide, and was subsequently used to identify a 1.4 kb cDNA clone from a fibroblast cDNA library. The clone contained a single open reading frame that encoded a polypeptide of 270 amino acid residues, but no translational stop sequence. The first 25 residues of the predicted protein were characteristic of a signal sequence, followed by the N-terminal sequence identified in the purified mature protein. A typical trans-membrane domain was predicted at residue 217-237, followed by 33 residues of a predicted cytoplasmic domain. In the putative external domain, four potential sites of N-linked glycosylation were identified, as well as four cysteine residues. Huang et al. identified a single major mRNA transcript (6.5 kb) and two minor transcripts (4.6 and 3.5 kb) by Northern analysis of fibroblast RNA, using the 1.4 kb c-kit cDNA as a probe. Anderson et al. [70] also used a degenerate oligonucleotide PCR strategy to clone a partial mouse cDNA from two cell lines known to produce MGF, +/+ and LDAl 1. The partial clone thus obtained was used to identify two additional clones (MGF4 and MGF-10) from a +/+ cDNA library. MGF-10 was approximately 2.1 kb in length, and contained a 819 bp orfcapable of encoding a protein of 273 amino acid residues. The protein encoded by this cDNA was the same as that reported by Huang et al., except for the additional final three residues of the cytoplasmic domain found to be missing from the Huang et al. clone. The clone MGF4 encoded a protein truncated by three residues at the C-terminus, identical to the Huang et al. clone. Anderson et al. [70] also reported the isolation of an additional MGF clone, MGF-94, that appeared to have an internal 48 bp deletion just 5’ of the predicted transmembrane domain. The significance of this clone was not addressed. Anderson et al. identified three hybridizing mRNAs of similar size to those described by Huang et al., but reported variable representation of the three species in different tissues and cell lines. Expression of the MGF-4 or MGF-10 cDNA clones in COS cells resulted in the production of membranebound activity that was able to support the proliferation of MC-6 cells. No bioactivity was identified in the supernatants of the COS cells expressing membranebound MGF. Since MGF was originally identified as a soluble factor, the cDNA clone was truncated 1 residue 5’ of the predicted trans-membrane domain, expressed in COS cells, and assayed for bioactivity. The activity of the purified recombinant, soluble protein was equivalent to purified natural material in the MC-6 proliferation assay. The purified natural material, as well as

10

yeast-expressed soluble protein, was assayed for effects on BFU-E and CFU-GEMM (Granulocyte, Erythroid, Macrophage, and Megakaryocyte) colony formation from murine bone marrow, in the presence of Epo. Either form of MGF was a potent inducer of BFU-E and CFU-GEMM, but the dose-response curves suggested that MGF was more effective at promoting CFU-GEMM formation. These results were consistent with the known phenotype of the SL defect. Martin et al. [71] also used mixed oligonucleotide PCR to isolate a rat SCF product from BRL-3A cDNA that was used to identify a partial cDNA clone from a BRL-3A cDNA library. Sequence analysis of the clone thus obtained revealed a predicted protein similar to those described above. The first residue of the mature rat protein was again predicted to be glutamate. Comparison of the predicted protein sequence with the deduced protein sequence of purified SCF suggested that the purified protein was post-translationally processed at the C-terminus. Interestingly, the C-terminus of the protein purified from BRL-3A cells corresponded to Ala-164 or Ala-165, precisely the 3’ boundary of the internal deletion in MGF-94 described by Anderson et al. Recombinant rat SCF was expressed in both COS cells and E. co/i, and the purified material subjected to assay of bioactivity. Either form of the recombinant protein was reported to have similar bioactivity to purified native material in the previously mentioned HPPCFC assay and in the stimulation of the MC/9 mast cell line. In addition, the E. coli material was assayed for an effect on the IL-7-dependent growth of preB (B220’, surface Ig-, cytoplasmic mu+) colonies from murine bone marrow in vitro, and was shown to act synergistically with IL-7 in this assay. Martin et al. [71] also described the isolation of cDNAs encoding the human homologue of rat SCF. Northern analysis indicated that SCF-related transcripts were present in mRNA from a human hepatocellular carcinoma (HepG2), a human bladder carcinoma (5637 or ATCC HTB9), and a human fibrosarcoma, HT-1080. Two 5.4 kb cDNA clones were isolated from HT-1080 and sequenced. The human sequence thus identified was 79% identical to the corresponding regions of the mature rat protein, but extended 3’, past the proteolytic cleavage site of the rat protein, to a C-terminus identical to that identified by Anderson et al. The human SCF homolog corresponding to the processed rat protein was expressed in COS cells and assayed for stimulation of HPP-CFC from human bone marrow. Human SCF was shown to promote erythroid burst formation from human bone marrow, when cultured in the presence of erythropoietin. E. coli-expressed mate-

rial was shown to have a similar effect, but was also shown to synergise with human G-CSF, GM-CSF, IL3, or Epo in stimulation of human colony-forming cells (CFCs) in vitro. Confirmation that the c-kit ligand was, in fact, encoded at the Steel locus was obtained shortly after the identification of cDNAs encoding the ligand [69,72-741. Following-up on the data of Fujita et al. [75], Flanagan and Leder [74] presented evidence that the c-kit ligand was expressed on the surface of normal murine fibroblasts, but not on the surface of fibroblasts derived from mice carrying a homozygous SL mutation. The data of Fujita et al. [75] indicated that normal mouse fibroblasts supported the growth of normal mast cells, but not mutant (W/W”) mast cells, suggesting that fibroblasts expressed the ligand for c-kit. Flanagan and Leder fused the extracellular domain of the c-kit receptor to placental alkaline phosphatase, producing a soluble receptor reagent (APtag-KIT) that could be enzymatically traced. They used the affinity reagent APtag-KIT to study expression of a putative ligand on normal mouse fibroblast cell lines, and correlated binding of the affinity reagent with stimulation of mast cell growth, suggesting that the reagent was binding to a biologicallyactive c-kit ligand. Fibroblasts derived from SL mutants, however, bound the affinity reagent weakly, at levels corresponding to non-specific background binding. These results demonstrated that a c-kit ligand could be expressed as a cell surface molecule, and suggested the possibility that the ligand was encoded at the SL locus. Zsebo et al. [72] assigned mouse SCF to chromosome 10, using a panel of hamster/mouse somatic cell hybrids. Southern analysis of genomic DNA from each member of this panel resulted in concordant segregation of SCF bands with mouse chromosome 10, consistent with the hypothesis that SL encoded SCF. Southern analysis of genomic DNA from normal and mutant SL/SL embryos demonstrated that, while the normal DNA exhibited multiple SCF-hybridizing bands, the SL/SL DNA did not. This result suggested that the SCF locus was grossly deleted in the SL/SL genome. Copeland et al. [73] and Huang et al. [69] used interspecific backcross mapping panels (C57/B16 x M. spretus) to map the MGF and KL genes near SL on mouse chromosome 10. Copeland et al. used a panel, described by Buchberg et al. [76], with an average map resolution approaching 3 CM. The position of MGF was determined by comparing the segregation pattern of species-specific MGF restriction- fragment length polymorphisms (RFLPs) in backcross mice to the more than 500 genes already mapped in this panel. Comparison of the deduced MGE map locus on the distal region of chromosome 10 with

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the composite mouse linkage map maintained at The Jackson Laboratory, Bar Harbor, Maine demonstrated that MGF mapped near the SL locus. Southern analysis of genomic DNA from several different SL alleles demonstrated gross rearrangements or deletions in some (SL, SLJ, SLgh, SLaH, SLloH, SLIBH), but undetectable rearrangement in others (SL’7H, SLp”“, SLd). The most severe alleles were all demonstrated to result from deletions or rearrangements, while the less severe, viable alleles were suggested to result from point mutations or other, intragenic alterations. Huang et al. [69] mapped KL with respect to a transgene insertion site tightly linked to SL in an interspecific backcross panel (C57/BI6 x M. spretus). Linkage between a KL probe and a probe from the transgene insertion site was assessed by scoring for concordance of inheritance of their respective TaqI RFLPs. A single recombinant between KL and the transgene was identified in 53 backcross progeny, corresponding to a recombination percentage of 1.9 5 1.9. This value is close to the genetic distance measured between the transgene and SL (0.8 + 0.8), suggesting that KL maps near SL. When Southern analysis of SL/SL embryos was performed, using the KL cDNA as a probe, no hybridization was observed, suggesting a gross deletion of KL in SL/SL mutants. This finding correlates well with those of Copeland et al. and Zsebo et al., and is further suggestive that MGF/SCF/KL is encoded at the Steel locus. Flanagan et al. [77] and Brannan et al. [78] reported further analysis of the SLd allele, and identified an internal deletion in the SLd gene that removed the transmembrane and intracellular domains from the expressed MGF/KL protein. As such, the product of the SLd allele was not retained in the membrane, but recombinant protein produced from the SLd cDNA exhibited biological activity equivalent to full-length MGF. De Vries et al. [79] reported that purified recombinant MGF supported the proliferation of purified murine hematopoietic progenitor cells (spleen colony-forming cells, CFU-S) in culture, both alone and in combination with IL-3 or IL-la. MGF alone, however, could not prevent the loss of the most primitive CFU-S assayed in these cultures (day 14 post-transplant CFU-S or CFU-S,,), but when combined with IL-3 or IL-la, resulted in a net increase in CFU-S,, in vitro. Dolci et al. [80] reported that membrane-bound MGF was required for the survival of murine primordial germ cells (PGC) in vitro, but did not directly support their proliferation. Soluble MGF was effectively unable to do either, a result in agreement with the sterile phenotype of SLd/SLdanimals. .Godin et al. [81] reported essentially identical results to those of Dolci et al. Interestingly, each of these groups used feeder layers of murine

ST0 cells to demonstrate survival and proliferation of primordial germ cells. While ST0 cells are known to produce membrane-bound MGF, an additional activity was also being provided by the ST0 cells because NIH3T3 cells (that also secrete membrane-bound MGF) could only promote survival (not proliferation) of primordial germ cells. Matsui et al. [82] provided a potential candidate for the additional activity produced by ST0 cells with their finding that MGF/SCF/KL could act in synergy with the leukemia inhibitory factor (LIF), a cytokine known to be produced by ST0 cells, to support PGC survival and proliferation in vitro. Fletcher et al. suggested a similar combined effect of MGF and LIF acting to support the survival of murine hematopoietic stem cells [83] and CFU-S,, [84] in vitro. Several groups have begun to characterize the expression of the c-kit and SL gene products [85,86], revealing high levels of expression in the embryonic brain. Despite the apparent co-ordinate regulation of receptor/ ligand expression in the developing floor plate and neural crest cell migration pathways, no neurological phenotype has been associated with any of the W or SL mutant alleles, It is possible that these phenotypes have been masked by the severe hematological disorders that result in embryonic death of homozygous W and SL animals, but it is more likely that functional redundancy in the developing nervous system compensates for a mutation at W or SL. III-C. PIXY321

PIXY 32 1 is a so-called ‘second-generation’ cytokine that combines functional epitopes of individual cytokines into a single, multi-functional protein. Curtis et al. [87] reported that the PIXY321 molecule consisted of the entire cDNAs for human GM-CSF and IL-3. joined by a flexible ‘linker’ molecule. The linker (GGGGS), was chosen to contain as little structure as possible in the hopes that each cytokine domain would fold in the native conformation. The protein was produced in yeast and purified to homogeneity. The fusion protein was shown to bind efficiently to cell lines that expressed either IL-3 receptors alone (JM-1) or GM-CSF receptors alone (HL-60), at an affinity approximately the same as either IL-3 or GM-CSF alone, thus demonstrating functional binding of the fusion protein through either of its domains. On cell lines that expressed receptors for both IL-3 and GM-CSF (KG-l and AML-193) the fusion protein exhibited enhanced binding affinity relative to IL-3. This improved binding was shown to be of functional relevance by measuring 3H-incorporation in AML-193 after stimulation with either cytokine alone, the combination of IL-3+GM-

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CSF or PIXY321 alone. IL-3, GM-CSF or IL-3+GMCSF were shown to be equipotent, but PIXY321 was approximately IO-fold more active than an equimolar mixture of the two individual cytokines. PIXY321 was also demonstrated to be significantly more active than either cytokine alone, or in combination, in assays of BFU-E, CFU-GM, and CFU-GEMM formation from T cell- and adherent cell-depleted human bone marrow. The mechanism of improved activity of PIXY321 is unknown. It was postulated that, since the GM-CSF and IL-3 receptors share a common /I subunit [88], the binding of PIXY321 might create a novel receptor complex consisting of one each a subunit, capable of binding GM-CSF or IL-3, and two identical B subunits. In effect, a novel cross-competable receptor. A recent report by Cannistra et al. [89] suggested that such a receptor might exist on a primitive myeloid progenitor, and be associated with early proliferative events. Affinity crosslinking experiments (Park LS and Williams DE, unpublished data) have failed to show evidence for such a complex, but have revealed the presence of a unique cell-surface protein to which PIXY321 preferentially binds. The size of this protein does not correspond to either the GM-CSF or IL-3 CI- or p-subunits, and the nature of this putative PIXY321 receptor remains unknown. In vivo studies of PIXY321 in normal and irradiated rhesus monkeys have demonstrated an effect on both the neutrophil and platelet compartments [90]. PIXY321 stimulated both the production and functional activation of circulating neutrophils, in accordance with the predicted function of GM-CSF portion of the hybrid molecule. In addition, PIXY321 accelerated regeneration of the circulating platelet compartment following sub-lethal irradiation, an effect consistent with the predicted function of the IL3 portion of PIXY321. IV. Conclusion

Characterization of recently identified hematopoietic cytokines supports a hypothesis of a functionally redundant network of cytokines, capable of interacting in a cascade of effects, whose action can result in differential stimulation of the immunological and hematopoietic systems. This interaction of cytokines is perhaps best exemplified in the present context by the proposed interaction between Tnl and Tn2 cell subsets, mediated in part by IL-IO, that influences the type of primary immune response to a non-self antigen. IL- 10 shares activities with other interleukins, including IL-2, IL-4. and IL-9, but also exhibits a novel activity that acts to inhibit the synthesis of cytokines by T, 1 cells. As we have seen, functional inactivation of T, 1 cells by IL-10 activ-

ity should favor antigen presentation by B cells, resulting in preferential activation of a humoral immune response to those antigens that manage to stimulate IL-10 production from monocytes, B cells, or Tn2 cells. The overlap of function is also noted for those recently identified cytokines that act primarily at the level of hematopoiesis. As we have seen, the activities of IL-l 1 closely parallel those of IL-6, with the possible exception of the BFU-MK reported for IL-l 1. It is possible that IL-l 1 is a member of the IL-6/LIF/Oncostatin M/G-CSF group of cytokines. As such, a logical prediction would be that the un-identified IL-l 1 receptor is a novel member of the hematopoietin receptor family; and that it shares a common high-affinityconverting receptor subunit, gp130, with the IL-6 and LIF low affinity receptors [91,92]. This prediction could account for the similar functions of IL-l 1 and IL-6. At the level of a specific target cell population, functional redundancy is also apparent. IL-9, IL-10 and MGF/ SCF/KL all share activity on activation and proliferation of mast cells. The mast cell proliferative activity also seems to be correlated with an erythroid burstpromoting activity for these, and other cytokines (e.g., IL-3). A notable exception to this correlation is IL-lo, but it is not clear whether a BPA assay has been conducted in this case. There is little question that the pace of cytokine discovery has quickened. It is inevitable that, as more cytokines are characterized, the functional interactions that describe the extent of the cytokine cascade will be revealed. The multiplicity of activities found in native cytokines, however, might soon be rivalled by synthetic hybrids, such as PIXY321. These second-generation cytokines promise to allow novel combinations of existing activities, and perhaps even novel activities, not found in individual native cytokines. The advent of such multifunctional recombinant molecules could be useful for the clinical and experimental manipulation of the immune and hematopoietic systems. Biographies Douglas E. Williams received his Ph.D. from the Roswell Park Memorial Institute Division of SUNY Buffalo. Dr. Williams is currently Vice President and Director of Experimental Hematology at Immunex Research and Development Corporation. Frederick A. Fletcher received his Ph.D. from Baylor College of Medicine and is currently an Assistant Staff Scientist in Experimental Hematology at Immunex. Reviewer This paper was reviewed by Hal Broxmeyer, Ph.D., Scientific Director. The Walther Oncology Center, Indianapolis, IN 46202-5121, USA.

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References 1 Arai K-I, Lee F. Miyajima A, Miyatake S, Arai N, Yokota T. Cytokines: Coordinators of immune and inflammatory responses. In: Richardson CC. Abelson JN, Meister A, Walsh CT, eds. Annual review of biochemistry. Palo Alto: Annual Reviews Inc., 1990; 783-836. 2 Nicolai NA. Hemopoietic cell growth factors and their receptors. In: Richardson CC, Abelson JN. Boyer PD, Meister A, eds. Annual review of biochemistry. Palo Alto: Annual Reviews Inc., 1989; 45578. 3 Stout RD. Bottomly K. Antigen-specific activation of effector macrophages by IFN-y-producing (Tnl) T cell clones. Failure of IL4-producing (Tn2) T cell clones to activate effector function in macrophages. J Immunol 142:76&765, 1989. 4 Cher DJ, Mosmann TR. Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by Tnl clones. J Immunol 138:3688-3694, 1987. 5 Killar L. MacDonald G, West J, Woods A, Bottomly K. Cloned. Id-restricted T cells that do not produce interleukin 4 (IL-4)/B cell stimuiatory factor I (BSFI) fail to help antigen-specific B cells. J Immunol 138:16741679. 1987. 6 Boom WH. Liano D, Abbas AK. Heterogeneity of helper/inducer T lymphocytes. II. Effects of interleukin 4- and interleukin-2-producing T cell clones on resting B lymphocytes. J Exp Med 167:1352-1363, 1988. 7 Uyttenhove C, Simpson R. van Snick J. Functional and structural characterization of P40, a mouse glycoprotein with T-cell growth factor activity. Proc Nat1 Acad Sci USA 85:6934-6938. 1988. 8 van Snick J. Goethals A, Renauld J-C et al. Cloning and characterization of a cDNA for a new mouse T cell growth factor (P40). J Exp Med 169:363-368, 1989. 9 Yang Y-C, Ricciardi S, Ciarletta A, Calvetti J, Kelleher K, Clark SC. Expression cloning of a cDNA encoding a novel human hematopoietic growth factor: human homologue of murine T-cell growth factor P40. Blood 74:1880-1884. 1989. IO Renauld J-C, Goethals A, Houssiau F. van Roost R. van Snick J. Cloning and expression of a cDNA for the human homolog of mouse T cell and mast cell growth factor P40. Cytokine 2:9-12. 1990. 1I Arya SK. Wong-Staal F. Gallo RC. T-cell growth factor gene: lack of expression in human T-cell leukemia-lymphoma virus-infected ceils. Science 223:1086-1087. 1984. 12 Renauld J-C. Goethals A, Houssiau F. Merz H, van Roost E. van Snick J. Human P4OiIL-9: expression in activated CD4’ T cells, genomic organization and comparison with the mouse gene. J Immunol 144:4235-4241, 1990. 13 Schmitt E. van Brandwijk R. van Snick J, Siebold B. Ruede E. TCGF III/P40 is produced by naive murine CD4’ T-cells but is not a general T-cell growth factor. Eur J 1mmunol19:216772170.1989. 14 Suda T, Murray R, Fischer M, Yokota T. Zlotnik A. Tumor necrosis factor-a and P40 induce day 15 murine fetal thymocyte proliferation in combination with IL-2. J Immunol 144:178331787, 1990. 15 Hueltner L. Druez C. Moeller J et al. Mast cell growth enhancing activity (MEA) is structurally related and functionally identical to the novel mouse T cell growth factor P40/TCGFIII. Eur J Immuno1 20:1413~1416, 1990. 16 Donahue RE. Yang Y-C, Clark SC. Human P40 T-cell growth factor (interleukin-9) supports erythroid colony formation. Blood 7512271-2275. 1990. 17 Williams DE, Morrissey PJ, Mochizuki DY et al. T-cell growth factor P40 promotes the proliferation of myeloid ceil lines and enhances erythroid burst formation by normal murine bone marrow cells in vitro. Blood 76:906911, 1990. 18 Mock BA. Krall M, Kozak CA et al. IL9 maps to mouse chromosome 13 and human chromosome 5. Immunogenetics 3 1:2655270, 1990. 19 Kelleher K. Bean K, Clark SC et al. Human interleukin-9: genomic

sequence, chromosomal location, and sequences essential for its expression in human T-cell leukemia virus (HTLV)-I-transformed human T cells. Blood 77:143&1441, 1991. 20 Druez C, Coulie P, Uyttenhove C, van Snick J. Functional and biochemical characterization of mouse P4O/IL-9 receptors. J Immunol 145:249&2499, 1990. 21 Katsura Y. Cell-mediated and humoral immune responses in mice. III. Dynamic balance between delayed-type hypersensitivity and antibody response. Immunology 32:227, 1977. 22 Parish CR. The relationship between humoral and cell-mediated immunity. Transplant Rev 13:35, 1972. 23 Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 136:2348-2357, 1986. 24 Coffman RL. Seymour BW, Lebman DA et al. The role of helper T cell products in mouse B cell differentiation and isotype regulation. Immunol Rev 102:5, 1988. 25 Stevens TL. Bossie A, Sanders VM et al. Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells. Nature 334:255-258, 1988. 26 Romagnani S. Human Tnl and T,2 subsets: doubt no more. Immunol Today 12:256-257, 1991. 27 Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper cell. IV. Tn2 clones secrete a factor that inhibits cytokine production by T,l clones. J Exp Med 170:2081-2095, 1989. 28 Fiorentino DF, Zlotnik A, Vieira Pet al. IL-10 acts on the antigenpresenting cell to inhibit cytokine production by Thl cells. J Immunol 146:344&3451, 1991. 29 Weaver CT, Hawrylowicz CM, Unanue ER. T helper cell subsets require the expression of distinct costimulatory signals by antigenpresenting cells. Proc Nat1 Acad Sci USA 85:8181-8185, 1988. 30 Go NF, Castle BE, Barrett R et al. Interleukin 10, a novel B cell stimulatory factor: unresponsiveness of X-chromosome-linked immunodeficiency B cells. J Exp Med 172:1625-1631, 1990. 31 Moore KW. Vieira P, Fiorentino DF. Trounstine ML, Khan TA. Mosmann TR. Homology of cytokine synthesis inhibitory factor (IL-IO) to the Epstein-Barr virus gene BCRFI. Science 248:12301234, 1990. 32 Thompson-Snipes L, Dhar V, Bond MW, Mosmann TR, Moore KW. Rennick DM. Interleukin-10: a novel stimulatory factor for mast cells and their progenitors. J Exp Med 173:507-510, 1991. 33 MacNeil IA, Suda T, Moore KW, Mosmann TR, Zlotnik A. IL-IO. a novel growth cofactor for mature and immature T cells. J Immunol 145:41674173. 1990. 34 Suds T, O’Garra A. MacNeil I. Fischer M, Bond M, Zlotnik A. Identification of a novel thymocyte growth promoting factor derived from B cell lymphomas. Cell Immunol 129:228-240, 1990. 35 Chen W-F, Zlotnik A. IL-IO: A novel cytotoxic T cell differentiation factor. J Immunol 147:528-534, 1991. 36 Hsu D-H, Malefyt RdW, Fiorentino DF et al. Expression of interleukin-IO activity by Epstein-Barr virus protein BCRFl. Science 250:830-832, 1990. 37 Slovin SF. Schooley RT, Thorley-Lawson DA. Analysis of cellular immune response to EBV by using cloned T cell lines. J Immunol 130:2127-2132, 1983. 38 Hudson GS, Bankier AT, Satchwell SC, Barrel1 BG. Virology 147:81. 1985. 39 Gately MK, Wilson DE, Wong HL. Synergy between recombinant interleukin-2 (rIL2) and IL-2-depleted lymphokine-containing supernatants in facilitating allogeneic human cytolytic T lymphocyte responses in vitro. J Immunol 136:1274-1282, 1986. 40 Wong HL, Wilson DE, Jenson JC, Familetti PC, Stremlo DL, Gately MK. Characterization of a factor(s) which synergizes with recombinant interleukin-2 in promoting allogeneic human cytolytic T-lymphocyte responses in vitro. Cell Immunol 111:39-54, 1988. 41 Stern AS. Podlaski FJ, Hulmes JD et al. Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells. Proc Nat1 Acad Sci USA 87:6808-6812, 1990.

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42 Gately MK, Desai BB, Wolitzky AG et al. Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-l 2 (cytotoxic lymphocyte maturation factor). J Immunol 147:874-882. 1991. 43 Kobayashi M, Fitz L, Ryan M et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 170:827-845, 1989. 44 Chan SH, Perussia B, Gupta JW et al. Induction of interferon-y production by natural killer cell stimulatory factor: Characterization of the responder cells and synergy with other inducers. J Exp Med 173:869-879, 1991. 45 Gubler U, Chua AO, Schoenhaut DS et al. Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor. Proc Nat1 Acad Sci USA 88:41434147, 1991. 46 Paul SR, Bennett F, Calvetti JA et al. Molecular cloning of a cDNA encoding interleukin 11,a stromal cell-derived lymphopoietic and hematopoietic cytokine. Proc Nat1 Acad Sci USA 87:7512- 7516, 1990. 47 Kawashima I, Ohsumi J, Mita-Honjo K et al. Molecular cloning of cDNA encoding adipogenesis inhibitory factor and identity with interleukin- 1I. FEBS Letters 283: 199-202, 199 1. 48 Bruno E, Briddell RA, Cooper RJ, Hoffman R. Effects of recombinant interleukin-1 1 on human megakaryocyte progenitor cells, Exp Hematol 19:378-381, 1991. 49 Metcalf C. Actions and interactions of G-CSF, LIF and IL-6 on normal and leukemic murine cells. Leukemia 3:3499355, 1989. 50 Musashi M, Yang Y-C, Paul SR, Clark SC, Sudo T. Ogawa M. Direct and synergistic effects of interleukin-1 1 on murine hemopoiesis in culture. Proc Nat1 Acad Sci USA 88:765-769, 1991. 51 Ikebuchi K, Wong GG, Clark SC, Ihle JN, Hirai Y, Ogawa M. Interleukin-6 enhancement of interleukin-3-dependent proliferation ofmultipotential hemopoietic progenitors. Proc Nat1 Acad Sci USA 84:9035-9039, 1987. 52 Leary AG, Wong GG, Clark SC, Ogawa M. Leukemia inhibitory factor (LIF)/differentiation-inducing activity (DIA) is a synergistic factor for human hemopoietic stem cells. Exp Hematol 17:524, 1989. 53 Russell ES. Hereditary anemias of the mouse: a review for geneticists. Adv Genet 20~357459. 1979. 54 Mintz B, Russell ES. Gene-induced embryological modifications of primordial germ cells in the mouse. J Exp 2001 134:207-237. 1957. 55 Mayer TC, Green TC. An experimental analysis of the pigment defect caused by mutations at the Wand SL loci in mice. Dev Biol 18:62-75, 1968. 56 Russell ES, Smith LJ. Lawson FA. Implantation of normal bloodforming tissue in irradiated genetically anemic hosts. Science 124:10761077, 1956. 57 Russell ES, Bernstein SE. Proof of whole cell implant in therapy of W series anemia. Arch Biochem Biophys 125:594-597. 1968. 58 Bernstein SE. Tissue transplantation as an analytic and therdpeutic tool in hereditary anemias. Am J Surg 119:448451, 1970. 59 Harrison DE, Russell ES. The response of W/w and SL/SL” anemic mice to hematopoietic stimuli. Br J Haematol 22: 155-l 68, 1972. 60 Fried W, Chamberlin W, Knopse WH, Husseini S. Trobaug FE, Jr. Studies on the defective hepatopoietic microenvironment of SL/ SLd mice. Br J Haematol 24:643-650, 1973. 61 Chabot B, Stephenson DA, Chapman VM, Besmer P, Bernstein A. The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature 335:88-89. 1988. 62 Geissler EN, Ryan MA, Housman DE. The dominant-white spotting (w) locus of the mouse encodes the c-kit proto-oncogene. Cell 55:185-192. 1988. 63 Yarden Y, Escobedo JA, Kuang W-J et al. Structure of the recep-

64

65 66

67

68

69

70

71

72

73

74 75

76

77

78

79

80

81

82

83

84

tor for platelet derived growth factor helps define a family of closely related growth factor receptors. Nature 3232266232. 1986. Qui F. Ray P, Brown K et al. Primary structure of c-kit: Relationship with the CSF- IiPDGF receptor kinase family-oncogenic activation of v&r involves deletion of extracellular domain and Cterminus. EMBO J 7:100331011, 1988. Williams DE, Eisenman J, Baird A et al. Identification of a ligand for the c-kit proto-oncogene. Cell 63:1677174. 1990. Zsebo KM, Wypych J, McNiece IK et al. Identification. purification, and biological characterization of hematopoietic stem cell factor from Bufalo rat liver-conditioned medium. Cell 63:195%201. 1990. Nocka K, Buck J. Levi E, Besmer P. Candidate ligand for the c-hir transmembrane kinase receptor: KL. a fibroblast-derived growth factor stimulates mast cells and erythroid progenitors. EMBO J 913287.-3294, 1990. Bose11 HS. Mochizuki DY, Burgess GS et al. A novel mast cell growth factor (MCGF-3) produced by marrow-adherant cells that synergizes with interleukin-3 and interleukin-4. Exp Hematol 18:794-800, 1990. Huang E. Nocka K. Beier DR et al. The hematopoietic growth factor KL is encoded by the SL locus and is the ligdnd of the c-h-d receptor, the gene product of the Wlocus. Cell 63:225 233, 1990. Anderson DM. Lyman SD, Baird A et al. Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell 63:235-~243. 1990. Martin FE. Suggs SV, Langley KE et al. Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 63:203321 I. 1990. Zsebo KM. Williams DA, Geissler EN et al. Stem cell factor is encoded at the SL locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63:213-224. 1990. Copeland NG, Gilbert DJ. Cho BC et al. Mast cell growth factor maps near the Steel locus on mouse chromosome 10 and is deleted in a number of &eel alleles. Cell 63: 175 183. 1990. Flangan JG, Leder P. The h-ir ligand: a cell surface molecule altered in S/eel mutant fibroblasts. Cell 63: 185- 194, 1990. Fujita J. Nakayama H. Onoue H et al. Failure of lv/IP mousederived cultured mast cells to enter S phase upon contact with NIHi3T3 fibroblasts. Blood 724633468. 1988. Buchberg AM, Bedigian HG. Taylor BA et al. Localization of Eoi-2 to chromosome 11: linkage to other proto-oncogene and growth factor loci using interspecific backcross mice. Oncogene Res 2:1499165, 1988. Flanagan JG, Chan DC. Leder P. Transmembrane form of the hit ligdnd growth factor is determined by alternative splicing and is missing in the S’ mutant. Cell 64:1025 -1035, 1991. Brannan CI, Lyman SD. Williams DE et al. Steel-Dickie mutation encodes c-kit ligand lacking trdnsmembrane and cytoplasmic domains. Proc Nat1 Acad Sci USA 88:4671 4674. 1991. de Vries P. Brasel KA, Eisenman JR. Alpert AR. Williams DE. The effect of recombinant mast cell growth factor on purified murine hematopoietic stem cells, J Exp Med 173: I205 13 I I, 199 I, Dolci S. Williams DE. Ernst MK et al. Requirement for mast cell growth factor for primordial germ cell survical in culture. Nature 352:809-811. 1991. Godin I. Deed R. Cooke J, Zsebo K. Dexter M, Wylie CC. Effects of the steel gene product on mouse primordial germ cells in culture. Nature 352:8077808. 199 1. Matsui Y. Toksoz D, Nishikawa S et al. Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture. Nature 353:750-752. 1991 Fletcher FA, Moore KA. Ashkenazi M et al. Leukemia inhibitory factor improves survival of retroviral vector-infected hematopoietic stem cells in vitro. allowing efficient long-term expression of vector-encoded human adenosine deaminase in vivo. J Exp Med 174:837-845, 1991. Fletcher FA, Williams DE, Maliszewski C, Anderson D. Rives M, Belmont JW. Murine leukemia inhibitory factor enhances retrovi-

15

85

86

87

88

ral-vector infection efficiency of hematopoietic progenitors. Blood 76:1098-l 103. 1990. Matsui Y, Zsebo KM, Hogan BL. Embryonic expression of a haematopoietic growth factor encoded by the SL locus and the ligand for c-kit. Nature 347:667-669, 1990. Keshet E, Lyman SD, Williams DE et al. Embryonic RNA expression patterns of the c-kit receptor and its cognate ligand suggest multiple functional roles in mouse development. EMBO J 10:2425-2435, 1991. Curtis BM, Williams DE, Broxmeyer HE et al. Enhanced-hematopoietic activity of a human granulocyte/macrophage colony-stimulating factor-interleukin-3 fusion protein. Proc Nat1 Acad Sci USA 88:5809-5813, 1991. Kitamura T. Sato N, Arai K. Miyajima A. Expression cloning of the human IL-3 receptor cDNA reveals a shared /I subunit for the human IL-3 and GM-CSF receptors. Cell 66:116551174, 1991.

89 Cannistra SA, Koenigsmann M, DiCarlo J. Groshek P, Griffin JD. Differentiation-associated expression of two functionally distinct classes of granulocyte-macrophage colony-stimulating factor receptors by human myeloid cells. J Biol Chem 265:12656-12663, 1990. 90 Williams D. unpublished observation. 91 Gearing DP, Bruce GA. Oncostatin M binds the high-affinity leukemia inhibitory factor receptor. The New Biologist in press, 1992. 92 Gearing DP. Comeau MR, Friend DJ et al. The IL-6 signal transfucer, gp130, is an Oncostatin M receptor and affinity convertor for the LIF receptor. Science, in press. 1992.