SHP-1 phosphatase activity counteracts increased T cell receptor affinity

Anti-self/tumor T cell function can be improved by increasing TCR-peptide MHC (pMHC) affinity within physiological limits, but paradoxically further increases (K_d < 1 μM) lead to drastic functional declines. Using human CD8⁺ T cells engineered with TCRs of incremental affinity for the tumor antigen HLA-A2/NY-ESO-1, we investigated the molecular mechanisms underlying this high-affinity–associated loss of function. As compared with cells expressing TCR affinities generating optimal function (K_d = 5 to 1 μM), those with supraphysiological affinity (K_d = 1 μM to 15 nM) showed impaired gene expression, signaling, and surface expression of activatory/costimulatory receptors. Preferential expression of the inhibitory receptor programmed cell death-1 (PD-1) was limited to T cells with the highest TCR affinity, correlating with full functional recovery upon PD-1 ligand 1 (PD-L1) blockade. In contrast, upregulation of the Src homology 2 domain-containing phosphatase 1 (SHP-1/PTPN6) was broad, with gradually enhanced expression in CD8⁺ T cells with increasing TCR affinities. Consequently, pharmacological inhibition of SHP-1 with sodium stibogluconate augmented the function of all engineered T cells, and this correlated with the TCR affinity–dependent levels of SHP-1. These data highlight an unexpected and global role of SHP-1 in regulating CD8⁺ T cell activation and responsiveness and support the development of therapies inhibiting protein tyrosine phosphatases to enhance T cell–mediated immunity.     

Potentiating Functional Antigen-specific CD8+ T Cell Immunity by a Novel PD1 Isoform-based Fusion DNA Vaccine

Understanding and identifying new ways of mounting an effective CD8⁺ T cell immune response is important for eliminating infectious pathogens. Although upregulated programmed death-1 (PD1) in chronic infections (such as HIV-1 and tuberculosis) impedes T cell responses, blocking this PD1/PD-L pathway could functionally rescue the “exhausted” T cells. However, there exists a number of PD1 spliced variants with unknown biological function. Here, we identified a new isoform of human PD1 (Δ42PD1) that contains a 42-nucleotide in-frame deletion located at exon 2 domain found expressed in peripheral blood mononuclear cells (PBMCs). Δ42PD1 appears to function distinctly from PD1, as it does not engage PD-L1/PD-L2 but its recombinant form could induce proinflammatory cytokines. We utilized Δ42PD1 as an intramolecular adjuvant to develop a fusion DNA vaccine with HIV-1 Gag p24 antigen to immunize mice, which elicited a significantly enhanced level of anti-p24 IgG1/IgG2a antibody titers, and important p24-specific and tetramer⁺CD8⁺ T cells responses that lasted for ≥7.5 months. Furthermore, p24-specific CD8⁺ T cells remain functionally improved in proliferative and cytolytic capacities. Importantly, the enhanced antigen-specific immunity protected mice against pathogenic viral challenge and tumor growth. Thus, this newly identified PD1 variant (Δ42PD1) amplifies the generation of antigen-specific CD8⁺ T cell immunity when used in a DNA vaccine.     

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.     

Polyethylenimine-based siRNA nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to elicit therapeutic antitumor immunity

The success of clinically relevant immunotherapies requires reversing tumor-induced immunosuppression. Here we demonstrated that linear polyethylenimine-based (PEI-based) nanoparticles encapsulating siRNA were preferentially and avidly engulfed by regulatory DCs expressing CD11c and programmed cell death 1–ligand 1 (PD-L1) at ovarian cancer locations in mice. PEI-siRNA uptake transformed these DCs from immunosuppressive cells to efficient antigen-presenting cells that activated tumor-reactive lymphocytes and exerted direct tumoricidal activity, both in vivo and in situ. PEI triggered robust and selective TLR5 activation in vitro and elicited the production of hallmark TLR5-inducible cytokines in WT mice, but not in Tlr5^–/– littermates. Thus, PEI is a TLR5 agonist that, to our knowledge, was not previously recognized. In addition, PEI-complexed nontargeting siRNA oligonucleotides stimulated TLR3 and TLR7. The nonspecific activation of multiple TLRs (specifically, TLR5 and TLR7) reversed the tolerogenic phenotype of human and mouse ovarian tumor–associated DCs. In ovarian carcinoma–bearing mice, this induced T cell–mediated tumor regression and prolonged survival in a manner dependent upon myeloid differentiation primary response gene 88 (MyD88; i.e., independent of TLR3). Furthermore, gene-specific siRNA-PEI nanocomplexes that silenced immunosuppressive molecules on mouse tumor-associated DCs elicited discernibly superior antitumor immunity and enhanced therapeutic effects compared with nontargeting siRNA-PEI nanocomplexes. Our results demonstrate that the intrinsic TLR5 and TLR7 stimulation of siRNA-PEI nanoparticles synergizes with the gene-specific silencing activity of siRNA to transform tumor-infiltrating regulatory DCs into DCs capable of promoting therapeutic antitumor immunity.     

Attraction and Activation of Dendritic Cells at the Site of Tumor Elicits Potent Antitumor Immunity

Tumor cells harbor unique genetic mutations, which lead to the generation of immunologically foreign antigenic peptide repertoire with the potential to induce individual tumor-specific immune responses. Here, we developed an in situ tumor vaccine with the ability to elicit antitumor immunity. This vaccine comprised an E1B-deleted oncolytic adenovirus expressing β-defensin-2 (Ad-BD2-E1A) for releasing tumor antigens, recruiting and activating plasmacytoid dendritic cells (pDCs). Intratumoral injections of Ad-BD2-E1A vaccine inhibited primary breast tumor growth and blocked naturally occurring metastasis in mice. Ad-BD2-E1A vaccination induced potent tumor-specific T-cell responses. Splenic and intratumoral DCs isolated from Ad-BD2-E1A-immunized mice were able to stimulate or promote the differentiation of naive T cells into tumor-specific cytotoxic T cells. We further found that the increased numbers of mature CD45RA⁺CD8α⁺CD40⁺ pDCs infiltrated into Ad-BD2-E1A-treated tumors. The antitumor effect of Ad-BD2-E1A vaccination was abrogated in toll-like receptor 4 (TLR4) deficient mice, suggesting the critical role of TLR4 in the induction of antitumor immunity by Ad-BD2-E1A. The results of this study indicate that in situ vaccination with the oncolytic BD2-expressing adenovirus preferentially attracts pDCs and promotes their maturation, and thus elicits potent tumor-specific immunity. This vaccine represents an attractive therapeutic strategy for the induction of individualized antitumor immunity.     

Ad.Egr-TNF and Local Ionizing Radiation Suppress Metastases by Interferon-��-Dependent Activation of Antigen-specific CD8+ T Cells

Ad.Egr-TNF is a radioinducible adenovector currently in phase 3 trials for inoperable pancreatic cancer. The combination of Ad.Egr-TNF and ionizing radiation (IR) contributes to local tumor control through the production of tumor necrosis factor-α (TNFα) in the tumor microenvironment. Moreover, clinical and preclinical studies with Ad.Egr-TNF/IR have suggested that this local approach suppresses the growth of distant metastatic disease; however, the mechanisms responsible for this effect remain unclear. These studies have been performed in wild-type (WT) and TNFR1,2^−/− mice to assess the role of TNFα-induced signaling in the suppression of draining lymph node (DLN) metastases. The results demonstrate that production of TNFα in the tumor microenvironment induces expression of interferon (IFNβ). In turn, IFNβ stimulates the production of chemokines that recruit CD8⁺ T cells to the tumor. The results further demonstrate that activation of tumor antigen–specific CD8⁺ CTLs contributes to local antitumor activity and suppression of DLN metastases. These findings support a model in which treatment of tumors with Ad.Egr-TNF and IR is mediated by local and distant immune-mediated antitumor effects that suppress the development of metastases.     

Irradiation and anti–PD-L1 treatment synergistically promote antitumor immunity in mice

High-dose ionizing irradiation (IR) results in direct tumor cell death and augments tumor-specific immunity, which enhances tumor control both locally and distantly. Unfortunately, local relapses often occur following IR treatment, indicating that IR-induced responses are inadequate to maintain antitumor immunity. Therapeutic blockade of the T cell negative regulator programmed death–ligand 1 (PD-L1, also called B7-H1) can enhance T cell effector function when PD-L1 is expressed in chronically inflamed tissues and tumors. Here, we demonstrate that PD-L1 was upregulated in the tumor microenvironment after IR. Administration of anti–PD-L1 enhanced the efficacy of IR through a cytotoxic T cell–dependent mechanism. Concomitant with IR-mediated tumor regression, we observed that IR and anti–PD-L1 synergistically reduced the local accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs), which suppress T cells and alter the tumor immune microenvironment. Furthermore, activation of cytotoxic T cells with combination therapy mediated the reduction of MDSCs in tumors through the cytotoxic actions of TNF. Our data provide evidence for a close interaction between IR, T cells, and the PD-L1/PD-1 axis and establish a basis for the rational design of combination therapy with immune modulators and radiotherapy.     

Inhibiting the MNK1/2-eIF4E axis impairs melanoma phenotype switching and potentiates antitumor immune responses

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti–PD-1 immunotherapy. We showed that phospho-eIF4E–deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E–mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8⁺ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1⁺PD-1⁺CD8⁺ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti–PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti–PD-1 immunotherapy.     

JAML promotes CD8 and γδ T cell antitumor immunity and is a novel target for cancer immunotherapy

T cells are critical mediators of antitumor immunity and a major target for cancer immunotherapy. Antibody blockade of inhibitory receptors such as PD-1 can partially restore the activity of tumor-infiltrating lymphocytes (TILs). However, the activation signals required to promote TIL responses are less well characterized. Here we show that the antitumor activity of CD8 and γδ TIL is supported by interactions between junctional adhesion molecule–like protein (JAML) on T cells and its ligand coxsackie and adenovirus receptor (CXADR) within tumor tissue. Loss of JAML through knockout in mice resulted in accelerated tumor growth that was associated with an impaired γδ TIL response and increased CD8 TIL dysfunction. In mouse tumor models, therapeutic treatment with an agonistic anti-JAML antibody inhibited tumor growth, improved γδ TIL activation, decreased markers of CD8 TIL dysfunction, and significantly improved response to anti–PD-1 checkpoint blockade. Thus, JAML represents a novel therapeutic target to enhance both CD8 and γδ TIL immunity.     

Therapeutic Immunity by Adoptive Tumor-primed CD4+ T-cell Transfer in Combination With In Vivo GITR Ligation

Tumor-primed CD4⁺ T cells from splenocytes of tumor-rejection mice in combination with in vivo glucocorticoid-induced tumor necrosis factor receptor (GITR) ligation (the combination therapy) elicited effective host CD8⁺ T cell–dependent therapeutic immunity against a murine breast tumor. GITR ligation in vitro enhanced tumor-primed CD4⁺ T-cell activity and partially abrogated regulatory T cells (Treg) suppressor function. Dendritic cells (DCs) from tumor-draining lymph nodes (TDLNs) of tumor-bearing mice treated by the combination therapy stimulated Ag-specific T cells and produced interleukin (IL)-12 ex vivo. Whereas tumor-primed CD4⁺ T cells or in vivo GITR ligation alone induced a tumor-specific interferon (IFN)-γ-producing cellular response, the combination therapy enhanced and sustained it. Furthermore, the combination therapy in vivo attenuated Treg''s ability to suppress IL-12 production by DCs and IFN-γ production by effectors ex vivo. Importantly, tumor-primed CD4⁺ CD25⁻ T cells from splenocytes of untreated tumor-bearing mice in combination with in vivo GITR ligation also elicited an effective therapeutic effect in this model. These data suggest that the combination therapy may improve DC function, accentuate tumor-specific T-cell responses, and attenuate Treg suppressor function, thereby eliciting effective therapeutic immunity.     

Th9 cells promote antitumor immune responses in vivo

Th9 cells are a subset of CD4⁺ Th cells that produce the pleiotropic cytokine IL-9. IL-9/Th9 can function as both positive and negative regulators of immune response, but the role of IL-9/Th9 in tumor immunity is unknown. We examined the role of IL-9/Th9 in a model of pulmonary melanoma in mice. Lack of IL-9 enhanced tumor growth, while tumor-specific Th9 cell treatment promoted stronger antitumor responses in both prophylactic and therapeutic models. Th9 cells also elicited strong host antitumor CD8⁺ CTL responses by promoting Ccl20/Ccr6-dependent recruitment of DCs to the tumor tissues. Subsequent tumor antigen delivery to the draining LN resulted in CD8⁺ T cell priming. In agreement with this model, Ccr6 deficiency abrogated the Th9 cell–mediated antitumor response. Our data suggest a distinct role for tumor-specific Th9 cells in provoking CD8⁺ CTL-mediated antitumor immunity and indicate that Th9 cell–based cancer immunotherapy may be a promising therapeutic approach.     

CD73 has distinct roles in nonhematopoietic and hematopoietic cells to promote tumor growth in mice

CD73 is overexpressed in many types of human and mouse cancers and is implicated in the control of tumor progression. However, the specific contribution from tumor or host CD73 expression to tumor growth remains unknown to date. Here, we show that host CD73 promotes tumor growth in a T cell–dependent manner and that the optimal antitumor effect of CD73 blockade requires inhibiting both tumor and host CD73. Notably, enzymatic activity of CD73 on nonhematopoietic cells limited tumor-infiltrating T cells by controlling antitumor T cell homing to tumors in multiple mouse tumor models. In contrast, CD73 on hematopoietic cells (including CD4⁺CD25⁺ Tregs) inhibited systemic antitumor T cell expansion and effector functions. Thus, CD73 on hematopoietic and nonhematopoietic cells has distinct adenosinergic effects in regulating systemic and local antitumor T cell responses. Importantly, pharmacological blockade of CD73 using its selective inhibitor or an anti-CD73 mAb inhibited tumor growth and completely restored efficacy of adoptive T cell therapy in mice. These findings suggest that both tumor and host CD73 cooperatively protect tumors from incoming antitumor T cells and show the potential of targeting CD73 as a cancer immunotherapy strategy.     

Tim-3 mediates T cell trogocytosis to limit antitumor immunity

T cell immunoglobulin mucin domain-containing protein 3 (Tim-3) negatively regulates innate and adaptive immunity in cancer. To identify the mechanisms of Tim-3 in cancer immunity, we evaluated the effects of Tim-3 blockade in human and mouse melanoma. Here, we show that human programmed cell death 1–positive (PD-1⁺) Tim-3⁺CD8⁺ tumor-infiltrating lymphocytes (TILs) upregulate phosphatidylserine (PS), a receptor for Tim-3, and acquire cell surface myeloid markers from antigen-presenting cells (APCs) through transfer of membrane fragments called trogocytosis. Tim-3 blockade acted on Tim-3⁺ APCs in a PS-dependent fashion to disrupt the trogocytosis of activated tumor antigen–specific CD8⁺ T cells and PD-1⁺Tim-3⁺ CD8⁺ TILs isolated from patients with melanoma. Tim-3 and PD-1 blockades cooperated to disrupt trogocytosis of CD8⁺ TILs in 2 melanoma mouse models, decreasing tumor burden and prolonging survival. Deleting Tim-3 in dendritic cells but not in CD8⁺ T cells impeded the trogocytosis of CD8⁺ TILs in vivo. Trogocytosed CD8⁺ T cells presented tumor peptide–major histocompatibility complexes and became the target of fratricide T cell killing, which was reversed by Tim-3 blockade. Our findings have uncovered a mechanism Tim-3 uses to limit antitumor immunity.     

Therapeutic potential of 4-1BB (CD137) as a regulator for effector CD8(+) T cells

A fundamental problem of antitumor immunity is tumor-induced immunosuppression. Tumor cells often down-regulate expression of co-stimulatory molecules, tumor antigens, and major histocompatibility complex (MHC) molecules on tumor cells, secrete immunosuppressive substance such as transforming growth factor-beta (TGF-beta) or interleukin-4 (IL-4), and induce apoptosis of effector T cells to escape surveillance. A major goal of antitumor or antivirus immunotherapy is to generate long-lived protective T cells that enable killing of target cells. In this review, we discuss the importance of 4-1BB for development or survival of functionally active effector CD8(+) T cells against tumors, virus infection, and allogeneic immune responses and for potential therapeutic application.     

PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8+ T cells in mice

Viral vector–based vaccines that induce protective CD8⁺ T cell immunity can prevent or control pathogenic SIV infections, but issues of preexisting immunity and safety have impeded their implementation in HIV-1. Here, we report the development of what we believe to be a novel antigen-targeting DNA vaccine strategy that exploits the binding of programmed death-1 (PD1) to its ligands expressed on dendritic cells (DCs) by fusing soluble PD1 with HIV-1 GAG p24 antigen. As compared with non–DC-targeting vaccines, intramuscular immunization via electroporation (EP) of the fusion DNA in mice elicited consistently high frequencies of GAG-specific, broadly reactive, polyfunctional, long-lived, and cytotoxic CD8⁺ T cells and robust anti-GAG antibody titers. Vaccination conferred remarkable protection against mucosal challenge with vaccinia GAG viruses. Soluble PD1–based vaccination potentiated CD8⁺ T cell responses by enhancing antigen binding and uptake in DCs and activation in the draining lymph node. It also increased IL-12–producing DCs and engaged antigen cross-presentation when compared with anti-DEC205 antibody-mediated DC targeting. The high frequency of durable and protective GAG-specific CD8⁺ T cell immunity induced by soluble PD1–based vaccination suggests that PD1-based DNA vaccines could potentially be used against HIV-1 and other pathogens.     

TNF signaling drives myeloid-derived suppressor cell accumulation

TNF, an inflammatory cytokine that is enriched in the tumor microenvironment, promotes tumor growth and subverts innate immune responses to cancer cells. We previously reported that tumors implanted in TNF receptor–deficient (Tnfr^–/–) mice are spontaneously rejected; however, the molecular mechanisms underlying this rejection are unclear. Here we report that TNF signaling drives the peripheral accumulation of myeloid-derived suppressor cells (MDSCs). MDSCs expand extensively during inflammation and tumor progression in mice and humans and can enhance tumor growth by repressing T cell–mediated antitumor responses. Peripheral accumulation of MDSCs was drastically impaired in Tnfr^–/– mice. Signaling of TNFR-2, but not TNFR-1, promoted MDSC survival through upregulation of cellular FLICE-inhibitory protein (c-FLIP) and inhibition of caspase-8 activity. Loss of TNFRs impaired the induction of MDSCs from bone marrow cells, but this could be reversed by treatment with caspase inhibitors. These results demonstrate that TNFR-2 signaling promotes MDSC survival and accumulation and helps tumor cells evade the immune system.     

CHI3L1 regulates PD-L1 and anti–CHI3L1–PD-1 antibody elicits synergistic antitumor responses

Evasion of the immune response is a hallmark of cancer, and programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) are major mediators of this immunosuppression. Chitinase 3–like 1 (CHI3L1) is induced in many cancers, where it portends a poor prognosis and contributes to tumor metastasis and spread. However, the mechanism(s) that CHI3L1 uses in metastasis have not been defined. Here we demonstrate that CHI3L1 regulates the expression of PD-L1, PD-L2, PD-1, LAG3, and TIM3 and plays a critical role in melanoma progression and lymphatic spread. CHI3L1 also contributed to IFN-γ–stimulated macrophage PD-L1 expression, and RIG-like helicase innate immunity suppressed CHI3L1, PD-L1, and melanoma progression. Individual antibodies against CHI3L1 or PD-1 had discrete antitumor effects and additive antitumor responses in metastasis models and T cell–tumor cell cocultures when administered simultaneously. Synergistic cytotoxic tumor cell death was seen in T cell–tumor cell cocultures, and significantly enhanced antitumor responses were seen in in vivo tumor models treated with bispecific antibodies that simultaneously target CHI3L1 and PD-1. CHI3L1 contributes to tumor progression by stimulating the PD-1/PD-L1 axis and other checkpoint molecules. The simultaneous targeting of CHI3L1 and the PD-1/PD-L1 axis with individual and, more powerfully, with bispecific antibodies represents a promising therapy for pulmonary metastasis and progression.     

Identification of heme oxygenase-1–specific regulatory CD8+ T cells in cancer patients

Treg deficiencies are associated with autoimmunity. Conversely, CD4⁺ and CD8⁺ Tregs accumulate in the tumor microenvironment and are associated with prevention of antitumor immunity and anticancer immunotherapy. Recently, CD4⁺ Tregs have been much studied, but little is known about CD8⁺ Tregs and the antigens they recognize. Here, we describe what we believe to be the first natural target for CD8⁺ Tregs. Naturally occurring HLA-A2–restricted CD8⁺ T cells specific for the antiinflammatory molecule heme oxygenase-1 (HO-1) were able to suppress cellular immune responses with outstanding efficacy. HO-1–specific CD8⁺ T cells were detected ex vivo and in situ among T cells from cancer patients. HO-1–specific T cells isolated from the peripheral blood of cancer patients inhibited cytokine release, proliferation, and cytotoxicity of other immune cells. Notably, the inhibitory effect of HO-1–specific T cells was far more pronounced than that of conventional CD4⁺CD25⁺CD127^– Tregs. The inhibitory activity of HO-1–specific T cells seemed at least partly to be mediated by soluble factors. Our data link the cellular stress response to the regulation of adaptive immunity, expand the role of HO-1 in T cell–mediated immunoregulation, and establish a role for peptide-specific CD8⁺ T cells in regulating cellular immune responses. Identification of potent antigen-specific CD8⁺ Tregs may open new avenues for therapeutic interventions in both autoimmune diseases and cancer.