CD4+ T helper cells use CD154–CD40 interactions to counteract T reg cell–mediated suppression of CD8+ T cell responses to influenza

CD4⁺ T cells promote CD8⁺ T cell priming by licensing dendritic cells (DCs) via CD40–CD154 interactions. However, the initial requirement for CD40 signaling may be replaced by the direct activation of DCs by pathogen-derived signals. Nevertheless, CD40–CD154 interactions are often required for optimal CD8⁺ T cell responses to pathogens for unknown reasons. Here we show that CD40 signaling is required to prevent the premature contraction of the influenza-specific CD8⁺ T cell response. CD40 is required on DCs but not on B cells or T cells, whereas CD154 is required on CD4⁺ T cells but not CD8⁺ T cells, NKT cells, or DCs. Paradoxically, even though CD154-expressing CD4⁺ T cells are required for robust CD8⁺ T cell responses, primary CD8⁺ T cell responses are apparently normal in the absence of CD4⁺ T cells. We resolved this paradox by showing that the interaction of CD40-bearing DCs with CD154-expressing CD4⁺ T cells precludes regulatory T cell (T reg cell)–mediated suppression and prevents premature contraction of the influenza-specific CD8⁺ T cell response. Thus, CD4⁺ T helper cells are not required for robust CD8⁺ T cell responses to influenza when T reg cells are absent.Primary CD8⁺ T cell responses often require help from CD4⁺ T cells, which produce cytokines and provide co-stimulation, including the engagement of CD40 by its ligand CD154 (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998). In one model, CD4⁺ T cells engage CD40 on DCs and license them to become efficient antigen-presenting cells for naive CD8⁺ T cells (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998). However, other models suggest that CD4⁺ T cells provide help to CD8⁺ T cells by activating B cells and promoting CD40-dependent antibody responses (Bachmann et al., 2004) or that they engage CD40 on CD8⁺ T cells (Bourgeois et al., 2002) and directly promote CD8⁺ T cell activation or survival.Interestingly, CD4⁺ T cell help is not required to prime all CD8⁺ T cells responses. Whereas CD8⁺ T cell responses to noninflammatory antigens are impaired in the absence of CD4⁺ T cells or CD40 signaling (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998; Feau et al., 2011), primary responses to some pathogens occur independently of CD4⁺ T cells or CD40 signaling (Whitmire et al., 1996, 1999; Shedlock and Shen, 2003; Shedlock et al., 2003; Sun and Bevan, 2003), possibly because of the direct activation of DCs through pathogen recognition receptors (Hamilton et al., 2001). Curiously, primary CD8⁺ T cell responses to influenza virus require CD40 signaling (Lee et al., 2003a) but not CD4⁺ T cells (Belz et al., 2002), suggesting that other cell types may express CD154 and license CD40-expressing targets in the absence of CD4⁺ T cells. Consistent with this view, activated CD8⁺ T cells (Hernandez et al., 2007; Wong et al., 2008) and natural killer T cells (NKT) express CD154 (Tomura et al., 1999) and may license DCs (Hernandez et al., 2007, 2008; Wong et al., 2008) and help B cells (Chang et al., 2012) in the absence of CD4⁺ T cells. In addition, CD154 is expressed on activated DCs (Johnson et al., 2009) and may directly activate CD40-expressing CD8⁺ T cells. However, the actual role of CD40 signaling and the cellular basis of CD40-mediated help to CD8⁺ T cells help are not fully understood.Whereas helper CD4⁺ T cells promote T and B cell responses, FoxP3-expressing CD4⁺ regulatory T cells (T reg cells) suppress them (Kim et al., 2007; Campbell and Koch, 2011; Chung et al., 2011; Dietze et al., 2011; Linterman et al., 2011). Although the potent suppressive activity of T reg cells is neutralized during infection to allow robust immune responses to pathogens, T reg cells are also involved in the late stages of immune responses to resolve inflammation and curtail immunopathology (Suvas et al., 2003; Fulton et al., 2010; McNally et al., 2011). However, the relationship between CD40-mediated CD4⁺ T cell help and the immunosuppressive activity of T reg cells in CD8⁺ T cell responses to pathogens remains unexplored.Here we determined what cells use CD40–CD154 interactions and how CD40 signaling promotes CD8⁺ T cell responses to influenza. We found that CD4⁺ T cells were the only cells to functionally express CD154 and that DCs were the only cells that required CD40 for optimal CD8⁺ T cell responses to influenza. However, rather than licensing DCs to prime naive CD8⁺ T cells, CD40 signaling was required to prevent the early contraction of the CD8⁺ T cell response. Despite the necessity for CD154 on CD4⁺ T cells, we also observed apparently normal CD8⁺ T cell responses in the absence of CD4⁺ T cells. Finally, we showed that CD8⁺ T cell responses were normal or even enhanced when T reg cells were depleted and that additional CD40 blockade did not change the CD8⁺ T cell response. Thus, our data demonstrate that CD154-expressing CD4⁺ T cells stimulate DCs through CD40 to counteract T reg cell–mediated suppression of the CD8⁺ T cell response during the contraction phase of the immune response.     

STIM1 controls T cell–mediated immune regulation and inflammation in chronic infection

Chronic infections induce a complex immune response that controls pathogen replication, but also causes pathology due to sustained inflammation. Ca²⁺ influx mediates T cell function and immunity to infection, and patients with inherited mutations in the gene encoding the Ca²⁺ channel ORAI1 or its activator stromal interaction molecule 1 (STIM1) are immunodeficient and prone to chronic infection by various pathogens, including Mycobacterium tuberculosis (Mtb). Here, we demonstrate that STIM1 is required for T cell–mediated immune regulation during chronic Mtb infection. Compared with WT animals, mice with T cell–specific Stim1 deletion died prematurely during the chronic phase of infection and had increased bacterial burdens and severe pulmonary inflammation, with increased myeloid and lymphoid cell infiltration. Although STIM1-deficient T cells exhibited markedly reduced IFN-γ production during the early phase of Mtb infection, bacterial growth was not immediately exacerbated. During the chronic phase, however, STIM1-deficient T cells displayed enhanced IFN-γ production in response to elevated levels of IL-12 and IL-18. The lack of STIM1 in T cells was associated with impaired activation-induced cell death upon repeated TCR engagement and pulmonary lymphocytosis and hyperinflammation in Mtb-infected mice. Chronically Mtb-infected, STIM1-deficient mice had reduced levels of inducible regulatory T cells (iTregs) due to a T cell–intrinsic requirement for STIM1 in iTreg differentiation and excessive production of IFN-γ and IL-12, which suppress iTreg differentiation and maintenance. Thus, STIM1 controls multiple aspects of T cell–mediated immune regulation to limit injurious inflammation during chronic infection.     

CD4+ and CD8+ T cell–dependent antiviral immunity requires STIM1 and STIM2

Calcium signaling is critical for lymphocyte function, and intracellular Ca²⁺ concentrations are regulated by store-operated Ca²⁺ entry (SOCE) through Ca²⁺ release–activated Ca²⁺ (CRAC) channels. In patients, loss-of-function mutations in CRAC channel components ORAI1 and STIM1 abolish SOCE and are associated with recurrent and chronic viral infections. Here, using mice with conditional deletion of Stim1 and its homolog Stim2 in T cells, we determined that both components are required for the maintenance of virus-specific memory CD8⁺ T cells and recall responses following secondary infection. In the absence of STIM1 and STIM2, acute viral infections became chronic. Early during infection, STIM1 and STIM2 were required for the differentiation of naive CD8⁺ T cells into fully functional cytolytic effector cells and mediated the production of cytokines and prevented cellular exhaustion in viral-specific CD8⁺ effector T cells. Importantly, memory and recall responses by CD8⁺ T cells required expression of STIM1 and STIM2 in CD4⁺ T cells. CD4⁺ T cells lacking STIM1 and STIM2 were unable to provide “help” to CD8⁺ T cells due to aberrant regulation of CD40L expression. Together, our data indicate that STIM1, STIM2, and CRAC channel function play distinct but synergistic roles in CD4⁺ and CD8⁺ T cells during antiviral immunity.     

Absence of mouse 2B4 promotes NK cell–mediated killing of activated CD8+ T cells,leading to prolonged viral persistence and altered pathogenesis

Persistent viral infections are often associated with inefficient T cell responses and sustained high-level expression of inhibitory receptors, such as the NK cell receptor 2B4 (also known as CD244), on virus-specific T cells. However, the role of 2B4 in T cell dysfunction is undefined, and it is unknown whether NK cells contribute to regulation of these processes. We show here that persistent lymphocytic choriomeningitis virus (LCMV) infection of mice lacking 2B4 resulted in diminished LCMV-specific CD8⁺ T cell responses, prolonged viral persistence, and spleen and thymic pathologies that differed from those observed in infected wild-type mice. Surprisingly, these altered phenotypes were not caused by 2B4 deficiency in T cells. Rather, the entire and long-lasting pathology and viral persistence were regulated by 2B4-deficient NK cells acting early in infection. In the absence of 2B4, NK cells lysed activated (defined as CD44^hi) but not naive (defined as CD44^lo) CD8⁺ T cells in a perforin-dependent manner in vitro and in vivo. These results illustrate the importance of NK cell self-tolerance to activated CD8⁺ T cells and demonstrate how an apparent T cell–associated persistent infection can actually be regulated by NK cells.     

Neuroprotective intervention by interferon-γ blockade prevents CD8+ T cell–mediated dendrite and synapse loss

Neurons are postmitotic and thus irreplaceable cells of the central nervous system (CNS). Accordingly, CNS inflammation with resulting neuronal damage can have devastating consequences. We investigated molecular mediators and structural consequences of CD8⁺ T lymphocyte (CTL) attack on neurons in vivo. In a viral encephalitis model in mice, disease depended on CTL-derived interferon-γ (IFN-γ) and neuronal IFN-γ signaling. Downstream STAT1 phosphorylation and nuclear translocation in neurons were associated with dendrite and synapse loss (deafferentation). Analogous molecular and structural alterations were also found in human Rasmussen encephalitis, a CTL-mediated human autoimmune disorder of the CNS. Importantly, therapeutic intervention by IFN-γ blocking antibody prevented neuronal deafferentation and clinical disease without reducing CTL responses or CNS infiltration. These findings identify neuronal IFN-γ signaling as a novel target for neuroprotective interventions in CTL-mediated CNS disease.In many inflammatory diseases of the central nervous system (CNS) neuronal damage determines permanent neurological deficits. Novel strategies for neuroprotection are urgently sought and require a detailed understanding of the mechanisms underlying neuronal damage. The key contribution of cytotoxic CD8⁺ T lymphocytes (CTLs) to this process has become increasingly appreciated in recent years (Neumann et al., 2002). CTLs are commonly recruited to the brain in viral infections, paraneoplastic disorders (Albert and Darnell, 2004), and autoimmune diseases such as multiple sclerosis (MS) and Rasmussen’s encephalitis (RE; Hauser et al., 1986; Bien et al., 2005; Friese and Fugger, 2005; Goverman, 2009). Upon engagement of their cognate peptide–MHC class I (MHC-I) complex on target cells, CTLs activate an array of effector functions. Cytotoxicity is typically mediated by perforin-dependent mechanisms and Fas–FasL (CD95/CD95L) interactions (Stinchcombe and Griffiths, 2007), but CTLs also secrete cytokines including IFN-γ and TNF. The relative contribution of each of these pathways to tissue damage varies greatly and depends on the target cell type and tissue (Guidotti et al., 1996; Kägi et al., 1996; Medana et al., 2000). At the same time, CTL cytotoxicity and cytokine secretion can also both contribute to virus control in the CNS (Binder and Griffin, 2001; Shrestha and Diamond, 2007; Pinschewer et al., 2010b).Neurons show limited turnover and regenerative capacity yet serve essential functions. Classical concepts have therefore suggested that neurons are spared from CTL attack. This “immune privilege” has been accredited to limited MHC-I expression (Joly et al., 1991), secretion of immunomodulatory TGF-β (Liu et al., 2006), and expression of Fas-L (Medana et al., 2001a). The same mechanisms are also thought to represent an evolutionary reason why neurons serve as a sanctuary for several RNA and DNA viruses, namely members of the Herpes, Paramyxo-, and Arenavirus families (Brown et al., 1979; Sequiera et al., 1979; ter Meulen et al., 1984; Joly et al., 1991). Recent studies have shown, however, that infected neurons do not escape CTL recognition altogether (McDole et al., 2010). For example, CNS-infiltrating CTLs established stable peptide/MHC-I–specific contacts with Herpes simplex virus– or Borna disease virus–infected neurons, respectively (Khanna et al., 2003; Chevalier et al., 2011). In the Theiler’s murine encephalomyelitis virus model, depletion of CTLs or genetic deficiency in MHC-I or perforin preserved axon integrity and prevented neurological deficits (Murray et al., 1998; Deb et al., 2009, 2010). CTLs can attack neuronal somata (Manning et al., 1987) and axons (Medana et al., 2001b) in primary neuronal cultures and explants, and cultured neurons are sensitive to lysis or silencing by perforin (Rensing-Ehl et al., 1996; Meuth et al., 2009). The Fas/Fas-L pathway can cause cytoskeleton breaks and membrane disruption which eventually cause neuronal death (Medana et al., 2000). However, the morphology, electrical activity, and glial cell environment of neurons differ considerably between in vitro culture conditions and the natural tissue habitat, and all of these factors can affect susceptibility to CTL attack (Neumann et al., 1995). Hence, we still lack a clear understanding of how CTLs damage neurons in vivo and which alterations result from such damage.We have recently established the viral déjà vu model, allowing us to study CTL-mediated neuronal damage and the resulting disease in vivo (Merkler et al., 2006). Neonatal intracranial (i.c.) infection of mice with an attenuated lymphocytic choriomeningitis virus (LCMV) variant (rLCMV/INDG) results in viral persistence selectively in CNS neurons (a status referred to as carrier mice). rLCMV/INDG is not cytolytic and carrier mice are therefore clinically healthy, but they express viral nonself-antigens in neurons. Notably, they are free of CNS-infiltrating T cells, and viral epitope-specific CD8⁺ T cell frequencies in peripheral blood remain below detection limits of peptide/MHC-I tetramer measurements (Merkler et al., 2006). This indicates that neonatal rLCMV/INDG infection fails to trigger a clinically significant CTL response. Upon adult infection with LCMV WT (LCMVwt; referred to as challenge), carrier mice mount vigorous CTL responses against the immunodominant H-2D^b–restricted nucleoprotein-derived epitope NP396 that is shared between rLCMV/INDG and LCMVwt. These CTLs infiltrate the CNS gray matter, attack NP396-expressing rLCMV/INDG-infected neurons, and cause severe disease within 7–10 d after challenge. The topographical distribution and the composition of inflammatory infiltrates in viral déjà vu disease recreate histopathological hallmarks of RE. In this human autoimmune disease, oligoclonal and therefore putatively antigen-specific CD8⁺ T cell populations dominate the histological picture and are typically found in direct contact with neurons (Li et al., 1997; Bien et al., 2005). The clinical presentation of RE is characterized by treatment-refractory epilepsy with consequent intellectual decline and hemiparesis, thus necessitating surgical resection of affected brain regions. A viral contribution to RE pathogenesis has long been suspected but remains to be substantiated (Friedman et al., 1977; Walter and Renella, 1989; Farrell et al., 1991).Here, we show that CTL attack on neurons in vivo induces rapid loss of dendrites and synapses rather than neuronal depletion. This deafferentation and the resulting disease depend on IFN-γ signaling from CTLs to neurons but neither on Fas- nor on perforin-dependent pathways. Accordingly, STAT1 phosphorylation and nuclear translocation together with deafferentation represented hallmarks of both the murine viral déjà vu model and human RE. These findings provide important new insights into the molecular mechanisms and cellular consequences of CTL–neuron interactions in viral and autoimmune CNS disorders. This delineates a promising new strategy for neuroprotective intervention in immune-mediated CNS disease.     

CD19 CAR–T cells of defined CD4+:CD8+ composition in adult B cell ALL patients

BACKGROUND. T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR–T cell products were prepared from unselected T cells.METHODS. We conducted a clinical trial to evaluate CD19 CAR–T cells that were manufactured from defined CD4⁺ and CD8⁺ T cell subsets and administered in a defined CD4⁺:CD8⁺ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy.RESULTS. The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR–T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR–T cell dosing based on BM disease burden decreased toxicity. CD8⁺ T cell–mediated anti-CAR transgene product immune responses developed after CAR–T cell infusion in some patients, limited CAR–T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR–T cell persistence and disease-free survival.CONCLUSION. Immunotherapy with a CAR–T cell product of defined composition enabled identification of factors that correlated with CAR–T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR–T cell dosing strategies that mitigated toxicity and improved disease-free survival.TRIAL REGISTRATION. ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01865617","term_id":"NCT01865617"}}NCT01865617.FUNDING. R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.     

CD4+ CD25+调节性T细胞

机体的免疫系统维持着对感染性抗原的反应和自身耐受的平衡,而对自身抗原产生免疫耐受则是防止发生自身免疫病的关键。阴性选择中的克隆清除、免疫无能和克隆忽略被认为是控制自身反应性T细胞的主要机制。近些年随着研究的深入,CD4^＋ CD25^＋调节性T细胞（Treg细胞）在维持自身耐受中的作用得到进一步的认识。     

TIGIT and PD-1 impair tumor antigen–specific CD8+ T cells in melanoma patients

T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen–specific (TA-specific) CD8⁺ T cells and CD8⁺ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8⁺ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8⁺ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1⁺TIM-3⁺ TA-specific CD8⁺ T cells. PD-1⁺TIGIT⁺, PD-1^–TIGIT⁺, and PD-1⁺TIGIT^– CD8⁺ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand–expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8⁺ T cells and CD8⁺ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8⁺ T cells and CD8⁺ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8⁺ T cell responses in patients with advanced melanoma.     

Up-regulation of a death receptor renders antiviral T cells susceptible to NK cell–mediated deletion

Antiviral T cell responses in hepatotropic viral infections such as hepatitis B virus (HBV) are profoundly diminished and prone to apoptotic deletion. In this study, we investigate whether the large population of activated NK cells in the human liver contributes to this process. We show that in vitro removal of NK cells augments circulating CD8⁺ T cell responses directed against HBV, but not against well-controlled viruses, in patients with chronic hepatitis B (CHB). We find that NK cells can rapidly eliminate HBV-specific T cells in a contact-dependent manner. CD8⁺ T cells in the liver microcirculation are visualized making intimate contact with NK cells, which are the main intrahepatic lymphocytes expressing TNF-related apoptosis-inducing ligand (TRAIL) in CHB. High-level expression of the TRAIL death receptor TRAIL-R2 is found to be a hallmark of T cells exposed to the milieu of the HBV-infected liver in patients with active disease. Up-regulation of TRAIL-R2 renders T cells susceptible to caspase-8–mediated apoptosis, from which they can be partially rescued by blockade of this death receptor pathway. Our findings demonstrate that NK cells can negatively regulate antiviral immunity in chronic HBV infection and illustrate a novel mechanism of T cell tolerance in the human liver.T cell responses are tightly regulated to maintain immune homeostasis and limit damage to vital organs. T cells in the liver, in particular, are subjected to potent tolerizing mechanisms. Although these mechanisms prevent overzealous responses causing tissue injury, they may be exploited by hepatotropic pathogens to subvert antiviral immunity (Protzer et al., 2012). There have been major recent advances in our understanding of the multiple co-inhibitory pathways driving T cell exhaustion in the liver and perpetuating persistent viral infections (Protzer et al., 2012). However, the potential for NK cells to regulate T cell immunity has not been defined in human viral infections.NK cells can contribute to the containment of many infections by intracellular pathogens (Orange et al., 2002; Khakoo et al., 2004; Lodoen and Lanier, 2006; Alter et al., 2011), acting though cytolytic or noncytolytic effects on target cells or by promoting adaptive immunity (Vivier et al., 2008). Accumulating data highlight the capacity of NK cells to also exert a negative regulatory effect on T cells (Su et al., 2001) through inhibition of antigen presentation (Andrews et al., 2010), production of IL-10 (Lee et al., 2009), or direct killing of T cells. Several receptor–ligand interactions between NK cells and T cells have been found to be capable of leading to autologous lysis of activated T cells (Rabinovich et al., 2003; Cerboni et al., 2007; Lu et al., 2007; Soderquest et al., 2011). More recently, NK cells have been shown to limit T cell immunity in a mouse model of chronic viral infection (Waggoner et al., 2010; Lang et al., 2012; Waggoner et al., 2012).In this study, we sought to investigate the impact of NK cells on antiviral T cell responses in the setting of persistent infection with a human hepatotropic virus. Activated NK cells are markedly enriched in the liver microcirculation, where we hypothesized they would come into prolonged, close contact with infiltrating T cells. Although NK cells in patients with chronic hepatitis B (CHB) infection have impaired noncytolytic antiviral function, we have previously shown that they maintain their cytotoxic potential and up-regulate the death ligand TRAIL, particularly in the intrahepatic compartment (Dunn et al., 2007; Peppa et al., 2010). HBV-specific CD8⁺ T cells, which are essential for viral control, are profoundly depleted in these patients (Maini et al., 2000; Boni et al., 2007). Here, we demonstrate that hepatitis B virus–specific T cells up-regulate a death receptor for TRAIL and become susceptible to NK cell–mediated killing, thereby contributing to the failure of antiviral immunity in CHB.     

ER stress regulates myeloid-derived suppressor cell fate through TRAIL-R–mediated apoptosis

Myeloid-derived suppressor cells (MDSCs) dampen the immune response thorough inhibition of T cell activation and proliferation and often are expanded in pathological conditions. Here, we studied the fate of MDSCs in cancer. Unexpectedly, MDSCs had lower viability and a shorter half-life in tumor-bearing mice compared with neutrophils and monocytes. The reduction of MDSC viability was due to increased apoptosis, which was mediated by increased expression of TNF-related apoptosis–induced ligand receptors (TRAIL-Rs) in these cells. Targeting TRAIL-Rs in naive mice did not affect myeloid cell populations, but it dramatically reduced the presence of MDSCs and improved immune responses in tumor-bearing mice. Treatment of myeloid cells with proinflammatory cytokines did not affect TRAIL-R expression; however, induction of ER stress in myeloid cells recapitulated changes in TRAIL-R expression observed in tumor-bearing hosts. The ER stress response was detected in MDSCs isolated from cancer patients and tumor-bearing mice, but not in control neutrophils or monocytes, and blockade of ER stress abrogated tumor-associated changes in TRAIL-Rs. Together, these data indicate that MDSC pathophysiology is linked to ER stress, which shortens the lifespan of these cells in the periphery and promotes expansion in BM. Furthermore, TRAIL-Rs can be considered as potential targets for selectively inhibiting MDSCs.     

Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen–specific CD8+ T cell dysfunction in melanoma patients

The paradoxical coexistence of spontaneous tumor antigen–specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction. In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1–specific CD8⁺ T cells that up-regulate PD-1 expression. We also observed that PD-1 regulates NY-ESO-1–specific CD8⁺ T cell expansion upon chronic antigen stimulation. In the present study, we show that a fraction of PD-1⁺ NY-ESO-1–specific CD8⁺ T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3⁺PD-1⁺ NY-ESO-1–specific CD8⁺ T cells are more dysfunctional than Tim-3⁻PD-1⁺ and Tim-3⁻PD-1⁻ NY-ESO-1–specific CD8⁺ T cells, producing less IFN-γ, TNF, and IL-2. Tim-3–Tim-3L blockade enhanced cytokine production by NY-ESO-1–specific CD8⁺ T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity. In addition, Tim-3–Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1–specific CD8⁺ T cells upon prolonged antigen stimulation and acted in synergy with PD-1–PD-L1 blockade. Collectively, our findings support the use of Tim-3–Tim-3L blockade together with PD-1–PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.There is ample evidence that patients with melanoma can develop immune responses directed against antigens expressed by their own tumor (Boon et al., 2006). Among these antigens, cancer-germline antigens (CGAs) are expressed by tumors of many different histological types, including melanoma, but not by normal tissues, except testis. Because germ cells in testis do not express HLA molecules on their surface (Haas et al., 1988), CGAs represent strictly tumor-specific T cell targets (Boon et al., 2006). Among CGAs, NY-ESO-1 has been shown to stimulate spontaneous cellular and humoral responses that are detectable only in patients with advanced NY-ESO-1–expressing cancer (Stockert et al., 1998; Jäger et al., 2000; Mandic et al., 2005; Fourcade et al., 2008). Understanding the failure of spontaneous NY-ESO-1–specific T cell responses to promote regression of NY-ESO-1⁺ tumors is therefore critical for the design of novel therapeutic interventions aimed at overcoming tumor-induced immune escape.We have previously shown that the large majority of spontaneous NY-ESO-1–specific CD8⁺ T cells up-regulates programmed death 1 (PD-1) expression (Fourcade et al., 2009), which appears to be associated with T cell exhaustion/dysfunction in chronic viral infections in animals and humans (Barber et al., 2006; Day et al., 2006; Petrovas et al., 2006; Trautmann et al., 2006). We observed that PD-1 up-regulation on spontaneous NY-ESO-1–specific CD8⁺ T cells occurs along with T cell activation and is not directly associated with an inability to produce cytokines ex vivo upon stimulation with cognate antigen. Blockade of the PD-1–programmed death ligand 1 (PD-L1) pathway in combination with prolonged antigen stimulation with PD-L1⁺ APCs or melanoma cells augmented the frequencies of cytokine-producing, proliferating, and total NY-ESO-1–specific CD8⁺ T cells. Our findings are in line with previous studies of PD-1 expression by HIV- and SIV-specific CD8⁺ T cells, demonstrating that PD-1 is a regulator of antigen-specific CD8⁺ T cell expansion in the context of chronic antigen exposure, although it does not exhibit a major impact upon their functionality on a cell-per-cell basis (Petrovas et al., 2006, 2007). To further determine whether other molecular pathways are involved in tumor antigen–specific T cell dysfunction, we studied T cell immunoglobulin and mucin-domain–containing molecule 3 (Tim-3) expression on spontaneous NY-ESO-1–specific CD8⁺ T cells from patients with advanced melanoma and investigated whether Tim-3 up-regulation defines a subgroup of dysfunctional tumor antigen–specific CD8⁺ T cells. Tim-3 is a transmembrane protein constitutively expressed on Th1/Tc1 cells in mice and humans (Monney et al., 2002). Several lines of evidence support the role of Tim-3 as an inhibitory molecule that down-regulates effector Th1/Tc1 cell responses. In mice, blocking the Tim-3–Tim-3L pathway resulted in hyperproliferation of Th1-type cells and abrogated the induction of peripheral and transplantation tolerance (Sabatos et al., 2003; Sánchez-Fueyo et al., 2003). Tim-3 interacts with its ligand galectin-9 to induce cell death in Th1 cells (Zhu et al., 2005). In humans, Tim-3 expression is defective in CD4⁺ T cells producing high levels of IFN-γ, as well as those isolated from cerebrospinal fluid of patients with multiple sclerosis (Koguchi et al., 2006). Recently, Tim-3 up-regulation has been reported in HIV-specific and HCV-specific CD8⁺ T cells in patients with progressive HIV infection and chronic hepatitis C, respectively (Jones et al., 2008; Golden-Mason et al., 2009). Tim-3⁺ HIV- and HCV-specific CD8⁺ T cells were distinct from the PD-1⁺ CD8⁺ T cells and exhibited T cell dysfunction. However, it is unknown whether tumor antigen–specific CD8⁺ T cells in patients with advanced cancers express Tim-3.In this study, we show that a fraction of PD-1⁺ NY-ESO-1–specific CD8⁺ T cells, which represents the large majority of circulating NY-ESO-1–specific CD8⁺ T cells in patients with advanced melanoma, up-regulates Tim-3 expression. Tim-3⁺PD-1⁺ NY-ESO-1–specific CD8⁺ T cells are highly dysfunctional compared with Tim-3⁻PD-1⁺ and Tim-3⁻PD-1⁻ NY-ESO-1–specific CD8⁺ T cells. Tim-3–Tim-3L pathway blockade alone or in combination with PD-1–PD-L1 pathway blockade enhanced NY-ESO-1–specific CD8⁺ T cell numbers and functions. Collectively, our findings support the use of Tim-3–Tim-3L blockade in association with PD-1–PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.     

Thromboxane A2 acts as tonic immunoregulator by preferential disruption of low-avidity CD4+ T cell–dendritic cell interactions

Interactions between dendritic cells (DCs) and T cells control the decision between activation and tolerance induction. Thromboxane A2 (TXA2) and its receptor TP have been suggested to regulate adaptive immune responses through control of T cell–DC interactions. Here, we show that this control is achieved by selectively reducing expansion of low-avidity CD4⁺ T cells. During inflammation, weak tetramer-binding TP-deficient CD4⁺ T cells were preferentially expanded compared with TP-proficient CD4⁺ T cells. Using intravital imaging of cellular interactions in reactive peripheral lymph nodes (PLNs), we found that TXA2 led to disruption of low- but not high-avidity interactions between DCs and CD4⁺ T cells. Lack of TP correlated with higher expression of activation markers on stimulated CD4⁺ T cells and with augmented accumulation of follicular helper T cells (T_FH), which correlated with increased low-avidity IgG responses. In sum, our data suggest that tonic suppression of weak CD4⁺ T cell–DC interactions by TXA2–TP signaling improves the overall quality of adaptive immune responses.T cells have evolved to quickly react to potentially dangerous microbes by recognizing pathogen-derived peptide (p)-MHC complexes displayed on antigen-presenting cells, in particular DCs. Because T cells are selected in the thymus for their ability to recognize self-pMHC complexes (Morris and Allen, 2012) and numerous self-reactive T cells are released into the periphery (Su et al., 2013), peripheral tolerance education is critical to avoid activation of autoreactive T cells. Studies using intravital two-photon microscopy (2PM) of reactive PLNs have shed light on the dynamic T cell–DC interactions and their correlation with full versus curtailed T cell activation and tolerance induction. The amount of cognate pMHC complexes on activated DCs is critical in determining the transition of a highly motile scanning-mode T cell to an immotile, stably interacting one (Cahalan and Parker, 2006; Henrickson and von Andrian, 2007; Bajénoff and Germain, 2007). Such stable T cell–DC interactions (>8h) are a prerequisite for full effector T cell differentiation (Rachmilewitz and Lanzavecchia, 2002). Thus, in presence of high amounts of cognate pMHC on activated DCs, T cells decelerate rapidly, whereas T cells show a motile DC sampling behavior when cognate pMHC levels are low. Altered peptide ligands (APLs) with reduced affinity for a given TCR also decrease the length of T cell–DC interactions, limiting T cell activation. Under tolerogenic conditions (i.e., in the absence of co-stimulation), 2PM studies uncovered shortened T cell–DC interactions (Hugues et al., 2004) although this is still controversial (Shakhar et al., 2005). Similarly, the presence of regulatory T (T reg) cells reduces T cell–DC interactions and subsequent T cell activation (Tadokoro et al., 2006; Tang et al., 2006).A perhaps counterintuitive recent finding has revealed a significant increase in CD8⁺ T cell immune response avidity in presence of T reg cells (Pace et al., 2012). This is due to T reg cell–mediated suppression of excessive interactions between DCs and CD8⁺ T cells bearing TCRs with low avidity for pMHC complexes. In the absence of T reg cells, uncontrolled CCR5 ligand secretion by activated DCs induces attraction of bystander TCR clones with low affinity for pMHC complexes, which decreases overall avidity and memory T cell generation of the resulting immune response. Whether a comparable mechanism also exists to selectively support activation of high avidity CD4⁺ T cells by immunoregulatory factors is currently unknown.The short-lived arachidonic acid–derived lipid thromboxane A2 (TXA2) has been suggested to regulate adaptive immune responses (Kabashima et al., 2003). Activated DCs and other cell types produce TXA2, which binds its G-protein coupled receptor TP expressed in thymocytes and naive but not effector/memory CD4⁺ and CD8⁺ T cells. Addition of high amounts of the TP agonist I-BOP induces chemokinesis in naive T cells and decreases in vitro aggregate formation between T cells and DCs, causing reduced T cell activation (Kabashima et al., 2003). Combined with the observation that TXA2 levels rapidly rise in reactive PLN during immune responses (Moore et al., 1989), these data suggest a model where TXA2 may act as a general suppressor of T cell–DC interactions. In line with this hypothesis, aged TP-deficient T cells develop lymphoid hyperplasia and high antibody titers (Kabashima et al., 2003). Yet, it has remained unknown how TXA2 signaling affects dynamic CD4⁺ T cell interactions with DC displaying varying pMHC abundance and affinity in vivo, and how this impacts avidity patterns of responding T cells.Here, we show that during sterile and microbial inflammation, absence of TP resulted in increased expansion of low-avidity CD4⁺ T cells. Using 2PM imaging of cellular interactions in reactive PLNs, we report that paracrine TXA2 signaling preferentially disrupted low-avidity interactions between DCs and OT-II CD4⁺ T cells induced by low cognate pMHC levels or low-affinity peptide. As a consequence, TP^−/− OT-II CD4⁺ T cells show increased expression of early activation markers, as well as augmented accumulation of follicular helper T cells (T_FH) compared with WT OT-II CD4⁺ cells. High numbers of TP^−/− T_FH correlated with increased low-avidity IgG production, thus thwarting the overall quality of the adaptive immune response. In sum, our data uncover a previously unappreciated contribution of a tolerance-inducing mechanism for preferential activation of high avidity CD4⁺ T cells.     

CD4+CD25+调节性T细胞的研究进展

CD4^＋CD25^＋调节性T细胞（regulatory T cell,Treg）具有维持自身免疫耐受和调节免疫应答的功能,其功能紊乱或数目下降是导致自身免疫性疾病的重要原因之一。近年来,研究发现Foxp3在调控CD4^＋CD25^＋Treg细胞的发育和功能上起着重要作用。本文就CD4^＋CD25^＋Treg细胞的免疫抑制机制、Foxp3在其发育和功能上的作用、IL-2和细胞毒性T淋巴细胞相关抗原4（CTLA-4）等对其产生、维持及活化的作用等方面作一综述。     

CD4+CD25+调节性T细胞与GVHD

源于胸腺的表达转录因子Foxp3的CD4＋CD25＋调节性T细胞具有免疫调节功能,在外周免疫耐受中有重要作用,可控制异基因造血干细胞移植（allo—HSCT）的移植物抗宿主病（GVHD）,而不影响移植物抗白血病作用（GVL）,许多移植模型和临床试验均在探索中。植入CD4＋CD25＋调节性T细胞后所获得的抗GVHD作用的动物移植模型及早期HSCT临床研究结果表明,CD4＋CD25＋调节性T细胞输注将能极大改变allo—HSCT的现状。     

移植患者CD4+CD25+CD127low调节性T细胞、TGF-β1及IL-10的表达

目的观察肝肾移植患者外周血中调节性T细胞(Treg细胞)的百分比与转化生长因子β1(TGF-β1)及白细胞介素10(IL-10)水平的表达情况及其相关性。方法15例移植患者的外周静脉血,采用流式细胞术检测CD4^+CD25^+CD127^low Treg细胞的百分比,并以20例体检健康者为对照组;用酶联免疫吸附试验法检测移植患者血清中细胞因子TGF-β1、IL-10的水平,分析这2种细胞因子水平与CD4^+CD25^+CD127^low Treg细胞百分比的关系及移植前后TGF-β1、IL-10水平的差异。结果与对照组比较,移植患者移植前CD4^+CD25^+CD127^low Treg细胞占CD4+T淋巴细胞的百分比明显升高,差异有统计学意义(P<0.05);肝肾移植后CD4^+CD25^+CD127^low Treg细胞百分比下降为0。肝肾移植前CD4^+CD25^+CD127^low Treg细胞百分比与TGF-β1、IL-10的水平呈正相关(P<0.05)。TGF-β1、IL-10的水平移植后低于移植前,差异无统计学意义(P>0.05)。结论CD4^+CD25^+CD127^low Treg的百分比与TGF-β1、IL-10的水平存在一定相关性;移植后CD4^+CD25^+CD127^low Tre细胞百分比下降为0,可能与应用大量免疫抑制剂有关。     

Intratumoral IL-12 combined with CTLA-4 blockade elicits T cell–mediated glioma rejection

Glioblastomas (GBs) are the most aggressive form of primary brain cancer and virtually incurable. Accumulation of regulatory T (T reg) cells in GBs is thought to contribute to the dampening of antitumor immunity. Using a syngeneic mouse model for GB, we tested whether local delivery of cytokines could render the immunosuppressive GB microenvironment conducive to an antitumor immune response. IL-12 but not IL-23 reversed GB-induced immunosuppression and led to tumor clearance. In contrast to models of skin or lung cancer, IL-12–mediated glioma rejection was T cell dependent and elicited potent immunological memory. To translate these findings into a clinically relevant setting, we allowed for GB progression before initiating therapy. Combined intratumoral IL-12 application with systemic blockade of the co-inhibitory receptor CTLA-4 on T cells led to tumor eradication even at advanced disease stages where monotherapy with either IL-12 or CTLA-4 blockade failed. The combination of IL-12 and CTLA-4 blockade acts predominantly on CD4⁺ cells, causing a drastic decrease in FoxP3⁺ T reg cells and an increase in effector T (T eff) cells. Our data provide compelling preclinical findings warranting swift translation into clinical trials in GB and represent a promising approach to increase response rates of CTLA-4 blockade in solid tumors.Glioblastoma (GB) is among the most aggressive cancers known. Current treatment options are limited and the clinical prognosis is poor. Patients diagnosed with GB show a median survival of little more than a year despite aggressive surgery, radiation therapy, and chemotherapy (Weller et al., 2013). Moreover, GBs induce a highly immunosuppressive microenvironment, characterized by the presence of T reg cells (Grauer et al., 2007; Jacobs et al., 2010). Given the failure of conventional therapy in GBs, the most promising treatment option may thus rely on the exploration of immunotherapeutic strategies. IL-12 is the prototype member of a group of heterodimeric cytokines with predominantly proinflammatory properties. IL-12 polarizes naive helper T cells (T_H) to adopt a T_H1 phenotype and stimulates cytotoxic T cells, NK T (NKT) cells, and conventional NK cells. The therapeutic success of application of IL-12 in various preclinical animal models of cancer is compelling (Colombo and Trinchieri, 2002). However, in humans, systemic delivery of IL-12 evoked serious adverse events such as leukopenia and thrombocytopenia, including fatalities in two patients, at moderately effective doses (Atkins et al., 1997; Leonard et al., 1997). Thus, local rather than systemic delivery of IL-12 represents the only viable option for using IL-12 in cancer immunotherapy in humans. IL-12 appears to exert its cancer-suppressive properties through different effector cells in a tissue-specific manner. In the B16 melanoma model, IL-12–mediated suppression of s.c. tumor growth is mediated by a small population of IL-12–responsive, Rorγt-dependent innate lymphoid cells (ILCs; Eisenring et al., 2010). On the other hand, B16-derived lung tumors are controlled through IL-12–activated NK cells (Kodama et al., 1999; Eisenring et al., 2010). Conversely, IL-12–mediated glioma control has been attributed to T cells and NK cells, but open questions remain about which cell types indeed are the precise cellular targets of IL-12, consequently mediating anti-GB immunity (Vetter et al., 2009; Yamanaka et al., 2002, 2003). IL-23 is another member of the IL-12 family and also has potent pro-inflammatory properties. Several groups reported potent antitumor activity in various experimental settings including brain tumors (Lo et al., 2003; Hu et al., 2006). Others have reported a protumorigenic effect of IL-23 (Langowski et al., 2006). The goal of this study was to systematically analyze whether and how IL-12 and IL-23 induce an antitumor immune response in a syngeneic murine model of GB.     

CD4+T细胞的研究进展

近年来,随着对CD4+T细胞研究的深入,对Th1和Th2细胞的免疫生物学活性及免疫调节也有了进一步的认识.CD4+T细胞新亚型CD4+CD25+调节性T细胞(Treg)及Th17细胞受到了人们广泛的关注,尤其是细胞的分化调节、功能及相关疾病的发病机制.本文就CD4+T细胞各亚群的生物学特征及分工、亚群间的相互调节、免疫应答与疾病的关系等作一综述.     

Toll-like receptor 4–mediated lymphocyte influx induces neonatal necrotizing enterocolitis

The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1^–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3⁺CD4⁺IL-17⁺ and reduced tolerogenic Foxp3⁺ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification.