MicroRNA-155 is required for effector CD8+ T cell responses to virus infection and cancer.

MicroRNAs (miRNAs) regulate the function of several immune cells, but their role in promoting CD8(+) T cell immunity remains unknown. Here we report that miRNA-155 is required for CD8(+) T cell responses to both virus and cancer. In the absence of miRNA-155, accumulation of effector CD8(+) T cells was severely reduced during acute and chronic viral infections and control of virus replication was impaired. Similarly, Mir155(-/-) CD8(+) T cells were ineffective at controlling tumor growth, whereas miRNA-155 overexpression enhanced the antitumor response. miRNA-155 deficiency resulted in accumulation of suppressor of cytokine signaling-1 (SOCS-1) causing defective cytokine signaling through STAT5. Consistently, enforced expression of SOCS-1 in CD8(+) T cells phenocopied the miRNA-155 deficiency, whereas SOCS-1 silencing augmented tumor destruction. These findings identify miRNA-155 and its target SOCS-1 as key regulators of effector CD8(+) T cells that can be modulated to potentiate immunotherapies for infectious diseases and cancer.

PD-1 Blockade Unleashes Effector Potential of Both High- and Low-Affinity Tumor-Infiltrating T Cells.

Antitumor T cell responses involve CD8+ T cells with high affinity for mutated self-antigen and low affinity for nonmutated tumor-associated Ag. Because of the highly individual nature of nonsynonymous somatic mutations in tumors, however, immunotherapy relies often on an effective engagement of low-affinity T cells. In this study, we studied the role of T cell affinity during peripheral priming with single-peptide vaccines and during the effector phase in the tumor. To that end, we compared the antitumor responses after OVA257-264 (N4) peptide vaccination of CD8+ T cells carrying TCRs with high (OT-1) and low (OT-3) avidity for the N4 peptide in B16.N4 tumor-bearing C57BL/6 mice. Additionally, we assessed the response of OT-1 cells to either high-affinity (B16.N4) or low-affinity (B16.T4) Ag-expressing tumors after high-affinity (N4) or low-affinity (T4) peptide vaccination. We noticed that although low-affinity tumor-specific T cells expand less than high-affinity T cells, they express lower levels of inhibitory receptors and produce more cytokines. Interestingly, tumor-infiltrating CD8+ T cells show similar in vivo re-expansion capacity to their counterparts in secondary lymphoid organs when transferred to tumor-free hosts, suggesting that T cells in tumors may be rekindled upon relief of tumor immunosuppression. Moreover, our results show that αPD-1 treatment enhances tumor control of high- and low-affinity ligand-expressing tumors, suggesting that combination of high-affinity peripheral priming by altered peptide ligands and checkpoint blockade may enable tumor control upon low-affinity Ag recognition in the tumor.

Mammalian target of rapamycin complex 1 orchestrates invariant NKT cell differentiation and effector function.

Invariant NKT (iNKT) cells play critical roles in bridging innate and adaptive immunity. The Raptor containing mTOR complex 1 (mTORC1) has been well documented to control peripheral CD4 or CD8 T cell effector or memory differentiation. However, the role of mTORC1 in iNKT cell development and function remains largely unknown. By using mice with T cell-restricted deletion of Raptor, we show that mTORC1 is selectively required for iNKT but not for conventional T cell development. Indeed, Raptor-deficient iNKT cells are mostly blocked at thymic stage 1-2, resulting in a dramatic decrease of terminal differentiation into stage 3 and severe reduction of peripheral iNKT cells. Moreover, residual iNKT cells in Raptor knockout mice are impaired in their rapid cytokine production upon αGalcer challenge. Bone marrow chimera studies demonstrate that mTORC1 controls iNKT differentiation in a cell-intrinsic manner. Collectively, our data provide the genetic evidence that iNKT cell development and effector functions are under the control of mTORC1 signaling.

CD1d-antibody fusion proteins target iNKT cells to the tumor and trigger long-term therapeutic responses.

Despite the well-established antitumor activity of CD1d-restricted invariant natural killer T lymphocytes (iNKT), their use for cancer therapy has remained challenging. This appears to be due to their strong but short-lived activation followed by long-term anergy after a single administration of the CD1d agonist ligand alpha-galactosylceramide (αGC). As a promising alternative, we obtained sustained mouse iNKT cell responses associated with prolonged antitumor effects through repeated administrations of tumor-targeted recombinant sCD1d-antitumor scFv fusion proteins loaded with αGC. Here, we demonstrate that CD1d fusion proteins bound to tumor cells via the antibody fragment specific for a tumor-associated antigen, efficiently activate human iNKT cell lines leading to potent tumor cell lysis. The importance of CD1d tumor targeting was confirmed in tumor-bearing mice in which only the specific tumor-targeted CD1d fusion protein resulted in tumor inhibition of well-established aggressive tumor grafts. The therapeutic efficacy correlated with the repeated activation of iNKT and natural killer cells marked by their release of TH1 cytokines, despite the up-regulation of the co-inhibitory receptor PD-1. Our results demonstrate the superiority of providing the superagonist αGC loaded on recombinant CD1d proteins and support the use of αGC/sCD1d-antitumor fusion proteins to secure a sustained human and mouse iNKT cell activation, while targeting their cytotoxic activity and cytokine release to the tumor site.

Combination of lentivector immunization and low-dose chemotherapy or PD-1/PD-L1 blocking primes self-reactive T cells and induces anti-tumor immunity.

In the last two decades, anti-cancer vaccines have yielded disappointing clinical results despite the fact that high numbers of self/tumor-specific T cells can be elicited in immunized patients. Understanding the reasons behind this lack of efficacy is critical in order to design better treatment regimes. Recombinant lentivectors (rLVs) have been successfully used to induce antigen-specific T cells to foreign or mutated tumor antigens. Here, we show that rLV expressing a murine nonmutated self/tumor antigen efficiently primes large numbers of self/tumor-specific CD8(+) T cells. In spite of the large number of tumor-specific T cells, however, no anti-tumor activity could be measured in a therapeutic setting, in mice vaccinated with rLV. Accumulating evidence shows that, in the presence of malignancies, inhibition of T-cell activity may predominate overstimulation. Analysis of tumor-infiltrating lymphocytes revealed that specific anti-tumor CD8(+) T cells fail to produce cytokines and express high levels of inhibitory receptors such as programmed death (PD)-1. Association of active immunization with chemotherapy or antibodies that block inhibitory pathways often leads to better anti-tumor effects. We show here that combining rLV vaccination with either cyclophosphamide or PD-1 and PD-L1 blocking antibodies enhances rLV vaccination efficacy and improves anti-tumor immunity.

SITC 26th annual meeting--summary.

The 26th annual meeting of the Society for Immunotherapy of Cancer took place in Bethesda on November 4 to 6, 2011 and was organized by Charles G. Drake (Johns Hopkins University) Dolores J. Schendel (Helmholtz Zentrum Muenchen - German Research Center for Environmental Health Institute of Molecular Immunology), Jeffrey Schlom (National Cancer Institute, National Institutes of Health), and Jedd D. Wolchok (Memorial Sloan-Kettering Cancer Center). It was an event marked by a number of extraordinary circumstances: it attracted a record attendance of 805 participants from 24 different countries. The gathering came in the wake of great as well as very sad news for the tumor immunology community. Good news included the approval of anti-CTLA-4 as a therapy for metastatic melanoma in April and the announcement in early October of the Nobel Prize in Physiology and Medicine awarded to pioneering studies in the field of immunology. Indeed, one part of the prize went to Dr. Bruce Beutler, Scripps Research Institute, La Jolla, USA and Dr. Jules Hoffman, Institute for Molecular Cell Biology, Strasbourg, France, for their discoveries in innate immunity and the other part to Dr. Ralph Steinman, The Rockfeller University, New York, for his discovery of dendritic cells. Sad news was the losses of two giants in the field. Jürg Tschopp of the University of Lausanne in March and Ralph Steinman, who passed away just three days before his Nobel Prize announcement. The loss of these two charismatic scientific leaders was particularly sad for the Annual Meeting as both J. Tschopp and R. Steinman were confirmed speakers at this meeting: the former to deliver the keynote lecture and the latter as recipient of the Richard V. Smalley prize.

The mitochondrial pyruvate carrier regulates memory T cell differentiation and antitumor function.

Glycolysis, including both lactate fermentation and pyruvate oxidation, orchestrates CD8+ T cell differentiation. However, how mitochondrial pyruvate metabolism and uptake controlled by the mitochondrial pyruvate carrier (MPC) impact T cell function and fate remains elusive. We found that genetic deletion of MPC drives CD8+ T cell differentiation toward a memory phenotype. Metabolic flexibility induced by MPC inhibition facilitated acetyl-coenzyme-A production by glutamine and fatty acid oxidation that results in enhanced histone acetylation and chromatin accessibility on pro-memory genes. However, in the tumor microenvironment, MPC is essential for sustaining lactate oxidation to support CD8+ T cell antitumor function. We further revealed that chimeric antigen receptor (CAR) T cell manufacturing with an MPC inhibitor imprinted a memory phenotype and demonstrated that infusing MPC inhibitor-conditioned CAR T cells resulted in superior and long-lasting antitumor activity. Altogether, we uncover that mitochondrial pyruvate uptake instructs metabolic flexibility for guiding T cell differentiation and antitumor responses.

Sustained activation and tumor targeting of NKT cells using a CD1d-anti-HER2-scFv fusion protein induce antitumor effects in mice.

Invariant NKT (iNKT) cells are potent activators of DCs, NK cells, and T cells, and their antitumor activity has been well demonstrated. A single injection of the high-affinity CD1d ligand alpha-galactosylceramide (alphaGalCer) leads to short-lived iNKT cell activation followed, however, by long-term anergy, limiting its therapeutic use. In contrast, we demonstrated here that when alphaGalCer was loaded on a recombinant soluble CD1d molecule (alphaGalCer/sCD1d), repeated injections led to sustained iNKT and NK cell activation associated with IFN-gamma secretion as well as DC maturation in mice. Most importantly, when alphaGalCer/sCD1d was fused to a HER2-specific scFv antibody fragment, potent inhibition of experimental lung metastasis and established s.c. tumors was obtained when systemic treatment was started 2-7 days after the injection of HER2-expressing B16 melanoma cells. In contrast, administration of free alphaGalCer at this time had no effect. The antitumor activity of the CD1d-anti-HER2 fusion protein was associated with HER2-specific tumor localization and accumulation of iNKT, NK, and T cells at the tumor site. Targeting iNKT cells to the tumor site thus may activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy.

Redirecting iNKT Cell Antitumor Immunity with α-GalCer/CD1d-scFv Fusion Proteins.

Invariant natural killer T (iNKT) cells display important properties that could bridge the innate and adaptive immunity, and they have been shown to play key roles in cancer immunotherapy. However, administration of iNKT cell agonist αGalCer fails to induce sustained antitumor immunity due to the rapid anergy induction after an initial strong activation. To this end, we have designed a recombinant CD1d protein that is fused to an anti-TAA scFv, which is able to recruit iNKT cells to the tumor site and induce tumor regression. Importantly, recombinant CD1d fusion proteins loaded with α-GalCer demonstrated sustained activation of iNKT cells upon repeated injections and superior tumor control, as compared to α-GalCer treatment.

Enforced PGC-1α expression promotes CD8 T cell fitness, memory formation and antitumor immunity.

Memory CD8 T cells can provide long-term protection against tumors, which depends on their enhanced proliferative capacity, self-renewal and unique metabolic rewiring to sustain cellular fitness. Specifically, memory CD8 T cells engage oxidative phosphorylation and fatty acid oxidation to fulfill their metabolic demands. In contrast, tumor-infiltrating lymphocytes (TILs) display severe metabolic defects, which may underlie their functional decline. Here, we show that overexpression of proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the master regulator of mitochondrial biogenesis (MB), favors CD8 T cell central memory formation rather than resident memory generation. PGC-1α-overexpressing CD8 T cells persist and mediate more robust recall responses to bacterial infection or peptide vaccination. Importantly, CD8 T cells with enhanced PGC-1α expression provide stronger antitumor immunity in a mouse melanoma model. Moreover, TILs overexpressing PGC-1α maintain higher mitochondrial activity and improved expansion when rechallenged in a tumor-free host. Altogether, our findings indicate that enforcing mitochondrial biogenesis promotes CD8 T cell memory formation, metabolic fitness, and antitumor immunity in vivo.

Serial Stimulation of Invariant Natural Killer T Cells with Covalently Stabilized Bispecific T-cell Engagers Generates Antitumor Immunity While Avoiding Anergy.

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. SIGNIFICANCE: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.

Recombinant fusion proteins for targeting dendritic cell subsets in therapeutic cancer vaccine.

Dendritic cells (DCs) are professional antigen-presenting cells, which are optimal for the priming of a T cell response against pathogens and tumors. Therefore, many efforts are made to develop therapeutic cancer vaccines which preferentially target the antigen to DC subsets. To this aim, we developed two types of recombinant fusion proteins, which favor antigen delivery to pro-inflammatory DCs as well as the crosstalk between specialized subpopulations of DCs. The first approach combines peptide/CpG vaccination with the recruitment of iNKT cells to the tumor site via CD1d-antitumor scFv fusion proteins. The second approach is targeting the tumor antigen to cross-presenting Xcr1+ DCs via a fusion protein made of Xcl1 fused to a synthetic long peptide followed by an IgG1 Fc fragment. Both strategies allow a potent tumor-specific CD8 T cell response associated with tumor regression or tumor growth delay depending on the model. In the case of iNKT cell activation, the strategy relies on a strong IL-12 release by splenic DCs, while in the second case, the T cell response is strictly dependent on the presence of Xcr1+ cross-presenting DCs.

An Immunomodulatory Gallotanin-Rich Fraction From Caesalpinia spinosa Enhances the Therapeutic Effect of Anti-PD-L1 in Melanoma.

PD-1/PD-L1 pathway plays a role in inhibiting immune response. Therapeutic antibodies aimed at blocking the PD-1/PD-L1 interaction have entered clinical development and have been approved for a variety of cancers. However, the clinical benefits are reduced to a group of patients. The research in combined therapies, which allow for a greater response, is strongly encouraging. We previously characterized a polyphenol-rich extract from Caesalpinia spinosa (P2Et) with antitumor activity in both melanoma and breast carcinoma, as well as immunomodulatory activity. We hypothesize that the combined treatment with P2Et and anti-PD-L1 can improve the antitumor response through an additive antitumor effect. We investigated the antitumor and immunomodulatory activity of P2Et and anti-PD-L1 combined therapy in B16-F10 melanoma and 4T1 breast carcinoma. We analyzed tumor growth, hematologic parameters, T cell counts, cytokine expression, and T cell cytotoxicity. In the melanoma model, combined P2Et and anti-PD-L1 therapy has the following effects: decrease in tumor size; increase in the number of activated CD4+ and CD8+ T cells; decrease in the number of suppressor myeloid cells; increase in PD-L1 expression; decrease in the frequency of CD8+ T cell expressing PD-1; improvement in the cytotoxic activity of T cells; and increase in the IFN γ secretion. In the breast cancer model, P2Et and PD-L1 alone or in combination show antitumor effect with no clear additive effect. This study shows that combined therapy of P2Et and anti-PD-L1 can improve antitumor response in a melanoma model by activating the immune response and neutralizing immunosuppressive mechanisms.

miR-155 Overexpression in OT-1 CD8+ T Cells Improves Anti-Tumor Activity against Low-Affinity Tumor Antigen.

Therapy by adoptive transfer of ex vivo-expanded tumor-infiltrating or genetically modified T cells may lead to impressive clinical responses. However, there is a need to improve in vivo persistence and functionality of the transferred T cells, in particular, to face the highly immunosuppressive environment of solid tumors. Here, we investigate the potential of miR-155, a microRNA known to play an important role in CD8+ T cell fitness. We show that forced expression of miR-155 in tumor antigen-specific T cells improves the tumor control of B16 tumors expressing a low-affinity antigen ligand. Importantly, miR-155-transduced T cells exhibit increased proliferation and effector functions associated with a higher glycolytic activity independent of exogenous glucose. Altogether, these data suggest that miR-155 may optimize the antitumor activity of adoptively transferred low-affinity tumor-infiltrating lymphocytes (TILs), in particular, by rendering them more resistant to the glucose-deprived environment of solid tumors. Thus, transgenic expression of miR-155 may enable therapeutic targeting of self-antigen-specific T cells in addition to neoantigen-specific ones.

Combination of Synthetic Long Peptides and XCL1 Fusion Proteins Results in Superior Tumor Control.

Cross-presenting Xcr1+CD8α DCs are attractive APCs to target for therapeutic cancer vaccines, as they are able to take up and process antigen from dying tumor cells for their MHCI-restricted presentation to CD8 T cells. To this aim, we developed fusion proteins made of the Xcr1 ligand Xcl1 fused to an OVA synthetic long peptide (SLP) and IgG1 Fc fragment. We demonstrated the specific binding and uptake of the Xcl1 fusion proteins by Xcr1+ DCs. Most importantly, their potent adjuvant effect on the H-2Kb/OVA specific T cell response was associated with a sustained tumor control even against the poorly immunogenic B16-OVA melanoma tumor. The increased tumor protection correlated with higher tumor infiltration of antigen-specific CD8+ T cells, increased IFNγ production and degranulation potential. Altogether, these results demonstrate that therapeutic cancer vaccines may be greatly improved by the combination of SLP antigen and Xcl1 fusion proteins.

MicroRNA-155 Expression Is Enhanced by T-cell Receptor Stimulation Strength and Correlates with Improved Tumor Control in Melanoma.

microRNAs are short noncoding RNAs that regulate protein expression posttranscriptionally. We previously showed that miR-155 promotes effector CD8+ T-cell responses. However, little is known about the regulation of miR-155 expression. Here, we report that antigen affinity and dose determine miR-155 expression in CD8+ T cells. In B16 tumors expressing a low-affinity antigen ligand, tumor-specific infiltrating CD8+ T cells showed variable miR-155 expression, whereby high miR-155 expression was associated with more cytokine-producing cells and tumor control. Moreover, anti-PD-1 treatment led to both increased miR-155 expression and tumor control by specific CD8+ T cells. In addition, miR-155 overexpression enhanced exhausted CD8+ T-cell persistence in the LCMV cl13 chronic viral infection model. In agreement with these observations in mouse models, miR-155 expression in human effector memory CD8+ T cells positively correlated with their frequencies in tumor-infiltrated lymph nodes of melanoma patients. Low miR-155 target gene signature in tumors was associated with prolonged overall survival in melanoma patients. Altogether, these results raise the possibility that high miR-155 expression in CD8+ tumor-infiltrating T cells may be a surrogate marker of the relative potency of in situ antigen-specific CD8+ T-cell responses.

Lymphocyte-Derived Exosomal MicroRNAs Promote Pancreatic ß Cell Death and May Contribute to Type 1 Diabetes Development.

Type 1 diabetes is an autoimmune disease initiated by the invasion of pancreatic islets by immune cells that selectively kill the ß cells. We found that rodent and human T lymphocytes release exosomes containing the microRNAs (miRNAs) miR-142-3p, miR-142-5p, and miR-155, which can be transferred in active form to ß cells favoring apoptosis. Inactivation of these miRNAs in recipient ß cells prevents exosome-mediated apoptosis and protects non-obese diabetic (NOD) mice from diabetes development. Islets from protected NOD mice display higher insulin levels, lower insulitis scores, and reduced inflammation. Looking at the mechanisms underlying exosome action, we found that T lymphocyte exosomes trigger apoptosis and the expression of genes involved in chemokine signaling, including Ccl2, Ccl7, and Cxcl10, exclusively in ß cells. The induction of these genes may promote the recruitment of immune cells and exacerbate ß cell death during the autoimmune attack. Our data point to exosomal-miRNA transfer as a communication mode between immune and insulin-secreting cells.

Prophylactic vs. Therapeutic Treatment With P2Et Polyphenol-Rich Extract Has Opposite Effects on Tumor Growth.

Polyphenols have tumoricidal effects via anti-proliferative, anti-angiogenic and cytotoxic mechanisms and have recently been demonstrated to modulate the immune response through their anti- or pro- oxidant activity. Nevertheless, it remains controversial whether antioxidant-rich supplements have real beneficial effects on health, especially in complex diseases such as cancer. We previously identified a polyphenol-rich extract obtained from Caesalpinia spinosa (P2Et) with anti-tumor activity in both breast carcinoma and melanoma. The present work evaluated the ability of P2Et extract to modulate the immune system in either the steady state or following tumor challenge. We found that the prophylactic treatment of healthy mice increased the number of CD4+ and CD8+ activated T, NK, regulatory T, dendritic and myeloid-derived suppressor cells in lymphoid organs together with a significant increase in plasma IL-6. Interestingly, this pre-conditioning of the host immune system with P2Et did not involve a protective effect against the control of tumor growth and metastasis in transplantable models of melanoma (B16) and breast cancer (4T1), but in contrast, a detrimental effect was observed in both models. We further demonstrated that this effect was at least partly due to an increase in regulatory T cells, myeloid-derived suppressor cells, and proinflammatory cytokines, with a concomitant decrease in CD4+ and CD8+ T cells. Taken together, these results suggest that the anti-tumor and immunomodulation properties of the P2Et extract critically depend on the presence of the tumor and might be mediated by the complex interactions between the tumor cells and the other components of the tumor microenvironment.

CART cells are prone to Fas- and DR5-mediated cell death.

Adoptive transfer of T cells transduced with Chimeric Antigen Receptors (CAR) are now FDA-approved for the treatment of B-cell malignancies. Yet, the functionality of the endogenous TCR in CART cells has not been fully assessed. Here, we demonstrate that CART cells progressively upregulate Fas, FasL, DR5 and TRAIL, which result in their programmed cell death, independently of antigen-mediated TCR or CAR activation. CART cell apoptosis occurs even when the CAR contains a single (co-)activatory domain such as CD3ζ, CD28 or 4-1BB. Importantly, the dominant role of the Fas and DR5 pathways in CART cell apoptosis is demonstrated by the significant rescue of CART cells upon in vivo blockade by combined Fas-Fc and DR5-Fc recombinant proteins. These observations are of crucial importance for the long-term persistence of CART cells and for the development of new applications including the combined TCR and CAR activation against solid tumors.

Correction to: CART cells are prone to Fas- and DR5-mediated cell death.

After publication of this article [1], it was noticed that 3 authors were missed from the author list.