SLAM receptors foster iNKT cell development by reducing TCR signal strength after positive selection.

Invariant natural killer T cells (iNKT cells) develop through an incompletely understood process that requires positive selection by CD4+CD8+ double-positive thymocytes and SLAM family receptors (SFRs). Here we found that SFRs promoted the development of iNKT cells by reducing the strength of the T cell antigen receptor (TCR) signal after positive selection. This effect improved the survival of iNKT cells and their responses to antigen. Loss of SFRs upregulated the expression of inhibitory receptors, including PD-1, on iNKT cells to mitigate the deleterious effect of SFR deficiency. The role of SFRs could be mimicked by expression of SLAMF6 alone in SFR-deficient mice, and this involved the adaptor SAP-kinase Fyn complex and the phosphatase SHP-1. Thus, SFRs foster iNKT cell development by attenuating TCR signal strength after positive selection.

Severe adult hemophagocytic lymphohistiocytosis (HLHa) correlates with HLH-related gene variants.

BACKGROUND: The contribution of genetic factors to the severity of adult hemophagocytic lymphohistiocytosis (HLHa) remains unclear. OBJECTIVE: We sought to assess a potential link between HLHa outcomes and HLH-related gene variants. METHODS: Clinical characteristics of 130 HLHa patients (age ≥ 18 years and HScore ≥ 169) and genotype of 8 HLH-related genes (LYST, PRF1, UNC13-D, STX11, STXBP2, RAB27A, XIAP, and SAP) were collected. A total of 34 variants found in only 6 genes were selected on the basis of their frequency and criteria predicted to impair protein function. Severity was defined by refractory disease to HLH treatment, death, or transfer to an intensive care unit. RESULTS: HLHa-associated diseases (ADs) were neoplasia (n = 49 [37.7%]), autoimmune/inflammatory disease (n = 33 [25.4%]), or idiopathic when no AD was identified (n = 48 [36.9%]). Infectious events occurred in 76 (58.5%) patients and were equally distributed in all ADs. Severe and refractory HLHa were observed in 80 (61.5%) and 64 (49.2%) patients, respectively. HScore, age, sex ratio, AD, and infectious events showed no significant association with HLHa severity. Variants were identified in 71 alleles and were present in 56 (43.1%) patients. They were distributed as follows: 44 (34.4%), 9 (6.9%), and 3 (2.3%) patients carrying 1, 2, and 3 variant alleles, respectively. In a logistic regression model, only the number of variants was significantly associated with HLHa severity (1 vs 0: 3.86 [1.73-9.14], P = .0008; 2-3 vs 0: 29.4 [3.62-3810], P = .0002) and refractoriness (1 vs 0: 2.47 [1.17-5.34], P = .018; 2-3 vs 0: 13.2 [2.91-126.8], P = .0003). CONCLUSIONS: HLH-related gene variants may be key components to the severity and refractoriness of HLHa.

Overexpression of SH2D1A promotes cancer progression and is associated with immune cell infiltration in hepatocellular carcinoma via bioinformatics and in vitro study.

BACKGROUND: SH2 domain containing 1A (SH2D1A) expression has been linked to cancer progression. However, the functions of SH2D1A in hepatocellular carcinoma (HCC) have not been reported. METHODS: The effects of SH2D1A on the proliferation, migration, and invasion of HCC cells and the related pathways were re-explored in cell models with SH2D1A overexpression using the CCK-8, migration and invasion assays and western blotting. The functions and mechanisms of genes co-expressed with SH2D1A were analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The relationship between SH2D1A expression and immune microenvironment features in HCC was explored. RESULTS: Elevated SH2D1A expression promoted cell proliferation, migration, and invasion, which was related to the overexpression of p-Nf-κB and BCL2A1 protein levels in HCC. SH2D1A expression was related to the immune, stromal, and ESTIMATE scores, and the abundance of immune cells, such as B cells, CD8+ T cells, and T cells. SH2D1A expression was significantly related to the expression of immune cell markers, such as PDCD1, CD8A, and CTLA4 in HCC. CONCLUSION: SH2D1A overexpression was found to promote cell growth and metastasis via the Nf-κB signaling pathway and may be related to the immune microenvironment in HCC. The findings indicate that SH2D1A can function as a biomarker in HCC.

Overexpression of the PTPN22 Autoimmune Risk Variant LYP-620W Fails to Restrain Human CD4+ T Cell Activation.

A missense mutation (R620W) of protein tyrosine phosphatase nonreceptor type 22 (PTPN22), which encodes lymphoid-tyrosine phosphatase (LYP), confers genetic risk for multiple autoimmune diseases including type 1 diabetes. LYP has been putatively demonstrated to attenuate proximal T and BCR signaling. However, limited data exist regarding PTPN22 expression within primary T cell subsets and the impact of the type 1 diabetes risk variant on human T cell activity. In this study, we demonstrate endogenous PTPN22 is differentially expressed and dynamically controlled following activation. From control subjects homozygous for the nonrisk allele, we observed 2.1- (p < 0.05) and 3.6-fold (p < 0.001) more PTPN22 transcripts in resting CD4+ memory and regulatory T cells (Tregs), respectively, over naive CD4+ T cells, with expression peaking 24 h postactivation. When LYP was overexpressed in conventional CD4+ T cells, TCR signaling and activation were blunted by LYP-620R (p < 0.001) but only modestly affected by the LYP-620W risk variant versus mock-transfected control, with similar results observed in Tregs. LYP overexpression only impacted proliferation following activation by APCs but not anti-CD3- and anti-CD28-coated microbeads, suggesting LYP modulation of pathways other than TCR. Notably, proliferation was significantly lower with LYP-620R than with LYP-620W overexpression in conventional CD4+ T cells but was similar in Treg. These data indicate that the LYP-620W variant is hypomorphic in the context of human CD4+ T cell activation and may have important implications for therapies seeking to restore immunological tolerance in autoimmune disorders.

Allosteric inhibition of SHP2 rescues functional T-cell abnormalities in SAP deficiency.

BACKGROUND: X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency arising from SH2D1A mutations leading to loss of SLAM-associated protein (SAP). SAP is an intracellular adaptor protein that binds to SLAM family receptors and is expressed in specific lymphoid lineages. In T cells, SAP relays activatory signals from the T-cell receptor but in its absence SH2 containing protein tyrosine phosphase-1 (SHP1), SH2 containing protein tyrosine phosphase-2 (SHP2), and SH2 containing inositol 5'-phosphatase proteins (SHIP) induce T-cell inhibitory signals leading to abnormal T-cell responses. This results in severe clinical manifestations including immune dysregulation, dysgammaglobulinemia, lymphoma, and hemophagocytic lymphohistiocytosis. Current treatment relies on supportive therapies including immunoglobulin replacement and symptom-directed therapy, with hematopoietic stem cell transplant offering the only curative option. OBJECTIVES: As most XLP symptoms are due to defective T-cell function, this study investigated whether inhibition of SHP2 can restore cellular function in the absence of SAP. METHODS: Healthy donor and XLP patient T cells were activated with anti-CD3/CD28 in T-cell media supplemented with a SHP2 inhibitor (RMC-4550 in vitro for 24 hours) and functional assays were performed to assess follicular TH (TFH) cell function, CD8 cytotoxicity, and sensitivity to restimulation-induced cell death. Additionally, SAP-deficient (SAPy/-) mice were treated with RMC-4550 before T-cell mediated challenge with 4-hydroxy-3-nitrophenylacetly conjugated chicken gammaglobulin and subsequent assessment of humoral immunity analyzing TFH cell population, germinal center formation, and antigen-dependent immunoglobulin secretion. RESULTS: This study shows that the use of RMC-4550 restores T-cell function in XLP patient cells and a SAPy/- model, demonstrating restoration of TFH cell function through immunoglobulin and cytokine secretion analysis alongside rescue of cytotoxicity and restimulation-induced cell death. CONCLUSIONS: These data suggest that SHP2 inhibitors could offer a novel and effective targeted treatment approach for patients with XLP.

Pediatric hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome describing patients with severe systemic hyperinflammation. Characteristic features include unremitting fever, cytopenias, hepatosplenomegaly, and elevation of typical HLH biomarkers. Patients can develop hepatitis, coagulopathy, liver failure, central nervous system involvement, multiorgan failure, and other manifestations. The syndrome has a high mortality rate. More and more, it is recognized that while HLH can be appropriately used as a broad summary diagnosis, many pediatric patients actually suffer from an expanding spectrum of genetic diseases that can be complicated by the syndrome of HLH. Classic genetic diseases in which HLH is a typical and common manifestation include pathogenic changes in familial HLH genes (PRF1, UNC13D, STXBP2, and STX11), several granule/pigment abnormality genes (RAB27A, LYST, and AP3B1), X-linked lymphoproliferative disease genes (SH2D1A and XIAP), and others such as NLRC4, CDC42, and the Epstein-Barr virus susceptibility diseases. There are many other genetic diseases in which HLH is an infrequent complication of the disorder as opposed to a prominent manifestation of the disease caused directly by the genetic defect, including other primary immune deficiencies and inborn errors of metabolism. HLH can also occur in patients with underlying rheumatologic or autoinflammatory disorders and is usually designated macrophage activation syndrome in those settings. Additionally, HLH can develop in patients during infections or malignancies without a known (or as-yet-identified) genetic predisposition. This article will attempt to summarize current concepts in the pediatric HLH field as well as offer a practical diagnostic and treatment overview.

T-B Lymphocyte Interactions Promote Type 1 Diabetes Independently of SLAM-Associated Protein.

Signaling lymphocytic activation molecule-associated protein (SAP), a critical intracellular signaling molecule for T-B lymphocyte interactions, drives T follicular helper (Tfh) cell development in germinal centers (GCs). High-affinity islet autoantibodies predict type 1 diabetes (T1D) but do not cause ß cell destruction. This paradox intimates Tfh cells as key pathologic effectors, consistent with an observed Tfh signature in T1D. To understand how fully developed Tfh (GC Tfh) contribute to different autoimmune processes, we investigated the role of SAP in T1D and autoantibody-mediated arthritis. Whereas spontaneous arthritis depended on SAP in the autoantibody-mediated K/BxN model, organized insulitis and diabetes onset were unabated, despite a blocked anti-insulin vaccine response in SAP-deficient NOD mice. GC Tfh and GC B cell development were blocked by loss of SAP in K/BxN mice. In contrast, although GC B cell formation was markedly reduced in SAP-deficient NOD mice, T cells with a GC Tfh phenotype were found at disease sites. CXCR3+ CCR6- (Tfh1) subset bias was observed among GC Tfh cells infiltrating the pancreas of NOD mice, which was enhanced by loss of SAP NOD T cells override SAP requirement to undergo activation and proliferation in response to Ag presentation, demonstrating the potential for productive cognate T-B lymphocyte interactions in T1D-prone mice. We find that SAP is essential when autoantibody-driven immune complexes promote inflammation but is not required for effective organ-specific autoimmune attack. Thus, Tfh induced in classic GC reactions are dispensable for T1D, but the autoimmune process in the NOD model retains pathogenic Tfh without SAP.

Critical Role for SLAM/SAP Signaling in the Thymic Developmental Programming of IL-17- and IFN-γ-Producing γδ T Cells.

During thymic development, mouse γδ T cells commit to either an IFN-γ- or an IL-17-producing phenotype through mechanisms that remain unclear. In this study, we investigated the extent to which the SLAM/SAP signaling pathway regulates the functional programming of γδ T cells. Characterization of SLAM family receptor expression revealed that thymic γδ T cell subsets were each marked by distinct coexpression profiles of SLAMF1, SLAMF4, and SLAMF6. In the thymus, Vγ1 and Vγ4 T cells that exhibited an SLAMF1+SLAMF6+ double positive phenotype were largely contained within immature CD24+CD73- and CD24+CD73+ subsets, whereas SLAMF1 single positive, SLAMF6 single positive, or SLAMF1SLAMF6 double negative cells were found within mature CD24-CD73+ and CD24-CD73- subsets. In the periphery, SLAMF1 and SLAMF6 expression distinguished IL-17- and IFN-γ-producing γδ T cells, respectively. Disruption of SLAM family receptor signaling through deletion of SAP resulted in impaired thymic Vγ1 and Vγ4 T cell maturation at the CD24+CD73-SLAMF1+SLAMF6+ double positive stage that was associated with a decreased frequency of CD44+RORγt+ γδ T cells. Impaired development was in turn associated with decreased γδ T cell IL-17 and IFN-γ production in the thymus as well as in peripheral tissues. The role for SAP was subset-specific, as Vγ1Vδ6.3, Vγ4, Vγ5, but not Vγ6 subsets were SAP-dependent. Together, these data suggest that the SLAM/SAP signaling pathway plays a larger role in γδ T cell development than previously appreciated and represents a critical checkpoint in the functional programming of both IL-17- and IFN-γ-producing γδ T cell subsets.

Two Clonally Distinct B-Cell Lymphomas Reveal the Diagnosis of XLP1 in a Male Child and His Asymptomatic Male Relatives: Case Report and Review of the Literature.

X-linked lymphoproliferative disease type 1 (XLP1) is a primary immunodeficiency disorder caused by pathogenic variants in the SH2D1A gene (SH2 domain containing protein 1A). Patients with XLP1 may present acutely with fulminant infectious mononucleosis, hemophagocytic lymphohistiocytosis, and/or B-cell non-Hodgkin lymphoma (B-NHL). We report a boy who developed 2 clonally distinct B-NHL 4 years apart and was found to have previously unrecognized XLP1. The report highlights the importance of clonal analysis and XLP1 testing in males with presumed late recurrences of B-NHL, and the role of allogeneic stem cell transplant (allo-SCT) in XLP1 patients and their affected male relatives.

SAP interacts with CD28 to inhibit PD-1 signaling in T lymphocytes.

T cell co-stimulation is important for the maintenance of immunologic tolerance. Co-inhibitory receptors including programmed cell death-1 (PD-1) confer peripheral tolerance to prevent autoimmunity. SAP (SH2D1A) is an adaptor molecule that is important in T cell signaling and has been shown to interact with signaling lymphocytic activation molecule (SLAM) family receptors also in the context of self-tolerance. We recently reported that SAP interferes with PD-1 function. In the current study, we investigated the levels of SAP and PD-1 in patients with rheumatoid arthritis (RA) to further understand what role they play in disease activity. We observed increased SAP levels in lymphocytes of RA patients and found that PD-1 levels correlated positively with RA disease activity. Additionally, we found that SAP interacts with CD28 to inhibit T cell signaling in vitro. This work demonstrates a putative molecular mechanism for SAP mediated PD-1 inhibition.

Identification of amino acid residues involved in the interaction between peste-des-petits-ruminants virus haemagglutinin protein and cellular receptors.

Peste-des-petits-ruminants virus (PPRV) haemagglutinin (H) protein mediates binding to cellular receptors and then initiates virus entry. To identify the key residues of PPRV H (Hv) protein of the Nigeria 75/1 strain involved in binding to receptors, interaction of the Hv and mutated Hv (mHv) proteins with receptors (SLAM and Nectin 4) and their mutants (mSLAM1, mSLAM2, mSLAM3 and mNectin 4) was investigated using surface plasmon resonance imaging (SPRi) and coimmunoprecipitation (co-IP) assays. The results showed that the Hv protein failed to interact with mSLAM3, but interacted at a strong or medium intensity with SLAM, mSLAM2, Nectin 4 and mNectin 4, and at a low level with mSLAM1. The mHv protein was unable to interact with SLAM and its mutants, but bound to Nectin 4 and mNectin 4 with medium and weak intensity, respectively. Further analysis showed that the Hv protein could precipitate mSLAM1, mSLAM2 and mNectin 4, but not mSLAM3. The mHv protein failed to coprecipitate with SLAM and its mutants. The binding activities of mNectin 4 and Nectin 4 to mHv were less than 30.36 and 51.94 % of the wild-type levels, respectively. Based on the results obtained, amino acids at positions R389, L464, I498, R503, R533, Y541, Y543, F552 and Y553 of H protein and I61, H62, L64, K76, K78, E123, H130, I210, A211, S226 and R227 in SLAM were identified to be essential for the specificity of H-SLAM interaction, while the critical residues of H-Nectin 4 interaction require further study. These findings would improve our understanding of the invasive mechanisms of PPRV.

Diagnostic challenges for a novel SH2D1A mutation associated with X-linked lymphoproliferative disease.

Mutations in SH2D1A, encoding the intracellular adaptor signaling lymphocyte activation molecule associated protein (SAP), are associated with X-linked lymphoproliferative disease type 1 (XLP1). We identified a novel hemizygous SH2D1A c.49G > A (p.E17K) variant in a 21-year-old patient with fatal Epstein-Barr virus infection-associated hemophagocytic lymphohistiocytosis. Cellular and biochemical assays revealed normal expression of the SAP variant protein, yet binding to phosphorylated CD244 receptor was reduced by >95%. Three healthy brothers carried the SH2D1A c.49G > A variant. Thus, data suggest that this variant represents a pathogenic mutation, but with variable expressivity. Importantly, our results highlight challenges in the clinical interpretation of SH2D1A variants and caution in using functional flow cytometry assays for the diagnosis of XLP1.

X-linked lymphoproliferative syndrome in mainland China: review of clinical, genetic, and immunological characteristic.

X-linked lymphoproliferative syndrome (XLP) is a rare primary immunodeficiency disease that can be divided into two types: SAP deficiency (XLP1) and XIAP deficiency (XLP2), caused by mutations in the SH2D1A and XIAP genes, respectively. Few cases of XLP (particularly XIAP deficiency) have been reported in mainland China; hence, little is known about the characteristics of Chinese patients with XLP. We identified 13 and 7 patients with SAP and XIAP deficiency, respectively, in our center. Of our 20 patients, 19/20 (95%) presented with disease symptoms at a very early age: six in infancy and 13 in childhood. One XIAP- and three SAP-deficient patients died, while 3/7(42.9%) and 4/13(30.8%), respectively, developed hemophagocytic lymphohistiocytosis (HLH). Epstein-Barr virus (EBV) infection was significantly more common in SAP-deficient 10/13 (76.9%) than XIAP-deficient 2/7 (28.6%) patients, as was hypogammaglobulinemia (10/13 (76.9%) vs. 1/7 (14.3%)). None of the seven XIAP-deficient patients had colitis or lymphoma. Nine SAP-deficient patients and five XIAP-deficient patients showed markedly deficient SAP and XIAP expression, respectively, in lymphocytes. Significantly reduced levels of switched memory B cells were observed in six SAP-deficient patients with persistent hypogammaglobulinemia. One of 13 (7.7%) SAP-deficient patients and 1 of 7 (12.3%) XIAP-deficient patients have received HSCT treatment and are now alive and well; the other alive patients were waiting for HSCT. We also summarized clinical, genetic, and immunological characteristics of all 55 patients (including our 20 patients) reported in the literature in mainland China today.Conclusion: The overall characteristics of SAP deficiency in mainland China were consistent with those in previous reports, whereas manifestations of XIAP deficiency varied significantly. None of inflammatory bowel disease (IBD) has been reported among XIAP-deficient patients in our center; however, whether Chinese XIAP-deficient patients will develop colitis in the future warrants further investigation. HSCT is the only curative therapy for XLP and this therapy should be urgently considered.What is Known:• SAP and XIAP deficiencies share common clinical feature, HLH, whereas they also have their own specific manifestations.• IBD affects 25-30% of XIAP-deficient patients, which has been reported in other countries especially in European country and Japan.What is New:• This is the largest patient cohort study of XLP in China.• We firstly summarized the clinical features and outcomes of Chinese XIAP-deficient patients and found only 1 in 22 patients developed IBD and diet background may contribute to it; Asian SAP-deficient patients carrying SH2D1A R55X mutation were more prone to HLH.

Repression of miR-31 by BCL6 stabilizes the helper function of human follicular helper T cells.

Follicular helper T cells (TFHs) are a key component of adaptive immune responses as they help antibody production by B cells. Differentiation and function of TFH cells are controlled by the master gene BCL6, but it is largely unclear how this transcription repressor specifies the TFH program. Here we asked whether BCL6 controlled helper function through down-regulation of specific microRNAs (miRNAs). We first assessed miRNA expression in TFH cells and defined a TFH-specific miRNA signature. We report that hsa-miR-31-5p (miR-31) is down-regulated in TFH; we showed that BCL6 suppresses miR-31 expression by binding to its promoter; and we demonstrated that miR-31 inhibits the expression of molecules that control T-helper function, such as CD40L and SAP. These findings identify a BCL6-initiated inhibitory circuit that stabilizes the follicular helper T cell program at least in part through the control of miRNA transcription. Although BCL6 controls TFH activity in human and mouse, the role of miR-31 is restricted to human TFH cell differentiation, reflecting a species specificity of the miR-31 action. Our findings highlight miR-31 as a possible target to modulate human T cell dependent antibody responses in the settings of infection, vaccination, or immune dysregulation.

T Cells Regulate Peripheral Naive Mature B Cell Survival by Cell-Cell Contact Mediated through SLAMF6 and SAP.

The control of lymphoid homeostasis is the result of a very fine balance between lymphocyte production, proliferation, and apoptosis. In this study, we focused on the role of T cells in the maintenance/survival of the mature naive peripheral B cell population. We show that naive B and T cells interact via the signaling lymphocyte activation molecule (SLAM) family receptor, SLAMF6. This interaction induces cell type-specific signals in both cell types, mediated by the SLAM-associated protein (SAP) family of adaptors. This signaling results in an upregulation of the expression of the cytokine migration inhibitory factor in the T cells and augmented expression of its receptor CD74 on the B cell counterparts, consequently enhancing B cell survival. Furthermore, in X-linked lymphoproliferative disease patients, SAP deficiency reduces CD74 expression, resulting in the perturbation of B cell maintenance from the naive stage. Thus, naive T cells regulate B cell survival in a SLAMF6- and SAP-dependent manner.

Rare cause of Hemophagocytic Lymphohistiocytosis due to mutation in PRF1 and SH2D1A genes in two children - a case report with a review.

BACKGROUND: Hemophagocytic Lymphohistiocytosis (HLH) is a rare, complex, life-threatening hyper-inflammatory condition due to over activation of lymphocytes mediated secretory cytokines in the body. It occurs as a primary HLH due to genetic defect that mostly occurs in the childhood and associated with early neonatal death. Secondary HLH is triggered by secondary to infection and can occur at any age. CASE PRESENTATION: The current report presents two cases of HLH. Case 1, three-months-old boy born to second degree consanguineous parents was clinically suspected with HLH. A pathogenic variant in exon 2 of PRF1 gene [c.386G > C (p.Trp129Ser); FLH-type2] was detected. The parents and the fetus under investigation were shown to be heterozygous carriers, while Case-1 was homozygous for the said variant. Case 2, a one and half-year old male child referred for work-up was born to non-consanguineous young parents. His HLH suspicion was in accordance with HLH-2004 Revised diagnostic guidelines (fulfilling 5/8 criteria). Molecular study revealed hemizygous likely pathogenic variant c.138-3C > G in intron 1 of SH2D1A gene. Both the mother and younger sister were confirmed to be the carrier of the same variant. CONCLUSION: This study has represented two rare cases of HLH carrying missense variant in PRF1 and splice site variant in SH2D1A gene. Detailed molecular analysis has helped the families with precise genetic counselling and prenatal diagnosis during subsequent pregnancy. It is advocated that male patients presenting with EBV-associated HLH may be screened for XLP that may lead to early diagnosis and therapeutic implication if any.

Transfer of gene-corrected T cells corrects humoral and cytotoxic defects in patients with X-linked lymphoproliferative disease.

BACKGROUND: X-linked lymphoproliferative disease 1 arises from mutations in the SH2D1A gene encoding SLAM-associated protein (SAP), an adaptor protein expressed in T, natural killer (NK), and NKT cells. Defects lead to abnormalities of T-cell and NK cell cytotoxicity and T cell-dependent humoral function. Clinical manifestations include hemophagocytic lymphohistiocytosis, lymphoma, and dysgammaglobulinemia. Curative treatment is limited to hematopoietic stem cell transplantation, with outcomes reliant on a good donor match. OBJECTIVES: Because most symptoms arise from defective T-cell function, we investigated whether transfer of SAP gene-corrected T cells could reconstitute known effector cell defects. METHODS: CD3+ lymphocytes from Sap-deficient mice were transduced with a gammaretroviral vector encoding human SAP cDNA before transfer into sublethally irradiated Sap-deficient recipients. After immunization with the T-dependent antigen 4-hydroxy-3-nitrophenylacetly chicken gammaglobulin (NP-CGG), recovery of humoral function was evaluated through germinal center formation and antigen-specific responses. To efficiently transduce CD3+ cells from patients, we generated an equivalent lentiviral SAP vector. Functional recovery was demonstrated by using in vitro cytotoxicity and T follicular helper cell function assays alongside tumor clearance in an in vivo lymphoblastoid cell line lymphoma xenograft model. RESULTS: In Sap-deficient mice 20% to 40% engraftment of gene-modified T cells led to significant recovery of germinal center formation and NP-specific antibody responses. Gene-corrected T cells from patients demonstrated improved cytotoxicity and T follicular helper cell function in vitro. Adoptive transfer of gene-corrected cytotoxic T lymphocytes from patients reduced tumor burden to a level comparable with that seen in healthy donor cytotoxic T lymphocytes in an in vivo lymphoma model. CONCLUSIONS: These data demonstrate that autologous T-cell gene therapy corrects SAP-dependent defects and might offer an alternative therapeutic option for patients with X-linked lymphoproliferative disease 1.

FOXP3 renders activated human regulatory T cells resistant to restimulation-induced cell death by suppressing SAP expression.

Restimulation-induced cell death (RICD) is an apoptotic program that regulates effector T cell expansion, triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). Although CD4+ regulatory T cells (Tregs) consume IL-2 and experience frequent TCR stimulation, they are highly resistant to RICD. Resistance in Tregs is dependent on the forkhead box P3 (FOXP3) transcription factor, although the mechanism remains unclear. T cells from patients with X-linked lymphoproliferative disease (XLP-1), that lack the adaptor molecule SLAM-associated protein (SAP), are also resistant to RICD. Here we demonstrate that normal Tregs express very low levels of SAP compared to conventional T cells. FOXP3 reduces SAP expression by directly binding to and repressing the SH2D1A (SAP) promoter. Indeed, ectopic SAP expression restores RICD sensitivity in human FOXP3+ Tregs. Our findings illuminate the mechanism behind FOXP3-mediated RICD resistance in Tregs, providing new insight into their long-term persistence.

Identification of a novel nonsense mutation in SH2D1A in a patient with X-linked lymphoproliferative syndrome type 1: a case report.

BACKGROUND: X-linked lymphoproliferative syndrome type 1 (XLP1) is an X-linked recessive genetic disorder with a strong resemblance to hemophagocytic lymphohistiocytosis (HLH). Causative mutations for XLP1 have been identified in SH2D1A, located on chromosome Xq25. CASE PRESENTATION: We report a case of an 18-month-old male with a novel nonsense mutation in SH2D1A. The patient presented the typical phenotype of HLH, including splenomegaly and hemophagocytosis in the bone marrow. Thus, he was initially diagnosed with HLH based on HLH-2004 guidelines. High-throughput amplicon sequencing was performed to detect mutations in the most commonly reported causative genes of HLH, i.e., PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP. A likely pathogenic nonsense mutation was detected in SH2D1A (NM_002351.4:c.300T>A). The mutation was inherited from the patient's mother, and an X-linked recessive mode of inheritance was confirmed by a two-generation pedigree analysis based on Sanger sequencing results. CONCLUSIONS: The nonsense mutation in SH2D1A (NM_002351.4:c.300T>A) was reported for the first time in a case of XLP1 and was considered to be likely pathogenic based on the truncation of the mRNA sequence. This finding expands the spectrum of known XLP-related mutations in Chinese patients and indicates the utility of amplicon sequencing for XLP and HLH diagnosis.

Genetic variant spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: Molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP.

Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening hyperinflammatory disease. This study aimed to investigate the frequencies and distributions of inherited variants in PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP genes in Chinese patients with HLH. A total of 265 patients diagnosed with HLH from January, 2010 to December, 2016 were recruited and analyzed for the 6 genes. Genetic variants were observed in 87 (32.83%) patients. 36 (13.58%) exhibited variants in UNC13D, 18 (6.79%) exhibited PRF1 variants, 10 (3.77%) had variants in XIAP, 9 (3.40%) exhibited variants in STXBP2, 6 (2.26%) carried variants in SH2D1A, 1 (0.38%) had STX11 variant, and 7 (2.64%) exhibited digenic variants. Monoallelic variants were the most common, which accounted for 49.43% of all cases with variants. All variants were confirmed to be germline-derived. The present study describes a distinct variant spectrum in Chinese patients with HLH, whereby UNC13D is the most frequently mutated gene with missense variants that are the most common molecular defects. The variant profile of Chinese HLH patients is quite different from that of Western cohorts but similar to that of Korean patients, yet showing its own uniqueness. This racial difference shows the role of genetic background in the occurrence of HLH.