Characterising negative regulation of CD8+ T cell function in tolerance and exhaustion

CD8+ T cells play a vital role in the immune system by clearing pathogen-infected tissues, however self-reactive CD8+ T cells that escape thymic selection pose the danger of causing autoimmune disease. These self-reactive CD8+ T cells are controlled in the periphery by tolerance mechanisms, which inhibit their function (anergy) or induce apoptosis (deletion). However, the specific molecular pathways crucial for negatively regulating self-reactive CD8+ T cells are not well elucidated. A distinct form of negative regulation called "exhaustion" occurs within chronically stimulated effector CD8+ T cells during cancer and chronic infection. Due to the phenotypical similarities between CD8+ T cell tolerance and exhaustion, we aimed to understand if common underlying molecular pathways regulate these states. The pro-apoptotic protein BIM is important in deletion of self-reactive CD8+ T cells, however the transcriptional control of Bim induction has been unclear. In Chapter 2, we assessed the contribution of the transcription factor FOXO3 in deletion of self-reactive CD8+ T cells given its role in Bim induction and cell death in effector and exhausted CD8+ T cells. While FOXO3 protein underwent activatory dephosphorylation during tolerance, FOXO3-deficient CD8+ T cells maintained the ability to induce BIM expression and undergo deletion. This result indicated that FOXO3 plays distinct roles in cell death of tolerant versus effector CD8+ T cells. To further characterise CD8+ T cell tolerance pathways, in Chapter 3, we investigated whether the ubiquitin ligase adaptor NDFIP1, which is crucially required for CD4+ T cell anergy, influences CD8+ T cell tolerance. In a model of peptide-induced anergy, Ndfip1-deficient CD8+ T cells aberrantly expanded and differentiated into effector cells against high dose exogenous antigen, likely driven by increases in TCR signaling. In contrast, NDFIP1 was dispensable for peripheral deletion to low-dose exogenous antigen, and had little impact upon effector responses to acute infection. These results showed the importance of NDFIP1 in regulating CD8+ T cell tolerance and indicated that CD8+ T cell deletion and anergy are molecularly separable checkpoints. While CD8+ T cell exhaustion appears distinct from tolerance, the transcriptional regulator EGR2 is commonly expressed between these states. In Chapter 4, we showed that exhausted CD8+ T cells in chronic LCMV infection expressed elevated levels of EGR2 compared to functional effectors. Loss of Egr2 severely disrupted terminal CD8+ T cell exhaustion in a cell intrinsic manner, with RNA-Seq results indicating a global enrichment of the exhaustion "stem cell" gene set in Egr2-deficient cells. Strikingly, the genes regulated by Egr2 during exhaustion appeared distinct from those controlled by Egr2 during T cell tolerance, suggesting that EGR2 is repurposed during T cell exhaustion. These findings indicate that while there is molecular overlap between CD8+ T cell tolerance and exhaustion, shared proteins may differ in their mechanism of action. Such insight into the differences between tolerance and exhaustion checkpoints is important for refining future immunotherapies against cancer and autoimmune diseases

The Ubiquitin Ligase Adaptor NDFIP1 Selectively Enforces a CD8⁺ T Cell Tolerance Checkpoint to High-Dose Antigen

Escape from peripheral tolerance checkpoints that control cytotoxic CD8+ T cells is important for cancer immunotherapy and autoimmunity, but pathways enforcing these checkpoints are mostly uncharted. We reveal that the HECT-type ubiquitin ligase activator, NDFIP1, enforces a cell-intrinsic CD8+ T cell checkpoint that desensitizes TCR signaling during in vivo exposure to high antigen levels. Ndfip1-deficient OT-I CD8+ T cells responding to high exogenous tolerogenic antigen doses that normally induce anergy aberrantly expanded and differentiated into effector cells that could precipitate autoimmune diabetes in RIP-OVAhi mice. In contrast, NDFIP1 was dispensable for peripheral deletion to low-dose exogenous or pancreatic islet-derived antigen and had little impact upon effector responses to Listeria or acute LCMV infection. These data provide evidence that NDFIP1 mediates a CD8+ T cell tolerance checkpoint, with a different mechanism to CD4+ T cells, and indicates that CD8+ T cell deletion and anergy are molecularly separable checkpoints.This work was funded by NIH grant U19-AI100627, by an Australian Government Research Training Program Domestic Scholarship (to M.V.W.), by a Sydney Parker Smith Postdoctoral Research Fellowship from the Cancer Council of Victoria (to J.M.M.), and by the National Health and Medical Research Council (NHMRC) through Program Grants 1016953, 1113904, and 1054925, Australia Fellowship 585490 (to C.C.G.), Senior Principal Research Fellowship 1081858 (to C.C.G.), CJ Martin Early Career Fellowship 585518 (to I.A.P.), and Independent Research Institutes Infrastructure Support Scheme Grant 361646. Florey Institute of Neuroscience and Mental Health and WEHI acknowledge the strong support from the Victorian Government and in particular funding from the Operational Infrastructure Support Grant

The Ubiquitin Ligase Adaptor NDFIP1 Selectively Enforces a CD8+ T Cell Tolerance Checkpoint to High-Dose Antigen

Escape from peripheral tolerance checkpoints that control cytotoxic CD8+ T cells is important for cancer immunotherapy and autoimmunity, but pathways enforcing these checkpoints are mostly uncharted. We reveal that the HECT-type ubiquitin ligase activator, NDFIP1, enforces a cell-intrinsic CD8+ T cell checkpoint that desensitizes TCR signaling during in vivo exposure to high antigen levels. Ndfip1-deficient OT-I CD8+ T cells responding to high exogenous tolerogenic antigen doses that normally induce anergy aberrantly expanded and differentiated into effector cells that could precipitate autoimmune diabetes in RIP-OVAhi mice. In contrast, NDFIP1 was dispensable for peripheral deletion to low-dose exogenous or pancreatic islet-derived antigen and had little impact upon effector responses to Listeria or acute LCMV infection. These data provide evidence that NDFIP1 mediates a CD8+ T cell tolerance checkpoint, with a different mechanism to CD4+ T cells, and indicates that CD8+ T cell deletion and anergy are molecularly separable checkpoints.This work was funded by NIH grant U19-AI100627, by an Australian Government Research Training Program Domestic Scholarship (to M.V.W.), by a Sydney Parker Smith Postdoctoral Research Fellowship from the Cancer Council of Victoria (to J.M.M.), and by the National Health and Medical Research Council (NHMRC) through Program Grants 1016953, 1113904, and 1054925, Australia Fellowship 585490 (to C.C.G.), Senior Principal Research Fellowship 1081858 (to C.C.G.), CJ Martin Early Career Fellowship 585518 (to I.A.P.), and Indepen- dent Research Institutes Infrastructure Support Scheme Grant 361646. Florey Institute of Neuroscience and Mental Health and WEHI acknowledge the strong support from the Victorian Government and in particular funding from the Operational Infrastructure Support Grant

Systems-guided forward genetic screen reveals a critical role of the replication stress response protein ETAA1 in T cell clonal expansion

T-cell immunity requires extremely rapid clonal proliferation of rare, antigen-specific T lymphocytes to form effector cells. Here we identify a critical role for ETAA1 in this process by surveying random germ line mutations in mice using exome sequencing and bioinformatic annotation to prioritize mutations in genes of unknown function with potential effects on the immune system, followed by breeding to homozygosity and testing for immune system phenotypes. Effector CD8+ and CD4+ T-cell formation following immunization, lymphocytic choriomeningitis virus (LCMV) infection, or herpes simplex virus 1 (HSV1) infection was profoundly decreased despite normal immune cell development in adult mice homozygous for two different Etaa1 mutations: an exon 2 skipping allele that deletes Gly78-Leu119, and a Cys166Stop truncating allele that eliminates most of the 877-aa protein. ETAA1 deficiency decreased clonal expansion cell autonomously within the responding T cells, causing no decrease in their division rate but increasing TP53-induced mRNAs and phosphorylation of H2AX, a marker of DNA replication stress induced by the ATM and ATR kinases. Homozygous ETAA1-deficient adult mice were otherwise normal, healthy, and fertile, although slightly smaller, and homozygotes were born at lower frequency than expected, consistent with partial lethality after embryonic day 12. Taken together with recently reported evidence in human cancer cell lines that ETAA1 activates ATR kinase through an exon 2-encoded domain, these findings reveal a surprisingly specific requirement for this ATR activator in adult mice restricted to rapidly dividing effector T cells. This specific requirement may provide new ways to suppress pathological T-cell responses in transplantation or autoimmunity.This work was funded by National Institutes of Health Grant U19-AI100627; by the National Health and Medical Research Council through Program Grants 1016953 and 1113904, Australia Fellowship 585490, Senior Principal Research Fellowship 1081858, and C. J. Martin Early Career Fellowship 585518 (to I.A.P.); and by the National Collaborative Research Infrastructure Strategy

Effective priming of herpes simplex virusspecific CD8+ T cells in vivo does not require infected dendritic cells

Resolution of virus infections depends on the priming of virus-specific CD8+ T cells by dendritic cells (DC). While this process requires major histocompatibility complex (MHC) class I-restricted antigen presentation by DC, the relative contribution to CD8+ T cell priming by infected DC is less clear. We have addressed this question in the context of a peripheral infection with herpes simplex virus 1 (HSV). Assessing the endogenous, polyclonal HSV-specific CD8+ T cell response, we found that effective in vivo T cell priming depended on the presence of DC subsets specialized in cross-presentation, while Langerhans cells and plasmacytoid DC were dispensable. Utilizing a novel mouse model that allows for the in vivo elimination of infected DC, we also demonstrated in vivo that this requirement for cross-presenting DC was not related to their infection but instead reflected their capacity to cross-present HSV-derived antigen. Taking the results together, this study shows that infected DC are not required for effective CD8+ T cell priming during a peripheral virus infection. IMPORTANCE The ability of some DC to present viral antigen to CD8+ T cells without being infected is thought to enable the host to induce killer T cells even when viruses evade or kill infected DC. However, direct experimental in vivo proof for this notion has remained elusive. The work described in this study characterizes the role that different DC play in the induction of virus-specific killer T cell responses and, critically, introduces a novel mouse model that allows for the selective elimination of infected DC in vivo. Our finding that HSV-specific CD8+ T cells can be fully primed in the absence of DC infection shows that cross-presentation by DC is indeed sufficient for effective CD8+ T cell priming during a peripheral virus infection.Our research is supported by the National Health and Medical Research Council of Australia. P. Whitney is supported by an Overseas Biomedical Fellowship (NHMRC) and a MDHS Faculty Fellowship (University of Melbourne). T. Gebhardt is supported by a fellowship from the Sylvia and Charles Viertel Charitable Foundation. D. Tscharke is supported by a Senior Research Fellowship (NHMRC)

Antigen-driven EGR2 expression is required for exhausted CD8 + T cell stability and maintenance

Chronic stimulation of CD8 T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells. +This work was funded by National Institutes of Health Grant U19-AI100627, the Swiss National Science Foundation and the Novartis Foundation for Medical-Biological Research (S.S.G.), the Australian Cancer Research Foundation (for the Peter Mac Flow Cytometry and Molecular Genomics facilities) and by the National Health and Medical Research Council (NHMRC) through Program Grants 1016953 & 1113904, Ideas Grant APP2001719, Australia Fellowship 585490 (C.C.G.), Senior Principal Research Fellowships (1081858, C.C.G., 1139607, A.K.), and CJ Martin Early Career Fellowship 585518 (I.A.P.)

Up-regulation of LFA-1 allows liver-resident memory T cells to patrol and remain in the hepatic sinusoids

Liver-resident CD8+ T cells are highly motile cells that patrol the vasculature and provide protection against liver pathogens. A key question is: How can these liver CD8+ T cells be simultaneously present in the circulation and tissue-resident? Because liver-resident T cells do not express CD103—a key integrin for T cell residence in epithelial tissues—we investigated other candidate adhesion molecules. Using intravital imaging, we found that CD8+ T cell patrolling in the hepatic sinusoids is dependent on LFA-1–ICAM-1 (intercellular adhesion molecule–1) interactions. Liver-resident CD8+ T cells up-regulate LFA-1 compared with effector memory cells, presumably to facilitate this behavior. Last, we found that LFA-1–deficient CD8+ T cells failed to form substantial liver-resident memory populations after Plasmodium immunization or lymphocytic choriomeningitis virus infection. Collectively, our results demonstrate that it is adhesion through LFA-1 that allows liver-resident memory CD8+ T cells to patrol and remain in the hepatic sinusoids.This work was supported by start-up funds from the ANU (to I.A.C.) and grants from the Perpetual Foundation (to I.A.C., FR2014/1152), the Ian Potter Foundation (to I.A.C., grant number 32616), the Ramaciotti Foundation (to C.G.G. and A.E.), the NIH (to C.G.G., grant number U19 AI100627), and the National Health and Medical Research Council (to A.E., grant number GNT1035858)