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Appropriate control of apoptosis during T lymphocyte differentiation is critical for destruction of T cells bearing potentially autoreactive or useless immuno-receptors and for survival of those T cells bearing antigen receptors that may recognize foreign proteins. Despite the well-established importance of thymocyte survival, the exact signals regulating thymocyte apoptosis have not been fully elucidated. Here, we show that thymocytes lacking the endoplasmic reticulum protein calcium-modulating cyclophilin ligand (CAML) failed to undergo normal T-cell development and exhibited dramatically increased rates of apoptosis. In vitro, CAML-deficient thymocytes accumulated high levels of reactive oxygen species (ROS) and underwent abnormally accelerated death in response to several cytotoxic stimuli, including treatment with etoposide, cytokine deprivation, or Fas ligation. Although neither p53 deletion nor loss of Fas rescued the survival and continued development of CAML-deficient thymocytes, removal of the pro-apoptotic BH3-only Bcl-2 family member Bim significantly restored their survival. This work reveals CAML to be a critically important regulator of ROS- and Bim-dependent thymocyte death.

Approximately 40% of patients who survive acute episodes of thrombotic thrombocytopenic purpura (TTP) associated with severe acquired ADAMTS13 deficiency experience one or more relapses. Risk factors for relapse other than severe ADAMTS13 deficiency and ADAMTS13 autoantibodies are unknown. ADAMTS13 autoantibodies, TTP episodes following infection or type I interferon treatment and reported ensuing systemic lupus erythematosus in some patients suggest immune dysregulation. This cross-sectional study asked whether autoantibodies against RNA-binding proteins or peripheral blood gene expression profiles measured during remission are associated with history of prior relapse in acquired ADAMTS13-deficient TTP. Peripheral blood from 38 well-characterized patients with autoimmune ADAMTS13-deficient TTP in remission was examined for autoantibodies and global gene expression. A subset of TTP patients (9 patients, 24%) exhibited a peripheral blood gene signature composed of elevated ribosomal transcripts that associated with prior relapse. A non-overlapping subset of TTP patients (9 patients, 24%) displayed a peripheral blood type I interferon gene signature that associated with autoantibodies to RNA-binding proteins but not with history of relapse. Patients who had relapsed bimodally expressed higher HLA transcript levels independently of ribosomal transcripts. Presence of any one potential risk factor (ribosomal gene signature, elevated HLA-DRB1, elevated HLA-DRB5) associated with relapse (OR = 38.4; p = 0.0002) more closely than any factor alone or all factors together. Levels of immune transcripts typical of natural killer (NK) and T lymphocytes positively correlated with ribosomal gene expression and number of prior episodes but not with time since the most recent episode. Flow cytometry confirmed elevated expression of cell surface markers encoded by these transcripts on T and/or NK cell subsets of patients who had relapsed. These data associate elevated ribosomal and immune transcripts with relapse history in acquired, ADAMTS13-deficient TTP.

SDF-1alpha (CXCL12) signaling via its receptor, CXCR4, stimulates T cell chemotaxis and gene expression. The ZAP-70 tyrosine kinase critically mediates SDF-1alpha-dependent migration and prolonged ERK mitogen-activated protein (MAP) kinase activation in T cells. However, the molecular mechanism by which CXCR4 or other G protein-coupled receptors activate ZAP-70 has not been characterized. Here we show that SDF-1alpha stimulates the physical association of CXCR4 and the T cell receptor (TCR) and utilizes the ZAP-70 binding ITAM domains of the TCR for signal transduction. This pathway is responsible for several of the effects of SDF-1alpha on T cells, including prolonged ERK MAP kinase activity, increased intracellular calcium ion concentrations, robust AP-1 transcriptional activity, and SDF-1alpha costimulation of cytokine secretion. These results suggest new paradigms for understanding the effects of SDF-1alpha and other chemokines on immunity.

SummaryCalcium modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein implicated in T cell signaling, although its mechanism and physiologic role in the immune system are unknown. We show here that CAML is essential for peripheral T cell development. Inactivation of CAML in mouse thymocytes lowered the numbers of double-positive and single-positive thymocytes, concomitant with reduced positive and enhanced negative selection. We found that CAML interacts with p56Lck and appears to regulate subcellular localization of the kinase in both resting and T cell receptor (TCR)-stimulated cells. CAML-deficient cells displayed enhanced p56lck and ZAP-70 phosphorylation and increased IL2 production and cell death after TCR stimulation, suggesting that CAML may act as a negative regulator of p56lck. Our data establish a novel role for CAML as an essential mediator of T cell survival during thymopoiesis and indicate that its loss deregulates p56Lck signaling.

Abstract 3694 Background: Current treatments for Thrombotic Thrombocytopenic Purpura (TTP) fail to successfully control this life-threatening syndrome in up to 20% of patients, and of those patients who survive the first episode, a substantial fraction experience at least one relapse. Genetic mutation or autoantibody inhibition of the ultra-large von-Willebrand factor protease, A Disintegrin And Metalloproteinase with a Thrombospondin Type 1 Motif (ADAMTS13), reduces its enzymatic activity and is an important factor that promotes TTP development. Case reports of acute TTP episodes following infections or Type I interferon (IFN) treatment, a phenomenon also observed in systemic lupus erythematosus (SLE), suggest that inflammatory dysregulation could be important in this disease. SLE patients have elevated serum Type I IFN activity and a peripheral blood Type I IFN gene signature, which are associated with the presence of autoantibodies specific for RNA-binding nuclear antigens. Purpose: This study was undertaken to determine whether, in the absence of SLE, Type I IFN is elevated in acquired TTP patients, and/or is associated with autoantibodies to RNA-binding proteins. Study Participants: We obtained blood and serum samples from patients with acquired ADAMTS13-deficient TTP and matched healthy controls. Patient Inclusion Criteria: 1) ADAMTS13 activity Methods and Results: Blood samples were collected from 38 eligible TTP subjects during remission and from 38 age- (±5 years), race- and sex-matched healthy controls. While anti-nuclear autoantibody prevalence (titer ≥1:120) did not significantly differ between TTP patients and controls, specific autoantibodies against one or more of the following RNA-binding antigens: Ro, La, Sm or nRNP, were more frequent in TTP patients (31.6%) compared to controls (5.3%) (McNemar c2 p=0.0063). Using previously-banked serum samples from acute phase(s) of the disease obtained from the same patients, we observed no differences in the prevalence of these autoantibodies among TTP subjects in the acute phase of disease compared to remission. Serum Type I IFN activity was measured by the capacity of serum samples to induce expression of Type I IFN genes in the WISH epithelial cell line and was significantly increased in remission TTP samples compared to controls (McNemar c2 p=0.0313). Finally, global gene expression was examined (Illumina WG6 version 3 chips) in globin RNA-cleared total RNA taken from whole blood samples of the TTP patients in remission and controls. In initial analysis, 17 genes were found to be ≥ 1.5× differentially expressed in the peripheral blood of TTP patients compared to controls. Among the 7 genes exhibiting the greatest expression difference, 6 are recognized interferon-regulated genes. The relative expression of IFI44 effectively partitioned 13 of the TTP samples (34.2%) into a subset bearing a Type I interferon gene signature similar to that observed in SLE, compared to occurrence of a Type I interferon gene signature observed in 3 of the healthy controls (7.9%) (McNemar c2 p=0.0020). The Type I IFN gene signature was significantly associated with autoantibodies against specific RNA-binding nuclear antigens, defined as the presence of detectable IgG antibodies to one or more of the Ro, La, Sm, or nRNP antigens (Fisher p=0.0086) and correlated with serum Type I interferon activity in the WISH assay (r=.47, p=.0031). However, the Type I interferon gene signature did not associate with tendency of a patient to relapse after a first episode. Conclusion: From these studies, we propose a new subset of acquired, ADAMTS13–deficient TTP patients characterized by elevated serum Type I interferon activity, Type I interferon gene signature and IgG antibodies directed to RNA-binding proteins. Disclosures: Merrill: MedImmune: Consultancy, Honoraria; Genentech: Consultancy, Honoraria.