Project description:Checkpoint inhibitors targeting CTLA-4 and PD-1 revolutionized the treatment of cancer patients but their use is limited by the emergence of immune-related adverse events (irAE). We assessed autoreactive B cell frequencies in the blood of cancer patients before and after treatment with checkpoint inhibitors by testing the reactivity of recombinant antibodies cloned from single B cells. We found that anti-PD-1 and anti-CTLA-4 combination therapy induced the emergence of autoreactive mature naïve B cells, whereas central B-cell tolerance remained functional. In contrast, anti-PD-1 alone did not alter autoreactive B cell counterselection. Anti-CTLA-4 injections in humanized mice also resulted in the production of autoreactive B cells, whereas anti-PD-1 did not. We conclude that CTLA-4 but not PD-1 is required for the removal of developing autoreactive mature naïve B cells and that CTLA-4 blockade broadens the peripheral B cell repertoire which likely contains clones that promote not only irAEs but also anti-tumor responses.
Project description:We show that thymic epithelial cells in the medulla (mTECs) that present self-antigens (self-Ags) to developing thymocytes for establishing immunological self-tolerance also express CTLA-4 if Aire, loss of which is responsible for the hereditary autoimmunity, is nonfunctional. Upon binding with its ligand, CD80 and CD86, expressed on thymic DCs, CTLA-4/ligand complex was internalized by Aire-deficient mTECs. This attenuated the ability of DCs to provide costimulatory signals and to present self-Ags, resulting in the reduced production of Tregs.
Project description:The importance of CTLA-4 regulation of T cell function is well recognized. Studies here report the expression of this immune-regulator in murine B-1a cells, and demonstrate the critical roles CTLA-4 plays in maintaining self-tolerance through regulating these early-developing B cells that express a repertoire enriched for auto-reactivity. By selectively deleting CTLA-4 from B cells, we show that the conditional knockout mice spontaneously generate T follicular helper (Tfh) cells and germinal centers (GC) in spleen; produce autoantibodies; and, later in life, develop autoimmune pathologies. This impaired immune homeostasis results from B-1a dysfunction when they lose CTLA-4. Thus, CTLA-4-deficient B-1a cells up-regulate epigenetic and transcriptional activation programs and show increased self-replenishment. These activated cells further internalize surface IgM, differentiate to antigen-presenting cells and, when reconstituted in normal congenic recipients, induce GC responses and Tfh cells expressing a highly selected repertoire. These findings introduce CTLA-4 regulation of B-1a as a crucial immune-regulatory mechanism.
Project description:Loss of NBEAL2 function leads to grey platelet syndrome (GPS), a bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets. A proportion of patients with GPS develop autoimmunity through an unknown mechanism, which might be related to the proteins NBEAL2 interacts with, specifically in immune cells. Here we show a comprehensive interactome of NBEAL2 in primary T cells, based on mass spectrometry identification of altogether 74 protein association partners. These include LRBA, a member of the same BEACH domain family as NBEAL2, recessive mutations of which cause autoimmunity and lymphocytic infiltration through defective CTLA-4 trafficking. Investigating the potential association between NBEAL2 and CTLA-4 signalling that is suggested by the mass spectrometry results, we confirm by co-immunoprecipitation that CTLA-4 and NBEAL2 interact with each-other. Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T cells, while their regulatory T cells appear unaffected. Knocking- down NBEAL2 in healthy primary T cells recapitulates the low CTLA-4 expression observed in T cells of GPS patients. Our results thus show that NBEAL2 is involved in the regulation of CTLA-4 expression in conventional T cells and provide a rationale for considering CTLA-4- immunoglobulin therapy in patients with GPS and autoimmune disease.
Project description:Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) plays a pivotal role in preventing autoimmunity and fostering anticancer immunity by interacting with B7 proteins CD80 and CD86. CTLA-4 is the first immune checkpoint targeted with a monoclonal antibody inhibitor. Checkpoint inhibitors have generated durable responses in many cancer patients, representing a revolutionary milestone in cancer immunotherapy. However, therapeutic efficacy is limited to a small portion of patients, and immune-related adverse events are noteworthy, especially for monoclonal antibodies directed against CTLA-4. Previously, small molecules have been developed to impair the CTLA-4: CD80 interaction; however, they directly targeted CD80 and not CTLA-4. In this study, we performed artificial intelligence (AI)-powered virtual screening of approximately ten million compounds to target CTLA-4. We validated primary hits with biochemical, biophysical, immunological, and experimental animal assays. We then optimized lead compounds and obtained inhibitors with an inhibitory concentration of 1 micromole in disrupting the interaction between CTLA-4 and CD80. Unlike ipilimumab, these small molecules did not degrade CTLA-4. Several compounds inhibited tumor development prophylactically and therapeutically in syngeneic and CTLA-4-humanized mice. This project supports an AI-based framework in designing small molecules targeting immune checkpoints for cancer therapy.
Project description:CTLA-4 is thought to inhibit effector T cells both intrinsically, by competing with CD28 for B7 ligands, and extrinsically, through the action of regulatory T cells. We studied in vivo responses of normal and CTLA-4-deficient antigen-specific murine effector CD4+ T cells. In order to do these studies in a physiological model of immunity to foreign antigen, we transferred small numbers of congenically marked RAG2-deficient 5C.C7 T cells with either a normal or knockout allele of CTLA-4 into normal syngeneic B10.A recipient mice. The T cells were then activated by immunization with MCC peptide and LPS. To look for transcriptional signatures of negative regulation of T cell responses by CTLA-4, we used microarray analysis to compare transcripts in wild type and CTLA-4 KO 5C.C7 T cells four days after immunization. This is the first instance in which differences are observed in extent of accumulation of wild type and CTLA-4 KO 5C.C7 T cells. To compare the gene expression profile between wild type and CTLA-4 KO adoptively transferred T cells 4 days after immunization.
Project description:Single-cell immune repertoire sequencing (gene expression and immune receptor repertoire) was performed under various contexts. This includes CD4 T follicular cells (sorted by surface expression of PD1+ CXCR5+ CD4+) following high dose LCMV clone 13 infection on days 10, 25, and 50 and low dose clone 13 infection on day 10 post infection. We additionally profiled virus-specific CD8 T cells that are specific to either GP33 or NP396. Gp33-specific CD8 T cells were sorted 15 months post infection with either low dose clone 13 LCMV, MCMV-ie2-gp33 or MCMV-m45-gp33. NP396-specific CD8 T cells were isolated either 10 days post challenge or 25 days post challenge using the viral model of dejavu (https://pubmed.ncbi.nlm.nih.gov/16604192/). B and T cells were isolated 14 days post induction of Experimental autoimmune encephalomyelitis (EAE).
Project description:Presentation of tissue-restricted antigens (TRA) in the thymus is essentialfor establishing self-tolerancethrough elimination of autoreactive Tcells.It remains unclear why certain self T cells can bypassthymic selectionand become driversoftissue-specific autoimmunity. Herewe assessedthymic TRA presented by a major histocompatibility class II molecule (MHC-II) that confers genetic propensity to develop type 1 diabetes. This analysisestablished the thymic peptidome containing self-peptidesderivedfrom variousperipheral tissues,butalso revealed a disparity in the MHC-II epitoperepertoirebetween thymus and the target site of autoimmunity, the pancreatic islets.The thymic repertoire consistedof canonicalMHC-IIepitopes capable of enforcing central tolerance but lacked presentation of uniqueepitopes derived from unconventionalself-proteinprocessing orneoepitopes formed by post-translational modifications. Suchepitopes were noteworthily generatedin the isletsvia a tissue-intrinsic mechanism, crinophagy, and were recognized by CD4 T cells escaping central tolerance.These findings supportthat central tolerance mechanisms are effective but incomplete, as tissue-characteristic handling of self-antigens may broaden the peripheralrepertoire thatextends beyond the scope of thymic selection.