Oncolytic viruses alter the biogenesis of tumor extracellular vesicles and influence their immunogenicity.

Extracellular vesicles (EVs) are mediators of intercellular communication in the tumor microenvironment. Tumor EVs are commonly associated with metastasis, immunosuppression or drug resistance. Viral infections usually increase EV secretion, but little is known about the effect of oncolytic viruses (OVs) on tumor EVs. Here, we investigated the impact of oncolytic vesicular stomatitis virus (VSV) and vaccinia virus on EVs secreted by human melanoma and thoracic cancer cells. We found that OV infection increases the production of EVs by tumor cells. These EVs contain proteins of viral origin, such as VSV-G, thus creating a continuum of particles sharing markers of both canonical EVs and viruses. As such, the presence of VSV-G on EVs improves the transfer of their protein content to cell types commonly found in the tumor microenvironment. A proteomic analysis also revealed that EVs-OV secreted during VSV infection are enriched in immunity-related proteins. Finally, CD8+ T cells incubated with EVs-OV from infected cells display slightly enhanced cytotoxic functions. Taken together, these data suggest that OVs enhance the communication mediated by tumor EVs, which could participate in the therapeutic efficacy of OVs. These results also provide rationale for engineering OVs to exploit EVs and disseminate therapeutic proteins within the tumor microenvironment.

Differential impact of genetic deletion of TIGIT or PD-1 on melanoma-specific T-lymphocytes.

Immune checkpoint (IC) blockade and adoptive transfer of tumor-specific T-cells (ACT) are two major strategies to treat metastatic melanoma. Their combination can potentiate T-cell activation in the suppressive tumor microenvironment, but the autoimmune adverse effects associated with systemic injection of IC blockers persist with this strategy. ACT of tumor-reactive T-cells defective for IC expression would overcome this issue. For this purpose, PD-1 and TIGIT appear to be relevant candidates, because their co-expression on highly tumor-reactive lymphocytes limits their therapeutic efficacy within the tumor microenvironme,nt. Our study compares the consequences of PDCD1 or TIGIT genetic deletion on anti-tumor properties and T-cell fitness of melanoma-specific T lymphocytes. Transcriptomic analyses revealed down-regulation of cell cycle-related genes in PD-1KO T-cells, consistent with biological observations, whereas proliferative pathways were preserved in TIGITKO T-cells. Functional analyses showed that PD-1KO and TIGITKO T-cells displayed superior antitumor reactivity than their wild-type counterpart in vitro and in a preclinical melanoma model using immunodeficient mice. Interestingly, it appears that TIGITKO T-cells were more effective at inhibiting tumor cell proliferation in vivo, and persist longer within tumors than PD-1KO T-cells, consistent with the absence of impact of TIGIT deletion on T-cell fitness. Taken together, these results suggest that TIGIT deletion, over PD-1 deletion, in melanoma-specific T-cells is a compelling option for future immunotherapeutic strategies.

Oncolytic attenuated measles virus encoding NY-ESO-1 induces HLA I and II presentation of this tumor antigen by melanoma and dendritic cells.

Antitumor virotherapy stimulates the antitumor immune response during tumor cell lysis induced by oncolytic viruses (OVs). OV can be modified to express additional transgenes that enhance their therapeutic potential. In this study, we armed the spontaneously oncolytic Schwarz strain of measles viruses (MVs) with the gene encoding the cancer/testis antigen NY-ESO-1 to obtain MVny. We compared MV and MVny oncolytic activity and ability to induce NY-ESO-1 expression in six human melanoma cell lines. After MVny infection, we measured the capacity of melanoma cells to present NY-ESO-1 peptides to CD4 + and CD8 + T cell clones specific for this antigen. We assessed the ability of MVny to induce NY-ESO-1 expression and presentation in monocyte-derived dendritic cells (DCs). Our results show that MVny and MV oncolytic activity are similar with a faster cell lysis induced by MVny. We also observed that melanoma cell lines and DC expressed the NY-ESO-1 protein after MVny infection. In addition, MVny-infected melanoma cells and DCs were able to stimulate NY-ESO-1-specific CD4 + and CD8 + T cells. Finally, MVny was able to induce DC maturation. Altogether, these results show that MVny could be an interesting candidate to stimulate NY-ESO-1-specific T cells in melanoma patients with NY-ESO-1-expressing tumor cells.

A microbiota-modulated checkpoint directs immunosuppressive intestinal T cells into cancers.

Antibiotics (ABX) compromise the efficacy of programmed cell death protein 1 (PD-1) blockade in cancer patients, but the mechanisms underlying their immunosuppressive effects remain unknown. By inducing the down-regulation of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the ileum, post-ABX gut recolonization by Enterocloster species drove the emigration of enterotropic α4ß7+CD4+ regulatory T 17 cells into the tumor. These deleterious ABX effects were mimicked by oral gavage of Enterocloster species, by genetic deficiency, or by antibody-mediated neutralization of MAdCAM-1 and its receptor, α4ß7 integrin. By contrast, fecal microbiota transplantation or interleukin-17A neutralization prevented ABX-induced immunosuppression. In independent lung, kidney, and bladder cancer patient cohorts, low serum levels of soluble MAdCAM-1 had a negative prognostic impact. Thus, the MAdCAM-1-α4ß7 axis constitutes an actionable gut immune checkpoint in cancer immunosurveillance.

The DCMU Herbicide Shapes T-cell Functions By Modulating Micro-RNA Expression Profiles.

DCMU [N-(3,4-dichlorophenyl)-N-dimethylurea] or diuron is a widely used herbicide, which can cause adverse effects on human, especially on immune cells, due to their intrinsic properties and wide distribution. These cells are important for fighting not only against virus or bacteria but also against neoplastic cell development. We developed an approach that combines functional studies and miRNA and RNA sequencing data to evaluate the effects of DCMU on the human immune response against cancer, particularly the one carried out by CD8+ T cells. We found that DCMU modulates the expression of miRNA in a dose-dependent manner, leading to a specific pattern of gene expression and consequently to a diminished cytokine and granzyme B secretions. Using mimics or anti-miRs, we identified several miRNA, such as hsa-miR-3135b and hsa-miR-21-5p, that regulate these secretions. All these changes reduce the CD8+ T cells' cytotoxic activity directed against cancer cells, in vitro and in vivo in a zebrafish model. To conclude, our study suggests that DCMU reduces T-cell abilities, participating thus to the establishment of an environment conducive to cancer development.

Targeting NKG2A to boost anti-tumor CD8 T-cell responses in human colorectal cancer.

Recently, the inhibitory CD94/NKG2A receptor has joined the group of immune checkpoints (ICs) and its expression has been documented in NK cells and CD8+ T lymphocytes in several cancers and some infectious diseases. In colorectal cancer (CRC), we previously reported that NKG2A+ tumor-infiltrating lymphocytes (TILs) are predominantly CD8+ αß T cells and that CD94 overexpression and/or its ligand HLA-E were associated with a poor prognosis. This study aimed to thoroughly characterize the NKG2A+ CD8+ TIL subpopulation and document the impact of NKG2A on anti-tumor responses in CRC. Our findings highlight new features of this subpopulation: (i) enrichment in colorectal tumors compared to paired normal colonic mucosa, (ii) their character as tissue-resident T cells and their majority terminal exhaustion status, (iii) co-expression of other ICs delineating two subgroups differing mainly in the level of NKG2A expression and the presence of PD-1, (iv) high functional avidity despite reduced proliferative capacity and finally (v) inhibition of anti-tumor reactivity that is overcome by blocking NKG2A. From a clinical point of view, these results open a promising alternative for immunotherapies based on NKG2A blockade in CRC, which could be performed alone or in combination with other IC inhibitors, adoptive cell transfer or therapeutic vaccination.

hnRNP-A1 binds to the IRES of MELOE-1 antigen to promote MELOE-1 translation in stressed melanoma cells.

The major challenge in antigen-specific immunotherapy of cancer is to select the most relevant tumor antigens to target. To this aim, understanding their mode of expression by tumor cells is critical. We previously identified a melanoma-specific antigen, melanoma-overexpressed antigen 1 (MELOE-1)-coded for by a long noncoding RNA-whose internal ribosomal entry sequence (IRES)-dependent translation is restricted to tumor cells. This restricted expression is associated with the presence of a broad-specific T-cell repertoire that is involved in tumor immunosurveillance in melanoma patients. In the present work, we explored the translation control of MELOE-1 and provide evidence that heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) binds to the MELOE-1 IRES and acts as an IRES trans-activating factor (ITAF) to promote the translation of MELOE-1 in melanoma cells. In addition, we showed that endoplasmic reticulum (ER) stress induced by thapsigargin, which promotes hnRNP-A1 cytoplasmic translocation, enhances MELOE-1 translation and recognition of melanoma cells by a MELOE-1-specific T-cell clone. These findings suggest that pharmacological stimulation of stress pathways may enhance the efficacy of immunotherapies targeting stress-induced tumor antigens such as MELOE-1.

Phase I/II clinical trial of adoptive cell transfer of sorted specific T cells for metastatic melanoma patients.

Adoptive cell transfer (ACT) of tumor-specific T lymphocytes represents a relevant therapeutic strategy to treat metastatic melanoma patients. Ideal T-cells should combine tumor specificity and reactivity with survival in vivo, while avoiding autoimmune side effects. Here we report results from a Phase I/II clinical trial (NCT02424916, performed between 2015 and 2018) in which 6 metastatic HLA-A2 melanoma patients received autologous antigen-specific T-cells produced from PBMC, after peptide stimulation in vitro, followed by sorting with HLA-peptide multimers and amplification. Each patient received a combination of Melan-A and MELOE-1 polyclonal specific T-cells, whose specificity and anti-tumor reactivity were checked prior to injection, with subcutaneous IL-2. Transferred T-cells were also characterized in terms of functional avidity, diversity and phenotype and their blood persistence was evaluated. An increase in specific T-cells was detected in the blood of all patients at day 1 and progressively disappeared from day 7 onwards. No serious adverse events occurred after this ACT. Clinically, five patients progressed and one patient experienced a partial response following therapy. Melan-A and MELOE-1 specific T-cells infused to this patient were diverse, of high avidity, with a high proportion of T lymphocytes co-expressing PD-1 and TIGIT but few other exhaustion markers. In conclusion, we demonstrated the feasibility and safety of ACT with multimer-sorted Melan-A and MELOE-1 specific T cells to metastatic melanoma patients. The clinical efficacy of such therapeutic strategy could be further enhanced by the selection of highly reactive T-cells, based on PD-1 and TIGIT co-expression, and a combination with ICI, such as anti-PD-1.

Anti-Tumor Efficacy of PD-L1 Targeted Alpha-Particle Therapy in a Human Melanoma Xenograft Model.

PD-L1 (programmed death-ligand 1, B7-H1, CD274), the ligand for PD-1 inhibitory receptor, is expressed on various tumors, and its expression is correlated with a poor prognosis in melanoma. Anti-PD-L1 mAbs have been developed along with anti-CTLA-4 and anti-PD-1 antibodies for immune checkpoint inhibitor (ICI) therapy, and anti-PD-1 mAbs are now used as first line treatment in melanoma. However, many patients do not respond to ICI therapies, and therefore new treatment alternatives should be developed. Because of its expression on the tumor cells and on immunosuppressive cells within the tumor microenvironment, PD-L1 represents an interesting target for targeted alpha-particle therapy (TAT). We developed a TAT approach in a human melanoma xenograft model that stably expresses PD-L1 using a 213Bi-anti-human-PD-L1 mAb. Unlike treatment with unlabeled anti-human-PD-L1 mAb, TAT targeting PD-L1 significantly delayed melanoma tumor growth and improved animal survival. A slight decrease in platelets was observed, but no toxicity on red blood cells, bone marrow, liver or kidney was induced. Anti-tumor efficacy was associated with specific tumor targeting since no therapeutic effect was observed in animals bearing PD-L1 negative melanoma tumors. This study demonstrates that anti-PD-L1 antibodies may be used efficiently for TAT treatment in melanoma.

Low Levels of Natural Anti-α-N-Acetylgalactosamine (Tn) Antibodies Are Associated With COVID-19.

Human serum contains large amounts of anti-carbohydrate antibodies, some of which may recognize epitopes on viral glycans. Here, we tested the hypothesis that such antibodies may confer protection against COVID-19 so that patients would be preferentially found among people with low amounts of specific anti-carbohydrate antibodies since individual repertoires vary considerably. After selecting glycan epitopes commonly represented in the human anti-carbohydrate antibody repertoire that may also be expressed on viral glycans, plasma levels of the corresponding antibodies were determined by ELISA in 88 SARS-CoV-2 infected individuals, including 13 asymptomatic, and in 82 non-infected controls. We observed that anti-Tn antibodies levels were significantly lower in patients as compared to non-infected individuals. This was not observed for any of the other tested carbohydrate epitopes, including anti-αGal antibodies used as a negative control since the epitope cannot be synthesized by humans. Owing to structural homologies with blood groups A and B antigens, we also observed that anti-Tn and anti-αGal antibodies levels were lower in blood group A and B, respectively. Analyses of correlations between anti-Tn and the other anti-carbohydrates tested revealed divergent patterns of correlations between patients and controls, suggesting qualitative differences in addition to the quantitative difference. Furthermore, anti-Tn levels correlated with anti-S protein levels in the patients' group, suggesting that anti-Tn might contribute to the development of the specific antiviral response. Overall, this first analysis allows to hypothesize that natural anti-Tn antibodies might be protective against COVID-19.

PD-1 and TIGIT coexpression identifies a circulating CD8 T cell subset predictive of response to anti-PD-1 therapy.

BACKGROUND: Clinical benefit from programmed cell death 1 receptor (PD-1) inhibitors relies on reinvigoration of endogenous antitumor immunity. Nonetheless, robust immunological markers, based on circulating immune cell subsets associated with therapeutic efficacy are yet to be validated. METHODS: We isolated peripheral blood mononuclear cell from three independent cohorts of melanoma and Merkel cell carcinoma patients treated with PD-1 inhibitor, at baseline and longitudinally after therapy. Using multiparameter flow cytometry and cell sorting, we isolated four subsets of CD8+ T cells, based on PD-1 and TIGIT expression profiles. We performed phenotypic characterization, T cell receptor sequencing, targeted transcriptomic analysis and antitumor reactivity assays to thoroughly characterize each of these subsets. RESULTS: We documented that the frequency of circulating PD-1+TIGIT+ (DPOS) CD8+ T-cells after 1 month of anti-PD-1 therapy was associated with clinical response and overall survival. This DPOS T-cell population was enriched in highly activated T-cells, tumor-specific and emerging T-cell clonotypes and T lymphocytes overexpressing CXCR5, a key marker of the CD8 cytotoxic follicular T cell population. Additionally, transcriptomic profiling defined a specific gene signature for this population as well as the overexpression of specific pathways associated with the therapeutic response. CONCLUSIONS: Our results provide a convincing rationale for monitoring this PD-1+TIGIT+ circulating population as an early cellular-based marker of therapeutic response to anti-PD-1 therapy.

Selective SIRPα blockade reverses tumor T cell exclusion and overcomes cancer immunotherapy resistance.

T cell exclusion causes resistance to cancer immunotherapies via immune checkpoint blockade (ICB). Myeloid cells contribute to resistance by expressing signal regulatory protein-α (SIRPα), an inhibitory membrane receptor that interacts with ubiquitous receptor CD47 to control macrophage phagocytosis in the tumor microenvironment. Although CD47/SIRPα-targeting drugs have been assessed in preclinical models, the therapeutic benefit of selectively blocking SIRPα, and not SIRPγ/CD47, in humans remains unknown. We report a potent synergy between selective SIRPα blockade and ICB in increasing memory T cell responses and reverting exclusion in syngeneic and orthotopic tumor models. Selective SIRPα blockade stimulated tumor nest T cell recruitment by restoring murine and human macrophage chemokine secretion and increased anti-tumor T cell responses by promoting tumor-antigen crosspresentation by dendritic cells. However, nonselective SIRPα/SIRPγ blockade targeting CD47 impaired human T cell activation, proliferation, and endothelial transmigration. Selective SIRPα inhibition opens an attractive avenue to overcoming ICB resistance in patients with elevated myeloid cell infiltration in solid tumors.

Cross-reactivity between tumor MHC class I-restricted antigens and an enterococcal bacteriophage.

Intestinal microbiota have been proposed to induce commensal-specific memory T cells that cross-react with tumor-associated antigens. We identified major histocompatibility complex (MHC) class I-binding epitopes in the tail length tape measure protein (TMP) of a prophage found in the genome of the bacteriophage Enterococcus hirae Mice bearing E. hirae harboring this prophage mounted a TMP-specific H-2Kb-restricted CD8+ T lymphocyte response upon immunotherapy with cyclophosphamide or anti-PD-1 antibodies. Administration of bacterial strains engineered to express the TMP epitope improved immunotherapy in mice. In renal and lung cancer patients, the presence of the enterococcal prophage in stools and expression of a TMP-cross-reactive antigen by tumors correlated with long-term benefit of PD-1 blockade therapy. In melanoma patients, T cell clones recognizing naturally processed cancer antigens that are cross-reactive with microbial peptides were detected.

Tumor infiltrating lymphocytes as adjuvant treatment in stage III melanoma patients with only one invaded lymph node after complete resection: results from a multicentre, randomized clinical phase III trial.

BACKGROUND: Adoptive tumor-infiltrating lymphocytes (TIL) therapy and interleukin-2 (IL-2) have been investigated in melanoma. AIM: To confirm previously observed preventive effects of TIL + IL2 in a subgroup of patients with relapsing metastatic stage III melanoma. METHODOLOGY: Open-label, randomized two-group, multicenter five-year trial in adult stage III melanoma patients with only one invaded lymph node after complete resection. Patients received TIL + IL2 or abstention. TIL + IL2 was administered within 8 weeks after lymph node resection and 4 weeks after. Disease-free survival was assessed every 2 months up to month 18, every 3 months up to month 36 and every 4 months up to 5 years. A once-a-year follow-up was scheduled beyond the five-year follow-up. Safety was assessed throughout the trial. RESULTS: Overall, 49 patients accounted for the modified intent-to-treat and 47 for the PP. Slightly more male than female patients participated; mean age was 57.7 ± 11.4 years in the TIL + IL2 group and 53.5 ± 13.0 years in the abstention group. After 5 years of follow-up, 11/26 patients in the TIL + IL2 group and 13/23 in the abstention group had relapsed. There was no statistical difference between the groups (HR: 0.63 CI 95% [0.28-1.41], p = 0.258), nine patients in the TIL + IL2 and 11 in the abstention group died with no significant difference between the two groups (HR: 0.65 CI95% [0.27 - 1.59], p = 0.34). Safety was good. CONCLUSION: We did not confirm results of a previous trial. However, ulceration of the primary melanoma may be considered predictive of the efficacy of TIL in melanoma in adjuvant setting, in a manner similar to interferon α.

Increased antitumor efficacy of PD-1-deficient melanoma-specific human lymphocytes.

BACKGROUND: Genome editing offers unique perspectives for optimizing the functional properties of T cells for adoptive cell transfer purposes. So far, PDCD1 editing has been successfully tested mainly in chimeric antigen receptor T (CAR-T) cells and human primary T cells. Nonetheless, for patients with solid tumors, the adoptive transfer of effector memory T cells specific for tumor antigens remains a relevant option, and the use of high avidity T cells deficient for programmed cell death-1 (PD-1) expression is susceptible to improve the therapeutic benefit of these treatments. METHODS: Here we used the transfection of CAS9/sgRNA ribonucleoproteic complexes to edit PDCD1 gene in human effector memory CD8+ T cells specific for the melanoma antigen Melan-A. We cloned edited T cell populations and validated PDCD1 editing through sequencing and cytometry in each T cell clone, together with T-cell receptor (TCR) chain's sequencing. We also performed whole transcriptomic analyses on wild-type (WT) and edited T cell clones. Finally, we documented in vitro and in vivo through adoptive transfer in NOD scid gamma (NSG) mice, the antitumor properties of WT and PD-1KO T cell clones, expressing the same TCR. RESULTS: Here we demonstrated the feasibility to edit PDCD1 gene in human effector memory melanoma-specific T lymphocytes. We showed that PD-1 expression was dramatically reduced or totally absent on PDCD1-edited T cell clones. Extensive characterization of a panel of T cell clones expressing the same TCR and exhibiting similar functional avidity demonstrated superior antitumor reactivity against a PD-L1 expressing melanoma cell line. Transcriptomic analysis revealed a downregulation of genes involved in proliferation and DNA replication in PD-1-deficient T cell clones, whereas genes involved in metabolism and cell signaling were upregulated. Finally, we documented the superior ability of PD-1-deficient T cells to significantly delay the growth of a PD-L1 expressing human melanoma tumor in an NSG mouse model. CONCLUSION: The use of such lymphocytes for adoptive cell transfer purposes, associated with other approaches modulating the tumor microenvironment, would be a promising alternative to improve immunotherapy efficacy in solid tumors.

MicroRNAs in Tumor Exosomes Drive Immune Escape in Melanoma.

MicroRNAs (miRNA), small noncoding RNAs that regulate gene expression, exist not only in cells but also in a variety of body fluids. These circulating miRNAs could enable intercellular communication. miRNAs are packaged in membrane-encapsulated vesicles, such as exosomes, or protected by RNA-binding proteins. Here, we report that miRNAs included in human melanoma exosomes regulate the tumor immune response. Using microscopy and flow cytometry, we demonstrate that CD8+ T cells internalize exosomes from different tumor types even if these cells do not internalize vesicles as readily as other immune cells. We explored the function of melanoma-derived exosomes in CD8+ T cells and showed that these exosomes downregulate T-cell responses through decreased T-cell receptor (TCR) signaling and diminished cytokine and granzyme B secretions. The result reduces the cells' cytotoxic activity. Using mimics, we found that miRNAs enriched in exosomes-such as Homo sapiens (hsa)-miR-3187-3p, hsa-miR-498, hsa-miR-122, hsa-miR149, and hsa-miR-181a/b-regulate TCR signaling and TNFα secretion. Our observations suggest that miRNAs in melanoma-derived exosomes aid tumor immune evasion and could be a therapeutic target.

Viral and tumor antigen-specific CD8 T-cell responses in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous cancer, which is immunogenic, regardless of the presence of MCPyV (80% of cases). The identification of MCC-specific epitopes recognized by CD8 T cells is crucial to expand the arsenal of immunotherapeutic treatments. Until now, most efforts focused on the identification of virus-specific epitopes, whereas immune responses directed against shared cellular tumor-specific antigens have not been evidenced. In this study, we measured T-cell responses against viral (n = 3) and tumor antigens (n = 47) from TILs derived from 21 MCC tumors. Virus-specific CD8 T-cell responses dominated MCC-specific immune responses, and we identified two new HLA-peptide complexes derived from the LT antigen, located in a region encompassing 3 previously identified epitopes. Finally, we show that MAGE-A3 antigen, frequently expressed by MCC tumors, was recognized by CD8 TILs from a virus-negative MCC tumor and thus could be a target for immunotherapy in this setting.

Cancer vaccines: designing artificial synthetic long peptides to improve presentation of class I and class II T cell epitopes by dendritic cells.

There is now a consensus that efficient peptide vaccination against cancer requires that peptides should (i) be exclusively presented by professional APC and (ii) stimulate both CD4 and CD8-specific T cell responses. To this aim, in recent trials, patients were vaccinated with pools of synthetic long peptides (SLP) (15-30 aa long) composed of a potential class I epitope(s) elongated at both ends with native antigen sequences to also provide a potential class II epitope(s). Using MELOE-1 as a model antigen, we present an alternative strategy consisting in linking selected class I and class II epitopes with an artificial cathepsin-sensitive linker to improve epitope processing and presentation by DC. We provide evidence that some linker sequences used in our artificial SLPs (aSLPs) could increase up to 100-fold the cross-presentation of class I epitopes to CD8-specific T cell clones when compared to cross-presentation of the corresponding native long peptide. Presentation of class II epitopes were only slightly increased. We confirmed this increased cross-presentation after in vitro stimulation of PBMC from healthy donors with aSLP and assessment of CD8-specific responses and also in vivo following aSLP vaccination of HLA*A0201/HLA-DRB0101 transgenic mice. Finally, we provide some evidence that vaccination with aSLP could inhibit the growth of transplanted tumors in mice. Our data thus support the use of such aSLPs in future cancer vaccination trials to improve anti-tumor CD8 T cell responses and therapeutic efficacy.