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SUMMARYChronic viral infections disrupt B cell responses leading to impaired affinity maturation and delayed control of viremia. Previous studies have identified early pre-germinal center (GC) B cell attrition but the impact of chronic infections on B cell fate decisions in the GC remains poorly understood. To address this question, we used single-cell transcriptional profiling of virus-specific GC B cells to test the hypothesis that chronic viral infection disrupted GC B cell fate decisions leading to suboptimal humoral immunity. These studies revealed a critical GC differentiation checkpoint that is disrupted by chronic infection, specifically at the point of dark zone re-entry. During chronic viral infection, virus-specific GC B cells were shunted towards terminal plasma cell (PC) or memory B cell (MBC) fates at the expense of continued participation in the GC. Early GC exit was associated with decreased B cell mutational burden and antibody quality. Persisting antigen and inflammation independently drove facets of dysregulation, with a key role for inflammation in directing premature terminal GC B cell differentiation and GC exit. Thus, these studies define GC defects during chronic viral infection and identify a critical GC checkpoint that is short-circuited, preventing optimal maturation of humoral immunity.

TCF-1 is a key transcription factor in progenitor exhausted CD8 T cells (Tex). Moreover, this Tex cell subset mediates responses to PD-1 checkpoint pathway blockade. However, the role of the transcription factor TCF-1 in early fate decisions and initial generation of Tex cells is unclear. Single cell RNA-sequencing (scRNA-seq) and lineage tracing identified a TCF-1(+)Ly108(+)PD-1(+) CD8 T cell population early during chronic infection that seeds development of mature Tex cells. TCF-1 mediated the bifurcation between divergent fates, repressing development of terminal KLRG1(Hi) effectors while fostering KLRG1(Lo) Tex precursor cells, and PD-1 stabilized this TCF-1(+) Tex precursor cell pool. TCF-1 mediated a T-bet to Eomes transcription factor transition in Tex precursors by promoting Eomes expression and drove c-Myb expression that controlled Bcl-2 and survival. These data define a role for TCF-1 in early fate bifurcation driving Tex precursor cells, and also identify PD-1 as a protector of this early TCF-1 subset.

In the metastasis-targeted organs, angiogenesis is essential for the progression of dormant micrometastases to rapidly growing and clinically overt lesions. However, we observed changes suggesting angiogenic switching in the mouse lungs prior to arrival of tumor cells (i.e., in the premetastatic niche) in the models of breast carcinoma. This angiogenic switching appears to be caused by myeloid-derived suppressor cells recruited to the premetastatic lungs through complement C5a receptor 1 signaling. These myeloid cells are known to secrete several proangiogenic factors in tumors, including IL-1β and matrix metalloproteinase-9, and we found upregulation of these genes in the premetastatic lungs. Blockade of C5a receptor 1 synergized with antiangiogenic Listeria monocytogenes–based vaccines to decrease the lung metastatic burden by reducing vascular density and improving antitumor immunity in the lungs. This was mediated even when growth of primary breast tumors was not affected by these treatments. This work provides initial evidence that angiogenesis contributes to the premetastatic niche in rapidly progressing cancers and that inhibiting this process through immunotherapy is beneficial for reducing or even preventing metastasis.

CD8

Robust CD8

We discovered that 90.3% of patients with angiomyolipomas, LAM, and tuberous sclerosis complex (TSC) carry the arginine variant of codon 72 (R72) of TP53 and that R72 increases the risk for angiomyolipoma. R72 transactivates NOTCH1 and NODAL better than the proline variant of codon 72 (P72), therefore, the expression of NOTCH1 and NODAL is increased in angiomyolipoma cells that carry R72. The loss of Tp53 and Tsc1 within nestin expressing cells in mice resulted in development of RCC with high Notch1 and Nodal expression suggesting that similar downstream mechanisms contribute to tumorigenesis as a result of p53 loss in mice and p53 polymorphism in humans. The loss of murine Tp53 or expression of human R72 contribute to tumorigenesis via enhancing epithelial-to-mesenchymal transition and motility of tumor cells through the Notch and Nodal pathways. IMPLICATIONS: This work revealed unexpected contributions of the p53 polymorphism to the pathogenesis of TSC and established signaling alterations caused by this polymorphism as a target for therapy. We found that the codon 72 TP53 polymorphism contributes to TSC-associated tumorigenesis via Notch and Nodal signaling.

CD8 T cells are critical for clearing infections and immune surveillance of cancers. However, in the setting of chronic viral infections and tumor growth, CD8 T cells become exhausted. T cell exhaustion (TEX) is characterized by a progressive increase of inhibitory receptor expression and concomitant diminishment of effector function. This diminished function renders the host unable to clear the virus or cancer. The TEX population is heterogenous and can be divided broadly into TEX progenitor and TEX terminal populations. TEX progenitor are self-renewing, responsive to PD-1 blockade and give rise to the later TEX subsets, including short-lived TEX terminal cells. Control of DNA demethylation is required in multiple settings for maintenance or loss of stem-like characteristics. We report that TET2, a methylcytosine dioxygenase that mediates active DNA demethylation, is required for TEX progenitors to differentiate fully into TEX terminal cells. TET2-deficiency during persistent viral infection resulted in a numerical loss of TEX terminal cells, while maintaining the TEX progenitor population. The TEX terminal cells that escaped this differentiation block retained some features of TEX progenitors, including decreased expression of several inhibitory receptors. TET2-deficient TEX progenitors responded to PD-1 blockade but were unable to establish expansion of TEX cells thought to be required for viral control. These data highlight the role of DNA methylation and TET2 in establishing TEX diversity.

Exhaustion gradually establishes in chronically stimulated CD8s and this process is not reverted by current therapeutic approaches due to establishment of a stable epigenetic program. Recent advances have informed on the developmental process of exhaustion and highlighted TOX as a key lineage-defining TF in the process. Yet, little remains known on molecular pathways capable of antagonizing the TOX-dependent exhaustion program. By depicting transcriptional changes at key developmental steps of exhaustion, we demonstrate an antagonistic role for the TF STAT5 in the development of CD8 T cell exhaustion. STAT5 transcriptional network is heavily silenced upon chronic antigenic stimulation in a TOX-dependent manner which allows initiation of the exhaustion lineage. Increasing STAT5 activity abrogates establishment of the exhaustion lineage leading to the development of effector-like CD8s that acquire a unique transcriptional identity, distinct from exhausted cells, persist throughout chronicity and demonstrate higher protective capacity. Using temporal loss and gain of function approaches, we show that STAT5 triggers loss of progenitor identity by exhausted CD8s (Tex) and subsequent differentiation into the recently identified effector-like intermediate Tex subset. Temporal increase in STAT5 activity also robustly synergizes with PD-L1 blockade by further fostering intermediate Tex cells accumulation. Together, we show that modulating STAT5 activity may counteract the exhaustion process and favor instigation of effector-like characteristic in Tex cells suitable for optimal therapeutic efficacy. This work is supported by the Parker Institute for Cancer Immunotherapy (PICI). JC-Beltra is a PICI scholar awardee.

Although immune checkpoint blockade revolutionized cancer therapy, response rates have been mixed in gynecological malignancies. While uterine endometrial cancer with high microsatellite instability (MSI-HI) and high tumor mutational burden (TMB) respond robustly to checkpoint blockade, high-grade serous ovarian cancer (HGSOC) with low TMB respond modestly. Currently, there has been no known immune signature or T cell phenotype that predicts clinical response in gynecological tumors. To dissect the immune landscape and T cell phenotypes in gynecological cancer patients receiving PD-1 blockade, we used high-dimensional cytometry (flow cytometry and mass cytometry (CyTOF)). We performed longitudinal deep immune profiling of PBMC from patients with recurrent uterine endometrial cancer receiving single-arm nivolumab, and HSGOC patients receiving neoadjuvant nivolumab plus platinum-based chemotherapy prior to debulking surgery. Chemotherapy-resistant MSI-HI uterine cancer patients treated with nivolumab had a proliferative T cell response 2-4 weeks post PD-1 blockade, consistent with responses seen in high TMB melanoma and lung cancer. The responding Ki67+ CD8 T cell population was largely CD45RAloCD27hi or CD45RAloCD27lo and highly expressed PD1, CTLA-4, and CD39, consistent with the phenotype of exhausted T cells (TEX). These exhausted-like cells, especially expansion of T-bet+ subset, were enriched in responders, whereas early expansion of regulatory T cells (Treg) and enrichment of terminally differentiated effector/senescence-like T cells were observed in non-responders. Unlike patients with uterine endometrial cancer, patients with TMB-low ovarian cancer did not have a clear proliferative CD8 T cell response, but had an uncoupled proliferation of Treg, after neoadjuvant nivolumab plus chemotherapy treatment, suggesting systemic immune suppression in ovarian cancer patients. At baseline, ovarian cancer without recurrence had more terminally differentiated effector-like CD8 T cells, in contrast to TMB-high uterine cancer. Furthermore, in both cohorts, non-responders or patients with recurrence, had more naive-like cell population. Thus, both high and low TMB gynecological tumors have distinct immune landscapes associated with clinical response. Additionally, in MSI-HI uterine endometrial cancer patients, the length of time between the prior chemotherapy and the initiation of immunotherapy was negatively correlated with T cell reinvigoration and clinical response. This suggests that therapeutic timing and optimized strategies for combining chemotherapy and immunotherapy are still needed. Collectively, our immune profiling revealed the distinct immune signatures associated with clinical response to PD-1 blockade in gynecological cancers. Our results also suggest that high TMB (inflamed) versus low TMB (cold) tumor microenvironment, and timing of chemo/immunotherapy could impact differentiation and functions of T cells. Citation Format: Yuki Muroyama, Sasikanth Manne, Alexander C. Huang, Divij Mathew, Lakshmi Chilukuri, Allison R. Greenplate, Takuya Ohtani, Dmitriy Zamarin, Claire F. Friedman, E. John Wherry. Distinct immune signatures predicting clinical response to PD-1 blockade therapy in gynecological cancers revealed by high-dimensional immune profiling [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO068.

Over the past decade, blockade of the immune checkpoints CTLA-4, PD-1, and PD-L1 has been demonstrated to significantly extend survival of cancer patients across multiple tumor types, including metastatic melanoma, formally proving that immunotherapy is a viable option for the treatment of cancer. These successes have paved the way for the development of additional immune-modulatory antibodies, blocking alternative inhibitory receptors, or engaging costimulatory receptors such as the TNF receptor family member GITR. However, the clinical experience accumulated thus far with checkpoint blockade has clearly shown that only a limited fraction of patients achieve durable clinical benefit with these treatments. This highlights the need to deepen our understanding of the molecular mechanisms underlying response and resistance to immunotherapy and design more personalized and rational combinations based on these therapies. Immune-regulatory mechanisms are one of the major barriers limiting efficacy of immunotherapy. CTLA-4 blockade and GITR costimulation are two immunotherapeutic strategies known to interfere with conventional immunosuppressive regulatory T cells (Tregs). We thus investigated the effects of CTLA-4 blockade and GITR costimulation on suppressive T cells in in vivo mouse melanoma models resistant to these therapies with the aim to clarify the molecular mechanisms underlying refractoriness and provide the rationale to develop more effective therapeutic combinations. To understand the clinical relevance of these findings, we explored the same effects in cancer patients treated with CTLA-4 blockade or GITR costimulation. We found that CTLA-4 blockade, while counteracting conventional Tregs, promotes the expansion of a subset of CD4+Foxp3-T cells expressing high levels of PD-1 (4PD1hi), which constitute a new immunosuppressive T-cell population with T-follicular-helper-like features. Importantly, we observed that anti-CTLA-4 increases the frequency of circulating 4PD1hi in a dose-dependent manner. In contrast, PD-1 blockade decreases 4PD1hi in function of its clinical activity, underscoring the relevance of this cell subset as a pharmacodynamic and prognostic biomarker of checkpoint blockade. These findings indicate that optimizing checkpoint blockade doses and combination regimens so as to keep 4PD1hi in the right balance may favor a positive outcome. In mouse melanoma models of response (early tumors) and refractoriness (advanced tumors) to GITR agonism, we found that anti-GITR efficiently reduces Tregs and increases effector:Treg ratios in both curative and refractory treatment conditions. However, T-cell activation and cytotoxic functions are favored selectively in the presence of low tumor burden. Counteracting exhaustion with PD-1 blockade in combination with GITR agonism restored responsiveness of advanced tumors and CD8+ T-cell functionality. Aligned with the effects of anti-GITR in mice, we found that the agonist anti-human GITR antibody TRX518 decreases Tregs in peripheral blood and tumor to similar extents in patients treated in the first-in-human single-dose monotherapy trial (NCT01239134). However, coincident downregulation of Tregs in the peripheral blood and at the tumor site upon TRX518 was not sufficient to achieve substantial clinical responses in this population of advanced solid cancer patients. This suggests that Treg elimination from advanced tumors may not be sufficient to activate cytotoxic T-cell responses unless the T-cell exhaustion process is concurrently blocked. Based on these preclinical and clinical observations, we have started to explore anti-GITR in combination with PD-1 pathway blockade in patients with advanced solid tumors (NCT02628574). Taken as a whole, these findings illustrate the value of conventional and nonconventional immune-suppressive T cells as biomarkers of biologic and therapeutic activity of immunotherapy in melanoma and other tumor types. In addition, these results indicate that inhibition of immune-regulatory mechanisms, such as immunosuppressive T cells, may need to be associated with strategies able to positively activate T-cell responses to achieve significant clinical benefit. Citation Format: Roberta Zappasodi, Sadna Budhu, Cynthia Sirard, Jingjing Qi, Cailian Liu, Yanyun Li, Yasin Senbabaoglu, Sasikanth Manne, Billel Gasmi, Hong Zhong, Xia Yang, Moshen Abu-Akeel, David Schaer, Alexander Huang, Walter Newman, Philip Wong, Katherine S. Panageas, Michael A. Postow, Henry Koon, Vamsidhar Velcheti, Margaret K. Callahan, Matthew D. Hellmann, E. John Wherry, Taha Merghoub, Jedd D. Wolchok. Overcoming immune resistance with rationally designed combination immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA04.

Despite the high tumor mutational burden (TMB), immune checkpoint blockade (ICB) still fails in some microsatellite instability-high (MSI-H) tumors, suggesting the underlying T cell dysfunction. Immune profiling of PBMC from chemotherapy-resistant MSI-H uterine cancer patients treated with nivolumab identified the proliferative T cell response 2–4 weeks post PD-1 blockade. However, this “immunological response” to ICB was observed regardless of clinical response, suggesting the additional determinants of clinical outcome beyond the proliferative response or high TMB. To test the hypothesis that the gap between immunological and clinical response to ICB was related to T celI-intrinsic response to genotoxic and/or proliferative stress, a novel high dimensional cytometry platform was developed. The platform enables simultaneous analysis of T cell differentiation state and interrogation of multiple DNA damage and repair response (DDR) pathways at single cell resolution. This DDR-Immune platform revealed consistent T cell subset specific patterns of DDR, as well as specific DDR pathways induced by different types of DNA damage. Application of the DDR-Immune platform to this cohort revealed that, in clinical responders, Ki67+ CD8 T cells had rapid increase of pATM, presumably in response to proliferative stress induced by PD-1 blockade. This DDR was not clearly observed in clinical non-responders. In addition, Tregs from clinical non-responders had elevated DDRs compared to responders, suggesting their greater resistance to genotoxic/proliferative stress. Collectively, the new platform reveals previously unrecognized roles for T cell-intrinsic DDR as a novel determinant of immune responsiveness and clinical outcome.

Overcoming acquired adaptive immune resistance to anti-PD-1 therapy is imperative for enhancing the efficacy of immune checkpoint blockade (ICB) in solid tumors. Regulatory T cells (Tregs) play a prominent role in the suppressive tumor microenvironment (TME) and are major contributors to adaptive immune resistance. Tregs limit CD8+ T cell reinvigoration and are a promising target for combination therapy. While the clinical efficacy of anti-CTLA4 may be partially explained by restriction of Tregs, its co-administration with anti-PD1 causes significant toxicity. Thus, safer approaches to limit Treg activity are needed. To elucidate the dynamic changes in immuno-regulatory circuits within the TME during ICB, we performed deep immune profiling of peripheral blood and tumors from patients with advanced melanoma prior to (n=7) and after 1 cycle of anti-PD-1 therapy with pembrolizumab (n=9). Tregs were abundant in the TME and retained their immunosuppressive phenotype and functionality following anti-PD-1. Epigenetic, transcriptomic, and proteomic analysis of Tregs after ICB identified tumor necrosis factor receptor 2 (TNFR2) signaling as a possible driver of CD8+ T cell suppression. TNFR2 was preferentially expressed by Tregs in the TME (mean 18.03 %, SD +/− 10.13 %) relative to CD8+ T cells (mean 0.64 %, SD +/− 0.82 %) and peripheral Tregs (mean 3.16 %, SD +/− 3.21 %), suggesting it might be a safe and effective target for combination therapy. Indeed, dual blockade of TNFR2 and PD-1 led to potent CD8+ T cell expansion in two mouse tumor models, and restored sensitivity to ICB in a resistant murine model of melanoma. Our data suggest that anti-TNFR2 might synergize with current ICB by countering the development of adaptive immune resistance.

Targeting PD1 has yielded clinical success across many tumor types, yet a significant proportion of patients remain unresponsive to treatment. Co-expression of PD1 and LAG3 inhibitory receptors (IRs) on CD8+ tumor-infiltrating T cells (TIL) is associated with an exhausted phenotype and dual blockade synergistically limits tumor growth, greater than targeting PD1 alone. The cellular and mechanistic basis for PD1/LAG3 synergy is unknown. We have generated conditional knockin mice that facilitates the CD8+ T cell-restricted deletion of Pdcd1 and/or Lag3 when crossed to E8ICre.GFP. These mice have also been crossed with a pMEL TCR transgenic mouse, congenically marked to allow intrinsic analysis of PD1 and/or LAG3-deficient CD8+ T cells, and controls, in a ‘quad’ co-adoptive transfer system. Pdcd1L/L and/or Lag3L/L-yfp E8ICre.GFP mice show reduced B16-F10 tumor growth with improved survival, compared to Lag3L/L-yfp E8ICre.GFP mice and controls. CD8+ TIL frequency is increased with loss of both IRs, as a result of enhanced proliferation. Although expression of TIM3, TIGIT and 2B4 IRs is maintained, CD8+ TIL isolated from Pdcd1L/LE8ICre.GFP and Pdcd1L/ Lag3L/L-yfp E8ICre.GFP mice show increased polyfunctionality, by IFNg, TNFa and GzmB release. This was confirmed to be largely driven by effector and chemoattractive secretions by analysis with a 28-plex single-cell cytokine response panel (Isoplexis). Overall, these data suggest that PD1 and LAG3 synergize to have a dominant effect on CD8+ TIL and limit antitumor immune effects, as intrinsic removal of both IRs results in reduced tumor growth that substantially impacts anti-tumor immunity. These results are encouraging for the development of co-targeting LAG3 and PD1 in the clinic.

Exhausted CD8 T cells (TEX) are essential during chronic viral infections and cancer. Two TEX subpopulations including a progenitor and a more terminally exhausted subset cooperate to maintain an active immune response during antigen persistence. However, non-overlapping delineations of these populations have suggested a more complex developmental biology. Here, using the LCMV mouse model of chronic viral infection, we identify four distinct TEX subsets based on Ly108 (Slamf6) and CD69 expression revealing a novel stepwise developmental framework. We reveal the transcriptional and epigenetic control mechanisms and associated biological changes underlying each TEX subset transition. Two TCF1+ progenitors were identified along with a novel TCF1-intermediate subset that re-engaged some aspects of effector biology. This subset depended on T-bet and was re-invigorated upon PD-L1 blockade. Ultimately, Tox coordinated loss of T-bet and differentiation into a fourth, terminally exhausted subset. These data define a new developmental hierarchy of TEX and reveal distinct biological properties with direct relevance to immunotherapy. Defining the control mechanisms of this TEX subset hierarchy provides novel opportunities to manipulate TEX biology for clinical goals.

Summary CD8+ T cell exhaustion is a major barrier to current anti-cancer immunotherapies. Despite this, the developmental biology of exhausted CD8+ T cells (Tex) remains poorly defined, restraining improvement of strategies aimed at “re-invigorating” Tex cells. Here, we defined a four-cell-stage developmental framework for Tex cells. Two TCF1+ progenitor subsets were identified, one tissue restricted and quiescent and one more blood accessible, that gradually lost TCF1 as it divided and converted to a third intermediate Tex subset. This intermediate subset re-engaged some effector biology and increased upon PD-L1 blockade but ultimately converted into a fourth, terminally exhausted subset. By using transcriptional and epigenetic analyses, we identified the control mechanisms underlying subset transitions and defined a key interplay between TCF1, T-bet, and Tox in the process. These data reveal a four-stage developmental hierarchy for Tex cells and define the molecular, transcriptional, and epigenetic mechanisms that could provide opportunities to improve cancer immunotherapy.

DNA damage and repair (DDR) has been extensively studied in the context of inducing cell death of tumor cells. Tumor cells are often highly-proliferating and vulnerable to DNA damage, a foundation for many chemotherapy and radiotherapy approaches. Accumulating evidence indicates the improved clinical prognosis when immune checkpoint blockade (ICB) are combined with chemotherapy or radiotherapy. However, the paradox of the combinational benefit of these proliferation-targeting cytotoxic therapies and proliferation-inducing immunotherapies remains poorly understood. Here, we aimed to interrogate the impact of DNA damage on T cell differentiation and function to better understand the mechanism of the paradox, and to better interrogate how T cell-intrinsic DDR can impact clinical responses to ICBs. We hypothesized that T cell subsets have different DDR responses that contribute to distinct biological outcomes upon exposure to DNA damaging agents and/or proliferative stress induced by ICBs. To test this hypothesis, we developed a novel high dimensional cytometry platform that enables simultaneous analysis of T cell differentiation state and interrogation of multiple DDR pathways at single cell resolution. After introduction of DNA damage by γ-irradiation (IR), UV irradiation (UV) or proliferative stress (i.e. aCD3/28 stimulation) DNA damage accumulation and response varied depending on the state of T cell differentiation. For example, terminally differentiated effector cells (TEMRA) had significantly higher DNA damage accumulation and cell death. In contrast, stem cell memory (TSCM) and regulatory T cells (Treg) displayed high DDR with less cell death, suggesting perhaps better cell-intrinsic DDR against genotoxic stress. Overall, this platform revealed consistent, T cell subset specific patterns of DDR as well as specific DDR pathways induced by different types of DNA damage. DDR-Immune profiling of PBMC from uterine cancer patients treated with the combination of chemotherapy and ICB indicated the correlation between distinct patterns of DDR-Immune signatures and therapeutic response. Notably, in clinical responders, Ki67+ CD8 T cells responding to PD-1 blockade had increased pATM, perhaps in response to proliferative stress induced by PD-1 blockade. This increase in pATM was not observed in clinical non-responders. In addition, Treg from clinical non-responders had elevated DDR responses compared to responders, suggesting the possibility of greater resistance to genotoxic/proliferative stress. Collectively, our new platform shed lights on previously unrecognized roles of T cell-intrinsic DDR as a novel determinant of T cell differentiation and immune responsiveness. These findings may have relevance to apply to understanding the paradox of synergy of chemotherapy and radiotherapy with ICBs. Citation Format: Yuki Muroyama, Alexander C. Huang, Sasikanth Manne, Ramin S. Herati, Lakshmi Chilukuri, Caiyue Xu, Claire F. Friedman, Dmitriy Zamarin, E. John Wherry. High dimensional DNA damage and repair (DDR) cytometric profiling of human T cell subsets revealed distinct DDR signatures [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B80.

A significant proportion of cancer patients do not respond to immune checkpoint blockade. To better understand the molecular mechanisms underlying these treatments, we explored the role of CD4(+)Foxp3(−) T cells expressing PD-1 (4PD1(hi)) and observed that 4PD1(hi) accumulate intratumorally as a function of tumor burden. Interestingly, CTLA-4 blockade promotes intratumoral and peripheral 4PD1(hi) increases in a dose-dependent manner, while combination with PD-1 blockade mitigates this effect and improves anti-tumor activity. We found that lack of effective 4PD1(hi) reduction after anti-PD-1 correlates with poor prognosis. Mechanistically, we provide evidence that mouse and human circulating and intra-tumor 4PD1(hi) inhibit T cell functions in a PD-1/PD-L1 dependent fashion and resemble follicular-helper-T-cell(T(FH))-like cells. Accordingly, anti-CTLA-4 activity is improved in T(FH) deficient mice.

The gut microbiota is a key environmental determinant of mammalian metabolism. Regulation of white adipose tissue (WAT) by the gut microbiota is a process critical to maintaining metabolic fitness, and gut dysbiosis can contribute to the development of obesity and insulin resistance (IR). However, how the gut microbiota regulates WAT function remains largely unknown. Here, we show that tryptophan-derived metabolites produced by the gut microbiota controlled the expression of the miR-181 family in white adipocytes in mice to regulate energy expenditure and insulin sensitivity. Moreover, we show that dysregulation of the gut microbiota-miR-181 axis was required for the development of obesity, IR, and WAT inflammation in mice. Our results indicate that regulation of miR-181 in WAT by gut microbiota-derived metabolites is a central mechanism by which host metabolism is tuned in response to dietary and environmental changes. As we also found that MIR-181 expression in WAT and the plasma abundance of tryptophan-derived metabolites were dysregulated in a cohort of obese human children, the MIR-181 family may represent a potential therapeutic target to modulate WAT function in the context of obesity.

Current paradigms of CD8 + T cell–mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8 + T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (T RMs ). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8 + T cells in LTs also resemble T RMs . Moreover, high frequencies of HIV-specific CD8 + T RMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific T RMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-T RMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8 + T cell responses resident within LTs.

Exhausted CD8 T (Tex) cells are immunotherapy targets in chronic infection and cancer, but a comprehensive assessment of Tex cell diversity in human disease is lacking. Here, we developed a transcriptomic- and epigenetic-guided mass cytometry approach to define core exhaustion-specific genes and disease-induced changes in Tex cells in HIV and human cancer. Single-cell proteomic profiling identified 9 distinct Tex cell clusters using phenotypic, functional, transcription factor, and inhibitory receptor co-expression patterns. An exhaustion severity metric was developed and integrated with high-dimensional phenotypes to define Tex cell clusters that were present in healthy subjects, common across chronic infection and cancer or enriched in either disease, linked to disease severity, and changed with HIV therapy. Combinatorial patterns of immunotherapy targets on different Tex cell clusters were also defined. This approach and associated datasets present a resource for investigating human Tex cell biology, with implications for immune monitoring and immunomodulation in chronic infections, autoimmunity, and cancer. Exhausted T (Tex) cells have poor function in chronic infections and cancer but can be therapeutically re-invigorated. Bengsch et al. use genes modified epigenetically during exhaustion and high-dimensional CyTOF profiling to define Tex cell heterogeneity in humans with HIV or lung cancer and link Tex cell features to disease progression and response to immunotherapy.

SUMMARY The transcription factors T-bet and Eomesodermin (Eomes) regulate CD8 T cell exhaustion through undefined mechanisms. Here, we show that the subcellular localization of T-bet and Eomes dictate their regulatory activity in exhausted T cells (TEXs). TEXs had a higher ratio of nuclear Eomes:T-bet than memory T cells (TMEMs) during chronic lymphocytic choriomeningitis virus (LCMV) infection in preclinical cancer models and in human tumors. Biochemically, T-bet and Eomes compete for the same DNA sequences, including the Pdcd1 T-box. High nuclear T-bet strongly represses Pdcd1 transcription in TMEM, whereas low nuclear T-bet in TEX leads to a dominant effect of Eomes that acts as a weaker repressor of Pdcd1. Blocking PD-1 signaling in TEXs increases nuclear T-bet, restoring stronger repression of Pdcd1, and driving T-bet-associated gene expression programs of chemotaxis, homing, and activation. These data identify a mechanism whereby the T-bet-Eomes axis regulates exhaustion through their nuclear localization, providing insights into how these transcription factors regulate TEX biology., In brief McLane et al. demonstrate that T-bet and Eomes expression contributes to exhaustion, but also their nuclear localization, and therefore functional activity, plays a key role. PD-1 blockade restores nuclear T-bet and promotes T cell homing and activation through direct competition with Eomes at gene promoters, such as Pdcd1., Graphical Abstract

Functions of extracellular vesicles including exosomes in the pathogenesis of tuberous sclerosis complex (TSC) have not yet been studied. We report that the extracellular vesicles such as exosomes derived from tuberous sclerosis 1 (Tsc1)-null cells transform phenotypes of neighboring wild-type cells in vivo in such manner that they become functionally similar to Tsc1-null cells. The loss of Tsc1 in the mouse neural tube increases the number of the wild-type neuronal progenitors, which is followed by the precocious and transient acceleration of neuronal differentiation of these cells. The mechanisms regulating these changes involve the exosomal delivery of exosomal shuttle Notch1 and Rheb esRNA and component of γ-secretase complex presenilin 1 from Tsc1-null cells to wild-type cells leading to the activation of Notch and Rheb signaling in the recipient cells. The exosome-mediated mechanisms may also operate in the cells of angiomyolipoma (AML), which develops as a result of mutations in TSC1/TSC2 genes in TSC patients, because we observed the reactivation of mammalian target of rapamycin and Notch pathways, driven by the delivery of Rheb and Notch1 esRNA, in AML cells depleted of Rheb that were treated with the exosomes purified from AML cells with the constitutively high Rheb levels.

Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche.

Despite the success of monotherapies based on blockade of programmed cell death 1 (PD-1) in human melanoma, most patients do not experience durable clinical benefit. Pre-existing T-cell infiltration and/or the presence of PD-L1 in tumours may be used as indicators of clinical response; however, blood-based profiling to understand the mechanisms of PD-1 blockade has not been widely explored. Here we use immune profiling of peripheral blood from patients with stage IV melanoma before and after treatment with the PD-1-targeting antibody pembrolizumab and identify pharmacodynamic changes in circulating exhausted-phenotype CD8 T cells (Tex cells). Most of the patients demonstrated an immunological response to pembrolizumab. Clinical failure in many patients was not solely due to an inability to induce immune reinvigoration, but rather resulted from an imbalance between T-cell reinvigoration and tumour burden. The magnitude of reinvigoration of circulating Tex cells determined in relation to pretreatment tumour burden correlated with clinical response. By focused profiling of a mechanistically relevant circulating T-cell subpopulation calibrated to pretreatment disease burden, we identify a clinically accessible potential on-treatment predictor of response to PD-1 blockade.

Differentiation abnormalities are a hallmark of tuberous sclerosis complex (TSC) manifestations; however, the genesis of these abnormalities remains unclear. Here we report on mechanisms controlling the multi-lineage, early neuronal progenitor and neural stem-like cell characteristics of lymphangioleiomyomatosis (LAM) and angiomyolipoma cells. These mechanisms include the activation of a previously unreported Rheb-Notch-Rheb regulatory loop, in which the cyclic binding of Notch1 to the Notch-responsive elements (NREs) on the Rheb promoter is a key event. This binding induces the transactivation of Rheb. The identified NRE2 and NRE3 on the Rheb promoter are important to Notch-dependent promoter activity. Notch cooperates with Rheb to block cell differentiation via similar mechanisms in mouse models of TSC. Cell-specific loss of Tsc1 within nestin-expressing cells in adult mice leads to the formation of kidney cysts, renal intraepithelial neoplasia, and invasive papillary renal carcinoma., Tuberous sclerosis complex (TSC) is a rare genetic condition causing tumours with differentiation abnormalities; however the molecular mechanisms causing these defects are unclear. Here the authors show that Notch cooperates with Rheb to block cell differentiation forming a regulatory loop that could underlie TSC tumorigenesis.

A significant proportion of cancer patients do not respond to immune checkpoint blockade therapy. To deepen our understanding of the mechanisms of resistance to immunotherapy, we studied a population of CD4+Foxp3− T cells expressing PD-1 (4PD1hi), which we found to be up-regulated in B16-melanoma bearing mice after CTLA-4 blockade in association with limited response to treatment. We observed that 4PD1hi accumulate intratumorally as a function of tumor burden in untreated tumor-bearing hosts. Interestingly, CTLA-4 blockade promotes intratumoral and peripheral 4PD1hiincreases in a dose-dependent manner, while combination with PD-1 blockade mitigates this effect in both B16-bearing mice and melanoma patients and significantly improves anti-tumor activity. In addition, we found that persistence of high levels of 4PD1hi after PD-1 blockade correlates with poor prognosis in melanoma patients. Mechanistically, we show that mouse and human circulating and intra-tumor 4PD1hi inhibit T-cell functions in a PD-1/PD-L1 dependent fashion. In addition, we found that mouse and human 4PD1hi resemble follicular-helper-T-cell(TFH)-like cells and that CTLA-4 blockade activity is improved in TFH deficient mice. These findings broaden our understanding of the mechanisms that limit anti-tumor immunity, providing an additional explanation for the incremental activity of combined CTLA-4 and PD-1 blockade. Our study also defines 4PD1hi as a new prognostic and pharmacodynamic biomarker for the design of optimal checkpoint blockade combination schedules and dosage.

The impact of complement on cancer metastasis has not been well studied. In this report, we demonstrate in a preclinical mouse model of breast cancer that the complement anaphylatoxin C5a receptor (C5aR) facilitates metastasis by suppressing effector CD8+ and CD4+ T-cell responses in the lungs. Mechanisms of this suppression involve recruitment of immature myeloid cells to the lungs and regulation of TGFβ and IL10 production in these cells. TGFβ and IL10 favored generation of T regulatory cells (Treg) and Th2-oriented responses that rendered CD8+ T cells dysfunctional. Importantly, pharmacologic blockade of C5aR or its genetic ablation in C5aR-deficient mice were sufficient to reduce lung metastases. Depletion of CD8+ T cells abolished this beneficial effect, suggesting that CD8+ T cells were responsible for the effects of C5aR inhibition. In contrast to previous findings, we observed that C5aR signaling promoted Treg generation and suppressed T-cell responses in organs where metastases arose. Overall, our findings indicated that the immunomodulatory functions of C5aR are highly context dependent. Furthermore, they offered proof-of-concept for complement-based immunotherapies to prevent or reduce cancer metastasis. Cancer Res; 74(13); 3454–65. ©2014 AACR.

MicroRNAs play an important role in CD8+ T cell differentiation and anti-viral immune responses. However, how microRNAs regulate CD8+ T cell memory formation remains poorly defined. Here, by microRNA profiling of CD8+ T cell during acute or chronic LCMV infection, we identified microRNAs that are specifically enriched in memory CD8+ T cells comparing to naïve, effector or exhausted CD8+ T cells. MiR-150, a microRNA that has been shown to be important in multiple biological processes, was identified as a potential memory CD8+ T cell biased microRNA. Functional interrogation of the role of miR-150 demonstrated that absence of miR-150 in CD8+ T cells enhanced memory CD8+ T cell differentiation, by both increasing KLRG-CD127+ memory precursors population during effector phase and enhancing CD127+CXCR3+ central memory phenotype long-term. Overexpression of miR-150 in CD8+ T cells, in contrast, negatively regulated CD8+ T cell memory formation. Furthermore, we found that miR-150 KO CD8+ T cells had higher expression of c-Myb comparing to miR-150 WT CD8+ T cells, a result observed using both in vitro CD8+ T cell differentiation and following infection with LCMV-Armstrong in vivo. Gene expression analysis implicated a relationship between the level of c-Myb expression and CD8+ T cell memory formation through potential anti-apoptotic molecules such as bcl-2. Thus, our studies identify a key role for miR-150 in CD8+ T cell memory and implicate a mechanism of CD8+ T cell memory effect by this microRNA through regulation of c-Myb.