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MicroRNAs (miRNAs/miRs) are small, endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling, we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8+ T cells. We observed that out of 362 measurable mature miRNAs, 120 were differentially expressed by at least 2-fold in influenza-specific effector CD8+ CTLs compared with naive CD8+ T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139–mediated regulation of CTL effector responses, we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139−/− mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8+ T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8+ T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet, miR-139−/− CD8+ T cells are outcompeted by wild-type CD8+ T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes. Using an in vitro model of T cell exhaustion, we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation, miR-139 expression is dispensable for influenza-specific CTL responses.

SignificanceThe CD4

SignificanceThe CD4+Tregresponse following acuteListeriainfection is heterogeneous and deploys two distinct modes of suppression coinciding with initial pathogen exposure and resolution of infection. This bimodal suppression of CD8+T cells during priming and contraction is mediated by separate Treglineages. These findings make a significant contribution to our understanding of the functional plasticity inherent within Tregs, which allows these cells to serve as a sensitive and dynamic cellular rheostat for the immune system to prevent autoimmune pathology in the face of inflammation attendant to acute infection, enable expansion of the pathogen-specific response needed to control the infection, and reestablish immune homeostasis after the threat has been contained.

Summary/AbstractWe previously identified the adhesion molecule PSGL-1 as a T cell intrinsic immune checkpoint regulator of T cell exhaustion. Here we show that the ability of PSGL-1 to restrain TCR sginaling correlates with decreased expression of the Zap70 inhibitor Ubash3b (Sts-1) in PSGL-1-deficient T cells. PSGL-1-deficency in T cells supports antitumor responses to a PD-1 blockade resistant melanoma wherein tumor-specific CD8+ T cells sustain an enhanced metabolic state, with an elevated metabolic gene signature that promotes increased glycolysis and glucose uptake to support effector functions. In models of chronic virus infection and cancer, this outcome was associated with CD8+ T cell stemness, as PSGL-1 deficient CD8+ T cells displayed increased TCF-1 and decreased TOX expression, a phenotype shown to be crucial for responsiveness to checkpoint inhibition. Further, we demonstrate that PSGL-1 signaling promotes development of exhaustion in human CD8+ T cells. Finally, pharmacologic blockade of PSGL-1 was sufficient to curtail T cell exhaustion and enhance functionality both with melanoma tumors and chronic LCMV infection, demonstrating that PSGL-1 represents a therapeutic target for immunotherapy for PD-1/PD-L1 resistant tumors.

There is an unmet need for more effective treatment strategies for diffuse intrinsic pontine glioma (DIPG), a devastating brain tumour arising in children and young adults. While immunotherapy is emerging as a powerful approach to treatment of other cancers, clinical trials with immune checkpoint inhibitors have failed to show a survival benefit for DIPG patients. In this study, we analysed the expression of immune checkpoint molecules on the surface of human and murine DIPG cells by flow cytometry and identified CD155 and B7-H3 as the most highly expressed checkpoint molecules, with minimal expression of PD-L1, PD-L2, Galectin-9, CEACAM-1, CD86, CD252 and CD137. These findings were confirmed in primary patient samples from pediatric brain tumours, including high-grade gliomas, medulloblastomas and ependymomas. To test whether CD155 inhibition increases susceptibility to CD8+ T cell killing in vitro, we cultured DIPG cells expressing ovalbumin (OVA) with CD8+ T cells from OT-I mice, which express T cell receptors specific for OVA. Addition of CD155 blocking antibodies to these cultures increased expression of T cell activation markers (CD25, CD44 and CD69) as well as T cell-mediated tumour killing, supporting the notion that CD155 can function as an immune checkpoint in DIPG. In addition to its effects on T cells, CD155 also exerted direct effects on tumour cells: treatment with anti-CD155 antibodies led to impaired cell viability, and shRNA-mediated knockdown of CD155 resulted in reduced cell proliferation in vitro. Strikingly, knockdown of CD155 also led to reduced growth of DIPG cells in vivo, and mice transplanted with the CD155-deficient cells had a clear survival benefit compared to mice transplanted with wild type cells. These studies demonstrate that CD155 functions as an immune checkpoint and as a regulator of tumor growth in DIPG, and suggest that targeting CD155 could be a valuable therapeutic strategy for this devastating disease.

MicroRNAs (miRNAs/miRs) are small, endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling, we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8(+) T cells. We observed that out of 362 measurable mature miRNAs, 120 were differentially expressed by at least 2-fold in influenza-specific effector CD8(+) CTLs compared with naive CD8(+) T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139–mediated regulation of CTL effector responses, we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139(2/2) mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8(+) T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8(+) T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet, miR-139(2/2) CD8(+) T cells are outcompeted by wild-type CD8(+) T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes. Using an in vitro model of T cell exhaustion, we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation, miR-139 expression is dispensable for influenza-specific CTL responses.

Exhaustion is a dysfunctional state of cytotoxic CD8+ T cells (CTL) observed in chronic infection and cancer. Current in vivo models of CTL exhaustion using chronic viral infections or cancer yield very few exhausted CTL, limiting the analysis that can be done on these cells. Establishing an in vitro system that rapidly induces CTL exhaustion would therefore greatly facilitate the study of this phenotype, identify the truly exhaustion-associated changes and allow the testing of novel approaches to reverse or prevent exhaustion. Here we show that repeat stimulation of purified TCR transgenic OT-I CTL with their specific peptide induces all the functional (reduced cytokine production and polyfunctionality, decreased in vivo expansion capacity) and phenotypic (increased inhibitory receptors expression and transcription factor changes) characteristics of exhaustion. Importantly, in vitro exhausted cells shared the transcriptomic characteristics of the gold standard of exhaustion, CTL from LCMV cl13 infections. Gene expression of both in vitro and in vivo exhausted CTL was distinct from T cell anergy. Using this system, we show that Tcf7 promoter DNA methylation contributes to TCF1 downregulation in exhausted CTL. Thus this novel in vitro system can be used to identify genes and signaling pathways involved in exhaustion and will facilitate the screening of reagents that prevent/reverse CTL exhaustion., Author summary In this manuscript, we describe an in vitro method that rapidly establishes large numbers of exhausted CD8+ T cells. The exhaustion of CTL induced by this method has been fully validated by multiple approaches (cytokine production, polyfunctionality, cytotoxicity, in vivo proliferation, inhibitory receptors, transcription factors, RNAseq and DNA methylation). This method will facilitate not only the study of T cell exhaustion but also the screening of drugs. As proof of point, we use this method to show that TCF-1 downregulation in terminally exhausted T cells is accompanied by Tcf7 DNA promoter methylation and show that a transmethylase inhibitor can prevent TCF-1 downregulation. Our method presents a critical resource for the study of CTL exhaustion and the screening of drugs and interventions.

Persistent viral infections and tumors drive development of exhausted T (TEX) cells. In these settings, TEX cells establish an important host-pathogen or host-tumor stalemate. However, TEX cells erode over time, leading to loss of pathogen or cancer containment. We identified microRNA (miR)-155 as a key regulator of sustained TEX cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection. Genetic deficiency of miR-155 ablated CD8 T cell responses during chronic infection. Conversely, enhanced miR-155 expression promoted expansion and long-term persistence of TEX cells. However, rather than strictly antagonizing exhaustion, miR-155 promoted a terminal TEX cell subset. Transcriptional profiling identified coordinated control of cell signaling and transcription factor pathways, including the key AP-1 family member Fosl2. Overexpression of Fosl2 reversed the miR-155 effects, identifying a link between miR-155 and the AP-1 transcriptional program in regulating TEX cells. Thus, we identify a mechanism of miR-155 regulation of TEX cells and a key role for Fosl2 in T cell exhaustion. During persistent viral infections, exhausted T cells (TEX) erode quantitatively and qualitatively and therefore fail to provide protection. Stelekati et al. identified microRNA (miR)-155 as a key molecule that can enhance and sustain TEX responses long-term during chronic viral infection.

Immune responses to SARS-CoV-2 reveal regulation and dynamics of lymphocytes

The immune system, and in particular, cytotoxic CD8+ T cells (CTLs), plays a vital part in the prevention and elimination of tumors. In many patients, however, CTL-mediated tumor killing ultimately fails in the clearance of cancer cells resulting in disease progression, in large part due to the progression of effector CTL into exhausted CTL. While there have been major breakthroughs in the development of CTL-mediated “reinvigoration”-driven immunotherapies such as checkpoint blockade therapy, there remains a need to better understand the drivers behind the development of T cell exhaustion. Our study highlights the unique differences in T cell exhaustion development in tumor-specific CTL which arises over time in a mouse model of mesothelioma. Importantly, we also show that peripheral tumor-specific T cells have a unique expression profile compared to exhausted tumor-infiltrating CTL at a late-stage of tumor progression in mice. Together, these data suggest that greater emphasis should be placed on understanding contributions of individual microenvironments in the development of T cell exhaustion.

Summary Repetitive elements (REs) compose ∼50% of the human genome and are normally transcriptionally silenced, although the mechanism has remained elusive. Through an RNAi screen, we identified FBXO44 as an essential repressor of REs in cancer cells. FBXO44 bound H3K9me3-modified nucleosomes at the replication fork and recruited SUV39H1, CRL4, and Mi-2/NuRD to transcriptionally silence REs post-DNA replication. FBXO44/SUV39H1 inhibition reactivated REs, leading to DNA replication stress and stimulation of MAVS/STING antiviral pathways and interferon (IFN) signaling in cancer cells to promote decreased tumorigenicity, increased immunogenicity, and enhanced immunotherapy response. FBXO44 expression inversely correlated with replication stress, antiviral pathways, IFN signaling, and cytotoxic T cell infiltration in human cancers, while a FBXO44-immune gene signature correlated with improved immunotherapy response in cancer patients. FBXO44/SUV39H1 were dispensable in normal cells. Collectively, FBXO44/SUV39H1 are crucial repressors of RE transcription, and their inhibition selectively induces DNA replication stress and viral mimicry in cancer cells.

Functional T cell exhaustion develops with persistent antigen exposure during chronic viral infection and cancer. Greater insights into T cell exhaustion are needed to promote immunity to viruses and tumors. RNF5 (RING-finger protein 5) is an E3 ubiquitin ligase that functions in protein degradation. We previously found enhanced anti-tumor responses in RNF5−/− mice, which was coupled with greater recovery and function of CD8+ T cell in melanoma tumors. Tumor-infiltrating RNF5−/− CD8+ T cells expressed high levels of inhibitory receptors (IR), yet greater functionality compared to WT CD8+ T cells, suggesting greater activation. To further assess RNF5-dependent regulation of CD8+ T cell exhaustion, we used the chronic LCMV Cl13 infection that has defined states of progressive T cell exhaustion. Virus-specific CD8+ T cells were significantly increased in RNF5−/− mice leading to greater numbers of cytokine producers, which expressed high levels of IRs. They also expressed higher levels of TOX, a transcriptional regulator of T cell exhaustion and persistence. With low dose infection, RNF5−/− mice displayed decreased morbidity and CD8+ T cells acquired a highly functional memory phenotype rather than exhaustion. To identify T cell intrinsic role(s) of RNF5, we analyzed anti-viral responses in bone marrow chimeras lacking RNF5 only in T cells and used an in vitro T cell exhaustion assay. RNF5−/− CD8+ T cells also expressed high levels of IRs and TOX, as did the in vitro exhausted T cells yet importantly displayed enhanced cytokine production compared to WT cells. These data reveal a T cell-intrinsic role of RNF5 in promoting exhaustion in settings of chronic antigen stimulation.

We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates T cell function and exhaustion in responses to chronic viral infection and melanoma tumors. Subsequently, our studies have focused on investigating the mechanisms by which PSGL-1 regulates T cell activation and exhaustion and evaluating PSGL-1 as a target for cancer immunotherapy. Using the CD4cre system in combination with PSGL-1-floxed mice, we find that T cell intrinsic deletion of PSGL-1 is sufficient to promote enhanced tumor growth control of mesothelioma as well as melanoma tumors. We have found that in vivo PSGL-1 blockade recapitulates the enhanced melanoma tumor growth control observed with PSGL-1-/- CD8+ T cells and limits the development of T cell exhaustion during chronic LCMV Clone 13 viral infection. Conversely, ligation of PSGL-1 in vitro or in vivo drives enhanced upregulation T cell exhaustion markers, most notably PD-1 expression. Phosphoproteomic analysis of early T cell activation revealed the rapid upregulation of more than 30 phosphorylated proteins with significantly enhanced expression following PSGL-1 ligation during T cell activation than T cell activation alone. GSEA and western blot analysis confirmed the increased expression of several regulators of T cell signaling including pAkt1 and pErk in both activated PSGL-1-/- T cells and following PSGL-1 ligation during T cell activation. Single-cell RNA-sequencing of melanoma tumor-specific PSGL-1-/- CD8+ T cells identified differential modulation of genes associated with T cell metabolism and effector responses, including increased Mtor and Hif1a in tumors and Tcf7 in tumor-draining lymph nodes. The Seahorse glycolysis stress test identified that PSGL-1-/- T cells show increased glycolysis after 72 hours of in vitro activation at both sub-optimal and optimal levels of αCD3 stimulation, and 2-NBDG glucose uptake assays confirmed increased glucose uptake by PSGL-1-/- CD8+ T cells within two hours of stimulation. Further, 2-NBDG uptake was increased by PSGL-1-/- CD8+ T cells ex vivo from melanoma tumors. Importantly, this increased glycolytic phenotype does not come at the cost of CD8+ T cell stemness, as determined by TCF-1 staining. We hypothesize that PSGL-1 serves as a key regulator of early T cell activation and repeated signaling through PSGL-1 promotes differentiation into exhausted CD8+ T cells by sustaining TCR signaling. Conversely, we hypothesize that the absence of PSGL-1 allows for faster initiation and termination of TCR signaling, inhibiting differentiation into exhausted CD8+ T cells. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses and their metabolic profile which may drive their enhanced responses to tumors. Citation Format: Jennifer L. Hope, Dennis C. Otero, Eun-ah Bae, Christopher J Stairiker, Ashley B. Palete, Hannah A. Faso, Petrus de Jong, Garth Powis, Linda M. Bradley. PSGL-1 is an early T cell signaling regulator that drives immunometabolism and terminal differentiation in tumor-specific CD8 T cells [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 PO014.

We previously identified PSGL-1 (P-selectin glycoprotein-1) as a T cell-intrinsic, cell surface checkpoint inhibitor during chronic viral infection. In this model, we found that cross-linking PSGL-1 with antibody reduced signaling through the T cell receptor (TCR) in exhausted T cells and further upregulated PD-1 expression. However, the mechanism responsible for PD-1 regulation by PSGL-1 remained to be described. Here we show that, in naïve T cells, PSGL-1 ligation results in enhanced PD-1 expression following activation through the T cell receptor. Furthermore, naïve T cells lacking PSGL-1 fail to upregulate PD-1 to the same extent as wild type T cells following activation. Strikingly we have discovered that T cells lacking PSGL-1 showed reduced calcium signaling. In T cells, the calcium/calcineurin/NFAT pathway is important for induction of many genes including PD-1. Consequently, PSGL-1 deficient T cells also exhibit reduced nuclear translocation of NFAT explaining the reduced PD-1 expression. Conversely, we found that ligating PSGL-1 enhanced NFAT nuclear translocation with TCR signaling thus contributing to upregulation of PD-1. Therefore, by regulating calcium signaling through the T cell receptor, PSGL-1 modulates the extent of PD-1 expression early in activation and subsequently influencing the extent of T cell exhaustion. Further studies aim to elucidate how PSGL-1 regulates calcium signaling through the T cell receptor.

Endoplasmic reticulum (ER) stress is induced by the accumulation of misfolded proteins and other cellular stresses, which lead to the unfolded protein response to restore ER homeostasis. Previous studies have shown that the ER stress response is required for T cell activation and differentiation. However, recent studies demonstrated that inhibition or deletion of ER stress mediators in T cells actually promotes anti-tumor immunity. Therefore, this incongruity as to the role of ER stress response in effector T cell function warrants further investigation. Our lab previously identified that the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), acts as a T cell immune checkpoint regulator. PSGL-1-deficiency enhances T cell function, resulting in the clearance of chronic viral infection and dramatic tumor control. To better understand the contribution of ER stress to T cell function, we assessed T cell phenotype and function together with the ER stress mediators, XBP1s and ATF4, in highly-functional PSGL-1−/− T cells as well as exhausted wild-type T cells in both tumor and virus models. In melanoma models, ER stress was highly induced in activated tumor-infiltrating T cells (TILs). Effector CD8+ TILs producing IFNg displayed elevated XBP1s and ATF4 compared to IFNg− TILs. Upon chronic LCMV Cl13 viral infection in PSGL-1−/− mice, we found increased virus-specific T cells with enhanced multi-functional effector responses and increased memory/stem cell-like T cell populations characterized by the expression of the transcription factor TCF-1, as well as increased ER stress responses. Together, these data suggest that ER stress plays an essential role in effector T cell functions in settings of chronic antigen stimulation.

We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates T cell function and exhaustion in response to chronic viral infection and tumors. Subsequent studies have focused on investigating the role of PSGL-1 signaling in T cell responses, with an emphasis on understanding the mechanisms by which PSGL-1 regulates T cell exhaustion. Single-cell RNA-sequencing of tumor infiltrating PSGL-1−/− CD8+ T cells identified the upregulation and differential modulation of several genes associated with T cell metabolism and enhanced intratumoral responses, including Mtor, Hif1a, and Mki67 in Gzmb/Ifng double-positive cells from tumors and Tcf7 in both tumor draining and non-draining inguinal lymph nodes. These data suggest an important role for PSGL-1 signaling in the development and maintenance of effective anti-tumor T cell responses to melanoma. Using the Seahorse glycolysis stress test, we identified that both CD4+ and CD8+ PSGL-1−/− T cells demonstrate increased glycolysis after 72 hours of in vitro activation compared to wild-type T cells. In situ activation of PSGL-1−/− CD8+ T cells demonstrated that PSGL-1−/− CD8+ T cells have increased glycolysis and increased glycolytic capacity at both sub-optimal and optimal levels of α-CD3 stimulation, and 2-NBDG glucose uptake assays confirmed increased glucose uptake by PSGL-1−/− CD8+ T cells within two hours of stimulation. Importantly, this increased glycolytic phenotype does not come at the cost of CD8+ T cell stemness, as determined by TCF-1 staining. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses and their metabolic profile in response to melanoma tumors.

Cytotoxic CD8+ T lymphocytes (CTLs) play a major role in protection against chronic viral infections and tumors. Chronic antigen stimulation in both conditions leads to CTL exhaustion and failure of CTLs to efficiently eradicate tumors and virally infected cells. miRNAs are small, non-coding RNA molecules that participate in post-transcriptional gene regulation. This study aimed to determine the core miRNA signature of CTL exhaustion in mouse models of both tumors and chronic infection. Exhausted anti-tumor CTLs were isolated from tumors of mice that received 10^6 mouse mesothelioma cells (AE17sOVA) followed by an adoptive transfer of 10^4 CD45.1+ OT-I cells. Effector anti-viral CTLs were isolated from mice adoptively transferred with 10^4 CD45.1+ OT-I cells or P14 cells and infected with WSN-OVA or LCMV Armstrong respectively. For exhausted anti-viral CTLs, mice were adoptively transferred with 10^4 CD45.1+ P14 cells and infected with LCMV-Cl13. Single cell suspensions were prepared from the different tissues, live CTLs were FACS sorted, RNA isolated and small RNA-seq was performed. Principle component analysis (PCA) of miRNA expression revealed that exhausted anti-tumor CTLs and anti-LCMV Cl13 CTLs cluster apart from effector anti-viral CTLs (WSN-OVA and LCMV Armstrong). Differential expression analysis identified 73 miRNAs commonly shared between the exhausted day anti-tumor CTL and anti-viral CTL. Using RNA sequencing of two different models of exhaustion, distinct RNA profiles common in exhausted CTLs were found. This core miRNA signature can now be used to manipulate the expression of specific miRNA to potentially prevent or revert CTL exhaustion and improve the CTL response against tumors and/or chronic infections.

Currently, there is little consensus regarding the most appropriate animal model to study acute infection and the virus-specific CD8+ T cell (CTL) responses in neonates. TCRβ high-throughput sequencing in naive CTL of differently aged neonatal mice was performed, which demonstrated differential Vβ family gene usage. Using an acute influenza infection model, we examined the TCR repertoire of the CTL response in neonatal and adult mice infected with influenza type A virus. Three-day-old mice mounted a greatly reduced primary NP(366–374)–specific CTL response when compared with 7-d-old and adult mice, whereas secondary CTL responses were normal. Analysis of NP(366–374)-specific CTL TCR repertoire revealed different Vβ gene usage and greatly reduced public clonotypes in 3-d-old neonates. This could underlie the impaired CTL response in these neonates. To directly test this, we examined whether controlling the TCR would restore neonatal CTL responses. We performed adoptive transfers of both nontransgenic and TCR-transgenic OVA(257–264)-specific (OT-I) CD8+ T cells into influenza-infected hosts, which revealed that naive neonatal and adult OT-I cells expand equally well in neonatal and adult hosts. In contrast, nontransgenic neonatal CD8+ T cells when transferred into adults failed to expand. We further demonstrate that differences in TCR avidity may contribute to decreased expansion of the endogenous neonatal CTL. These studies highlight the rapid evolution of the neonatal TCR repertoire during the first week of life and show that impaired neonatal CTL immunity results from an immature TCR repertoire, rather than intrinsic signaling defects or a suppressive environment.

The 1st International Conference on Human &Translational Immunology convened in Rhodes, Greece, and provided a venue for stimulating scientific discussions on the human immune system.

In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2′ deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo . These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.

P-selectin glycoprotein ligand-1 (PSGL-1) is an adhesion molecule expressed on the surface of naïve, effector, and memory CD4+ and CD8+ T cells. We identified PSGL-1 to be an immune checkpoint inhibitor as antibody-mediated ligation of PSGL-1 promotes T cell exhaustion and deletion of PSGL-1 prevents chronic viral infection and inhibits tumor. To investigate the role of PSGL-1 signaling in the development of T cell responses, we assessed the differentiation state, expansion capacity, and glycolytic profile of PSGL-1-deficient T cells. PSGL-1+/− OT-II CD4+ T cells demonstrated increased skewing towards IL-17A+ T cells compared to wild-type OT-II CD4+ T cells under TH17 conditions, and reduced IFNg+ cells under both TH0 and TH1 conditions. In a B16-OVA tumor model, we observed increased IL-13+ CD4+ T cells among the intratumoral T cell compartment in PSGL-1−/− mice. In vitro activation with a sub-optimal dose of αCD3 of PSGL-1−/− OT-II CD4+ T cells demonstrated increased expansion and expression of CD25 compared to wild-type OT-II CD4+ T cells. Further, adoptively transferred in vitro-activated PSGL-1−/− CD4+ T cells demonstrated greater expansion in vivo upon adoptive transfer into RAG−/− host mice. Using the Seahorse glycolysis stress test, we identified that both CD4+ and CD8+ PSGL-1−/− T cells demonstrate increased glycolysis after 72 hours of in vitro activation compared to wild-type T cells. Further, in situ activation of PSGL-1−/− CD8+ T cells demonstrates that at both sub-optimal and optimal levels of αCD3 stimulation, PSGL-1−/− CD8+T cells have increased glycolysis and increased glycolytic capacity. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses.

Background Despite the important role that microRNAs (miRNAs) play in immunity and inflammation, their involvement in systemic sclerosis (SSc) remains poorly characterized. miRNA-155 (miR-155) plays a role in pulmonary fibrosis and its expression can be induced with interleukin (IL)-1β. SSc fibroblasts have activated inflammasomes that are integrally involved in mediating the myofibroblast phenotype. In light of this, we investigated whether miR-155 played a role in SSc and if its expression was dependent on inflammasome activation. Methods miR-155 expression was confirmed in SSc dermal and lung fibroblasts by quantitative polymerase chain reaction (PCR). Wild-type and NLRP3-deficient murine fibroblasts were utilized to explore the regulation of miR-155 during inflammasome activation. miR-155-deficient fibroblasts and retroviral transductions with a miR-155 expression or control vectors were used to understand the contribution of miR-155 in fibrosis. Results miR-155 was significantly increased and the highest expressing miRNA in SSc lung fibroblasts. Its expression was dependent on inflammasome activation as miR-155 expression could be blocked when inflammasome signaling was inhibited. In the absence of miR-155, inflammasome-mediated collagen synthesis could not be induced but was restored when miR-155 was expressed in miR-155-deficient fibroblasts. Conclusions miR-155 is upregulated in SSc. These results suggest that the inflammasome promotes the expression of miR-155 and that miR-155 is a critical miRNA that drives fibrosis.

Mouse models of cancer remain the preferred means to assess the efficacy of anti-cancer therapies, and several models have been used to address the effect of cancer drugs or in the discovery of biomarkers. The Yale University Mutant melanoma lines, YUMM1.5 and YUMMER1.7 murine tumor models were developed from the BrafV600E/Pten/−/−Cdkn2a−/− engineered mouse model to more closely reflect poorly immunogenic and highly mutated melanomas, respectively, that are driven by human disease-relevant mutations; however, the dominant T cell-recognized epitopes specific for these tumors remains unknown. We therefore sought to engineer OVA-expressing versions of the YUMM1.5 and YUMMER1.7 tumor lines, hereafter referred to as YUMM1.5-OVA and YUMMER1.7-OVA. Using stable transfection to insert a plasmid expressing full-length secretory ovalbumin, we have generated polyclonal and monoclonal cell lines that generate an in vivo OVA-specific response as validated by MHC class I tetramer staining and cytokine production following OVA peptide stimulation. Together, these two new models for melanoma tumors will allow us to assess intrinsic and extrinsic mechanisms regulating the generation of effective and recovery of exhausted T cell responses to melanoma in vivo.

The p110δ isoform of PI3K is known to play an important role in immunity, yet its contribution to CTL responses has not been fully elucidated. Using murine p110δ-deficient CD8+ T cells, we demonstrated a critical role for the p110δ subunit in the generation of optimal primary and memory CD8+ T cell responses. This was demonstrated in both acute viral and intracellular bacterial infections in mice. We show that p110δ signaling is required for CD8+ T cell activation, proliferation and effector cytokine production. We provide evidence that the effects of p110δ signaling are mediated via Akt activation and through the regulation of TCR-activated oxidative phosphorylation and aerobic glycolysis. In light of recent clinical trials that employ drugs targeting p110δ in certain cancers and other diseases, our study suggests caution in using these drugs in patients, as they could potentially increase susceptibility to infectious diseases. These studies therefore reveal a novel and direct role for p110δ signaling in in vivo CD8+ T cell immunity to microbial pathogens.

The increasing risk of incidental exposure to nanomaterials has led to mounting concerns regarding nanotoxicity. Zinc oxide nanoparticles (ZnO NPs) are produced in large quantities and have come under scrutiny due to their capacity to cause cytotoxicity in vitro and potential to cause harm in vivo. Recent evidence has indicated that ZnO NPs promote autophagy in cells; however, the signaling pathways and the role of ion release inducing toxicity remain unclear. In this study, we report that ZnO NPs are immunotoxic to primary and immortalized immune cells. Importantly, such immunotoxicity is observed in mice in vivo, since death of splenocytes is seen after intranasal exposure to ZnO NPs. We determined that ZnO NPs release free Zn(2+) that can be taken up by immune cells, resulting in cell death. Inhibiting free Zn(2+) ions in solution with EDTA or their uptake with CaCl2 abrogates ZnO NP-induced cell death. ZnO NP-mediated immune cell death was associated with increased levels of intracellular reactive oxygen species (ROS). ZnO NP death was not due to apoptosis, necroptosis or pyroptosis. Exposure of immune cells to ZnO NPs resulted in autophagic death and increased levels of LC3A, an essential component of autophagic vacuoles. Accordingly, ZnO NP-mediated upregulation of LC3A and induction of immune cell death were inhibited by blocking autophagy and ROS production. We conclude that release of Zn(2+) from ZnO NPs triggers the production of excessive intracellular ROS, resulting in autophagic death of immune cells. Our findings suggest that exposure to ZnO NPs has the potential to impact host immunity.

MicroRNAs (miRNAs) are small, single-stranded non-coding RNAs that play essential roles in regulating key cellular processes, and are highly conserved across species. miRNA are known to regulate immune cells and play an important role in effective CD8+ T cell (CTL) responses to viral infection and tumors. To understand the role of miRNA in CTL responses we performed microarray miRNA expression profiling and identified unique in vivo expression patterns of miRNAs in naïve and effector anti-viral CTL. In particular, miR-155, which we and others previously have demonstrated to regulate CTL responses, was significantly upregulated in effector CTL. We show that further increasing the levels of miR-155 by retroviral overexpression significantly augments anti-viral effector CTL and skews memory CD8+ T cells towards an effector memory phenotype. MiR-155 overexpression resulted in enhanced T-bet expression, which is known to be required for effector CTL and to promote effector memory cell formation. MiR-155 overexpression-induced T-bet expression was required for miR-155 mediated increases in effector CTL. Our studies have revealed an important and unexpected link between miR-155 and T-bet transcription factor in the context of in vivo CTL responses.

Neonates have an immunological immaturity which persists from fetal life into the newborn period and can result in a poorly protective response to intracellular pathogens. We have previously shown a less complex TCR repertoire may be responsible for reduced CD8+ T cell immunity in a neonatal murine model of influenza virus infection. Therefore, we sought to determine if there were similar differences in the TCR repertoire across the human life span. High-throughput sequencing of the TCRβ CDR3 regions was performed in sorted, naïve CD8+ T cells from extremely preterm (23–27 weeks gestation), term (39–40 weeks gestation), children aged 16 months to 4 years old, and adults aged 25–50 years (n=5 in each group). There were differences in the CDR3 length across the life span, with preterm infants having a shorter CDR3 length, which was due to fewer N1 and N2 additions (p

We found upregulation of expression of the microRNA miR-155 in primary effector and effector memory CD8(+) T cells, but low miR-155 expression in naive and central memory cells. Antiviral CD8(+) T cell responses and viral clearance were impaired in miR-155-deficient mice, and this defect was intrinsic to CD8(+) T cells, as miR-155-deficient CD8(+) T cells mounted greatly diminished primary and memory responses. Conversely, miR-155 overexpression augmented antiviral CD8(+) T cell responses in vivo. Gene-expression profiling showed that miR-155-deficient CD8(+) T cells had enhanced type I interferon signaling and were more susceptible to interferon's antiproliferative effect. Inhibition of the type I interferon-associated transcription factors STAT1 or IRF7 resulted in enhanced responses of miR-155-deficient CD8(+) T cells in vivo. We have thus identified a previously unknown role for miR-155 in regulating responsiveness to interferon and CD8(+) T cell responses to pathogens in vivo.

The role of Type I interferon (IFN) during pathogenic HIV and SIV infections remains unclear, with conflicting observations suggesting protective versus immunopathological effects. We therefore examined the effect of IFNα/β on T cell death and viremia in HIV infection. Ex vivo analysis of eight pro- and anti-apoptotic molecules in chronic HIV-1 infection revealed that pro-apoptotic Bak was increased in CD4+ T cells and correlated directly with sensitivity to CD95/Fas-mediated apoptosis and inversely with CD4+ T cell counts. Apoptosis sensitivity and Bak expression were primarily increased in effector memory T cells. Knockdown of Bak by RNA interference inhibited CD95/Fas-induced death of T cells from HIV-1-infected individuals. In HIV-1-infected patients, IFNα-stimulated gene expression correlated positively with ex vivo T cell Bak levels, CD95/Fas-mediated apoptosis and viremia and negatively with CD4+ T cell counts. In vitro IFNα/β stimulation enhanced Bak expression, CD95/Fas expression and CD95/Fas-mediated apoptosis in healthy donor T cells and induced death of HIV-specific CD8+ T cells from HIV-1-infected patients. HIV-1 in vitro sensitized T cells to CD95/Fas-induced apoptosis and this was Toll-like receptor (TLR)7/9- and Type I IFN-dependent. This sensitization by HIV-1 was due to an indirect effect on T cells, as it occurred in peripheral blood mononuclear cell cultures but not purified CD4+ T cells. Finally, peak IFNα levels and viral loads correlated negatively during acute SIV infection suggesting a potential antiviral effect, but positively during chronic SIV infection indicating that either the virus drives IFNα production or IFNα may facilitate loss of viral control. The above findings indicate stage-specific opposing effects of Type I IFNs during HIV-1 infection and suggest a novel mechanism by which these cytokines contribute to T cell depletion, dysregulation of cellular immunity and disease progression., Author Summary Type I interferons (IFNα/β) are innate immune mediators that are produced by cells in response to viral infections. Although the protective effects of IFNα/β are well-established, it is not clear whether these cytokines are beneficial or deleterious during HIV-1 infection. We report that HIV-1 infection renders T cells more prone to undergo programmed death and that this enhanced apoptosis susceptibility is associated with abnormal expression of pro- and anti-apoptotic molecules. Importantly, IFNα/β increases the expression level of the pro-apoptotic protein, Bak, a major gatekeeper of the mitochondrial apoptosis machinery. Exposure of healthy donor T cells to IFNα/β increased Bak expression and induced an apoptosis sensitivity that is similar to what is observed in HIV-1-infected patient T cells. Furthermore, elevated IFNα production and Bak expression correlated with heightened T cell apoptosis, low CD4+ T cell numbers and high viral loads in patients. In a primate model of HIV-1 infection, we observed that increased IFNα was associated with low peak viral loads in early infection, but high viremia and decreased CD4+ T cell counts during chronic infection. These studies identify a novel mechanism by which Type I IFN may induce immune dysfunction in HIV-1 infection.