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Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product

Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked... BRIEF DEFINITIVE REPORT Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product 1 4 1 1 Benoit Pasquier, Luo Yin, Marie-Claude Fondanèche, Francis Relouzat, 1 1,2 1,3 Coralie Bloch-Queyrat, Nathalie Lambert, Alain Fischer, 1 1 Geneviève de Saint-Basile, and Sylvain Latour 1 2 Laboratoire du Développement Normal et Pathologique du Système Immunitaire, Unité INSERM 429, Centre d’Étude des Déficits Immunitaires, and Unité d’Immunologie-Hématologie Pédiatrique, Hôpital Necker Enfants-Malades, 75015 Paris, France International Agency for Research on Cancer, 69372 Lyon, France SAP is an adaptor protein expressed in T cells and natural killer cells. It plays a critical role in immunity, as it is mutated in humans with X-linked lymphoproliferative syndrome (XLP), a fatal immunodeficiency characterized by an abnormal response to Epstein-Barr virus (EBV) infection. SAP interacts with the SLAM family receptors and promotes transduction signal events by these receptors through its capacity to recruit and activate the Src kinase FynT. Because it has been previously established that FynT is selectively required for the development of NKT cells, we examined NKT cells in SAP-deficient mice and in humans with XLP. In the absence of SAP, the development of NKT cells is severely impaired both in mice and in humans. These results imply that SAP is a potent regulator of NKT cell development. They also identify for the first time a defect in NKT cells associated with a human primary immunodeficiency, revealing a potential role of NKT cells in the immune response to EBV. SAP (also named SH2D1A) is a small adaptor survive this initial episode or are asymptomatic CORRESPONDENCE Sylvain Latour: protein that is composed of a unique SH2 do- they go on to develop hypogammaglobunemia latour@necker.fr main with a short COOH-terminal extension. and aggressive lymphoproliferative disorders It is expressed in T cells and NK cells where it such as lymphomas (5). However, the patho- has been found to be essential for the functions physiology of XLP remains poorly understood of the SLAM family of immune receptors (1, although studies using SAP-deficient mice 2). Through its SH2 domain, SAP associates showed that the lack of SAP principally leads to with tyrosine-based motifs located in the cyto- impaired Th2 cell responses and to a defect in plasmic domain of the SLAM family receptors. long-term humoral responses (6–8). SAP couples these receptors to intracellular sig- NKT cells represent a peculiar subpopulation naling pathways by its ability to interact simul- of  T cells with immunoregulatory properties taneously with the Src-related protein tyrosine by their ability to rapidly secrete large amounts kinase FynT (3). This interaction between SAP of cytokines such as IFN-, IL-4, IL-10, and and FynT is direct and involves a second bind- TGF- (9–11). NKT cells express in their ma- ing surface in the SH2 domain of SAP and the jority an invariant TCR (V14-J18/V8 in SH3 domain of FynT (4). The importance of mouse and V24-J18/V11 in humans) that SAP in immunity was provided by the finding reacts with the monomorphic MHC class I–like that the SAP/SH2D1A gene is mutated or de- molecule CD1d. They are also characterized by leted in humans with X-linked lymphoprolifer- expression of receptors of the NK lineage, in- ative disease (XLP), an inherited fatal immune cluding NK1.1 and NK cell inhibitory receptors. dysfunction characterized by a defective immune NKT cells recognized glycolipid antigens pre- response to EBV infection (5). In most of the sented by CD1d (9–11). The sponge-derived cases, affected young boys develop a fulminant glycolipid -galactosyl ceramide (GalCer) infectious mononucleosis with the features of when presented by CD1d selectively activates a hemophagocytic syndrome. When children NKT cells. In contrast to conventional T cells, JEM © The Rockefeller University Press $8.00 695 Vol. 201, No. 5, March 7, 2005 695–701 www.jem.org/cgi/doi/10.1084/jem.20042432 The Journal of Experimental Medicine NKT cells are not subjected to negative selection in the thy- role in NKT cell ontogeny is not known. Because SAP has the mus but are positively selected by CD1d expressed on CD4 / capacity to associate and to activate FynT (4), we examined CD8 thymocytes in the presence of the self-glycolipid iGb3 whether SAP is required for NKT cell development. In this (12, 13). It has been proposed that NKT cells might be im- study, we report that the NKT cell development is severely portant for the initiation and the regulation of immune re- impaired both in mice and humans lacking SAP. This is the sponses by interplaying with innate and adaptive immune re- first report to date describing an inherited fatal immunodefi- sponses (9–11). ciency condition in humans in which NKT cells are lacking. There is accumulating evidence supporting the notion that RESULTS AND DISCUSSION some of the signaling pathways driving NKT cell development are unique and differ from those involved in conventional T Lack of NKT cells in SAP-deficient mice cells and NK cells (11). The Src kinase FynT has been shown We first examined the amount of NKT cells in the different to be required for NKT cell development but not for T cell hemopoietic organs of SAP-deficient mice by flow cytomet- and NK cell differentiation (14, 15), even though its precise ric analysis. The percentages of NKT cells stained by anti- Figure 1. Defect in NKT cells in SAP-deficient mice. NKT cells were mononuclear cells from SAP (black histogram), SAP (white histogram), analyzed by flow cytometry in the liver, spleen, and thymus of wild-type and Fyn mice (gray histogram) stained for NK1.1 and TCR (B) or NK1.1 (SAP ), SAP-deficient (SAP ), Fyn-deficient (Fyn ), and CD1d-deficient and V8.2 TCR (C). Cells were counted in each organ and absolute numbers (CD1d ) mice. (A) Liver lymphocytes and splenocytes were stained with of NKT cells were determined based on their proportion (gated on double low anti-CD19 and thymocytes with anti-HSA. After gating on CD19 or HSA positive cells). Numbers are mean  SD of three mice per group. (D) Same cells, dot plots were constructed. Representative two-color dot plots show experiment as A. Representative two-color dot plots show the staining with staining with anti-TCR versus anti-NK1.1 antibodies. The percentage of anti-TCR antibodies versus GalCer-loaded CD1d tetramers or unloaded NKT cells corresponding to double positive cells in the circle gate is indicated CD1d tetramers (right). The percentage of NKT cells corresponding to double in each plot. The data are representative of at least five mice per group. positive cells in the circle gate is indicated in each plot. The data are repre- (B and C) Absolute numbers of NKT cells in the liver, spleen, and thymus sentative of at least five mice per group. 696 REGULATION OF NKT CELL DEVELOPMENT BY SAP | Pasquier et al. BRIEF DEFINITIVE REPORT TCR and anti-NK1.1 antibodies in the liver, spleen, and thymus of SAP-deficient mice were severely decreased when compared with wild-type (SAP ) mice (Fig. 1 A). This de- crease was similar to that found in Fyn-deficient mice (Fig. 1 A). Similar results were obtained with anti-NK1.1 plus anti- V8.2 antibodies (not depicted). However, the percentages of conventional T cells (TCR NK1.1 ) and NK cells (TCR NK1.1 ) in SAP-deficient and Fyn-deficient mice were comparable to those observed in wild-type mice (Fig. 1 A). Consistent with the decreased proportions of NKT cells in SAP-deficient mice, absolute numbers of these cells in the liver, spleen, and thymus were found to be severely reduced in SAP-deficient and Fyn-deficient mice relative to wild- type mice (Fig. 1, B and C). Moreover, very low amounts of transcripts encoding the V14-J18 TCR rearrangement were detected by semiquantitative RT-PCR in the spleen of SAP-deficient and Fyn-deficient animals when compared with wild-type animals (not depicted). Next, we examined CD1d-restricted NKT cells using CD1d tetramers loaded with GalCer (Fig. 1 D). Although CD1d-restricted NKT cells were easily detected in wild-type (SAP ) mice, a dra- matic reduction of their frequency was observed in SAP- Figure 2. Absence of GalCer-dependent NKT cell responses in SAP-deficient mice. (A) Proliferation of splenic NKT cells in response to deficient, Fyn-deficient, and CD1d-deficient mice. Impor- GalCer or anti-CD3 plus IL-2. Splenocytes of wild-type (SAP ), SAP-deficient tantly, most of the residual TCR GalCer-loaded CD1d (SAP ), and Fyn-deficient (Fyn ) mice were cultured at various cell con- cells found in SAP-deficient, Fyn-deficient, and CD1d-defi- centrations with 100 ng/ml GalCer or in the presence of 3 g/ml of cient mice appeared to be nonspecific staining, as a close pro- immobilized anti-CD3 antibodies plus IL-2. Proliferation of cells was portion of these cells were detected with unloaded CD1d assessed by [ H]thymidine incorporation. Spontaneous proliferation in the tetramers (Fig. 1 D, right, and not depicted). These data absence of Galcer was similar with SAP , SAP , and Fyn splenocytes. indicate that SAP-deficient and Fyn-deficient mice lack Data are presented as a mean  SD of one representative experiment out CD1d-restricted NKT cells. 6 of three. (B and C) IFN- and IL-4 production by splenic NKT cells (10 sple- NKT cells are known to proliferate and produce IFN- nocytes) in response to GalCer or anti-CD3 plus IL-2. Cells were stimu- lated similarly as in A. The presence of IFN- and IL-4 in the supernatants and IL-4 upon engagement of their invariant TCR with was detected by ELISA. In some cases, IFN- and IL-4 were not detectable CD1d-presented GalCer (11). Stimulation of wild-type (*). Data are from one representative experiment representative of three. splenocytes with GalCer resulted in a robust cell prolifera- tion and production of IFN- and IL-4 (Fig. 2, A–C). By contrast, no significant cell proliferation and no production mus, CD1d-restricted NKT cell precursors primarily acquire of IFN- and IL-4 were observed with SAP-deficient and the V14-J18/V8 TCR that allows their subsequent Fyn-deficient splenocytes cultured in the presence of Gal- selection by CD1d-presented self-glycolipid expressed by Cer (Fig. 2, A–C). As control, stimulation with anti-CD3 CD4 CD8 thymocytes (12, 13). Then, NKT cell precur- plus IL-2 induced a strong cell proliferation by wild-type, sors up-regulate CD44 and lastly acquire NK1.1 expression SAP-deficient, and Fyn-deficient splenocytes (Fig. 2 A). during their final maturation (17). Because CD1d-deficient IFN- production in these conditions was found to be com- mice exhibit a profound defect in NKT cell development parable between wild-type and SAP-deficient splenocytes, (18), we ascertained that the defect of NKT cells in the ab- whereas it was slightly decreased with Fyn-deficient spleno- sence of SAP was not caused by a defective CD1d expres- cytes. However, SAP-deficient and Fyn-deficient spleno- sion. Expression of CD1d by thymocytes (Fig. 3 A) and cytes failed to produce IL-4 upon anti-CD3 plus IL-2 splenic T and B cells (Fig. 3 A and not depicted) in SAP-defi- stimulation. These data were consistent with recent studies cient mice was found to be equivalent to that of wild-type showing that activated T lymphocytes from SAP- and Fyn- (SAP ) and Fyn-deficient mice, excluding that a loss of deficient mice have a defect in IL-4 production (6, 16). CD1d expression accounts for the defect of NKT cells ob- Taken together, these results indicate that the compartment served in SAP-deficient mice. Next, we investigated the ex- of NKT cells is selectively and severely impaired in the ab- pression of CD44 and NK1.1 by thymocytes of SAP-defi- sence of the SAP protein. cient mice that were positive for GalCer-loaded CD1d tetramers. As shown above in Fig. 1, only a few thymocytes Early block in NKT cell development in SAP-deficient mice were positive for GalCer-loaded CD1d tetramers in the Next, we examined the developmental steps of NKT cells in SAP-deficient mice in comparison with wild-type (SAP ) SAP-deficient mice. During their development in the thy- mice. Nonetheless, when examined for CD44 and NK1.1 JEM VOL. 201, March 7, 2005 697 Figure 3. Impaired NKT cell development in SAP-deficient mice. histogram) were constructed. Data are from one experiment representative (A) Expression of CD1d on CD4 /CD8 thymocytes (thymus) and spleno- of three. (B) Expression of CD44 and NK1.1 on NKT cell precursors. Thy- cytes (spleen) of wild-type (SAP ), SAP-deficient (SAP ), Fyn-deficient mocytes were stained with anti-NK1.1, anti-CD44, and GalCer-loaded (Fyn ) and CD1d-deficient (CD1d ) mice. After gating on CD4 /CD8 CD1d tetramers. After gating on GalCer-loaded CD1d tetramer cells, thymocytes or TCR splenic cells, histograms corresponding to CD1d two-color dot plots corresponding to the staining of CD44 and NK1.1 were staining (gray histogram) and isotype-matched irrelevant antibodies (white constructed. The percentage of cells in each quadrant is indicated. expression, most of these residual CD1d tetramer cells in detected in the blood of patients with XLP. XLP patients SAP-deficient mice did not up-regulate CD44 (94%), and carrying different mutations in the SAP gene were analyzed none of them expressed NK1.1 in contrast to wild-type and compared with healthy age-matched individuals as well (SAP ) cells (0 vs. 31%, respectively; Fig. 3 B). To confirm as the mother of one patient. None of the XLP patients ex- the developmental arrest of NKT cells in absence of SAP, cept one (patient 4; see Materials and methods) tested in this thymocytes from SAP-deficient mice were compared with study expressed the SAP protein in his PBLs as shown by those of CD1d-deficient mice. The proportions of residual Western blotting of cell lysates performed with anti-SAP an- low CD1d tetramer cells that are CD44 were found to be tibodies (Fig. 4 B and not depicted). The presence of NKT similar in both mice (94 vs. 93%, respectively; Fig. 3 B), sug- cells within the PBLs of an XLP patient (patient 5), his gesting that the block of NKT cell development occurs at a mother, and a healthy age-matched donor was assessed by related stage in SAP-deficient and CD1d-deficient mice. flow cytometry by staining with anti-V24 TCR and anti- Together, these results suggest that the absence of SAP V11 TCR antibodies (Fig. 4 A, left) or with anti-V24 leads to a severe defect in the early steps of NKT cell develop- TCR antibodies and GalCer-loaded CD1d tetramers (Fig. ment before they up-regulate CD44. SAP could be involved 4 A, middle). In the PBLs from the healthy individual and in the intrinsic maturation of NKT cell precursors, the devel- the mother of the XLP patient, NKT cells were significantly opment of the thymic microenvironment, or both. However, detected with both staining reagents. As a control of specific- we could not strictly exclude that the defect may occur in ity, unloaded CD1d tetramers did not identify NKT cells in more mature cells by activation-induced cell death upon the healthy individual nor in the mother of the XLP patient physiological antigen encounter. It is proposed that the main (Fig. 4 A, right). In striking contrast, no NKT cells were function of SAP is to recruit the Src kinase FynT to SLAM found in the PBLs of the XLP patient. To confirm this re- family receptors, allowing their coupling to intracellular path- sult, PBLs from three additional patients with XLP and six ways (19). Thus, one or several members of the SLAM family healthy age-matched donors were analyzed. The proportion receptors might be required for normal NKT cell develop- of NKT cells that were positive for both GalCer-loaded ment. Further studies will be needed to test these possibilities. CD1d tetramers and anti-V24 TCR antibodies ranged from 0.08 to 0.18% (0.11  0.04%) in control donors, Absence of NKT cells in humans with an XLP whereas no detectable NKT cells were observed in XLP pa- Because the lack of SAP is responsible for XLP in humans tients (0.01  0.01%; P 0.001; Fig. 4 C). Similar results (1, 2), we investigated whether NKT cells could be normally were found by using anti-V24 TCR and anti-V11 TCR 698 REGULATION OF NKT CELL DEVELOPMENT BY SAP | Pasquier et al. BRIEF DEFINITIVE REPORT Figure 4. Lack of NKT cells in XLP patients. (A) Representative dot gate is indicated on each dot plot. (B) The absence of SAP protein expression plots showing NKT cells in PBLs from an XLP patient (XLP), his mother, and in cells from an XLP patient. Cell lysates from the same individuals repre- an age-matched healthy donor (Control). After gating on CD3 cells, two- sented in A were analyzed by Western blotting with anti-SAP or anti-Fyn color dot plots showing the staining with anti-V24 TCR and anti-V11 antibodies as loading controls. (C) Percentage of NKT cells (CD3 V24 TCR (left) or anti-V24 TCR antibodies and GalCer-loaded CD1d tetramers TCR GalCer-CD1d tetramer ) in the PBLs of blood samples from four (middle) or unloaded CD1d tetramers (right) were constructed. All of the patients with XLP (XLP) and six age-matched healthy donors (Ctr.). The cells that were V24 TCR GalCer-CD1d tetramer were also found to be bars corresponding to the means of percentages are indicated and * indi- V11 TCR . The percentage of NKT cells (double positive cells) in the circle cates P 0.01. staining (control donors: 0.18  0.08%, n 8; XLP pa- tive syndromes, the Chédiak-Higashi syndrome (CHS), and tients: 0.00%, n 6; P 0.0002; Fig. 5 A). The six XLP the familial hemophagocytic lymphohistiocytosis syndrome patients tested had clinical manifestations that were diverse (FHL; reference 20). In these patients, V24 /V11 TCR but typical of XLP (i.e, fulminant mononucleosis, hemo- NKT cells were significantly detectable (0.09  0.06%, n 3) phagocytic syndrome, hypogammaglobunemia, and lym- compared with XLP patients (0.00%, n 6; P 0.009) and phoma). Whatever these differences, all XLP patients were were found to be similar or slightly reduced relative to found to have this common lack of NKT cells. healthy donors (0.18  0.08%, n 8; P 0.12; Fig. 5 A). To point out that the absence of NKT cells is restricted Thus, the absence of NKT cells in XLP patients appears to to XLP, we further examined NKT cells from patients af- be specific of this immunodeficiency condition. In addition, fected with other primary immunodeficiencies such as the the absence of SAP seems to selectively impair NKT cell de- closely related inherited hemophagocytic lymphoprolifera- velopment because the proportions of NK cells in the PB- Figure 5. Specificity of the NKT cell defect in XLP. (A) Percentages centages of NK cells (CD56 /CD3 ) in the PBMCs of blood samples from of NKT cells (CD3 V24 TCR V11 TCR ) in the PBLs of blood samples the same individuals as in A. The bars corresponding to the means of per- from six patients with XLP (XLP) and eight age-matched healthy donors centages are indicated and * indicates P 0.01. (Ctr.) and three patients with CHS and FHL syndromes (Others). (B) Per- JEM VOL. 201, March 7, 2005 699 cells were first preincubated with anti-FcRII/III antibodies (2.4G2) to MCs of XLP patients (2.9  2.8%, n 6) were comparable block Fc receptors before staining. Finally, cells were analyzed using a to those observed in patients with other immune defects FACSCalibur and CELLQuest software (Becton Dickinson). (4.0  2.0%, n 3; P 0.55) or in healthy individuals (4.6  3.6%, n 7; P 0.34; Fig. 5 B). This is consistent Cell proliferation and cytokine production. Spleen cell suspensions with the normal development of NK cells found in SAP- were incubated in complete medium supplemented or not with 100 ng/ml deficient mice (Fig. 1). GalCer or stimulated with 3g/ml of immobilized anti-CD3 (145-2C11) in the presence of 100 IU/ml of recombinant IL-2. After 36 h in culture, Altogether, these data indicate that in mice and in hu- cells were labeled with [ H]thymidine for 12 h, harvested, and counted in a mans, SAP is required for normal NKT cell development. microbetaplate counter (Wallac). Supernatants were collected after 48 h of NKT cells have been proposed to play critical roles in a vari- stimulation with GalCer and were tested for IL-4 and IFN- contents by ety of immune responses, including host defense against ELISA according to the manufacturer’s instructions (R&D Systems). All as- pathogens, regulation of autoimmunity, and tumor surveil- says were performed in duplicate. lance (10, 11, 21, 22). In this report, we showed that patients Western blot. Immunoblots were performed as described previously (3). suffering from XLP are devoid of NKT cells, and because Polyclonal antibodies to human SAP were produced by immunizing rabbits XLP is a severe immunodeficiency characterized by an ex- with a bacterial fusion protein containing the entire human SAP protein. treme sensitivity to EBV infection, it is tempting to speculate that NKT cells may play an essential function in the control Semiquantitative RT-PCR. The transcripts encoding the V14-J18 of EBV infection. Future studies should be aimed at address- TCR rearrangement were detected by RT-PCR. In brief, 5g of total ing this important issue. RNA was reverse transcripted using random hexamers and Superscript II reverse transcriptase (Invitrogen), and the cDNAs were amplified by PCR MATERIALS AND METHODS using specific primers as described previously (18). Patients. XLP patients have been genotyped for SAP/SH2D1A and were found to be mutated in SAP resulting in the following amino acid changes Statistical analysis. Student’s t tests were performed with InStat software. in SAP: SAP X129R (patient 1), SAP R55X (patient 2), SAP E67G (patient We thank Patrick Revy and Agnès Lehuen for discussions and for their critical 3), and SAP R55P (patient 4). Patient 5 had a deletion of the third exon of reading of the manuscript. We also acknowledge Albert Bendelac and Kamel SAP and patient 6 had a single nucleotide insertion causing a frameshift that Benlagha for the generous gift of reagents. leads to a stop codon. The following are the clinical features of the XLP pa- This work was supported by grants from the Institut National de la Santé et de tients: patient 1 developed hypogammaglobunemia; patients 2, 4, and 6 had la Recherche Médicale, GIS-Institut des Maladies Rares, and the Association pour la a fulminant infectious mononucleosis with a hemophagocytic syndrome; Recherche contre le Cancer (France). S. Latour is a scientist from the Centre National and patients 3 and 5 had lymphoma with hypogammaglobunemia. Two pour la Recherche Scientifique (France). B. Pasquier is a recipient of a fellowship patients with CHS and one patient with FHL who developed hemophago- from La Ligue Contre le Cancer. cytic syndrome were also analyzed. Ages of the individuals ranged from 1 to The authors have no conflicting financial interests. 27 yr old for healthy age-matched donors, 4 to 20 yr old for XLP patients, and 2 mo to 3 yr old for CHS and FHL patients. The mother of patient 5 Submitted: 29 November 2004 was 42 yr old. Patients or families provided informed consent for the study Accepted: 31 January 2005 in accordance with the Declaration of Helsinki. This study was approved by the INSERM Institutional Review Board. REFERENCES 1. Latour, S., and A. Veillette. 2003. Molecular and immunological basis Animals. SAP-deficient (SAP ) mice, Fyn-deficient (Fyn ) mice, and of X-linked lymphoproliferative disease. Immunol. Rev. 192:212–224. CD1d-deficient (CD1d ) mice have been described elsewhere (15, 23, 2. Engel, P., M.J. Eck, and C. Terhorst. 2003. The SAP and SLAM fam- 24). Male SAP-deficient mice and male wild-type (SAP ) littermates were ilies in immune responses and X-linked lymphoproliferative disease. typed by PCR. 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Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product

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Copyright © 2005, The Rockefeller University Press
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0022-1007
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10.1084/jem.20042432
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Abstract

BRIEF DEFINITIVE REPORT Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product 1 4 1 1 Benoit Pasquier, Luo Yin, Marie-Claude Fondanèche, Francis Relouzat, 1 1,2 1,3 Coralie Bloch-Queyrat, Nathalie Lambert, Alain Fischer, 1 1 Geneviève de Saint-Basile, and Sylvain Latour 1 2 Laboratoire du Développement Normal et Pathologique du Système Immunitaire, Unité INSERM 429, Centre d’Étude des Déficits Immunitaires, and Unité d’Immunologie-Hématologie Pédiatrique, Hôpital Necker Enfants-Malades, 75015 Paris, France International Agency for Research on Cancer, 69372 Lyon, France SAP is an adaptor protein expressed in T cells and natural killer cells. It plays a critical role in immunity, as it is mutated in humans with X-linked lymphoproliferative syndrome (XLP), a fatal immunodeficiency characterized by an abnormal response to Epstein-Barr virus (EBV) infection. SAP interacts with the SLAM family receptors and promotes transduction signal events by these receptors through its capacity to recruit and activate the Src kinase FynT. Because it has been previously established that FynT is selectively required for the development of NKT cells, we examined NKT cells in SAP-deficient mice and in humans with XLP. In the absence of SAP, the development of NKT cells is severely impaired both in mice and in humans. These results imply that SAP is a potent regulator of NKT cell development. They also identify for the first time a defect in NKT cells associated with a human primary immunodeficiency, revealing a potential role of NKT cells in the immune response to EBV. SAP (also named SH2D1A) is a small adaptor survive this initial episode or are asymptomatic CORRESPONDENCE Sylvain Latour: protein that is composed of a unique SH2 do- they go on to develop hypogammaglobunemia latour@necker.fr main with a short COOH-terminal extension. and aggressive lymphoproliferative disorders It is expressed in T cells and NK cells where it such as lymphomas (5). However, the patho- has been found to be essential for the functions physiology of XLP remains poorly understood of the SLAM family of immune receptors (1, although studies using SAP-deficient mice 2). Through its SH2 domain, SAP associates showed that the lack of SAP principally leads to with tyrosine-based motifs located in the cyto- impaired Th2 cell responses and to a defect in plasmic domain of the SLAM family receptors. long-term humoral responses (6–8). SAP couples these receptors to intracellular sig- NKT cells represent a peculiar subpopulation naling pathways by its ability to interact simul- of  T cells with immunoregulatory properties taneously with the Src-related protein tyrosine by their ability to rapidly secrete large amounts kinase FynT (3). This interaction between SAP of cytokines such as IFN-, IL-4, IL-10, and and FynT is direct and involves a second bind- TGF- (9–11). NKT cells express in their ma- ing surface in the SH2 domain of SAP and the jority an invariant TCR (V14-J18/V8 in SH3 domain of FynT (4). The importance of mouse and V24-J18/V11 in humans) that SAP in immunity was provided by the finding reacts with the monomorphic MHC class I–like that the SAP/SH2D1A gene is mutated or de- molecule CD1d. They are also characterized by leted in humans with X-linked lymphoprolifer- expression of receptors of the NK lineage, in- ative disease (XLP), an inherited fatal immune cluding NK1.1 and NK cell inhibitory receptors. dysfunction characterized by a defective immune NKT cells recognized glycolipid antigens pre- response to EBV infection (5). In most of the sented by CD1d (9–11). The sponge-derived cases, affected young boys develop a fulminant glycolipid -galactosyl ceramide (GalCer) infectious mononucleosis with the features of when presented by CD1d selectively activates a hemophagocytic syndrome. When children NKT cells. In contrast to conventional T cells, JEM © The Rockefeller University Press $8.00 695 Vol. 201, No. 5, March 7, 2005 695–701 www.jem.org/cgi/doi/10.1084/jem.20042432 The Journal of Experimental Medicine NKT cells are not subjected to negative selection in the thy- role in NKT cell ontogeny is not known. Because SAP has the mus but are positively selected by CD1d expressed on CD4 / capacity to associate and to activate FynT (4), we examined CD8 thymocytes in the presence of the self-glycolipid iGb3 whether SAP is required for NKT cell development. In this (12, 13). It has been proposed that NKT cells might be im- study, we report that the NKT cell development is severely portant for the initiation and the regulation of immune re- impaired both in mice and humans lacking SAP. This is the sponses by interplaying with innate and adaptive immune re- first report to date describing an inherited fatal immunodefi- sponses (9–11). ciency condition in humans in which NKT cells are lacking. There is accumulating evidence supporting the notion that RESULTS AND DISCUSSION some of the signaling pathways driving NKT cell development are unique and differ from those involved in conventional T Lack of NKT cells in SAP-deficient mice cells and NK cells (11). The Src kinase FynT has been shown We first examined the amount of NKT cells in the different to be required for NKT cell development but not for T cell hemopoietic organs of SAP-deficient mice by flow cytomet- and NK cell differentiation (14, 15), even though its precise ric analysis. The percentages of NKT cells stained by anti- Figure 1. Defect in NKT cells in SAP-deficient mice. NKT cells were mononuclear cells from SAP (black histogram), SAP (white histogram), analyzed by flow cytometry in the liver, spleen, and thymus of wild-type and Fyn mice (gray histogram) stained for NK1.1 and TCR (B) or NK1.1 (SAP ), SAP-deficient (SAP ), Fyn-deficient (Fyn ), and CD1d-deficient and V8.2 TCR (C). Cells were counted in each organ and absolute numbers (CD1d ) mice. (A) Liver lymphocytes and splenocytes were stained with of NKT cells were determined based on their proportion (gated on double low anti-CD19 and thymocytes with anti-HSA. After gating on CD19 or HSA positive cells). Numbers are mean  SD of three mice per group. (D) Same cells, dot plots were constructed. Representative two-color dot plots show experiment as A. Representative two-color dot plots show the staining with staining with anti-TCR versus anti-NK1.1 antibodies. The percentage of anti-TCR antibodies versus GalCer-loaded CD1d tetramers or unloaded NKT cells corresponding to double positive cells in the circle gate is indicated CD1d tetramers (right). The percentage of NKT cells corresponding to double in each plot. The data are representative of at least five mice per group. positive cells in the circle gate is indicated in each plot. The data are repre- (B and C) Absolute numbers of NKT cells in the liver, spleen, and thymus sentative of at least five mice per group. 696 REGULATION OF NKT CELL DEVELOPMENT BY SAP | Pasquier et al. BRIEF DEFINITIVE REPORT TCR and anti-NK1.1 antibodies in the liver, spleen, and thymus of SAP-deficient mice were severely decreased when compared with wild-type (SAP ) mice (Fig. 1 A). This de- crease was similar to that found in Fyn-deficient mice (Fig. 1 A). Similar results were obtained with anti-NK1.1 plus anti- V8.2 antibodies (not depicted). However, the percentages of conventional T cells (TCR NK1.1 ) and NK cells (TCR NK1.1 ) in SAP-deficient and Fyn-deficient mice were comparable to those observed in wild-type mice (Fig. 1 A). Consistent with the decreased proportions of NKT cells in SAP-deficient mice, absolute numbers of these cells in the liver, spleen, and thymus were found to be severely reduced in SAP-deficient and Fyn-deficient mice relative to wild- type mice (Fig. 1, B and C). Moreover, very low amounts of transcripts encoding the V14-J18 TCR rearrangement were detected by semiquantitative RT-PCR in the spleen of SAP-deficient and Fyn-deficient animals when compared with wild-type animals (not depicted). Next, we examined CD1d-restricted NKT cells using CD1d tetramers loaded with GalCer (Fig. 1 D). Although CD1d-restricted NKT cells were easily detected in wild-type (SAP ) mice, a dra- matic reduction of their frequency was observed in SAP- Figure 2. Absence of GalCer-dependent NKT cell responses in SAP-deficient mice. (A) Proliferation of splenic NKT cells in response to deficient, Fyn-deficient, and CD1d-deficient mice. Impor- GalCer or anti-CD3 plus IL-2. Splenocytes of wild-type (SAP ), SAP-deficient tantly, most of the residual TCR GalCer-loaded CD1d (SAP ), and Fyn-deficient (Fyn ) mice were cultured at various cell con- cells found in SAP-deficient, Fyn-deficient, and CD1d-defi- centrations with 100 ng/ml GalCer or in the presence of 3 g/ml of cient mice appeared to be nonspecific staining, as a close pro- immobilized anti-CD3 antibodies plus IL-2. Proliferation of cells was portion of these cells were detected with unloaded CD1d assessed by [ H]thymidine incorporation. Spontaneous proliferation in the tetramers (Fig. 1 D, right, and not depicted). These data absence of Galcer was similar with SAP , SAP , and Fyn splenocytes. indicate that SAP-deficient and Fyn-deficient mice lack Data are presented as a mean  SD of one representative experiment out CD1d-restricted NKT cells. 6 of three. (B and C) IFN- and IL-4 production by splenic NKT cells (10 sple- NKT cells are known to proliferate and produce IFN- nocytes) in response to GalCer or anti-CD3 plus IL-2. Cells were stimu- lated similarly as in A. The presence of IFN- and IL-4 in the supernatants and IL-4 upon engagement of their invariant TCR with was detected by ELISA. In some cases, IFN- and IL-4 were not detectable CD1d-presented GalCer (11). Stimulation of wild-type (*). Data are from one representative experiment representative of three. splenocytes with GalCer resulted in a robust cell prolifera- tion and production of IFN- and IL-4 (Fig. 2, A–C). By contrast, no significant cell proliferation and no production mus, CD1d-restricted NKT cell precursors primarily acquire of IFN- and IL-4 were observed with SAP-deficient and the V14-J18/V8 TCR that allows their subsequent Fyn-deficient splenocytes cultured in the presence of Gal- selection by CD1d-presented self-glycolipid expressed by Cer (Fig. 2, A–C). As control, stimulation with anti-CD3 CD4 CD8 thymocytes (12, 13). Then, NKT cell precur- plus IL-2 induced a strong cell proliferation by wild-type, sors up-regulate CD44 and lastly acquire NK1.1 expression SAP-deficient, and Fyn-deficient splenocytes (Fig. 2 A). during their final maturation (17). Because CD1d-deficient IFN- production in these conditions was found to be com- mice exhibit a profound defect in NKT cell development parable between wild-type and SAP-deficient splenocytes, (18), we ascertained that the defect of NKT cells in the ab- whereas it was slightly decreased with Fyn-deficient spleno- sence of SAP was not caused by a defective CD1d expres- cytes. However, SAP-deficient and Fyn-deficient spleno- sion. Expression of CD1d by thymocytes (Fig. 3 A) and cytes failed to produce IL-4 upon anti-CD3 plus IL-2 splenic T and B cells (Fig. 3 A and not depicted) in SAP-defi- stimulation. These data were consistent with recent studies cient mice was found to be equivalent to that of wild-type showing that activated T lymphocytes from SAP- and Fyn- (SAP ) and Fyn-deficient mice, excluding that a loss of deficient mice have a defect in IL-4 production (6, 16). CD1d expression accounts for the defect of NKT cells ob- Taken together, these results indicate that the compartment served in SAP-deficient mice. Next, we investigated the ex- of NKT cells is selectively and severely impaired in the ab- pression of CD44 and NK1.1 by thymocytes of SAP-defi- sence of the SAP protein. cient mice that were positive for GalCer-loaded CD1d tetramers. As shown above in Fig. 1, only a few thymocytes Early block in NKT cell development in SAP-deficient mice were positive for GalCer-loaded CD1d tetramers in the Next, we examined the developmental steps of NKT cells in SAP-deficient mice in comparison with wild-type (SAP ) SAP-deficient mice. During their development in the thy- mice. Nonetheless, when examined for CD44 and NK1.1 JEM VOL. 201, March 7, 2005 697 Figure 3. Impaired NKT cell development in SAP-deficient mice. histogram) were constructed. Data are from one experiment representative (A) Expression of CD1d on CD4 /CD8 thymocytes (thymus) and spleno- of three. (B) Expression of CD44 and NK1.1 on NKT cell precursors. Thy- cytes (spleen) of wild-type (SAP ), SAP-deficient (SAP ), Fyn-deficient mocytes were stained with anti-NK1.1, anti-CD44, and GalCer-loaded (Fyn ) and CD1d-deficient (CD1d ) mice. After gating on CD4 /CD8 CD1d tetramers. After gating on GalCer-loaded CD1d tetramer cells, thymocytes or TCR splenic cells, histograms corresponding to CD1d two-color dot plots corresponding to the staining of CD44 and NK1.1 were staining (gray histogram) and isotype-matched irrelevant antibodies (white constructed. The percentage of cells in each quadrant is indicated. expression, most of these residual CD1d tetramer cells in detected in the blood of patients with XLP. XLP patients SAP-deficient mice did not up-regulate CD44 (94%), and carrying different mutations in the SAP gene were analyzed none of them expressed NK1.1 in contrast to wild-type and compared with healthy age-matched individuals as well (SAP ) cells (0 vs. 31%, respectively; Fig. 3 B). To confirm as the mother of one patient. None of the XLP patients ex- the developmental arrest of NKT cells in absence of SAP, cept one (patient 4; see Materials and methods) tested in this thymocytes from SAP-deficient mice were compared with study expressed the SAP protein in his PBLs as shown by those of CD1d-deficient mice. The proportions of residual Western blotting of cell lysates performed with anti-SAP an- low CD1d tetramer cells that are CD44 were found to be tibodies (Fig. 4 B and not depicted). The presence of NKT similar in both mice (94 vs. 93%, respectively; Fig. 3 B), sug- cells within the PBLs of an XLP patient (patient 5), his gesting that the block of NKT cell development occurs at a mother, and a healthy age-matched donor was assessed by related stage in SAP-deficient and CD1d-deficient mice. flow cytometry by staining with anti-V24 TCR and anti- Together, these results suggest that the absence of SAP V11 TCR antibodies (Fig. 4 A, left) or with anti-V24 leads to a severe defect in the early steps of NKT cell develop- TCR antibodies and GalCer-loaded CD1d tetramers (Fig. ment before they up-regulate CD44. SAP could be involved 4 A, middle). In the PBLs from the healthy individual and in the intrinsic maturation of NKT cell precursors, the devel- the mother of the XLP patient, NKT cells were significantly opment of the thymic microenvironment, or both. However, detected with both staining reagents. As a control of specific- we could not strictly exclude that the defect may occur in ity, unloaded CD1d tetramers did not identify NKT cells in more mature cells by activation-induced cell death upon the healthy individual nor in the mother of the XLP patient physiological antigen encounter. It is proposed that the main (Fig. 4 A, right). In striking contrast, no NKT cells were function of SAP is to recruit the Src kinase FynT to SLAM found in the PBLs of the XLP patient. To confirm this re- family receptors, allowing their coupling to intracellular path- sult, PBLs from three additional patients with XLP and six ways (19). Thus, one or several members of the SLAM family healthy age-matched donors were analyzed. The proportion receptors might be required for normal NKT cell develop- of NKT cells that were positive for both GalCer-loaded ment. Further studies will be needed to test these possibilities. CD1d tetramers and anti-V24 TCR antibodies ranged from 0.08 to 0.18% (0.11  0.04%) in control donors, Absence of NKT cells in humans with an XLP whereas no detectable NKT cells were observed in XLP pa- Because the lack of SAP is responsible for XLP in humans tients (0.01  0.01%; P 0.001; Fig. 4 C). Similar results (1, 2), we investigated whether NKT cells could be normally were found by using anti-V24 TCR and anti-V11 TCR 698 REGULATION OF NKT CELL DEVELOPMENT BY SAP | Pasquier et al. BRIEF DEFINITIVE REPORT Figure 4. Lack of NKT cells in XLP patients. (A) Representative dot gate is indicated on each dot plot. (B) The absence of SAP protein expression plots showing NKT cells in PBLs from an XLP patient (XLP), his mother, and in cells from an XLP patient. Cell lysates from the same individuals repre- an age-matched healthy donor (Control). After gating on CD3 cells, two- sented in A were analyzed by Western blotting with anti-SAP or anti-Fyn color dot plots showing the staining with anti-V24 TCR and anti-V11 antibodies as loading controls. (C) Percentage of NKT cells (CD3 V24 TCR (left) or anti-V24 TCR antibodies and GalCer-loaded CD1d tetramers TCR GalCer-CD1d tetramer ) in the PBLs of blood samples from four (middle) or unloaded CD1d tetramers (right) were constructed. All of the patients with XLP (XLP) and six age-matched healthy donors (Ctr.). The cells that were V24 TCR GalCer-CD1d tetramer were also found to be bars corresponding to the means of percentages are indicated and * indi- V11 TCR . The percentage of NKT cells (double positive cells) in the circle cates P 0.01. staining (control donors: 0.18  0.08%, n 8; XLP pa- tive syndromes, the Chédiak-Higashi syndrome (CHS), and tients: 0.00%, n 6; P 0.0002; Fig. 5 A). The six XLP the familial hemophagocytic lymphohistiocytosis syndrome patients tested had clinical manifestations that were diverse (FHL; reference 20). In these patients, V24 /V11 TCR but typical of XLP (i.e, fulminant mononucleosis, hemo- NKT cells were significantly detectable (0.09  0.06%, n 3) phagocytic syndrome, hypogammaglobunemia, and lym- compared with XLP patients (0.00%, n 6; P 0.009) and phoma). Whatever these differences, all XLP patients were were found to be similar or slightly reduced relative to found to have this common lack of NKT cells. healthy donors (0.18  0.08%, n 8; P 0.12; Fig. 5 A). To point out that the absence of NKT cells is restricted Thus, the absence of NKT cells in XLP patients appears to to XLP, we further examined NKT cells from patients af- be specific of this immunodeficiency condition. In addition, fected with other primary immunodeficiencies such as the the absence of SAP seems to selectively impair NKT cell de- closely related inherited hemophagocytic lymphoprolifera- velopment because the proportions of NK cells in the PB- Figure 5. Specificity of the NKT cell defect in XLP. (A) Percentages centages of NK cells (CD56 /CD3 ) in the PBMCs of blood samples from of NKT cells (CD3 V24 TCR V11 TCR ) in the PBLs of blood samples the same individuals as in A. The bars corresponding to the means of per- from six patients with XLP (XLP) and eight age-matched healthy donors centages are indicated and * indicates P 0.01. (Ctr.) and three patients with CHS and FHL syndromes (Others). (B) Per- JEM VOL. 201, March 7, 2005 699 cells were first preincubated with anti-FcRII/III antibodies (2.4G2) to MCs of XLP patients (2.9  2.8%, n 6) were comparable block Fc receptors before staining. Finally, cells were analyzed using a to those observed in patients with other immune defects FACSCalibur and CELLQuest software (Becton Dickinson). (4.0  2.0%, n 3; P 0.55) or in healthy individuals (4.6  3.6%, n 7; P 0.34; Fig. 5 B). This is consistent Cell proliferation and cytokine production. Spleen cell suspensions with the normal development of NK cells found in SAP- were incubated in complete medium supplemented or not with 100 ng/ml deficient mice (Fig. 1). GalCer or stimulated with 3g/ml of immobilized anti-CD3 (145-2C11) in the presence of 100 IU/ml of recombinant IL-2. After 36 h in culture, Altogether, these data indicate that in mice and in hu- cells were labeled with [ H]thymidine for 12 h, harvested, and counted in a mans, SAP is required for normal NKT cell development. microbetaplate counter (Wallac). Supernatants were collected after 48 h of NKT cells have been proposed to play critical roles in a vari- stimulation with GalCer and were tested for IL-4 and IFN- contents by ety of immune responses, including host defense against ELISA according to the manufacturer’s instructions (R&D Systems). All as- pathogens, regulation of autoimmunity, and tumor surveil- says were performed in duplicate. lance (10, 11, 21, 22). In this report, we showed that patients Western blot. Immunoblots were performed as described previously (3). suffering from XLP are devoid of NKT cells, and because Polyclonal antibodies to human SAP were produced by immunizing rabbits XLP is a severe immunodeficiency characterized by an ex- with a bacterial fusion protein containing the entire human SAP protein. treme sensitivity to EBV infection, it is tempting to speculate that NKT cells may play an essential function in the control Semiquantitative RT-PCR. The transcripts encoding the V14-J18 of EBV infection. Future studies should be aimed at address- TCR rearrangement were detected by RT-PCR. In brief, 5g of total ing this important issue. RNA was reverse transcripted using random hexamers and Superscript II reverse transcriptase (Invitrogen), and the cDNAs were amplified by PCR MATERIALS AND METHODS using specific primers as described previously (18). Patients. XLP patients have been genotyped for SAP/SH2D1A and were found to be mutated in SAP resulting in the following amino acid changes Statistical analysis. Student’s t tests were performed with InStat software. in SAP: SAP X129R (patient 1), SAP R55X (patient 2), SAP E67G (patient We thank Patrick Revy and Agnès Lehuen for discussions and for their critical 3), and SAP R55P (patient 4). Patient 5 had a deletion of the third exon of reading of the manuscript. We also acknowledge Albert Bendelac and Kamel SAP and patient 6 had a single nucleotide insertion causing a frameshift that Benlagha for the generous gift of reagents. leads to a stop codon. The following are the clinical features of the XLP pa- This work was supported by grants from the Institut National de la Santé et de tients: patient 1 developed hypogammaglobunemia; patients 2, 4, and 6 had la Recherche Médicale, GIS-Institut des Maladies Rares, and the Association pour la a fulminant infectious mononucleosis with a hemophagocytic syndrome; Recherche contre le Cancer (France). S. Latour is a scientist from the Centre National and patients 3 and 5 had lymphoma with hypogammaglobunemia. Two pour la Recherche Scientifique (France). B. Pasquier is a recipient of a fellowship patients with CHS and one patient with FHL who developed hemophago- from La Ligue Contre le Cancer. cytic syndrome were also analyzed. Ages of the individuals ranged from 1 to The authors have no conflicting financial interests. 27 yr old for healthy age-matched donors, 4 to 20 yr old for XLP patients, and 2 mo to 3 yr old for CHS and FHL patients. The mother of patient 5 Submitted: 29 November 2004 was 42 yr old. Patients or families provided informed consent for the study Accepted: 31 January 2005 in accordance with the Declaration of Helsinki. This study was approved by the INSERM Institutional Review Board. REFERENCES 1. Latour, S., and A. Veillette. 2003. Molecular and immunological basis Animals. SAP-deficient (SAP ) mice, Fyn-deficient (Fyn ) mice, and of X-linked lymphoproliferative disease. Immunol. Rev. 192:212–224. CD1d-deficient (CD1d ) mice have been described elsewhere (15, 23, 2. Engel, P., M.J. Eck, and C. Terhorst. 2003. The SAP and SLAM fam- 24). Male SAP-deficient mice and male wild-type (SAP ) littermates were ilies in immune responses and X-linked lymphoproliferative disease. typed by PCR. 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The Journal of Experimental MedicinePubmed Central

Published: Mar 7, 2005

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