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The requirements for tonic T-cell receptor (TCR) signaling in CD8+memory T-cell generation and homeostasis are poorly defined. The SRC homology 2 (SH2)-domain–containing leukocyte protein of 76 kDa (SLP-76) is critical for proximal TCR-generated signaling. We used temporally mediated deletion of SLP-76 to interrupt tonic and activating TCR signals after clearance of the lymphocytic choriomeningitis virus (LCMV). SLP-76–dependent signals are required during the contraction phase of the immune response for the normal generation of CD8 memory precursor cells. Conversely, LCMV-specific memory CD8 T cells generated in the presence of SLP-76 and then acutely deprived of TCR-mediated signals persist in vivo in normal numbers for more than 40 weeks. Tonic TCR signals are not required for the transition of the memory pool toward a central memory phenotype, but the absence of SLP-76 during memory homeostasis substantially alters the kinetics. Our data are consistent with a model in which tonic TCR signals are required at multiple stages of differentiation, but are dispensable for memory CD8 T-cell persistence.

SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) nucleates a signaling complex critical for T-cell receptor (TCR) signal propagation. Mutations in the tyrosines of SLP-76 result in graded defects in TCR-induced signals depending on the tyrosine(s) affected. Here we use 2 strains of genomic knock-in mice expressing tyrosine to phenylalanine mutations to examine the role of TCR signals in the differentiation of effector and memory CD8+T cells in response to infection in vivo. Our data support a model in which altered TCR signals can determine the rate of memory versus effector cell differentiation independent of initial T-cell expansion. Furthermore, we show that TCR signals sufficient to promote CD8+T-cell differentiation are different from those required to elicit inflammatory cytokine production.

CD8+T cell-numbers rapidly expand and then contract after exposure to their cognate antigen. Here we show that the sustained frequencies of transgene product-specific CD8+T cells elicited by replication-defective adenovirus vectors are linked to persistence of low levels of transcriptionally active adenovirus vector genomes at the site of inoculation, in liver, and lymphatic tissues. Continuously produced small amounts of antigen maintain fully active effector CD8+T cells, while also allowing for their differentiation into central memory cells. The long-term persistence of adenoviral vectors may be highly advantageous for their use as vaccines against pathogens for which T-cell–mediated protection requires both fully activated T cells for immediate control of virus-infected cells and central memory CD8+T cells that, because of their higher proliferative capacity, may be suited best to eliminate cells infected by pathogens that escaped the initial wave of effector T cells.

The adoptive transfer of autologous T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD19 has shown remarkable activity for patients with B cell malignancies. Using a second- generation CAR signaling through 4-1BB/CD3ζ, (CTL019), over 90% of patients with relapsed or refractory (R/R) acute lymphoblastic leukemia (ALL) will achieve complete remission (Maude, S.L. et. al. New England Journal of Medicine, 2014). Similar therapy can induce long-term remissions for R/R chronic lymphocytic leukemia (CLL), but only in a subset of patients (Porter, D.L. et. al. Science Transl. Medicine, 2015). The ability to identify individuals most likely to respond will have a dramatic impact on patient selection and provide critical information that may allow immune modification to enhance responses using this approach. We here present updated analyses and functional validation of our previously reported biomarker profiles of CTL019 cells in CLL (Fraietta, J.A. et. al., Blood, 2016). Retrospective samples of clinically manufactured CAR T cells from forty-one patients with advanced, heavily pre-treated and high-risk CLL were evaluated for functional determinants of potency. Transcriptomic analysis of CTL019 cells from subjects who had complete responses (CR) to this therapy revealed enrichment in early memory T cells and IL-6/STAT3 gene signatures, while T cells from non-responding (NR) patients were enriched in genes belonging to known pathways of terminal differentiation, exhaustion, apoptosis and glycolysis. Accordingly, CAR-stimulated T cells from NR relative to CR patents demonstrated an increased uptake of a glucose analog; inhibition of glycolysis using 2-deoxy-D-glucose resulted in the generation of early memory CAR T cells in culture. Furthermore, CTL019 cells that were highly functional in CLL patients had increased levels of phosphorylated STAT3 (pSTAT3) upon activation compared to CAR T cells from NR patients with poor anti-tumor activity. These data were concordant with the highly significant direct correlation between serum IL-6 and the degree of CTL019 proliferative capacity in patients. Increasing pSTAT3 activity by addition of exogenous IL-6 to culture media elevated the absolute numbers of CAR T cells following repeated stimulation with CD19-expressing tumor targets, while pSTAT3 inhibition diminished CTL019 cell growth.

DNA methylation is one of the major epigenetic mechanisms that control T cell differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines, intermediates in active DNA demethylation. Here we demonstrate that TET2 regulates CD8+T cell differentiation in vivofollowing acute viral infection. At steady-state, mice with a T-cell specific deletion of TET2 have intact thymic and peripheral T cell populations. However, following acute viral infection with LCMV-Armstrong, TET2 loss promotes early acquisition of a memory CD8+T cell fate in a cell-intrinsic manner without disrupting antigen-driven cell expansion or effector function. Integration of genome-wide methylation analysis and expression data suggest that TET2 loss leads to hypermethyation of the PRDM1genomic locus (encoding Blimp-1) and alters the relative expression of Blimp-1 and Bcl-6, two antagonistic transcriptional repressors known to direct CD8+T cell memory differentiation. Together, our data indicate that TET2 is an important regulator of CD8+T cell fate decisions.

DNA methylation is one of the major epigenetic mechanisms that controls cellular differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases mediates active DNA demethylation through the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and subsequent intermediates. Here we demonstrate that TET2 regulates CD8+T cell differentiation in vivofollowing acute and chronic viral infection. At steady-state, mice with a T-cell specific deletion of TET2 have intact thymic and peripheral T cell populations. Following acute viral infection with LCMV-Armstrong, TET2 loss enhances LCMV-specific CD8+T cell memory differentiation in a cell-intrinsic manner without disrupting antigen-specific cell expansion or cytokine production. However, TET2-deficient memory CD8+T cells exhibit altered recall responses with blunted re-expansion, retained expression of phenotypic memory markers and restricted re-expression of activation markers. During chronic viral infection with LCMV-clone 13, TET2 controls CD8+T cell expansion and alters differentiation. Importantly, though mice with T-cell specific loss of TET2 developed similar levels of CD8+T cell exhaustion as wild-type mice, TET2 loss specifically reduced PD-1 expression suggesting that TET2 may direct DNA demethylation of the PD-1 locus. Together, our data indicate that TET2 is an important regulator of CD8+T cells following both acute and chronic viral infections and suggest targeting epigenetic regulators have potential for enhancing antiviral immunity.

Wherry: Genentech: Patents & Royalties.

Following activation by antigen, costimulation, and inflammation, naïve CD8+T cells initiate a program of clonal expansion and differentiation resulting in wide-spread changes in expression of genes involved in cell-cycle, metabolism, effector function, apoptosis, and homing. Although, several key transcription factors (TFs) have been shown to be important in effector CD8+T cell differentiation, the precise transcriptional regulation of this differentiation program remains poorly understood. The AP-1 family member BATF plays an important role in regulating differentiation and function in CD4+Th17 cells, CD4+follicular helper T cells, and in Ig class switching in B cells. We now show that BATF is also required for effector CD8+T cell differentiation and regulates a core program of genes involved in effector differentiation. We found that BATF expression is rapidly up-regulated during effector CD8+T cell differentiation in the mouse model of lymphocytic choriomeningitis virus (LCMV) infection. To examine the role of BATF in effector differentiation, we studied congenically distinct wild type (WT) and BATF knockout (KO) naïve P14 TCR transgenic CD8+T cells co- transferred into a WT host. Upon infection, the BATF KO cells exhibited a profound, cell-intrinsic defect in effector CD8+T cell differentiation, with a ∼400-fold decrease in peak number of effector cells. BATF KO effectors showed sustained activation and increased cell death by the mid-expansion phase of the immune response. To address the question of how loss of BATF causes such a severely diminished antigen-specific response, we profiled the binding sites of BATF throughout the genome by chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) in primary CD8+effector cells. We found that BATF bound to regulatory regions in many genes critical for effector differentiation, including transcription factors (e.g. Tbx21, Eomes, Prdm1), genes involved in cytokine signaling (e.g. Il12rb2, Il2ra), homing (e.g. Sell, Selp, Ccr9), effector function (e.g. Gzmb, Ifng, Il2), apoptosis (e.g. Bcl2, Bcl2l1, Mcl1), and T cell activation (e.g. Ctla4, Cd247, Tnfrsf4), suggesting a major role for BATF in effector CD8+T cell differentiation. Indeed, we found that genes bound by BATF were highly significantly overrepresented among genes that changed as a result of naïve CD8+T cells differentiating into effectors in vivo(P = 10-27). Comparison of gene expression in in vitroWT and BATF KO effectors confirmed that BATF bound genes were perturbed by BATF loss of function. Analysis of the kinetics of gene expression during the first 72 hours of effector differentiation showed that loss of BATF perturbed the temporal sequence of expression of critical transcription factors, such as T-bet and Eomes, and resulted in inappropriately early cytokine expression. This suggests that BATF may be required to coordinate the earliest events in CD8+T cell effector differentiation. To test this hypothesis, we used in vivoCFSE tracking to follow the early CD8+T cell response during LCMV infection. We found that while BATF KO CD8+T cells initiate cell division, there was a dramatic collapse in the ability to sustain proliferation and differentiation as early as day 3 post-infection. These results indicate that BATF ensures the orderly progression of a program of genes required by effector cells, restraining the expression of some and promoting the expression of others. More broadly, our results suggest that BATF may provide a common regulatory infrastructure for the development of effector cells in all T cell lineages.

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Abstract 2662