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The transcription factor BATF is required for differentiation of certain helper T cell subsets. Haining and colleagues show that BATF crucially regulates CD8 + effector cells by coordinating a transcription factor network. The transcription factor BATF is required for the differentiation of interleukin 17 (IL-17)-producing helper T cells (T H 17 cells) and follicular helper T cells (T FH cells). Here we identified a fundamental role for BATF in regulating the differentiation of effector of CD8 + T cells. BATF-deficient CD8 + T cells showed profound defects in effector population expansion and underwent proliferative and metabolic catastrophe early after encountering antigen. BATF, together with the transcription factors IRF4 and Jun proteins, bound to and promoted early expression of genes encoding lineage-specific transcription-factors (T-bet and Blimp-1) and cytokine receptors while paradoxically repressing genes encoding effector molecules (IFN-γ and granzyme B). Thus, BATF amplifies T cell antigen receptor (TCR)-dependent expression of transcription factors and augments the propagation of inflammatory signals but restrains the expression of genes encoding effector molecules. This checkpoint prevents irreversible commitment to an effector fate until a critical threshold of downstream transcriptional activity has been achieved.

Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

Lymph node fibroblastic reticular cells (FRCs) respond to signals from activated T cells by releasing nitric oxide, which inhibits T cell proliferation and restricts the size of the expanding T cell pool. Whether interactions with FRCs also support the function or differentiation of activated CD8 + T cells is not known. Here we report that encounters with FRCs enhanced cytokine production and remodeled chromatin accessibility in newly activated CD8 + T cells via interleukin-6. These epigenetic changes facilitated metabolic reprogramming and amplified the activity of pro-survival pathways through differential transcription factor activity. Accordingly, FRC conditioning significantly enhanced the persistence of virus-specific CD8 + T cells in vivo and augmented their differentiation into tissue-resident memory T cells. Our study demonstrates that FRCs play a role beyond restricting T cell expansion—they can also shape the fate and function of CD8 + T cells. Fibroblastic reticular cells (FRCs) are dynamic regulators of lymphoid tissue structure. Turley and colleagues show FRCs also support activated T cells by producing IL-6, which confers an advantage to CD8+ T cell memory responses.

Significance Effector CD8 ⁺ T-cell differentiation is essential for protective immunity. Investigating specific genes in this process by knockdown with RNAi is challenging because naive T cells are refractory to viral transduction. To overcome this obstacle, we developed a novel strategy to knock down genes in naive CD8 ⁺ T cells by creating bone marrow chimera from hematopoietic progenitors transduced with an inducible shRNA. This approach enabled inducible in vivo gene knockdown in any cell type developed from this progenitor pool. We applied this strategy to show that the transcription factor BATF is essential for initial commitment to effector differentiation but becomes dispensable by 72 h. This approach now enables the study of gene function in vivo in cells of hematopoietic origin otherwise refractory to viral transduction. The differentiation of effector CD8 ⁺ T cells is critical for the development of protective responses to pathogens and for effective vaccines. In the first few hours after activation, naive CD8 ⁺ T cells initiate a transcriptional program that leads to the formation of effector and memory T cells, but the regulation of this process is poorly understood. Investigating the role of specific transcription factors (TFs) in determining CD8 ⁺ effector T-cell fate by gene knockdown with RNAi is challenging because naive T cells are refractory to transduction with viral vectors without extensive ex vivo stimulation, which obscures the earliest events in effector differentiation. To overcome this obstacle, we developed a novel strategy to test the function of genes in naive CD8 ⁺ T cells in vivo by creating bone marrow chimera from hematopoietic progenitors transduced with an inducible shRNA construct. Following hematopoietic reconstitution, this approach allowed inducible in vivo gene knockdown in any cell type that developed from this transduced progenitor pool. We demonstrated that lentivirus-transduced progenitor cells could reconstitute normal hematopoiesis and develop into naive CD8 ⁺ T cells that were indistinguishable from wild-type naive T cells. This experimental system enabled induction of efficient gene knockdown in vivo without subsequent manipulation. We applied this strategy to show that the TF BATF is essential for initial commitment of naive CD8 ⁺ T cells to effector development but becomes dispensable by 72h. This approach makes possible the study of gene function in vivo in unperturbed cells of hematopoietic origin that are refractory to viral transduction.

Follicular Tregs (Tfr cells) inhibit antibody production, whereas follicular Th cells (Tfh cells) stimulate it. Tfr cells are found in blood; however, relatively little is known about the developmental signals for these cells or their functions. Here we demonstrated that circulating Tfr and Tfh cells share properties of memory cells and are distinct from effector Tfr and Tfh cells found within lymph nodes (LNs). Circulating memory-like Tfh cells were potently reactivated by DCs, homed to germinal centers, and produced more cytokines than did effector LN Tfh cells. Circulating memory-like Tfr cells persisted for long periods of time in vivo and homed to germinal centers after reactivation. Effector LN Tfr cells suppressed Tfh cell activation and production of cytokines, including IL-21, and inhibited class switch recombination and B cell activation. The suppressive function of this population was not dependent on specific antigen. Similar to LN effector Tfr cells, circulating Tfr cells also suppressed B and Tfh cells, but with a much lower capacity. Our data indicate that circulating memory-like Tfr cells are less suppressive than LN Tfr cells and circulating memory-like Tfh cells are more potent than LN effector Tfh cells; therefore, these circulating populations can provide rapid and robust systemic B cell help during secondary antigen exposure.

Blocking Programmed Death-1 (PD-1) can reinvigorate exhausted CD8 Tcells (TEX) and improve control of chronic infections and cancer. However, whether blocking PD-1 can reprogram TEX into durable memory Tcells (TMEM) is unclear. We found that reinvigoration of TEX in mice by PD-L1 blockade caused minimal memory development. After blockade, reinvigorated TEX became reexhausted if antigen concentration remained high and failed to become TMEM upon antigen clearance. TEX acquired an epigenetic profile distinct from that of effector Tcells (TEFF) and TMEM cells that was minimally remodeled after PD-L1 blockade. This finding suggests that TEX are a distinct lineage of CD8 T cells. Nevertheless, PD-1 pathway blockade resulted in transcriptional rewiring and reengagement of effector circuitry in the TEX epigenetic landscape. These data indicate that epigenetic fate inflexibility may limit current immunotherapies.

Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8⁺ T cells and show that it is distinct from functional memory CD8⁺ T cells. Exhausted CD8⁺ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing snows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8⁺ T cells.

NUT midline carcinoma (NMC) is an aggressive type of squamous cell carcinoma that is defined by the presence of BRD-NUT fusion oncogenes, which encode chimeric proteins that block differentiation and maintain tumor growth. BRD-NUT oncoproteins contain two bromodomains whose binding to acetylated histones is required for the blockade of differentiation in NMC, but the mechanisms by which BRD-NUT act remain uncertain. Here, we provide evidence that MYC is a key downstream target of BRD4-NUT. Expression profiling of NMCs shows that the set of genes whose expression is maintained by BRD4-NUT is highly enriched for MYC upregulated genes, and MYC and BRD4-NUT protein expression is strongly correlated in primary NMCs. More directly, we find that BRD4-NUT associates with the MYC promoter and is required to maintain MYC expression in NMC cell lines. Moreover, both siRNA knockdown of MYC and a dominant-negative form of MYC, omomyc, induce differentiation of NMC cells. Conversely, differentiation of NMC cells induced by knockdown of BRD4-NUT is abrogated by enforced expression of MYC. Together, these findings suggest that MYC is a downstream target of BRD4-NUT that is required for maintenance of NMC cells in an undifferentiated, proliferative state. Our findings support a model in which dysregulation of MYC by BRD-NUT fusion proteins has a central role in the pathogenesis of NMC.

Wucherpfennig and colleagues show that the microRNA miR-31 increases the sensitivity of T cells to type I interferons, which interferes with effector T cell function during chronic infection. During infection, antigen-specific T cells undergo tightly regulated developmental transitions controlled by transcriptional and post-transcriptional regulation of gene expression. We found that the microRNA miR-31 was strongly induced by activation of the T cell antigen receptor (TCR) in a pathway involving calcium and activation of the transcription factor NFAT. During chronic infection with lymphocytic choriomeningitis virus (LCMV) clone 13, miR-31-deficent mice recovered from clinical disease, while wild-type mice continued to show signs of disease. This disease phenotype was explained by the presence of larger numbers of cytokine-secreting LCMV-specific CD8 + T cells in miR-31-deficent mice than in wild-type mice. Mechanistically, miR-31 increased the sensitivity of T cells to type I interferons, which interfered with effector T cell function and increased the expression of several proteins related to T cell dysfunction during chronic infection. These studies identify miR-31 as an important regulator of T cell exhaustion in chronic infection.