The adjuvant effect of melanin is superior to incomplete Freund's adjuvant in subunit/peptide vaccines in mice.

Peptide vaccines represent an attractive alternative to conventional anti-tumor therapies, but have not yet achieved significant clinical efficacy with commonly used formulations. Combination of short antigenic peptides, synthetic melanin and TLR9 agonist (Toll-like receptor 9, CpG-28) was reported as highly efficient to trigger strong CD8 + T-cell responses. We compared this vaccine approach to the standard adjuvant formulation that combines the incomplete Freund's adjuvant (IFA) and CpG-28, using either an ovalbumin epitope (pOVA30) or a spontaneously occurring tumor neoepitope (mAdpgk).Melanin-based vaccine induced significantly higher cytotoxic T lymphocytes (CTL) responses than IFA-based vaccine in both pOVA30- and mAdpgk-targeted vaccines. The anti-tumor efficacy of melanin-based vaccine was further assessed in mice, grafted either with E.G7-OVA cells (E.G7 cells transfected with ovalbumin) or with MC38 cells that spontaneously express the mAdpgk neoepitope. Melanin-based vaccine induced a major inhibition of E.G7-OVA tumor growth when compared to IFA-based vaccine (p < 0.001), but tumors eventually relapsed from day 24. In the MC38 tumor model, no significant inhibition of tumor growth was observed. In both cases, tumor escape appeared related to the loss of antigen presentation by tumor cells (loss of ovalbumin expression in E.G7-OVA model; poor presentation of mAdpgk in MC38 model), although the CTL responses displayed an effector memory phenotype, a high cytolytic potential and low programmed cell death-1 (PD1) expression.In conclusion, synthetic melanin can be efficiently used as an adjuvant to enhance T-cells response against subunit vaccine antigens and compared favorably to the classic combination of IFA and TLR9 agonist in mice.

[Immune system and tumors]. / Système immunitaire et tumeurs.

Despite having been much debated, it is now well established that the immune system plays an essential role in the fight against cancer. In this article, we will highlight the implication of the immune system in the control of tumor growth and describe the major components of the immune system involved in the antitumoral immune response. The immune system, while exerting pressure on tumor cells, also will play a pro-tumoral role by sculpting the immunogenicity of tumors cells as they develop. Finally, we will illustrate the numerous mechanisms of immune suppression that take place within the tumoral microenvironment which allow tumor cells to escape control from the immune system. The increasingly precise knowledge of the brakes to an effective antitumor immune response allows the development of immunotherapy strategies more and more innovating and promising of hope.

A CCR4 antagonist combined with vaccines induces antigen-specific CD8+ T cells and tumor immunity against self antigens.

Regulatory T cells (Tregs) may impede cancer vaccine efficacy in hematologic malignancies and cancer. CCR4 antagonists, an emergent class of Treg inhibitor, have been shown to block recruitment of Tregs mediated by CCL22 and CCL17. Our aim was to demonstrate the ability of a CCR4 antagonist (a small chemical molecule identified in silico) when combined with vaccines to break peripheral tolerance controlled by Tregs, a prerequisite for the induction of CD8(+) T cells against self Ags. Immunization of transgenic or normal mice expressing tumor-associated self Ags (Her2/neu, OVA, gp100) with a CCR4 antagonist combined with various vaccines led to the induction of effector CD8(+) T cells and partial inhibition of tumor growth expressing self Ags in both prophylactic and therapeutic settings. The CCR4 antagonist was more efficient than cyclophosphamide to elicit anti-self CD8(+) T cells. We also showed that the population of Tregs expressing CCR4 corresponded to memory (CD44(high)) and activated (ICOS(+)) Tregs, an important population to be targeted to modulate Treg activity. CCR4 antagonist represents a competitive class of Treg inhibitor able to induce functional anti-self CD8(+) T cells and tumor growth inhibition when combined with vaccines. High expression of CCR4 on human Tregs also supports the clinical development of this strategy.

Modulation of immunity by antiangiogenic molecules in cancer.

In the last decades a new class of therapeutic drugs have been developed that block tumor angiogenesis. These antiangiogenic molecules, which target VEGF or VEGFR, PDGFR, and c-kit, can act not only on endothelial cells but also on immune cells. Some antiangiogenic molecules inhibit the development of immunosuppressive mechanisms developed by the tumors to escape the immune system (such as regulatory T cells, myeloid-derived suppressor cells, and immunosuppressive cytokines). These immunomodulatory effects must be characterized in detail to enable a better prescription of these treatments. In this paper we will focus on the impact of anti-angiogenic drugs on immunosuppression and their potential combination with immunotherapeutic strategies. Interestingly, immune parameters or their modulation during treatment could serve as potential biomarkers of response or resistance to anti-angiogenic therapies.

Synergistic effect of combining sunitinib with a peptide-based vaccine in cancer treatment after microenvironment remodeling.

Although it has proven difficult to demonstrate the clinical efficacy of therapeutic vaccination as a monotherapy in advanced cancers, its combination with an immunomodulatory treatment to reduce intra-tumor immunosuppression and improve vaccine efficacy is a very promising strategy. In this context, we are studying the combination of a vaccine composed of peptides of the tumor antigen survivin (SVX vaccine) with the anti-angiogenic agent sunitinib in a colorectal carcinoma model. To this end, we have been focusing on administration scheduling and have highlighted a therapeutic synergy between SVX vaccine and sunitinib when the vaccine was administered at the end of anti-angiogenic treatment. In this setting, a prolonged control of tumor growth associated with an important percentage of complete tumor regression was observed. Studying the remodeling induced by each therapy on the immunological and angiogenic tumor microenvironment over time we observed, during sunitinib treatment, a transient increase in polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and a decrease in NK cells in the tumor microenvironment. In contrast, after sunitinib treatment was stopped, a decrease in PMN-MDSC populations has been observed in the tumor, associated with an increase in NK cells, pericyte coverage of tumor vessels and CD8+ T cell population and functionality. In conclusion, sunitinib treatment results in the promotion of an immune-favorable tumor microenvironment that can guide the optimal sequence of vaccine and anti-angiogenic combination to reinforce their synergy.

Mild dyslipidemia accelerates tumorigenesis through expansion of Ly6Chi monocytes and differentiation to pro-angiogenic myeloid cells.

Cancer and cardiovascular disease (CVD) share common risk factors such as dyslipidemia, obesity and inflammation. However, the role of pro-atherogenic environment and its associated low-grade inflammation in tumor progression remains underexplored. Here we show that feeding C57BL/6J mice with a non-obesogenic high fat high cholesterol diet (HFHCD) for two weeks to induce mild dyslipidemia, increases the pool of circulating Ly6Chi monocytes available for initial melanoma development, in an IL-1ß-dependent manner. Descendants of circulating myeloid cells, which accumulate in the tumor microenvironment of mice under HFHCD, heighten pro-angiogenic and immunosuppressive activities locally. Limiting myeloid cell accumulation or targeting VEGF-A production by myeloid cells decrease HFHCD-induced tumor growth acceleration. Reverting the HFHCD to a chow diet at the time of tumor implantation protects against tumor growth. Together, these data shed light on cross-disease communication between cardiovascular pathologies and cancer.

Vaccination-based immunotherapy to target profibrotic cells in liver and lung.

Fibrosis is the final path of nearly every form of chronic disease, regardless of the pathogenesis. Upon chronic injury, activated, fibrogenic fibroblasts deposit excess extracellular matrix, and severe tissue fibrosis can occur in virtually any organ. However, antifibrotic therapies that target fibrogenic cells, while sparing homeostatic fibroblasts in healthy tissues, are limited. We tested whether specific immunization against endogenous proteins, strongly expressed in fibrogenic cells but highly restricted in quiescent fibroblasts, can elicit an antigen-specific cytotoxic T cell response to ameliorate organ fibrosis. In silico epitope prediction revealed that activation of the genes Adam12 and Gli1 in profibrotic cells and the resulting "self-peptides" can be exploited for T cell vaccines to ablate fibrogenic cells. We demonstrate the efficacy of a vaccination approach to mount CD8+ T cell responses that reduce fibroblasts and fibrosis in the liver and lungs in mice. These results provide proof of principle for vaccination-based immunotherapies to treat fibrosis.

Increased CD127 expression on activated FOXP3+CD4+ regulatory T cells.

Regulatory T cells (Treg) are commonly identified by CD25 (IL-2R alpha) surface expression and/or intracellular expression of the FOXP3 transcription factor. In addition, Treg are also characterized by low CD127 (IL-7R alpha) expression when compared to conventional T cells and their biology in the periphery is considered essentially independent of IL-7. We further investigated CD127 expression on Treg and we demonstrated differential CD127 expression depending on Treg subsets considered. Notably, we observed high CD127 expression on inducible costimulatory molecule (ICOS)- and CD103-expressing Treg subsets. Since these two markers reflect activation status, we addressed whether Treg activation modulated CD127 expression. We demonstrated that in contrast to conventional T cells, Treg significantly upregulated CD127 expression during in vitro and in vivo activation using adoptive transfer and contact dermatitis models. High CD127 expression on Treg was also predominantly detected ex vivo in some specific sites, notably bone marrow and skin. Importantly, higher CD127 expression on Treg correlated with higher phosphorylation of STAT5 upon IL-7 exposure. High CD127 expression on Treg also provided survival advantage upon in vitro incubation with IL-7. We thus demonstrated that low CD127 expression is not an intrinsic characteristic of Treg and we identified activated Treg as a potential target of endogenous or therapeutic IL-7.

Targeting HGF/c-Met Axis Decreases Circulating Regulatory T Cells Accumulation in Gastric Cancer Patients.

Elucidating mechanisms involved in tumor-induced immunosuppression is of great interest since it could help to improve cancer immunotherapy efficacy. Here we show that Hepatocyte Growth Factor (HGF), a pro-tumoral and proangiogenic factor, and its receptor c-Met are involved in regulatory T cells (Treg) accumulation in the peripheral blood of gastric cancer (GC) patients. We observed that c-Met is expressed on circulating monocytes from GC patients. The elevated expression on monocytes is associated with clinical parameters linked to an aggressive disease phenotype and correlates with a worse prognosis. Monocyte-derived dendritic cells from GC patients differentiated in the presence of HGF adopt a regulatory phenotype with a lower expression of co-stimulatory molecules, impaired maturation capacities, and an increased ability to produce interleukin-10 and to induce Treg differentiation in vitro. In the MEGA-ACCORD20-PRODIGE17 trial, GC patients received an anti-HGF antibody treatment (rilotumumab), which had been described to have an anti-angiogenic activity by decreasing proliferation of endothelial cells and tube formation. Rilotumumab decreased circulating Treg in GC patients. Thus, we identified that HGF indirectly triggers Treg accumulation via c-Met-expressing monocytes in the peripheral blood of GC patients. Our study provides arguments for potential alternative use of HGF/c-Met targeted therapies based on their immunomodulatory properties which could lead to the development of new therapeutic associations in cancer patients, for example with immune checkpoint inhibitors.

CXCR6 deficiency impairs cancer vaccine efficacy and CD8+ resident memory T-cell recruitment in head and neck and lung tumors.

BACKGROUND: Resident memory T lymphocytes (TRM) are located in tissues and play an important role in immunosurveillance against tumors. The presence of TRM prior to treatment or their induction is associated to the response to anti-Programmed cell death protein 1 (PD-1)/Programmed death-ligand 1 (PD-L1) immunotherapy and the efficacy of cancer vaccines. Previous work by our group and others has shown that the intranasal route of vaccination allows more efficient induction of these cells in head and neck and lung mucosa, resulting in better tumor protection. The mechanisms of in vivo migration of these cells remains largely unknown, apart from the fact that they express the chemokine receptor CXCR6. METHODS: We used CXCR6-deficient mice and an intranasal tumor vaccination model targeting the Human Papillomavirus (HPV) E7 protein expressed by the TC-1 lung cancer epithelial cell line. The role of CXCR6 and its ligand, CXCL16, was analyzed using multiparametric cytometric techniques and Luminex assays.Human biopsies obtained from patients with lung cancer were also included in this study. RESULTS: We showed that CXCR6 was preferentially expressed by CD8+ TRM after vaccination in mice and also on intratumoral CD8+ TRM derived from human lung cancer. We also demonstrate that vaccination of Cxcr6-deficient mice induces a defect in the lung recruitment of antigen-specific CD8+ T cells, preferentially in the TRM subsets. In addition, we found that intranasal vaccination with a cancer vaccine is less effective in these Cxcr6-deficient mice compared with wild-type mice, and this loss of efficacy is associated with decreased recruitment of local antitumor CD8+ TRM. Interestingly, intranasal, but not intramuscular vaccination induced higher and more sustained concentrations of CXCL16, compared with other chemokines, in the bronchoalveolar lavage fluid and pulmonary parenchyma. CONCLUSIONS: This work demonstrates the in vivo role of CXCR6-CXCL16 axis in the migration of CD8+ resident memory T cells in lung mucosa after vaccination, resulting in the control of tumor growth. This work reinforces and explains why the intranasal route of vaccination is the most appropriate strategy for inducing these cells in the head and neck and pulmonary mucosa, which remains a major objective to overcome resistance to anti-PD-1/PD-L1, especially in cold tumors.

Decrease of Pro-Angiogenic Monocytes Predicts Clinical Response to Anti-Angiogenic Treatment in Patients with Metastatic Renal Cell Carcinoma.

The modulation of subpopulations of pro-angiogenic monocytes (VEGFR-1+CD14 and Tie2+CD14) was analyzed in an ancillary study from the prospective PazopanIb versus Sunitinib patient preferenCE Study (PISCES) (NCT01064310), where metastatic renal cell carcinoma (mRCC) patients were treated with two anti-angiogenic drugs, either sunitinib or pazopanib. Blood samples from 86 patients were collected prospectively at baseline (T1), and at 10 weeks (T2) and 20 weeks (T3) after starting anti-angiogenic therapy. Various subpopulations of myeloid cells (monocytes, VEGFR-1+CD14 and Tie2+CD14 cells) decreased during treatment. When patients were divided into two subgroups with a decrease (defined as a >20% reduction from baseline value) (group 1) or not (group 2) at T3 for VEGFR-1+CD14 cells, group 1 patients presented a median PFS and OS of 24 months and 37 months, respectively, compared with a median PFS of 9 months (p = 0.032) and a median OS of 16 months (p = 0.033) in group 2 patients. The reduction in Tie2+CD14 at T3 predicted a benefit in OS at 18 months after therapy (p = 0.04). In conclusion, in this prospective clinical trial, a significant decrease in subpopulations of pro-angiogenic monocytes was associated with clinical response to anti-angiogenic drugs in patients with mRCC.

Cytotoxic CD8+ T cells promote granzyme B-dependent adverse post-ischemic cardiac remodeling.

Acute myocardial infarction is a common condition responsible for heart failure and sudden death. Here, we show that following acute myocardial infarction in mice, CD8+ T lymphocytes are recruited and activated in the ischemic heart tissue and release Granzyme B, leading to cardiomyocyte apoptosis, adverse ventricular remodeling and deterioration of myocardial function. Depletion of CD8+ T lymphocytes decreases apoptosis within the ischemic myocardium, hampers inflammatory response, limits myocardial injury and improves heart function. These effects are recapitulated in mice with Granzyme B-deficient CD8+ T cells. The protective effect of CD8 depletion on heart function is confirmed by using a model of ischemia/reperfusion in pigs. Finally, we reveal that elevated circulating levels of GRANZYME B in patients with acute myocardial infarction predict increased risk of death at 1-year follow-up. Our work unravels a deleterious role of CD8+ T lymphocytes following acute ischemia, and suggests potential therapeutic strategies targeting pathogenic CD8+ T lymphocytes in the setting of acute myocardial infarction.

Therapeutic Cancer Vaccine and Combinations With Antiangiogenic Therapies and Immune Checkpoint Blockade.

Considering the high importance of immune surveillance and immune escape in the evolution of cancer, the development of immunotherapeutic strategies has become a major field of research in recent decades. The considerable therapeutic breakthrough observed when targeting inhibitory immune checkpoint molecules has highlighted the need to find approaches enabling the induction and proper activation of an immune response against cancer. In this context, therapeutic vaccination, which can induce a specific immune response against tumor antigens, is an important approach to consider. However, this strategy has its advantages and limits. Considering its low clinical efficacy, approaches combining therapeutic cancer vaccine strategies with other immunotherapies or targeted therapies have been emphasized. This review will list different cancer vaccines, with an emphasis on their targets. We highlight the results and limits of vaccine strategies and then describe strategies that combine therapeutic vaccines and antiangiogenic therapies or immune checkpoint blockade. Antiangiogenic therapies and immune checkpoint blockade are of proven clinical efficacy for some indications, but are limited by toxicity and the development of resistance. Their combination with therapeutic vaccines could be a way to improve therapeutic outcome by specifically stimulating the immune system and considering a global approach to tumor microenvironment remodeling.

High Therapeutic Efficacy of a New Survivin LSP-Cancer Vaccine Containing CD4+ and CD8+ T-Cell Epitopes.

The efficacy of an antitumoral vaccine relies both on the choice of the antigen targeted and on its design. The tumor antigen survivin is an attractive target to develop therapeutic cancer vaccines because of its restricted over-expression and vital functions in most human tumors. Accordingly, several clinical trials targeting survivin in various cancer indications have been conducted. Most of them relied on short peptide-based vaccines and showed promising, but limited clinical results. In this study, we investigated the immunogenicity and therapeutic efficacy of a new long synthetic peptide (LSP)-based cancer vaccine targeting the tumor antigen survivin (SVX). This SVX vaccine is composed of three long synthetic peptides containing several CD4+ and CD8+ T-cell epitopes, which bind to various HLA class II and class I molecules. Studies in healthy individuals showed CD4+ and CD8+ T-cell immunogenicity of SVX peptides in human, irrespective of the individual's HLA types. Importantly, high frequencies of spontaneous T-cell precursors specific to SVX peptides were also detected in the blood of various cancer patients, demonstrating the absence of tolerance against these peptides. We then demonstrated SVX vaccine's high therapeutic efficacy against four different established murine tumor models, associated with its capacity to generate both specific cytotoxic CD8+ and multifunctional Th1 CD4+ T-cell responses. When tumors were eradicated, generated memory T-cell responses protected against rechallenge allowing long-term protection against relapses. Treatment with SVX vaccine was also found to reshape the tumor microenvironment by increasing the tumor infiltration of both CD4+ and CD8+ T cells but not Treg cells therefore tipping the balance toward a highly efficient immune response. These results highlight that this LSP-based SVX vaccine appears as a promising cancer vaccine and warrants its further clinical development.

Towards a cellular definition of CD8+ T-cell memory: the role of CD4+ T-cell help in CD8+ T-cell responses.

Whereas the definition of B-cell memory is based on well-known cellular properties and differentiation steps, the process of T-cell memory generation was, until recently, less well understood. A series of recent reports, however, have drastically modified our notion of CD8(+) memory T cells. They show that, in addition to division, the generation of efficient memory cells requires a previously unknown differentiation process. As a whole, the generation of CD8(+) memory T cells appears to mimic the generation of memory B cells. Both processes depend on the help of CD4(+) T cells, they are irreversible, they have the same mechanism, and they occur progressively during the late expansion phase of the primary immune response.

CD4/CD8/Dendritic cell complexes in the spleen: CD8+ T cells can directly bind CD4+ T cells and modulate their response.

CD4+ T cell help to CD8+ T cell responses requires that CD4+ and CD8+ T cells interact with the same antigen presenting dendritic cell (Ag+DC), but it remains controversial whether helper signals are delivered indirectly through a licensed DC and/or involve direct CD4+/CD8+ T cell contacts and/or the formation of ternary complexes. We here describe the first in vivo imaging of the intact spleen, aiming to evaluate the first interactions between antigen-specific CD4+, CD8+ T cells and Ag+DCs. We show that in contrast to CD4+ T cells which form transient contacts with Ag+DC, CD8+ T cells form immediate stable contacts and activate the Ag+DC, acquire fragments of the DC membranes by trogocytosis, leading to their acquisition of some of the DC properties. They express MHC class II, and become able to present the specific Marilyn peptide to naïve Marilyn CD4+ T cells, inducing their extensive division. In vivo, these CD8+ T cells form direct stable contacts with motile naïve CD4+ T cells, recruiting them to Ag+DC binding and to the formation of ternary complexes, where CD4+ and CD8+ T cells interact with the DC and with one another. The presence of CD8+ T cells during in vivo immune responses leads to the early activation and up-regulation of multiple functions by CD4+ T lymphocytes. Thus, while CD4+ T cell help is important to CD8+ T cell responses, CD8+ T cells can interact directly with naïve CD4+ T cells impacting their recruitment and differentiation.

FAM65B controls the proliferation of transformed and primary T cells.

Cell quiescence is controlled by regulated genome-encoded programs that actively express genes which are often down-regulated or inactivated in transformed cells. Among them is FoxO1, a transcription factor that imposes quiescence in several cell types, including T lymphocytes. In these cells, the FAM65B encoding gene is a major target of FOXO1. Here, we show that forced expression of FAM65B in transformed cells blocks their mitosis because of a defect of the mitotic spindle, leading to G2 cell cycle arrest and apoptosis. Upon cell proliferation arrest, FAM65B is engaged in a complex containing two proteins well known to be involved in cell proliferation i.e. the HDAC6 deacetylase and the 14.3.3 scaffolding protein. In primary T cells, FAM65B is down-regulated upon T cell receptor engagement, and maintaining its expression blocks their proliferation, establishing that the decrease of FAM65B expression is required for proliferation. Conversely, inhibiting FAM65B expression in naive T lymphocytes decreases their activation threshold. These results identify FAM65B as a potential new target for controlling proliferation of both transformed and normal cells.

VEGF-A modulates expression of inhibitory checkpoints on CD8+ T cells in tumors.

Immune escape is a prerequisite for tumor development. To avoid the immune system, tumors develop different mechanisms, including T cell exhaustion, which is characterized by expression of immune inhibitory receptors, such as PD-1, CTLA-4, Tim-3, and a progressive loss of function. The recent development of therapies targeting PD-1 and CTLA-4 have raised great interest since they induced long-lasting objective responses in patients suffering from advanced metastatic tumors. However, the regulation of PD-1 expression, and thereby of exhaustion, is unclear. VEGF-A, a proangiogenic molecule produced by the tumors, plays a key role in the development of an immunosuppressive microenvironment. We report in the present work that VEGF-A produced in the tumor microenvironment enhances expression of PD-1 and other inhibitory checkpoints involved in CD8(+) T cell exhaustion, which could be reverted by anti-angiogenic agents targeting VEGF-A-VEGFR. In view of these results, association of anti-angiogenic molecules with immunomodulators of inhibitory checkpoints may be of particular interest in VEGF-A-producing tumors.

Synergistic CD40 signaling on APCs and CD8 T cells drives efficient CD8 response and memory differentiation.

The role of CD4 help during CD8 response and memory differentiation has been clearly demonstrated in different experimental models. However, the exact mechanisms of CD4 help remain largely unknown and preclude replacement therapy to develop. Interestingly, studies have shown that administration of an agonist aCD40ab can substitute CD4 help in vitro and in vivo, whereas the targets of this antibody remain elusive. In this study, we address the exact role of CD40 expression on APCs and CD8 T cells using aCD40ab treatment in mice. We demonstrate that aCD40 antibodies have synergetic effects on APCs and CD8 T cells. Full efficiency of aCD40 treatment requires CD40 expression on both populations: if one of these cell populations is CD40-deficient, the CD8 T cell response is impaired. Most importantly, direct CD40 signaling on APCs and CD8 T cells affects CD8 T cell differentiation differently. In our model, CD40 expression on APCs plays an important but dispensable role on CD8 T cell expansion and effector functions during the early phase of the immune response. Conversely, CD40 on CD8 T cells is crucial and nonredundant for their progressive differentiation into memory cells. Altogether, these results highlight that CD40-CD40L-dependent and independent effects of CD4 help to drive a complete CD8 T cell differentiation.