Co-operative failure of Aire-mediated clonal deletion and Cblb-mediated clonal anergy in the pathogenesis of autoimmunity

Breakdown of self-tolerance in autoimmune diseases is poorly understood, as is the existence of so many different mechanisms for actively acquired tolerance. In this thesis, I find that failure of two tolerance mechanisms, thymic T cell clonal deletion to organ specific antigens mediated by Aire and the two-signal mechanisms of T cell tolerance in mature T cells imposed by the ubiquitin ligase Cblb, individually cause slow and subclinical autoimmunity but combine co-operatively to allow lethal T cell-mediated destruction of the exocrine tissue in the pancreas and salivary glands within weeks after T cells emerge from the thymus. Intriguingly, there was no evidence of autoimmunity in other tissues although Aire regulates deletion of T cells recognising huge numbers of self-antigens from multiple tissues. Varying the nascent T cell repertoire by substituting the B10.BR.H2k MHC in which the T cells were selected upon with the C57/B6.H2b MHC or introducing an unlimited source of transgenic T cells with a single specificity towards an alternative organ failed to redirect the autoimmunity in Aire-/-Cblb-/- mice to other organs, albeit these strategies slowed down the intensity and tempo of the disease. This indicated that there was a unique feature of the ""driver"" T cells that escaped tolerance in Aire-/-Cblb-/- mice. To illuminate the unique properties of the autoimmune driver cells in Aire-/-Cblb-/- mice, a new approach to isolate pancreas-specific T cell clones was successfully established. This involved enriching for the driver T cells by serial adoptive transfer, followed by RT-PCR amplification and sequencing of mRNAs encoding the paired TCRalpha and TCRbeta chains from single T cells sorted from the pancreas or spleen. A remarkably oligoclonal expansion of CD8 T cells within the pancreas was revealed, comprising several clones within an animal bearing highly related TCRs. The TCR from one of the expanded CD8 T cell clones, 3.5, was expressed in retroviral transgenic (retrogenic) bone marrow chimeras. T cells expressing TCR3.5 were positively selected into the CD8 T cell lineage, showed no evidence of negative selection in Aire-wild-type thymi, and accumulated as mature naive CD8 T cells in the axillary and inguinal lymph nodes. TCR3.5 CD8 T cells were nevertheless activated in the pancreatic lymph node and accumulated in the pancreas, and retrogenic expression of TCR3.5 was sufficient to precipitate extensive exocrine pancreatitis in animals with wild-type Aire and Cblb genes. These results suggest that Aire may not directly regulate the driver CD8 T cells, raising novel possibilities about how the defects in Aire and Cblb co-operate to precipitate organ-specific autoimmunity. These findings are the first to reveal a higher-level architecture assembling individual tolerance mechanisms for robust autoimmunity, establishing a multistep framework for understanding how individual mechanisms fit together for robust tolerance. Understanding how genetic defects in tolerance mechanism co-operate with each other is critical for interpreting patterns of genetic and phenotypic variability in human autoimmune diseases. -- provided by Candidate

Visualizing the role of Cbl-b in control of islet-reactive CD4 T cells and susceptibility to Type 1 Diabetes

The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against Type 1 diabetes, but its in vivo role in the process of self-tolerance has not been examined at the level of potentially auto-aggressive CD4+ T cells. Here we visualize the consequences of Cbl-b deficiency on self-tolerance to lysozyme antigen expressed in transgenic mice under control of the insulin promoter (insHEL). By tracing the fate of pancreatic islet-reactive CD4+ T cells in pre-diabetic 3A9-TCR x insHEL double-transgenic mice, we find that Cbl-b deficiency contrasts with AIRE or IL-2 deficiency because it does not affect thymic negative selection of islet-reactive CD4+ cells nor the numbers of islet-specific CD4+ or CD4+ FOXP3+ T cells in the periphery, although it decreased differentiation of inducible Treg (iTreg) cells from TGF-b treated 3A9-TCR cells in vitro. When removed from Tregs and placed in culture, Cblb-deficient islet-reactive CD4+ cells reveal a capacity to proliferate to HEL antigen that is repressed in wild-type cells. This latent failure of T cell anergy is nevertheless controlled in vivo in pre-diabetic mice, so that islet-reactive CD4+ cells in spleen and pancreatic lymph node of Cblb-deficient mice show no evidence of increased activation or proliferation in situ. Cblb-deficiency subsequently precipitated diabetes in most TCR:insHEL animals by 15 wks of age. These results reveal a role for peripheral T cell anergy in organ-specific self-tolerance, and illuminate the interplay between Cblb-dependent anergy and other mechanisms for preventing organ-specific autoimmunity

B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8-dendritic cells require the intramembrane endopeptidase SPPL2A

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.R01 AI052127/AI/NIAID NIH HHS/United States U19 AI100627/AI/NIAID NIH HHS/United States Medical Research Council/United Kingdom Wellcome Trust/United Kingdo

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

T-cell regulation by casitas B-lineage lymphoma (Cblb) is a critical failsafe against autoimmune disease due to autoimmune regulator (Aire) deficiency

Autoimmune polyendocrinopathy syndrome type 1 (APS1) results from homozygous Aire mutations that cripple thymic deletion of organ-specific T cells. The clinical course in man and mouse is characterized by high variability both in the latent period befor

T-cell regulation by casitas B-lineage lymphoma (Cblb) is a critical failsafe against autoimmune disease due to autoimmune regulator (Aire) deficiency

Autoimmune polyendocrinopathy syndrome type 1 (APS1) results from homozygous Aire mutations that cripple thymic deletion of organ-specific T cells. The clinical course in man and mouse is characterized by high variability both in the latent period before onset of autoimmune disease and in the specific organs affected, but the reasons for this are unknown. Here we test the hypothesis that the latent period reflects the failsafe action of discrete postthymic mechanisms for imposing self-tolerance in peripheral T cells. Aire-deficient mice were crossed with mice of a uniform major histocompatibility complex (MHC) haplotype and genetic background carrying specific genetic defects in one of four distinct peripheral tolerance mechanisms: activation-induced cell death (Faslgld/gld), anergy and requirement for CD28 costimulation (Cblb−/−), inhibition of ICOS and TFH cells (Rc3h1san/san), or decreased numbers of Foxp3+ T regulatory cells (Card11unm/unm). Cblb-deficiency was unique among these four in precipitating rapid clinical autoimmune disease when combined with Aire-deficiency, resulting in autoimmune exocrine pancreatitis with median age of survival of only 25 d. Massive lymphocytic infiltration selectively destroyed most of the exocrine acinar cells of the pancreas and submandibular salivary gland, and CD4+ and CD8+ subsets were necessary and sufficient to transfer the disease. Intrinsic regulation of peripheral T cells by CBL-B thus serves a uniquely critical role as a failsafe against clinical onset of autoimmune disease in AIRE deficiency, and multiple peripheral tolerance mechanisms may need to fail before onset of clinical autoimmunity to many organs

The transcription repressor, ZEB1, cooperates with CtBP2 and HDAC1 to suppress IL-2 gene activation in T cells

Activation of T cells leads to the induction of many cytokine genes that are required for appropriate immune responses, including IL-2, a key cytokine for T cell proliferation and homeostasis. The activating transcription factors such as nuclear factor of activated T cells, nuclear factor κB/Rel and activated protein-1 family members that regulate inducible IL-2 gene expression have been well documented. However, negative regulation of the IL-2 gene is less studied. Here we examine the role of zinc finger E-box-binding protein (ZEB) 1, a homeodomain/Zn finger transcription factor, as a repressor of IL-2 gene transcription. We show here that ZEB1 is expressed in non-stimulated and stimulated T cells and using chromatin immunoprecipitation assays we show that ZEB1 binds to the IL-2 promoter. Over-expression of ZEB1 can repress IL-2 promoter activity, as well as endogenous IL-2 mRNA production in EL-4 T cells, and this repression is dependent on the ZEB-binding site at -100. ZEB1 cooperates with the co-repressor C-terminal-binding protein (CtBP) 2 and with histone deacetylase 1 to repress the IL-2 promoter and this cooperation depends on the ZEB-binding site in the promoter as well as the Pro-X-Asp-Leu-Ser protein-protein interaction domain in CtBP2. Thus, ZEB1 may function to recruit a repressor complex to the IL-2 promoter

Visualizing the role of Cbl-b in control of islet-reactive CD4 T cells and susceptibility to type 1 diabetes

The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against type 1 diabetes, but its invivo role in the process of self-tolerance has not been examined at the level of potentially autoaggressive CD4+ T cells. In this study, we visualize the consequences of Cbl-b deficiency on self-tolerance to lysozyme Ag expressed in transgenic mice under control of the insulin promoter (insHEL). By tracing the fate of pancreatic islet-reactive CD4+ T cells in prediabetic 3A9-TCR x insHEL double-transgenic mice, we find that Cbl-b deficiency contrasts with AIRE or IL-2 deficiency, because it does not affect thymic negative selection of islet-reactive CD4+ cells or the numbers of islet-specific CD4+ or CD4+ Foxp3+ T cells in the periphery, although it decreased differentiation of inducible regulatory T cells from TGF-β-treated 3A9-TCR cells in vitro. When removed from regulatory T cells and placed in culture, Cblb-deficient islet-reactive CD4+ cells reveal a capacity to proliferate to HEL Ag that is repressed in wild-type cells. This latent failure of T cell anergy is, nevertheless, controlled in vivo in prediabetic mice so that islet-reactive CD4+ cells in the spleen and the pancreatic lymph node of Cblb-deficient mice show no evidence of increased activation or proliferation in situ. Cblb deficiency subsequently precipitated diabetes in most TCR:insHEL animals by 15 wk of age. These results reveal a role for peripheral T cell anergy in organ-specific self-tolerance and illuminate the interplay between Cblb-dependent anergy and other mechanisms for preventing organ-specific autoimmunity

Deletion of self-reactive CCR7– thymocytes in the absence of MHC expression on thymic epithelial cells

The selection of αβ T cells in the thymus is punctuated by checkpoints at which thymocytes differentiate or undergo apoptosis. Wave 1 deletion is defined as apoptosis within nascent αβ T-cell antigen receptor (TCR)-signalled thymocytes that lack CCR7 expression. The antigen-presenting cell (APC) types that mediate wave 1 deletion are unclear. To measure wave 1 deletion, we compared the frequencies of TCRβ + CD5 + Helios + CCR7– cells in nascent thymocyte cohorts in mice with normal or defective apoptosis. This thymocyte population is small in mice lacking major histocompatibility complex (MHC) expression. The scale of wave 1 deletion was increased by transgenic expression of the self-reactive Yae62 TCRβ chain, was almost halved when haemopoietic APCs lacked MHC expression and, surprisingly, was unchanged when epithelial cells lacked MHC expression. These findings demonstrate efficiency, and some redundancy, in the APC types that mediate wave 1 deletion in the normal mouse thymus.</p