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Patients with hemophagocytic lymphohistiocytosis (HLH) exhibit immune hyper-activation as a consequence of genetic defects in secretory granule proteins of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Murine models of HLH demonstrate significant activation of CTL as central to the disease pathogenesis, but evaluation of CTL and NK activation in children with HLH or inflammatory conditions is not well described. CD8 T cells only express granzyme B (GrB) following stimulation and differentiation into CTL; therefore, we reasoned that GrB expression may serve as a signature of CTL activation. It is unknown whether human NK cells are similarly activated in vivo, as marked by increased granule proteins. Perforin and GrB levels are measured in both CTL and NK cells by flow cytometry to diagnose perforin deficiency. We retrospectively compared GrB expression in blood samples from 130 children with clinically suspected and/or genetically defined HLH to age-matched controls. As predicted, CD8 expressing GrB cells were increased in HLH, regardless of genetic etiology. Remarkably, the GrB protein content also increased in NK cells in patients with HLH and decreased following immunosuppressive therapy. This suggests that in vivo activation of NK cells occurs in hyper-inflammatory conditions. We conclude that increased detection of GrB in CTL and NK are an immune signature for lymphocyte activation in HLH, irrespective of genetic subtype and may also be a useful measure of immune activation in other related conditions.

Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)–immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3+ regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.

X-linked lymphoproliferative disease (XLP) is a rare congenital immunodeficiency that leads to an extreme, usually fatal increase in the number of lymphocytes upon infection with EBV. It is most commonly defined molecularly by loss of expression of SLAM-associated protein (SAP). Despite this, there is little understanding of how SAP deficiency causes lymphocytosis following EBV infection. Here we show that T cells from individuals with XLP are specifically resistant to apoptosis mediated by TCR restimulation, a process that normally constrains T cell expansion during immune responses. Expression of SAP and the SLAM family receptor NK, T, and B cell antigen (NTB-A) were required for TCR-induced upregulation of key pro-apoptotic molecules and subsequent apoptosis. Further, SAP/NTB-A signaling augmented the strength of the proximal TCR signal to achieve the threshold required for restimulation-induced cell death (RICD). Strikingly, TCR ligation in activated T cells triggered increased recruitment of SAP to NTB-A, dissociation of the phosphatase SHP-1, and colocalization of NTB-A with CD3 aggregates. In contrast, NTB-A and SHP-1 contributed to RICD resistance in XLP T cells. Our results reveal what we believe to be novel roles for NTB-A and SAP in regulating T cell homeostasis through apoptosis and provide mechanistic insight into the pathogenesis of lymphoproliferative disease in XLP.

X-linked thrombocytopenia (XLT) is a mild form of the Wiskott-Aldrich syndrome (WAS) caused by mutations in the WAS gene. A recent retrospective study of the clinical outcome and molecular basis of a large cohort of XLT patients demonstrated that although overall survival is excellent, event free survival is severely affected with conservative treatment. To answer the question whether hematopoietic stem cell transplantation (HSCT) offers a viable alternative therapeutic option in XLT, we retrospectively investigated the outcome of HSCT in a cohort of 24 XLT patients who received HSCT between 1990 and 2011 at 14 transplant centers in the United States, Italy, Germany, Canada, and Japan. The engraftment rate was 100 % and the overall survival rate was 83.3 %. Of the four non-survivors, 2 underwent splenectomy prior to HSCT and died of sepsis, and two of aspergillus infections associated with severe GVHD. In all but one patient, pretransplant complications were resolved by HSCT. Our data indicate that HSCT following myeloablative conditioning is curative and associated with acceptable risks as a treatment option for XLT.

Missense mutations in perforin, a critical effector of lymphocyte cytotoxicity, lead to a spectrum of diseases, from familial hemophagocytic lymphohistiocytosis to an increased risk of tumorigenesis. Understanding of the impact of mutations has been limited by an inability to express human perforin in vitro. We have shown, for the first time to our knowledge, that recombinant human perforin is expressed, processed appropriately, and functional in rat basophilic leukemia (RBL) cells following retroviral transduction. Subsequently, we have addressed how perforin missense mutations lead to absent perforin detection and impaired cytotoxicity by analyzing 21 missense mutations by flow cytometry, immunohistochemistry, and immunoblot. We identified perforin missense mutations with partial maturation (class 1), no apparent proteolytic maturation (class 2), and no recognizable forms of perforin (class 3). Class 1 mutations exhibit lytic function when expressed in RBL cells and are associated with residual protein detection and variable cytotoxic function in affected individuals, suggesting that carriers of class 1 alleles may exhibit more subtle immune defects. By contrast, class 3 mutations cause severely diminished perforin detection and cytotoxicity, while class 2 mutations have an intermediate phenotype. Thus, the pathologic mechanism of perforin missense mutation likely involves a protein dosage effect of the mature protein.

Protein tyrosine kinases (PTKs) play an integral role in T cell activation and differentiation. Defects in the Src-family PTKs in mice and in T cell lines have resulted in variable defects in thymic development and in T cell antigen receptor (TCR) signal transduction. Here, three siblings are described with an autosomal recessive form of severe combined immunodeficiency disease (SCID) in which ZAP-70, a non-Src PTK, is absent as a result of mutations in the ZAP-70 gene. This absence is associated with defects in TCR signal transduction, suggesting an important functional role for ZAP-70.

An analysis of outcomes from 2000 through 2009 at 25 centers treating children with severe combined immunodeficiency (SCID) showed that early transplantation of hematopoietic cells in uninfected children had the best outcome, regardless of donor type. Severe combined immunodeficiency (SCID) is a genetically heterogeneous and lethal disorder of infancy. It is characterized by severe T-cell lymphocytopenia and a lack of antigen-specific T-cell and B-cell immune responses. 1 Allogeneic hematopoietic-cell transplantation with the use of bone marrow from an HLA-identical sibling 2 or an unrelated donor, 3 T-cell–depleted marrow or peripheral-blood stem cells from a haploidentical, related donor, 4 – 7 or umbilical-cord blood 8 – 10 can fully correct the T-cell deficiency and, less consistently, the B-cell deficiency in patients with SCID. 2 – 12 Expanded donor availability and advances in supportive care and treatment of infections have improved long-term outcomes after hematopoietic-cell transplantation. 10 , . . .

Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)-immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3(+) regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.