NRF2-dependent regulation of the prostacyclin receptor PTGIR drives CD8 T cell exhaustion.

The progressive decline of CD8 T cell effector function-also known as terminal exhaustion-is a major contributor to immune evasion in cancer. Yet, the molecular mechanisms that drive CD8 T cell dysfunction remain poorly understood. Here, we report that the Kelch-like ECH-associated protein 1 (KEAP1)-Nuclear factor erythroid 2-related factor 2 (NRF2) signaling axis, which mediates cellular adaptations to oxidative stress, directly regulates CD8 T cell exhaustion. Transcriptional profiling of dysfunctional CD8 T cells from chronic infection and cancer reveals enrichment of NRF2 activity in terminally exhausted (Texterm) CD8 T cells. Increasing NRF2 activity in CD8 T cells (via conditional deletion of KEAP1) promotes increased glutathione production and antioxidant defense yet accelerates the development of terminally exhausted (PD-1+TIM-3+) CD8 T cells in response to chronic infection or tumor challenge. Mechanistically, we identify PTGIR, a receptor for the circulating eicosanoid prostacyclin, as an NRF2-regulated protein that promotes CD8 T cell dysfunction. Silencing PTGIR expression restores the anti-tumor function of KEAP1-deficient T cells. Moreover, lowering PTGIR expression in CD8 T cells both reduces terminal exhaustion and enhances T cell effector responses (i.e. IFN-γ and granzyme production) to chronic infection and cancer. Together, these results establish the KEAP1-NRF2 axis as a metabolic sensor linking oxidative stress to CD8 T cell dysfunction and identify the prostacyclin receptor PTGIR as an NRF2-regulated immune checkpoint that regulates CD8 T cell fate decisions between effector and exhausted states.

FOXP1 and KLF2 reciprocally regulate checkpoints of stem-like to effector transition in CAR T cells.

In cancer and infections, self-renewing stem-like CD8+ T cells mediate the response of immunotherapies and replenish terminally exhausted T cells and effector-like T cells. However, the programs governing the lineage choice in chimeric antigen receptor (CAR) T cells are unclear. Here, by simultaneously profiling single-cell chromatin accessibility and transcriptome in the same CAR T cells, we identified heterogeneous chromatin states within CD8+ T cell subsets that foreshadowed transcriptional changes and were primed for regulation by distinct transcription factors. Transcription factors that controlled each CD8+ T cell subset were regulated by high numbers of enhancers and positioned as hubs of gene networks. FOXP1, a hub in the stem-like network, promoted expansion and stemness of CAR T cells and limited excessive effector differentiation. In the effector network, KLF2 enhanced effector CD8+ T cell differentiation and prevented terminal exhaustion. Thus, we identified gene networks and hub transcription factors that controlled the differentiation of stem-like CD8+ CAR T cells into effector or exhausted CD8+ CAR T cells.

Facts and Hopes for Immunotherapy in Renal Cell Carcinoma.

Immunotherapy has made a significant impact in many tumors, including renal cell carcinoma (RCC). RCC has been known to be immunoresponsive since the cytokine era of IFNα and IL2, but only a small number of patients had durable clinical benefit. Since then, discoveries of key tumor drivers, as well as an understanding of the contribution of angiogenesis and the tumor microenvironment (TME), has led to advances in drug development, ultimately transforming patient outcomes. Combinations of anti-angiogenic agents with immune checkpoint inhibitors are now standard of care. Current challenges include patient selection for immunotherapy combinations, resistance acquisition, and optimally sequencing therapies. Further discoveries about RCC biology, the TME, and resistance mechanisms will likely pave the way for the next generation of therapies.

The GPR171 pathway suppresses T cell activation and limits antitumor immunity.

The recently identified G-protein-coupled receptor GPR171 and its ligand BigLEN are thought to regulate food uptake and anxiety. Though GPR171 is commonly used as a T cell signature gene in transcriptomic studies, its potential role in T cell immunity has not been explored. Here we show that GPR171 is transcribed in T cells and its protein expression is induced upon antigen stimulation. The neuropeptide ligand BigLEN interacts with GPR171 to suppress T cell receptor-mediated signalling pathways and to inhibit T cell proliferation. Loss of GPR171 in T cells leads to hyperactivity to antigen stimulation and GPR171 knockout mice exhibit enhanced antitumor immunity. Blockade of GPR171 signalling by an antagonist promotes antitumor T cell immunity and improves immune checkpoint blockade therapies. Together, our study identifies the GPR171/BigLEN axis as a T cell checkpoint pathway that can be modulated for cancer immunotherapy.

PI3Kδ coordinates transcriptional, chromatin, and metabolic changes to promote effector CD8+ T cells at the expense of central memory.

Patients with activated phosphatidylinositol 3-kinase delta (PI3Kδ) syndrome (APDS) present with sinopulmonary infections, lymphadenopathy, and cytomegalvirus (CMV) and/or Epstein-Barr virus (EBV) viremia, yet why patients fail to clear certain chronic viral infections remains incompletely understood. Using patient samples and a mouse model (Pik3cdE1020K/+ mice), we demonstrate that, upon activation, Pik3cdE1020K/+ CD8+ T cells exhibit exaggerated features of effector populations both in vitro and after viral infection that are associated with increased Fas-mediated apoptosis due to sustained FoxO1 phosphorylation and Fasl derepression, enhanced mTORC1 and c-Myc signatures, metabolic perturbations, and an altered chromatin landscape. Conversely, Pik3cdE1020K/+ CD8+ cells fail to sustain expression of proteins critical for central memory, including TCF1. Strikingly, activated Pik3cdE1020K/+ CD8+ cells exhibit altered transcriptional and epigenetic circuits characterized by pronounced interleukin-2 (IL-2)/STAT5 signatures and heightened IL-2 responses that prevent differentiation to memory-like cells in IL-15. Our data position PI3Kδ as integrating multiple signaling nodes that promote CD8+ T cell effector differentiation, providing insight into phenotypes of patients with APDS.

BACH2 enforces the transcriptional and epigenetic programs of stem-like CD8+ T cells.

During chronic infection and cancer, a self-renewing CD8+ T cell subset maintains long-term immunity and is critical to the effectiveness of immunotherapy. These stem-like CD8+ T cells diverge from other CD8+ subsets early after chronic viral infection. However, pathways guarding stem-like CD8+ T cells against terminal exhaustion remain unclear. Here, we show that the gene encoding transcriptional repressor BACH2 is transcriptionally and epigenetically active in stem-like CD8+ T cells but not terminally exhausted cells early after infection. BACH2 overexpression enforced stem-like cell fate, whereas BACH2 deficiency impaired stem-like CD8+ T cell differentiation. Single-cell transcriptomic and epigenomic approaches revealed that BACH2 established the transcriptional and epigenetic programs of stem-like CD8+ T cells. In addition, BACH2 suppressed the molecular program driving terminal exhaustion through transcriptional repression and epigenetic silencing. Thus, our study reveals a new pathway that enforces commitment to stem-like CD8+ lineage and prevents an alternative terminally exhausted cell fate.

Single-cell RNA-seq reveals TOX as a key regulator of CD8+ T cell persistence in chronic infection.

Progenitor-like CD8+ T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including T cell-specific transcription factor 1 (TCF1), but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing the single-cell transcriptomes and epigenetic profiles of CD8+ T cells responding to acute and chronic viral infections, we found that progenitor-like CD8+ T cells became distinct from memory precursor cells before the peak of the T cell response. We discovered a coexpression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursor cells. Moreover, thymocyte selection-associated high mobility group box protein TOX (TOX) promoted the persistence of antiviral CD8+ T cells and was required for the programming of progenitor-like CD8+ T cells. Thus, long-term CD8+ T cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.

IL-27 promotes the expansion of self-renewing CD8+ T cells in persistent viral infection.

Chronic infection and cancer are associated with suppressed T cell responses in the presence of cognate antigen. Recent work identified memory-like CXCR5+ TCF1+ CD8+ T cells that sustain T cell responses during persistent infection and proliferate upon anti-PD1 treatment. Approaches to expand these cells are sought. We show that blockade of interferon type 1 (IFN-I) receptor leads to CXCR5+ CD8+ T cell expansion in an IL-27- and STAT1-dependent manner. IFNAR1 blockade promoted accelerated cell division and retention of TCF1 in virus-specific CD8+ T cells. We found that CD8+ T cell-intrinsic IL-27 signaling safeguards the ability of TCF1hi cells to maintain proliferation and avoid terminal differentiation or programmed cell death. Mechanistically, IL-27 endowed rapidly dividing cells with IRF1, a transcription factor that was required for sustained division in a cell-intrinsic manner. These findings reveal that IL-27 opposes IFN-I to uncouple effector differentiation from cell division and suggest that IL-27 signaling could be exploited to augment self-renewing T cells in chronic infections and cancer.

miR-155 harnesses Phf19 to potentiate cancer immunotherapy through epigenetic reprogramming of CD8+ T cell fate.

T cell senescence and exhaustion are major barriers to successful cancer immunotherapy. Here we show that miR-155 increases CD8+ T cell antitumor function by restraining T cell senescence and functional exhaustion through epigenetic silencing of drivers of terminal differentiation. miR-155 enhances Polycomb repressor complex 2 (PRC2) activity indirectly by promoting the expression of the PRC2-associated factor Phf19 through downregulation of the Akt inhibitor, Ship1. Phf19 orchestrates a transcriptional program extensively shared with miR-155 to restrain T cell senescence and sustain CD8+ T cell antitumor responses. These effects rely on Phf19 histone-binding capacity, which is critical for the recruitment of PRC2 to the target chromatin. These findings establish the miR-155-Phf19-PRC2 as a pivotal axis regulating CD8+ T cell differentiation, thereby paving new ways for potentiating cancer immunotherapy through epigenetic reprogramming of CD8+ T cell fate.

mTOR Promotes Antiviral Humoral Immunity by Differentially Regulating CD4 Helper T Cell and B Cell Responses.

mTOR has important roles in regulation of both innate and adaptive immunity, but whether and how mTOR modulates humoral immune responses have yet to be fully understood. To address this issue, we examined the effects of rapamycin, a specific inhibitor of mTOR, on B cell and CD4 T cell responses during acute infection with lymphocytic choriomeningitis virus. Rapamycin treatment resulted in suppression of virus-specific B cell responses by inhibiting proliferation of germinal center (GC) B cells. In contrast, the number of memory CD4 T cells was increased in rapamycin-treated mice. However, the drug treatment caused a striking bias of CD4 T cell differentiation into Th1 cells and substantially impaired formation of follicular helper T (Tfh) cells, which are essential for humoral immunity. Further experiments in which mTOR signaling was modulated by RNA interference (RNAi) revealed that B cells were the primary target cells of rapamycin for the impaired humoral immunity and that reduced Tfh formation in rapamycin-treated mice was due to lower GC B cell responses that are essential for Tfh generation. Additionally, we found that rapamycin had minimal effects on B cell responses activated by lipopolysaccharide (LPS), which stimulates B cells in an antigen-independent manner, suggesting that rapamycin specifically inhibits B cell responses induced by B cell receptor stimulation with antigen. Together, these findings demonstrate that mTOR signals play an essential role in antigen-specific humoral immune responses by differentially regulating B cell and CD4 T cell responses during acute viral infection and that rapamycin treatment alters the interplay of immune cell subsets involved in antiviral humoral immunity. IMPORTANCE: mTOR is a serine/threonine kinase involved in a variety of cellular activities. Although its specific inhibitor, rapamycin, is currently used as an immunosuppressive drug in transplant patients, it has been reported that rapamycin can also stimulate pathogen-specific cellular immunity in certain circumstances. However, whether and how mTOR regulates humoral immunity are not well understood. Here we found that rapamycin treatment predominantly inhibited GC B cell responses during viral infection and that this led to biased helper CD4 T cell differentiation as well as impaired antibody responses. These findings suggest that inhibition of B cell responses by rapamycin may play an important role in regulation of allograft-specific antibody responses to prevent organ rejection in transplant recipients. Our results also show that consideration of antibody responses is required in cases where rapamycin is used to stimulate vaccine-induced immunity.

The TCF1-Bcl6 axis counteracts type I interferon to repress exhaustion and maintain T cell stemness.

During chronic viral infections and in cancer, T cells become dysfunctional, a state known as T cell exhaustion. Although it is well recognized that memory CD8 T cells account for the persistence of CD8 T cell immunity after acute infection, how exhausted T cells persist remains less clear. Using chronic infection with lymphocytic choriomeningitis virus clone 13 and tumor samples, we demonstrate that CD8 T cells differentiate into a less exhausted TCF1high and a more exhausted TCF1low population. Virus-specific TCF1high CD8 T cells, which resemble T follicular helper (TFH) cells, persist and recall better than do TCF1low cells and act as progenitor cells to replenish TCF1low cells. We show that TCF1 is both necessary and sufficient to support this progenitor-like CD8 subset, whereas cell-intrinsic type I interferon signaling suppresses their differentiation. Accordingly, cell-intrinsic TCF1 deficiency led to a loss of these progenitor CD8 T cells, sharp contraction of virus-specific T cells, and uncontrolled viremia. Mechanistically, TCF1 repressed several pro-exhaustion factors and induced Bcl6 in CD8 T cells, which promoted the progenitor fate. We propose that the TCF1-Bcl6 axis counteracts type I interferon to repress T cell exhaustion and maintain T cell stemness, which is critical for persistent antiviral CD8 T cell responses in chronic infection. These findings provide insight into the requirements for persistence of T cell immune responses in the face of exhaustion and suggest mechanisms by which effective T cell-mediated immunity may be enhanced during chronic infections and cancer.

TCF1 Is Required for the T Follicular Helper Cell Response to Viral Infection.

T follicular helper (TFH) and T helper 1 (Th1) cells generated after viral infections are critical for the control of infection and the development of immunological memory. However, the mechanisms that govern the differentiation and maintenance of these two distinct lineages during viral infection remain unclear. We found that viral-specific TFH and Th1 cells showed reciprocal expression of the transcriptions factors TCF1 and Blimp1 early after infection, even before the differential expression of the canonical TFH marker CXCR5. Furthermore, TCF1 was intrinsically required for the TFH cell response to viral infection; in the absence of TCF1, the TFH cell response was severely compromised, and the remaining TCF1-deficient TFH cells failed to maintain TFH-associated transcriptional and metabolic signatures, which were distinct from those in Th1 cells. Mechanistically, TCF1 functioned through forming negative feedback loops with IL-2 and Blimp1. Our findings demonstrate an essential role of TCF1 in TFH cell responses to viral infection.

Cutting Edge: miR-17-92 Is Required for Both CD4 Th1 and T Follicular Helper Cell Responses during Viral Infection.

Viral infections induce the differentiation of naive CD4 T cells into two distinct lineages, Th1 cells and T follicular helper (TFH) cells. Two recent studies demonstrated that the microRNA cluster miR-17-92 selectively promotes CD4 TFH responses. However, we show in this study that miR-17-92 expression is required for the clonal expansion of both virus-specific Th1 and TFH cells. Upon viral infection, miR-17-92-deficient CD4 T cells showed impaired clonal expansion and subsequent memory formation. Although miR-17-92 deficiency impaired the clonal expansion of both Th1 and TFH cells, the expansion of Th1 cells was more affected. Overexpression of miR-17-92 in CD4 T cells resulted in increased expansion of both virus-specific Th1 and TFH cells but selectively enhanced the Th1 response. Taken together, our data suggest that miR-17-92 is necessary for both Th1 and TFH cells to respond efficiently to viral infections and that the Th1 response is more sensitive to the level of miR-17-92 expression.

Distinct memory CD4+ T cells with commitment to T follicular helper- and T helper 1-cell lineages are generated after acute viral infection.

CD4(+) T follicular helper (Tfh) cells provide the required signals to B cells for germinal center reactions that are necessary for long-lived antibody responses. However, it remains unclear whether there are CD4(+) memory T cells committed to the Tfh cell lineage after antigen clearance. By using adoptive transfer of antigen-specific memory CD4(+) T cell subpopulations in the lymphocytic choriomeningitis virus infection model, we found that there are distinct memory CD4(+) T cell populations with commitment to either Tfh- or Th1-cell lineages. Our conclusions are based on gene expression profiles, epigenetic studies, and phenotypic and functional analyses. Our findings indicate that CD4(+) memory T cells "remember" their previous effector lineage after antigen clearance, being poised to reacquire their lineage-specific effector functions upon antigen reencounter. These findings have important implications for rational vaccine design, where improving the generation and engagement of memory Tfh cells could be used to enhance vaccine-induced protective immunity.

TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation.

Tuberous sclerosis complex human disease gene products TSC1 and TSC2 form a functional complex that negatively regulates target of rapamycin (TOR), an evolutionarily conserved kinase that plays a central role in cell growth and metabolism. Here, we describe a novel role of TSC1/2 in controlling stem cell maintenance. We show that in the Drosophila ovary, disruption of either the Tsc1 or Tsc2 gene in germline stem cells (GSCs) leads to precocious GSC differentiation and loss. The GSC loss can be rescued by treatment with TORC1 inhibitor rapamycin, or by eliminating S6K, a TORC1 downstream effecter, suggesting that precocious differentiation of Tsc1/2 mutant GSC is due to hyperactivation of TORC1. One well-studied mechanism for GSC maintenance is that BMP signals from the niche directly repress the expression of a differentiation-promoting gene bag of marbles (bam) in GSCs. In Tsc1/2 mutant GSCs, BMP signalling activity is downregulated, but bam expression is still repressed. Moreover, Tsc1 bam double mutant GSCs could differentiate into early cystocytes, suggesting that TSC1/2 controls GSC differentiation via both BMP-Bam-dependent and -independent pathways. Taken together, these results suggest that TSC prevents precocious GSC differentiation by inhibiting TORC1 activity and subsequently differentiation-promoting programs. As TSC1/2-TORC1 signalling is highly conserved from Drosophila to mammals, it could have a similar role in controlling stem cell behaviour in mammals, including humans.