Immunodeficiencies of genetic origin

capillary bed. They then move along the chemoattractant gradient via chemotaxis to the site of infection or inflammation. Two major subfamilies of chemokines, with four conserved cysteine residues, have now been identified (S. Sozzani, Milan). The (Y subfamily (CXC) is typified by interleukin 8 (IL-S) and comprises nine known proteins, and the l3 subfamily (CC), typified by macrophage chemotactic protein 1 (MU-l), comprises seven known proteins. IL-8 is a neutrophil chemoattractant, whilst MCI’-1 is a major macrophage chemoattractant and is produced by some tumour cells. MCP-1 is also a chemoattractant for T cells and natural killer cells. In addition, a third type of chemokine (C) has recently been identified. The only example at present is lymphotactin, which is specific for T cells. Two different types of receptor for [Y chemokines exist, whilst there are at least four different receptors for p chemokines. These all possess seven transmembrane domains, are G-protein linked and are inhibited by pertussis toxin.

Wortmannin has no effect on chemotaxis induced by II.-8 or fMLI’, but it does inhibit chemotaxis induced by platelet-derived growth factor (PDGF) and transforming growth factor l3 (TGF-@) (M. Thelan. Berne). Chemotaxis in response to PDGF and TGF-l3 is slow and occurs after a lag phase of 1 h. Wortmannin can inhibit this chemotaxis when added at any time during the lag phase. The concentration of inhibitor required for SO’% inhibition of activity (IC,,,) for wortmannin is S-1 0 nM, which is the same as that obtained for inhibition of PI 3-kinase. As neutrophils migrate into skin chambers, they fully mobilize their secretory vesicles, but their three classes of granules are only partly mobilized in a hierarchical manner (H. Sengelov, Copenhagen). These granules are secreted in the order: gelatinase-containing granules > specific granules > azurophilic granules. Conclusions The work presented Workshop has provided

at

this

new

in-

sights into the molecular processes that regulate neutrophil activation in infections and inflammation (as described in the published abstracts)‘. No doubt many of these themes, particularly those involving neutrophil signal transduction systems and assembly of the NADPH oxidase, will be explored in even greater depth at the next Workshop.

Stelgen Edulards and Fiona Watson are at the Dept of Biochemistry, UtC:ersity of Liverpool, PO Box 147, Liverpool, UK L69 3BX. Reference 1 EUY. j. AN-.A33

Clin.

lm~est. 25 (Suppl. 2),

The next Phagocyte Workshop, at the 30th Annual Meeting of the European Society for Clinical Investigation, will take place in Interlaken, Switzerland, on 24-27 April 1996.

Immunodeficiencies of geneticorigin Alain Fischer and Antonio Arnaiz-Villena

Severe combined immunodeficiency (SCID) diseases are genetic disorders in which human lymphocyte development is dismantled. Studies of the murine scid defect have enabled immunologists to approach these diseases as starting points for the identification of key molecules involved in lymphocyte differentiation. Defects in gene rearrangement F. Ah (Boston) reviewed the recent data obtained from the study of the murine scid defect, and also from several cell lines that are unable to rearrange variable (V), diversity (D) and joining (J) segments while being abnormally sensitive to ionizing radiation. Murine scid cells do not properly rearrange

Progress in genetics and the use of new molecular biological tools have resulted in major advances in the field of primary immunodeficiencies over the past two years, including the discovery of new genes. Interest is focused on primary immunodeficiencies as a series of models for the study of development and function of the immune system, as discussed at a recent workshop *. V(D)J elements of T-cell receptor (TCR) and immunoglobulin (Ig) genes. Furthermore, coding joints are not formed whereas signal joints do appear to be formed. In addition, “The Juan March Foundation Meeting on ‘Immunodeficiencies of Genetic Origin’ was held in Madrid, Spain, on h-7’ March 1995.

these cells exhibit a defect in the repair of DNA double-strand breaks. It was previously found that expression of human chromosome 8 could restore DNA repair and the formation of V(D)J coding joints, and it was subsequently observed that scid cells lack a DNAdependent protein kinase (DNAPK,,) activity 1,2. The gene encoding DNA-PK,, is located on chromosome 8qll and its product binds broken ends of DNA following binding of the p86/p70 heterodimeric protein Ku. DNA-PK,, phosphorylates a number of substrates, including pS3 and the products of the recombination-activating genes (RAG), but its precise role in DNA repair and V(D)J rearrangement is

not known. Interestingly, the radiosensitive XTS and sx-1 hamster cell lines have been shown to have defects in the ~86 and p70 subunits of the Ku antigen, respectively. In addition, a gene product (from chromosome 5) that complements the defect of the XR-1 cell line has been identified (Ah). It is also abnormally radiosensitive and does not properly rearrange V(D)J elements after transfection of RAG-Z and RAG-2. A phenotypic equivalent of the murine scid defect seems to exist in humans since Schwartz and colleagues have shown illegitimate D-J rearrangements in pre-B cells3, while abnormal radiosensitivity was observed independently in other patients. The many genes and their products that are involved in the recombinase machinery represent potential candidates to account for such human disease(s). S&palling defects in immunodeficiency X-linked SCID (X-SCID) has been observed in humans and dogs. It is characterized by an early block in T- and natural killer (NK)-cell differentiation while B-cell maturation still proceeds, and results in increased numbers of immature B cells in which activation is partly defective. The gene encoding the y chain of the interleukin 2 (IL-2) receptor (IL-2Ry) is mutated in all X-SCID patients4 (W. Leonard, Bethesda). The phenotype of X-SCID results from defective signalling of several cytokines, including IL-2, IL-4, IL-7, IL-9 and IL-15, all of which act through receptor complexes that contain the y chain. Studies of particular X-SCID cases could be very informative for understanding the function of the y chain. Leonard reported a case where a missense mutation at amino acid residue 271 resulted in a significant reduction of interaction of the Janus-family kinase 3 (JAK3) with the y chain s. Phenotypic variability of X-SCID has been observed (G. de Saint Basile, Paris). An ‘attenuated’ phenotype with detectable and, in part, functional T cells was identified in a patient who had a missense mutation at position 38, resulting in frequent abnormal splicing of exon 2. There was approximately 20% of normal

expression of the y chain on the cells of this patient. In another case, a stop codon in the proximal part of the intracellular domain resulted in a y chain that was devoid of almost all of its intracellular region. Initially, this was diagnosed as a ‘typical’ X-SCID phenotype. However, following a haploidentical bone marrow transplantation that did not engraft, host T cells could be detected for a long period of time and possessed some ability to proliferate when stimulated with antigens. Other cases with the same mutation did not show a similarly variable phenotype. These observations, together with the findings that X-SCID in dogs results in some T-cell differentiation and that y-chain-knockout mice also have some T-cell development6.‘, show that the y-chaininduced phenotype may vary according to genetic background and environmental modifications. Interestingly, thymocytes from y-chain-knockout mice can proliferate in response to certain stimuli’. This raises the question of the existence of an alternative pathway of early T- and NK-cell proliferation. Signals initiated by IL-2, IL-7 and IL-15 involve the signal transducers and activators of transcription STAT-3 and STAT-5, and both IL-2RB and IL-7R share related sequences spanning tyrosines in their cytoplasmic domains that serve as docking regions for STAT proteinsa. The meeting provided the opportunity to compare phenotypes observed in y-chain-knockout mice with those of B-chain-knockout (T. Mak, Toronto) and o-chain-knockout mice (Alt). The y-chain-knockout mice develop massive proliferation of CD4’ T cells. Interestingly, (Ychain-knockout mice also develop lymphoproliferation associated with a severe gut disease and autoimmunity; B-chain-knockout mice have poorly functioning T cells with decreased B-cell counts and myelopoiesis. To evaluate differences and similarities, one needs to take into account not only the distinct spectrum of cytokine association to these receptors, but also the genetic background of individual knockout mice. A description of CD3y deficiency found in two siblings of one family was provided by J. Regueiro (Madrid). Of particular interest

was the reduction in the CD8 + T-cell subset, which suggests possible interaction between CDS and CD3y. By using CD3y- cells transformed by herpes saimiri virus, Regueiro also observed that CD4’ T cells, but not CDS+ T cells, grew in vitro. In addition, the T cells from the patients were mostly CD4+CD45RO+; the explanation for this tinding remains unclear, but it is possible that a majority of permanently activated (memory) T cells is necessary to maintain some functionality. Bruton’s tyrosine kinase and XLA The latest findings concerning the involvement of mutations in the Bruton’s tyrosine kinase (Btk) gene in X-linked agammaglobulinemia (XLA)9*‘0were described by E. Smith (Huddinge). The btk gene is a member of the tyrosine kinase family that also includes tee, itk and bmx. All of these genes are expressed in the haematopoietic system, and bmx is also expressed in endothelial cells, heart and muscle. The proteins possess a proline-rich tee-homology (TH) domain and a pleckstrinhomology (PH) domain in addition to src-homology 2 (SH2), SH3 and kinase domains found in the SYC family of tyrosine kinases. This molecular complexity provides many opportunities for protein-protein interactions. To date, 118 distinct mutations of btk have now been described in XLA (Ref. ll), but few correlations between genotypes and phenotypes have yet been established. Nevertheless, one patient was observed to have a mutation that was associated with typical XLA (i.e. agammaglobulinemia and absence of detectable mature B cells), while a first cousin had residual B-cell populations and some serum Igs. It appears that, in many cases, btk mutations may result in hypogammaglobulinemia with persistence of mature B cells12, and genetic background and environment may modify the XLA phenotype. Interestingly, Alt described the phenotypes of two different btkknockout mice in which either the first two exons (encoding the PH domain) or the kinase domain were inactivated. In both cases, no Btk protein was detectable and the

IYYF

phenotype was similar, comprising a deficiency of CDS- B ceils, low levels of serum IgM and IgG3, absence of an antibody response to thymus-independent type II antigens, while T-cell-dependent antibody responses were mildly impaired. The phenotype was very similar to the X-linked immune deficiency (xid) mouse, which has a missense mutation at amino acid residue 28 of Btk (Ref. 23). Thus, it appears that, for unknown reasons, the Btk- phenotype in the mouse is usually less severe than the human equivalent l‘+. Consequences of Btk deficiencies on the B-cell-maturation pathway have not yet been determined. Pre-B cells from the bone marrow of XLA patients have a reduced intensity of CD19 expression when compared with normal pre-B cells. Furthermore, IL-7 does not downregulate expression of terminal deoxynucleotide transferase, which is in contrast to observations in control pre-B cells (M. Cooper, Birmingham, AL). Since IL-7 was shown to increase expression of CD19 in CD34’ B-progenitor cells, these findings indicate that Btk could be involved in IL--/R-mediated signal transduction. Btk may also be required for CD19 responses, since anti-CD19 antibody blocks the downregulation by IL-7 of expression of RAG-Z and RAG-2 in normal pre-B cells but not in XLA pre-B cells. The list of receptors that may use Btk is thus becoming quite large, and includes FccRI (in mast cells), IgMa, IgMB, IL-SR and IL-1OR (in mice) in addition to IL-7R and CD19. Wiskott-Aldrich syndrome The defective gene in WiskottAldrich syndrome (WASP) has been recently identified by positional cloningls. It encodes a protein of 502 amino acid residues that has no known homologues. A key feature of WAS is the ‘baldness’ of the lymphocytes, which lack microvilli formed by actin bundles (F. Rosen, Boston). This finding was confirmed by I. Molina (Granada), who studied saimiri-virus-transformed T-cell clones from WAS patients. An additional unexplained observation is the instability of expression of sialoglycoproteins (i.e. CD43, CD75,

CD?& CD24, CD37, CD6 and CD23) bv WAS lymphocytes and platelets. Furthermore, proliferative responses to anti-CD3 stimulation of WAS T-cell clones are reduced and IL-2 production is diminished, although early signalling events, such as Cal+ flux, are normal (Molina). Studies of X-chromosome inactivation patterns in obligate carriers has indicated that the gene encoding the WASP defect is already activated in CD34+ haematopoietic progenitor cells, and that this is nonrandom in obligate carriers (Rosen 1. H. Ochs (Seattle) made an attempt to correlate the many tnutations of the WASP gene with WAS phenotypes. Severity of WAS was classified from l-5 depending on the occurrence of thrombocytopenia only (score l), of some mild infections with or without eczema, severe infections, or autoimmunity and lymphomas (score 5). In contrast to other X-linked immunodeficiencies (e.g. X-SCID and XLA), there is a suggestion of possible genotype-phenotype correlation: complex gene rearrangements mostly led to a severe phenotype, while missense mutations, particularly in exon 2, were associated with mild phenotypes. Missense mutations in exons 3-6, 9 and 11 are more often found in patients with severe phenotypes. This information, if confirmed, will help both in patient management and in delineating the functions of WASP, which are, as yet, little understood. WASP contains a proline-rich region that may interact with SH3 domains. By using a polyclonal rabbit anti-WASP antibody, it has been shown that WASP is expressed in the cytoplasm and possibly in the nucleus of thymocytes and B cells transformed by Epstein-Barr virus. Obviously, much more work will be needed to understand the fascinating pathophysiology of WAS, but the tools are now available. Ataxia telangiectasia The search for the defective gene(s) in ataxia telangiectasia (AT) is progressing. Collaborative efforts in genetic and physical mapping have now reduced the gene region at 1 lq23.3 to approximately 500 kb (R. Gatti, Los Angeles). Candidate cDNAs and exons from this region

arc being isolated and screened for mutations by single-stranded conformational polymorphism (SSCP) heteroduplexes and direct DNA sequencing, using RNA and DNA from over 200 patients worldwide. Gatti reminded us that the discovery of the gene(s) responsible for AT will be of interest not only to immunologists (illegitimate recombination of Ig and TCR genes) and neurologists (progressive loss of cerebellum Purkinje cells in AT), but also to radiobiologists and oncologists, since AT cells miss the Ci 1 checkpoint that stops DNA synthesis following irradiation damage and, more importantly, since AT carriers have an increased risk of breast cancer. It is estimated that one woman in five or six who develops breast cancer is a carrier of the defect. Since the meeting, a gene responsible for AT has been identified (ATM); it encodes a product similar to yeast and mammalian phosphatidylinositol 3-kinases, which are involved in signal transduction mediated by mitogens, meiotic recombination and cell-cycle control’“. fas defects in lymphoproliferation A. Fischer (Paris) reported on three patients from two families who exhibit a lymphoproliferative svndrome associated, in one case, with severe autoimmune manifestations. A majority of T cells from peripheral and lymphoid organs were CD4-CDV. This syndrome is strikingly analogous to that of lpr mice, which exhibit a {as deficiency, and a similar deficiency was detected in these patients”. One of the patients has a homozygous deletion in the {as gene that removes the end of the intracytoplasmic domain, while the other two patients have a heterozygous fas mutation inherited from the healthy mother. The father does not exhibit a fas mutation, but has a mild defect in Fas-mediated apoptosis. Hence, it is proposed that the children have double heterozygosity at the fus locus and at an unknown locus, the product of which is involved in Fas-mediated apoptosis. It is speculated that [as mutations could be one genetic factor (among many others) that create susceptibility to autoimmunity because of

IWi

abnormally lymphocytes

prolonged survival in the periphery.

of

Hyper-IgM syndrome CD40-knockout mice do not develop germinal centres, lack IgG2b, IgG2a, IgGl and IgE synthesis, and fail to make antibody to T-celldependent antigen. This is strongly reminiscent of the hyper-IgM syndrome (HIGMl) immunodeficiency, which is caused by deficiency in CD40 ligand (CD40L) expression and function (R. Geha, Boston). The promoter of the gene encoding CD4OL contains four repeated sequences to which the transcription complex NF-AT, with Fos and Jun, can bind. Therefore, it is likely that

NF-AT is a major element controlling CD40L expression. Several proteins are co-precipitated by antiCD40 antibody, including ~63, ~120 and p23 proteins, which are yet to be characterized. p63 may correspond to the recently described CD40-binding protein18, and the amino acid sequence of peptides derived from p23 indicates that it is a novel protein. Leukocyte-adhesion deficiency Available information on the leukocyte-adhesion deficiency (LAD) was summarized by A. Corbi (Madrid). LAD results from impairment of leukocyte adhesion to endothelial cells and possibly other

immunology

Today

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Vol.

target cells. In this disease, the gene encoding B,-integrin is mutated and, consequently, expression of leukocyte function-associated molecule 1 (LFA-l), Mac-l and pl50,95 adhesion molecules is impaired. To date, 18 different mutations have now been described, and these are mostly localized in exons 5 and 6. LAD mainly involves defective transendothelial migration of polymorphonuclear leukocytes (PMNs), while migration of lymphocytes and monocytes is partially preserved. This could be explained by the expression of the very late antigen 4 (VLA-4) integrin by lymphocytes and monocytes but not by PMNs. VLA-4 binds vascular cell

16 No. I I lYq5

adhesion molecule 1 (VCAM-1) on activated endothelial cells, and also mediates binding and transendothelial migration. Thus, functions of VLA-4 and Bz integrins are, in part, redundant. Papillon-Lefevre syndrome The immune deficiency PapillonLefevre syndrome is recessively inherited and is marked by hyperkeratosis of the palms and soles, periodontitis and sometimes susceptibility to infection (A. Arnaiz-Villena, Madrid). A markedly reduced density of CD18 (Bz. integrin), CD2, CD29 and CD45RO on leukocytes has been observed, but it is unknown whether this reflects a primary and common defect that is relevant to the etiology of immune deficiency19. C4A defects Two related conditions, IgA deficiency and common variable immunodeficiency (CVID), were also discussed at the meeting (Cooper; T. Espaiiol, Barcelona). Both conditions comprise a failure of final, selective or global B-cell differentiation. There is a spectrum of phenotypes with an underlying defect that is likely to be common to both diseases. Gene-linkage analysis showed strongest association with the complement 4A (C4A) locus, indicating that the gene encoding C4A (or a nearby gene) is involved in IgA deficiency and CVID. Bare lymphocyte syndrome The elegant work of B. Mach and colleagues (Geneva) on the delineation of the molecular defects underlying deficient expression of HLA class II molecules in immunodeficient patients is reviewed later in this issue of Immunology Todayzo. Readers are referred to this discussion for an update on the major histocompatibility complex (MHC) class II tramactivator (CIITA) and the X-box DNA-binding protein RFXS. First steps in gene therapy Gene therapy trials have been performed in patients with adenosine deaminase (ADA) deficiency (M. Blaese, Bethesda). Two patients had repeated infusions of transduced autologous T cells (11 and 12 infusions, respectively, over a 19-month period). In the first pa-

tient, assessed two years after the last lymphocyte infusion, there is a sustamed number of circulating T cells with an average ADA expression that is half the heterozygous value. This shows persistence of transduced T cells for over two years. Polyclonal integration of the defective gene in ADA was demonstrated, with an average of one proviral copy per cell. ADA’ T-cell clones have also been grown from the patient. Significant improvement in immune function was observed: antigen-specific T-cell proliferation, delayed hypersensitivity skin tests and T-cell cytotoxicity were detected, whereas no adverse effects occurred. However, it is still difficult to draw a firm conclusion on efficacy, since treatment with ADA coupled to polyethyleneglycol is still maintained. In the second patient, results are less impressive since ADA + cells represent less than 1% of total lymphocytes. A second trial was initiated two years ago. Blood from the umbilical cord of three ADA-deficient neonates were used for gene transfer, after isolation of CD34’ cells. After 12-15 months, integration of the provirus is detectable in a low fraction of leukocytes using the same retroviral vector (also containing a gene encoding neomycin resistance). It seems that a detectable fraction of marrow colonies is neomycin resistant, indicating integration and expression. However, it is too early to evaluate the outcome and to know whether integration of the provirus in cord-blood haematopoietic stem cells did occur. Blaese also discussed using foreskin keratinocytes as an ‘enzyme sink’ in an alternative strategy for treating ADA deficiency. Transduction of ADAkeratinocytes in vitro results in massive expression of ADA (500 times that of normal cells), which could serve to detoxify ADA-deficient patients from toxic deoxyadenosine. In summary, although it is likely that primary immunodeficiencies cannot yet be cured by gene transfer, significant results have been achieved in the first studies in viva. Concluding

remarks

The genes responsible for clinically (but not etiologically) characterized immunodeficiencies are

being identified at a remarkable rate (Table 1). Even though the precise functions of many of these remain unknown, this is certainly an important development in the study of primary immunodeficiencies. Alain Fischer is at INSERM U429, HBpital des Enfants-Malades, 149 we de S&es, 75015 Paris, France; Antonio Arnaiz-Villena is at Immunologia, Hospital ‘12 de Octubre’, Universidad Complutense, Ctra. Andalucia, 28041 Madrid, Spain. References 1 Kirchgessner, C.U., Patil, C.K., Evans, J.W. et al. (1995) Science 267, 1178-1183 2 Blunt, T., Finnie, N.J., Taccioli, G.E. et al. (1995) Cell 80, 813-823 3 Schwartz, K., Hansen-Hagge, T.E., Knobloch, C. et al. (1991) J. Exp. Med. 174,1039-1045 4 Noguchi, M., Yi, H., Rosenblatt, H.M. et al. (1993) Cell 3, 147-157 5 Russell, S.M., Johnston, J.A., Noguchi, M. et al. (1994) Science 266, 1042-1045 6 Disanto, J.P., Muller, W., Guy-Grand, D. et al. (1995) Proc. Nat1 Acad. Sci. USA 92,377-381 7 Cao, X., Shores, E.W., Hu-Li, J. et al. (1995) Immunity 2,223-238 8 Lin, J.X., Migone, T.S., Tsang, M. et al. (1995) immunity 2,331-339 9 Vetrie, D., Vorechovsky, I., Sideras, I? et al. (1993) Nature 361,226-233 10 Tsukada, S., Saffran, D., Rawlings, S.D.J. et al. (1993) Cell 72, 279-290 11 Vihinen, M. et al. (1995) Immunol. Today 16,460-465 12 Saffran, D.C., Parolini, O., Fitch-Hilgenberg, M.E. et al. (1994) New Engl. J. Med. 330,1488-1491 13 Thomas, J.D., Sideras, P., Smith, C.I.E. et al. (1993) Science 261, 355-358 14 Rawlings, D.J., Saffran, D.C., Tsukada, S.T. et al. (1993) Science 261, 358-361 15 Derry, J.M.J., Ochs, H.D. and Francke, U. (1994) Cell 78, 635-644 16 Savitsky, K., Bar-Shira, A., Gilad, S. et al. (1995) Science 268, 1749-1753 17 Rieux-Laucat, F., Le Deist, F., Hivroz, C. et al. (1995) Science 268, 1347-I 349 18 Hu, H.M., O’Rourke, K., Boguski, MS. and Dixit, V.M. (1994) /. Biol. Chem. 269,30069-30072 19 Gongora, R., CoreII, A., Regueiro, J.R. et al. (1994) Hum. lmmunol. 41,185-192 20 Reith, W., Steimle, V. and Mach, B. (1995) lmmunol. Today 16,539-546 21 Macchi, I’., Villa, A., Giliani, S. et al. (1995) Nature 377, 65-68