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Summary: PSGL-1 (P-selectin glycoprotein-1) is a T cell-intrinsic checkpoint regulator of exhaustion with an unknown mechanism of action. Here, we show that PSGL-1 acts upstream of PD-1 and requires co-ligation with the T cell receptor (TCR) to attenuate activation of mouse and human CD8+ T cells and drive terminal T cell exhaustion. PSGL-1 directly restrains TCR signaling via Zap70 and maintains expression of the Zap70 inhibitor Sts-1. PSGL-1 deficiency empowers CD8+ T cells to respond to low-affinity TCR ligands and inhibit growth of PD-1-blockade-resistant melanoma by enabling tumor-infiltrating T cells to sustain an elevated metabolic gene signature supportive of increased glycolysis and glucose uptake to promote effector function. This outcome is coupled to an increased abundance of CD8+ T cell stem cell-like progenitors that maintain effector functions. Additionally, pharmacologic blockade of PSGL-1 curtails T cell exhaustion, indicating that PSGL-1 represents an immunotherapeutic target for PD-1-blockade-resistant tumors.

Abstract 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.

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.

Effective adaptive immune responses are characterized by stages of development and maturation of T and B cell populations that respond to disturbances in the host homeostasis in cases of both infections and cancer. For the T cell compartment, this begins with recognition of specific peptides by naïve, antigen-inexperienced T cells that results in their activation, proliferation, and differentiation, which generates an effector population that clears the antigen. Loss of stimulation eventually returns the host to a homeostatic state, with a heterogeneous memory T cell population that persists in the absence of antigen and is primed for rapid responses to a repeat antigen exposure. However, in chronic infections and cancers, continued antigen persistence impedes a successful adaptive immune response and the formation of a stereotypical memory population of T cells is compromised. With repeated antigen stimulation, responding T cells proceed down an altered path of differentiation that allows for antigen persistence, but much less is known regarding the heterogeneity of these cells and the extent to which they can become “memory-like,” with a capacity for self-renewal and recall responses that are characteristic of bona fide memory cells. This review focuses on the differentiation of CD4+ and CD8+ T cells in the context of chronic antigen stimulation, highlighting the central observations in both human and mouse studies regarding the differentiation of memory or “memory-like” T cells. The importance of both the cellular and molecular drivers of memory T cell development are emphasized to better understand the consequences of persisting antigen on T cell fates. Integrating what is known and is common across model systems and patients can instruct future studies aimed at further understanding T cell differentiation and development, with the goal of developing novel methods to direct T cells toward the generation of effective memory populations.

We report here that the expression of the transcription factor T-bet, which is known to be required for effector cytotoxic CD8+ T lymphocytes (CTL) generation and effector memory cell formation, is regulated in CTL by microRNA-155 (miR-155). Importantly, we show that the proliferative effect of miR-155 on CD8+ T cells is mediated by T-bet. T-bet levels in CTL were controlled in vivo by miR-155 via SH2 (Src homology 2)-containing inositol phosphatase-1 (SHIP-1), a known direct target of miR-155, and SHIP-1 directly downregulated T-bet. Our studies reveal an important and unexpected signaling axis between miR-155, T-bet, and SHIP-1 in in vivo CTL responses and suggest an important signaling module that regulates effector CTL immunity.

Respiratory support improvements have aided survival of premature neonates, but infection susceptibility remains a predominant problem. We previously reported that neonatal mice have a rapidly evolving T-cell receptor (TCR) repertoire that impairs CD8+ T cell immunity. To understand the impact of prematurity on the human CD8+ TCR repertoire, we performed next-generation sequencing of the complementarity-determining region 3 (CDR3) from the rearranged TCR variable beta (Vβ) in sorted, naïve CD8+ T cells from extremely preterm neonates (23–27 weeks gestation), term neonates (37–41 weeks gestation), children (16–56 months), and adults (25–50 years old). Strikingly, preterm neonates had an increased frequency of public clonotypes shared between unrelated individuals. Public clonotypes identified in preterm infants were encoded by germline gene sequences, and some of these clonotypes persisted into adulthood. The preterm neonatal naïve CD8+ TCR repertoire exhibited convergent recombination, characterized by different nucleotide sequences encoding the same amino acid CDR3 sequence. As determined by Pielou’s evenness and iChao1 metrics, extremely preterm neonates have less clonality, and a much lower bound for the number of unique TCR within an individual preterm neonate, which indicates a less rich and diverse repertoire, as compared to term neonates, children, and adults. This suggests that T cell selection in the preterm neonate may be less stringent or different. Our analysis is the first to compare the TCR repertoire of naïve CD8+ T cells between viable preterm neonates and term neonates. We find preterm neonates have a repertoire immaturity which potentially contributes to their increased infection susceptibility. A developmentally regulated, evenly distributed repertoire in preterm neonates may lead to the inclusion of public TCR CDR3β sequences that overlap between unrelated individuals in the preterm repertoire.

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.

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.