Immunodeficiencies caused by genetic defects in protein kinases

448

lmmunodeficiencies caused by genetic defects in protein kinases Luigi D Notarangelo The recognition

that defects

of JAK3 kinase

in humans

immunodeficiency, these

proteins

and, more recently,

and the demonstration

and other protein-kinase

immunodeficiency,

of ZAP-70

result in severe combined genes

have highlighted

play in T-cell differentiation

that targeting

of

in mice also leads to the crucial

role that these

and activation.

Addresses Department of Pediatrics, University of Brescia, c/o Spedali Civili 25123, Brescia, Italy; e-mail: [email protected] Current Opinion

in Immunology 1996, 8448-453

0 Current Biology Ltd ISSN 0952-7915 Abbreviations D diversity BCR B-cell receptor dsb double strand break IL interleukin IR ionizing radiation ITAM immunoreceptor tyrosine-based associated motif J joining JAK homology domain JH JAK Janus associated kinase natural killer NK Pl3-K phosphatidylinositol 3-kinase PK protein kinase PTK protein tyrosine kinase SCID severe combined immunodeficiency STAT signal transducers and activators of transcription TCR T-cell receptor V variable

Introduction Protein kinases play a crucial role in regulating T-cell differentiation and activation processes. Antigen-receptor signalling involves activation of the src related protein tyrosine kinases (PTKs) Ick and fyn, as well as of ZAP-70 and Syk (reviewed in [l-3]), whereas T-cell activation induced by cytokines is mediated by recruitment of a novel class of protein kinases termed JAKs (Janus associated kinases) [4,5]. More recently, a critical role for protein kinases has also been discovered in the repair of double-strand breaks of the DNA and correct V(D)J recombination (reviewed in [6]). This review will focus on the characterization (mostly achieved during the past year) of T cell protein kinase defects, illustrated by naturally occurring immunodeficiencies in humans or by gene-targeting models in mice.

lmmunodeficiencies resulting from defects of PTKs involved in antigen receptor signalling During T-cell differentiation and activation processes, signalling via the TCR-CD3 complex activates the CD4- (or CD%) associated PTK p561ck; this catalyzes

tyrosine-phosphorylation based chains, PTKs, T-cell

of the immunoreceptor

tyrosine-

associated motifs (ITAMs) of the CD3& and 5 creating docking sites for the SHZ-containing Syk and ZAP-70 [l-3]. The crucial role of Ick in differentiation and activation has been addressed

through gene-targetted l’ckl- mice, that show a severe and early block in thymocyte differentiation, with arrested transition from double negative to double positive cells [7]. Similar defects have been observed in transgenic mice that express a dominant negative form of lck [8]. The proliferative response to TCR cross-linking is marginally affected in the small number of peripheral T cells that are generated, but cytotoxic T lymphocyte responses are impaired [9]. Furthermore, activation of T cells recognizing class I- and class II-restricted antigens is severely defective [lo]. These data indicate a critical role for Ick both in thymocyte differentiation and in mature T-cell function. p59fyn has also been implicated in CD3-TCR signalling [ 111. Maturation of T cells is, however, marginally affected in jjn-/mice [l&13], and the proliferative response to TCR cross-linking is reduced in thymocytes, but largely normal in mature T cells. Tyrosine phosphorylation of the antigen receptor ITAM by src PTKs creates docking sites for Syk and ZAP-70. The interaction of ZAP-70 with the 5 chain requires that both SH2 domains of ZAP-70 bind to the doubly tyrosine-phosphorylated ITAMs [ 14’1. Defective expression or function of ZAP-70 in humans results in a severe T-cell defect, with a lack of mature CD8+ T cells [15-171. In the thymus, migration of CD8+ thymocytes into the medulla is absent, reflecting a defect in positive selection. Although peripheral blood CD4+ lymphocytes are present and their TCR repertoire is not restricted, they are unresponsive to mitogens and to anti-CD3 plus anti-CD4 cross-linking. The preserved generation of CD4 single positive thymocytes has been attributed to the compensatory role of Syk in the thymus; however, expression of Syk is drastically reduced in mature T cells [18], thus preventing functional compensation in peripheral CD4+ T cells. Zap-7&l- mice, generated by gene targeting [19”], present a severe defect in intrathymic differentiation and positive selection, with inability to generate both CD4 and CD8 single positive cells, indicating that defects in ZAP-70 cannot be compensated for by Syk in this system. In addition, the negative selection process is also disturbed, as double positive thymocytes from zap-70-imice that carry a transgene for the chicken ovalbumin peptide are not deleted in the presence of the specific peptide. Finally, both generation and function of natural

Genetic defects in protein kinases Notarangelo

killer (NK) cells are preserved deficient in ZAP-70 [17,19”].

in both

humans

and mice

The effects of disruption of the Qk gene have recently been addressed in gene-targeted mice [ZO*,Zl*]The phenotype is characterized by severe, although transient, embryonal haemorrhages and perinatal death. To assess the impact of Syk deficiency on lymphocyte development and function, radiation chimeric mice have been generated by injecting fetal liver cells of sykl- mice into lethally, or sub-lethally, irradiated RAG-deficient mice: B-cell development was found to be severely affected, with arrest at the late pro-B stage, and lack of positive selection and expansion of cells that productively rearrange heavy chain genes, expressed in association with variable (V),,,_B and h5 [Zl’]. This suggests that Syk is involved in pre-BCR signalling. In contrast, no effects on T-cell development have been observed, arguing for a secondary role of Syk in the thymus, as opposed to ZAP-70. Finally, other PTKs are also involved in T-cell signalling. Itk belongs to the Btk family of non-src PTKs [ZZ]; it is induced by IL-Z stimulation, and is phosphorylated and activated following T-cell stimulation with anti-CD3 or anti-CD28 antibodies [23]. I@mice [24*] show a decreased number of CD4 single positive thymocytes; a low CD4+:CD8+ ratio is also observed in the spleen. The proliferative response to mitogens and anti-CD3 is reduced, but can be increased by exogenous IL-Z, and is normal following stimulation with phorbol esters and ionomycin. These data suggest that Itk plays a role in the generation of single positive (mainly CD4+) cells in the thymus, and in the membrane-proximal signal transduction events in mature T cells.

lmmunodeficiencies defects

resulting from JAK3

Cytokines are crucial molecules that are involved in the regulation of cell growth and differentiation and mediate their activity through interaction with cytokine receptors and subsequent cellular protein phosphorylation. Members of the cytokine receptor superfamily do not have intrinsic kinase activity, but recruit and activate intracellular kinases following cytokine binding [4,5]. The tyrosine kinases that couple cytokine binding to tyrosine phosphorylation of protein substrates, and eventually to cell growth and differentiation, are members of the JAK family of protein kinases. At present, four members of the JAK family are known in mammals: JAKl, JAKZ, JAK3 and Tyk2. JAKs share a similar structure, with a carboxy-terminal kinase domain (JAK homology domain 1, JHl), a kinase-like domain (JHZ) and five other regions of homology (JH3-JH7) whose function is unknown. In contrast to the other JAKs, JAK3 is largely restricted to the hematopoietic system [ZS], although a splice variant devoid of kinase activity has recently been identified in epithelial cells [26]. Receptors for IL-Z, IL-4, IL-7, IL-9 and IL-15 share a common y chain (yc). JAKl and JAK3

449

are functionally coupled to these yc user receptors; in particular, JAK3 is associated with the yc chain [27,28], and plays a primary role in IL-Z induced cell growth [27,29*]. Upon cytokine binding, the cytoplasmic tail of the cytokine receptor dimerizes, and brings the JAKs into close proximity, allowing for their cross-phosphorylation. JAKs also mediate tyrosine phosphorylation of the cytokine receptor chains, generating docking sites for SHZ-containing proteins. Several signalling pathways can be elicited following activation of JAK [4,5], including: first, the recruitment and activation of the SHC-GrbZ-Sos-Ras-Rafl-MAPK signalling pathway, leading to transcription of early/immediate genes (c-fos, cjwz); second, the recruitment and activation of the signal transducers and activators of transcription (STATS). Following JAKs activation, STATS bind to the phosphorylated cytokine receptor, are themselves phosphorylated, they then dimerize, and are translocated to the nucleus, where they bind to the enhancer elements of target genes, driving gene transcription and cell differentiation [30-341: third, phosphorylation of insulin receptor substrates (IRSs), followed by recruitment and activation of phosphatidylinositol 3-kinase (PI3-K), thus providing a crucial signal for cell growth [35]. The need for JAK3 in cell proliferation induced by IL-2 has been illustrated by the observation that in the NIH3T3aPy cell line (expressing the three IL-2 receptor chains, JAKl and JAK2, but not JAK3) IL-2 induces progression to the S phase of the cell cycle only if JAK3 is also cotransfected [4,27]. The requirement for integrity of JAK3 structure in regulating cell growth and differentiation has been indicated by the fact that JAK3 mutants lacking the JH3-JH7 domains are unable to interact with yc, whereas mutants that lack the JHl kinase domain may bind to the yc, but are unable to transduce activation signals in response to IL-2 [4]. Mutations of the IL2RG (encoding the yc) are responsible for X-linked severe combined immunodeficiency (X-SCID) in humans [36], characterized by lack of circulating T cells and a normal to increased number of B lymphocytes (T-B+SCID), and targeting of the murine homolog gene also results in SCID, albeit with a different phenotype [37**,38”]. Milder forms of X-SCID in humans were shown to result because of point mutations in the cytoplasmic tail of the yc, that diminish, but not abolish, interaction with JAK3, leading to the hypothesis that mutations of JAK3 might account for autosomal T-B+SCID [28]. This hypothesis has proven correct. To date, three children with T-B+ autosomal SCID resulting from defects in JAK3 have been reported in unrelated families (Fig. 1); in all cases, mutation of the JAK3 gene resulted in absent or markedly reduced levels of JAK3 protein [39”,40”]. Patients deficient in JAK3 have almost undetectable T cell levels and lack NK lymphocytes in the periphery; proliferative responses to mitogens in vitro, anti-CD3, and allogenic cells are also severely reduced or absent. Similar to X-SCID, the

450

Genetic effects on immunity

discrepancy in the B-cell defect between JAK3- (or F-) deficient humans versus mice suggests that integrity of the yclJAK3 signalling pathway is more crucial to murine than to human B-cell differentiation, perhaps reflecting a more critical role for IL-7 in the murine system.

of B cells is normal or even increased; however, severe hypogammaglobulinemia is present. Although this may also reflect lack of T-cell help, a primary B-cell defect has been indicated by the inability of IL-4 to induce activation of STAT6 in the Epstein-Barr virus (EBV) transformed B-cell line from one such patient [40”]. proportion

Immunodeficiencies defects

J&3-/- mice have been recently generated [41**43**], and their phenotype resembles that observed in yc-/’ mice. The thymus is hypoplastic, but, despite the severe cellularity defect, all thymocyte subpopulations are recognizable, suggesting that defect in JAK3 does not result in ablation of intrathymic differentiation once thymocyte progenitors are seeded [43**]. Similar to ye-/y mice [38”], an excess of CD4+ versus CDS+ single positive thymocytes is observed with time, suggesting that JAK3 is less critical for maturation and proliferation of the CD4 subset than it is for CD8+ thymocytes. In spite of an overall reduced T-cell cellularity, an excess of CD4+ T cells is also observed in the spleen; these cells may co-express activation antigens (e.g. CD44, CD69 and CD25 [41”]) and respond poorly to mitogen stimulation, thus behaving as anergic cells. This observation is interesting in view of the evidence that stimulation of T cells through their CD3-TCR complex results solely in anergy or unresponsiveness, and that activation of JAK3 is required for the prevention of anergy [44]. Peripheral lymph nodes are nearly undetectable in J&3-/-‘ mice, and lack of dendritic epidermal T cells, intraepithelial lymphocytes and NK cells is also observed. Similar to ye-m mice, and in contrast to X-SCID and JAK3-deficient humans, Jailmice have a profound defect in B-cell differentiation, as revealed by a greatly reduced number of B lymphocytes and pre-B (B220+ CD43-) cells in the bone marrow, in spite of a normal number of pro-B cells. Severe B-cell defect is also observed in the spleen; although mature B lymphocytes may occasionally be seen in older mice, yet no STAT6 binding activity is induced in response to phorbol esters and IL-4 [42”]. The

resulting from DNA-PK

Repair of DNA double strand breaks (dsbs) is a crucial cellular event, as dsbs are highly recombinogenic and potentially mutagenic during DNA synthesis and cell mitosis processes [6]. However, the generation of dsbs is a necessary step during TCR (or Ig) gene rearrangement. A crucial example of the importance of dsb repair mechanisms was offered by the characterization of SCID mice which lack mature B and T cells because of defective V(D)J recombination present increased sensitivity to ionizing radiations (IRS), and are deficient in dsb repair [45]. In addition, a number of mutant rodent cell lines have been reported that associate defects of V(D)J rearrangement with increased sensitivity to IRS [6]. On the basis of cell fusion experiments, different complementation groups have been established for these mutants: moreover, transfer of human chromosomes into the mutant rodent cells has defined that distinct chromosomes can complement the defect in specific complementation groups [6]. DNA-PK is a heterotrimeric complex consisting of two smaller subunits of 70 and 80 kDa (collectively named Ku), that bind the DNA ends, and of a larger catalytic subunit of 450 kDa [6]. The gene encoding ~450 DNA-PK has been cloned recently [46*,47], and shares homology with PI3-K. In contrast to PI3-K, however, the ~450 subunit of DNA-PK is predicted not to have lipid, but rather serine/threonine protein kinase activity. The ~450 subunit catalyzes phosphorylation of p70 and ~80 Ku proteins, and might also recruit other proteins (e.g. RAG-l, RAG-Z and DNA ligases) involved in dsb repair and V(D)J recombination [6].

Figure 1 Representation of JAKB mutations in three patients with SCID resulting from JAK3 deficiency. Patients 1 and 2 are reported in [39”1, and were homozygous for the respective mutation; patient 3 is reported in [40”1. For each mutation, the nucleotide changes in the JAKB cDNA (as compared to the sequence deposited in GenBank) are indicated on the left of the arrow, and the corresponding effects at the protein level are shown on the right of the arrow. The JAK homology (JH) domains along the JAKB protein are also shown and are labelled beneath the figure. The three-letter code for amino acids is used. (Ins, insertion; Fs, frameshift; Del nt, nucleotide deletion).

Patient 1 A394G

+Tyrl

Patient 2

OOCys

Del nt 2295-2445

+Fs

Patient 3 Ins G1269

+Fs

/

Cl 790A Xys565stop

JH7

JH6

JH5

JH4

JH3

JH2

JHl Q 1996 Current Op~mon tn Immunology

Genetic defects in protein kinases Notarangelo

It has been shown that cells from SCZD mice have defective phosphorylation of Ku proteins; human chromosome 8, which contains the ~4.50 DNA-PK gene, can reconstitute Ku phosphorylation and V(D)J recombination in murine SC/D cells [48’,49*]. Furthermore, yeast artificial chromosomes containing the ~450 DNA-PK gene can complement sensitivity to IRS and V(D)J recombination in the mutant rodent cell line V-3, that belongs to the same complementation group that SCZD mice have been assigned to [SO’]. Finally, the murine homolog of human ~4.50 DNA-PK has been assigned to chromosome 16, to which the murine SCID locus had been mapped previously [Sl]. Taken together, these data support the notion that defects in the ~450 DNA-PK are responsible for murine SC/D.

In addition, deficiency of Ku DNA-end-binding has been reported in some other IR sensitive mutant rodent cell lines [6,52,53]; introduction of the Ku p80 gene complements all dsb repair defects in these mutants [53,54]. Within the same complementation group, some mutant cell lines were shown to express a mutated Ku ~80, whereas others failed to produce the specific transcript. Therefore, it is likely that mutations in the various subunits of DNA-PK may also lead to human immunodeficiencies with a similar phenotype of defective V(D)J recombination and increased sensitivity to IR. Indeed, abnormalities of V(D)J recombination have been reported in B-negative SCID [SS]; furthermore, some variants of autosomal recessive SCID and Omenn syndrome present increased sensitivity to IR [56].

Acknowledgements I wish to thank manuscript.

Raffaele

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Badolato for thoughtful

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Conclusions During the past year, identification of JAK3 mutations in humans with autosomal T-B+SCID, and generation of JAK3 gene-targeted SCID mice, have illustrated the crucial and non-redundant role that this kinase plays in cytokine-induced lymphocyte differentiation and activation. However, recognition that not all forms of T-B+ autosomal SCID in humans result from JAK3 defects (LD Notarangelo, unpublished data), suggests that other genes, possibly along the same signalling pathway (e.g. IL-7 and STATS) may be involved. Furthermore, as gene targeting of some T cell protein kinases in mice results in milder immunological phenotype (as for itkl- mice), it is possible that similar abnormalities may account for combined immunodeficiencies in humans (as opposed to SCID). Finally, it is now possible to assess if and which forms of T-BSCID in humans result from defects of dsb repair genes (including DNA-PK), or of other genes involved in V(D)J recombination, such as RAG-l and RAG-Z. Therefore, it can be expected that the molecular pathogenesis of novel immunodeficiencies in humans will be unravelled over the next months, further adding to the knowledge of the critical role that protein kinases play in T-cell development and function.

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of

Genetic

phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product Cell 1995, 82:849-856. This paper illustrates cloning of the human ~450 DNA-PK gene, which shares homology with the ATM gene (mutated in ataxia-telangiectasia) and with the gene encoding Pl3-K. Biochemical studies demonstrate that ~450 DNA-PK is devoid of lipid kinase activity, whereas it has protein kinase activity. 47.

Sipley JD, Menninger JC, Hartley KO, Ward DC, Jackson SP, Anderson CW: Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8. Proc Nat/ Acad Sci USA 1995, 92:7515-7519.

46. .

Boubnov NV, Weaver DT: SC/D cells are deficient in Ku and replication protein A phosphorylation by the DNA-dependent protein kinase. MO/ Cell Biol 1995, 15:5700-5706. The authors demonstrate that murine SC/D cells are deficient in the ability to phosphorylate Ku and replication protein A, two events dependent on DNAPK. Introduction of human chromosome 6, expressing the DNA-PK catalytic subunit, reverts the phenotype. 49. .

Kirchgessner CU, Patil CK, Evans JW, Cuomo CA, Fried LM, Carter T, Oettinger MA, Brown JM: DNA-dependent kinase (~350) as a candidate gene for the murine SCID defect. Science 1995, 287:i 176-l 182. This paper describes that ~450 DNA-PK protein levels are diminished in cells from SC/D mice; complementation studies showed that somatic-cell hybrids retaining human chromosome 8 (containing the ~450 DNA-PK locus) were restored in V(D)J recombination and sensitivity to radiation. Blunt T, Finnie NJ, Taccioli GE, Smith GCM, Demengeot J, Gottlieb TM, Mizuta R, Varghese Al, Alt FW, Jeggo PA, Jackson SP: Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 1995, 80:813-823. Defective activity of DNA-PK in SC/D mice and in the V3 mutant hamster ceil line results from deficiency in the DNA-PK catalytic subunit; addition

defects

in protein

kinases

Notarangelo

453

of purified DNA-PK catalytic subunit to V3 or SC/D cell extracts increased DNA-PK activity. Furthermore, yeast artificial chromosomes containing the DNA-PK catalytic subunit complemented DNA repair and recombination defects of V3 cells. 51.

Mil!er RD, Hogg J, Ozaki JH, Gell D, Jackson SP, Riblet R: Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the SCID locus. Proc Nat/ Acad Sci USA 1995, 92:i 0792-l 0795.

52.

Finnie NJ, Gottlieb TM, Blunt T, Jeggo PA, Jackson SP: DNAdependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA doublestrand break repair. Proc Nat/ Acad Sci USA 1995, 92:320-324.

53.

Boubnov NV, Hall KT, Wills Z, Lee SE, He DM, Benjamin DM, Pulaski CR, Band H, Reeves W, Hendrickson EA, Weaver DT: Complementation of the ionizing radiation sensitivity, DNA-end binding, and V(D)J recombination defects of double-strand break repair mutants by the p88 Ku autoantigen. Proc Nat/ Acad Sci USA 1995, 92:890-894.

54.

Smider V, Rathmell WK, Lieber MR, Chu G: Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science 1994, 266:288-291.

55.

Schwarz K, Hansen-Hugge TE, Knobloch C, Friedrich W, Kleihauer E, Bartram C: Severe combined immunodeficiency (SCID) in man: B cell-negative (B-j SCID patients exhibit an irregular recombination pattern at the JH locus. J Ocp Med 1991, 174:1039-1048.

56.

Cavazzana-Calve M, Le Deist F, De Saint Basile G, Papadopoulo D, De Villartay JP, Fischer A: Increased radiosensitivity of granulocyte macrophage colony-forming units and skin fibroblasts in human autosomal recessive severe combined immunodeficiency. J C/in /west 1993, 91 :1214-l 218.

50. .