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Myeloid-derived suppressor cells (MDSC) promote immunosuppression and are a target in the field of immuno-oncology. Accumulation of MDSCs is associated with poor prognosis and resistance to immunotherapy for several cancers. Here, we describe an accumulation of a subset of circulating monocytic MDSCs (M-MDSC) overexpressing TIE2, the receptor for angiopoietin-2 (ANGPT2), in patients with non–small cell lung cancer (NSCLC). Greater numbers of circulating TIE2+ M-MDSCs were detected in patients with NSCLC compared with healthy subjects, and this accumulation correlated with ANGPT2 concentration in blood. The presence of an ANGPT2-rich environment was associated with impairment of preexisting T-cell responses against tumor-associated antigens (TAA) in patients with NSCLC. We demonstrated that ANGPT2 sensitizes TIE2+ M-MDSCs such that these cells suppress TAA-specific T cells. In patients with NSCLC, upregulation of the ANGPT2/TIE2+ M-MDSC signature in blood was associated with a poor prognosis. Our results identify the ANGPT2/TIE2+ M-MDSC axis as a participant in tumor immune evasion that should be taken into account in future cancer immunotherapy.

Biochemical recurrence (BCR) occurs in up to 40% of prostate cancer patients after prostatectomy. In our study, we performed an immune monitoring study in 20 prostate cancer patients with BCR previously treated with metronomic cyclophosphamide (mCTX). We observed a decrease of regulatory T cells (Tregs) from 2 months and this was more pronounced after 6 months of mCTX treatment. This drop of Tregs was associated with increased level of activated HLADR+ CD45R0+ T cells in peripheral blood. Furthermore, a reactivation of Th1 polarized anti-PSA T-cell response was detected in BCR patients treated with mCTX. However, dendritic cell subsets counts and activation were not influenced by the treatment. In the clinical setting, we found that PSA level control was observed in 82% (9/11) of patients with a significant diminution of Tregs after mCTX compared to 33% (3/9) in patients without Tregs decrease. In addition, 30% (6/20) of patients previously treated with mCTX remained free for androgen deprivation therapy. In conclusion, Tregs diminution and immune activation associated with PSA level control occurred after mCTX in prostate cancer patients with BCR.

Exosomes are involved in modulating the immune system and mediating communication between cells. The aim of this study was to investigate the involvement of exosomes in psoriasis. Exosomes from patients with psoriasis were analysed by nanoparticle tracking analysis and protein expression was analysed by western blotting. The concentration of HSP70 was determined by an enzyme-linked immunosorbent assay, and concentrations of interleukin (IL)-1β, IL-2, IL-6, IL-10, IL-17A and tumour necrosis factor alpha (TNF-α) were determined by flow cytometry. Based on the severity of psoriasis, evaluated by body surface area (≤ 10% vs. > 10%), 2 groups of patients were compared (49 with mild psoriasis and 71 with moderate-to-severe psoriasis). The number (2.52×1011 ± 2.29×1010 vs. 1.79×1011 ± 1.93×1010, p = 0.19) and size (94.44 ± 22.00 nm vs. 96.87 ± 28.30 nm, p = 0.72) of exosomes and the concentration of HSP70 in the exosomes were not significantly different in the 2 groups of patients. IL-17A exosome levels were significantly higher in patients with moderate-to-severe psoriasis compared with those with mild psoriasis (p = 0.02). There were no significant differences in levels of TNF-α, IL-1, IL-2, IL-6 and IL-10. This study shows, for the first time, the presence of circulating exosomes in patients with psoriasis. These data confirm the involvement of circulating exosomes in psoriasis, in particular in moderate-to-severe psoriasis, through IL-17A-producing exosomes.

mTOR pathway inhibitors such as rapalogs represent a promising tool to induce functional memory CD8 T cells. In our study, we investigated the combination of temsirolimus with anticancer vaccines. Using various designs of cancer vaccines (short and long peptides or the B subunit of Shiga toxin as an antigen delivery vector) and tumor models (melanoma, lung and colon cancer), we showed that the administration of temsirolimus efficiently decreased tumor growth and enhanced tumor-specific CD8 T-cell responses induced by vaccination. Furthermore, tumor-specific CD8 T cells induced by the bi-therapy (vaccine + temsirolimus) exhibit phenotypic characteristics of central memory (CD127+ CD62L+ ) CD8 T cells compared to vaccination alone. We demonstrated that regulatory CD4 T cells (Tregs ) expansion in vivo limits the efficacy of the bi-therapy by altering the antitumor CD8 T-cell responses. Finally, the use of a small molecule CCR4 antagonist to prevent Tregs induction considerably improved the efficacy of the bi-therapy by enhancing CD8 T cells-mediated antitumor immunity. Taken together, our study highlights the potential interest of combining cancer vaccines with drugs that promote memory CD8 T cells and inhibit Tregs .

Natural killer (NK) cells are innate effector lymphocytes widely involved in cancer immunosurveillance. In this study, we described three circulating NK cell subsets in patients with non-small cell lung cancer (NSCLC). Compared to healthy donors (HD), lower rate of the cytotoxic CD56dim CD16+ NK cells was found in NSCLC patients (76.1% vs 82.4%, P = 0.0041). In contrast, the rate of CD56bright NK cells was similar between patients and HD. We showed in NSCLC patients a higher rate of a NK cell subset with CD56dim CD16− phenotype (16.7% vs 9.9% P = 0.0001). The degranulation property and cytokines production were mainly drive by CD56dim CD16− NK cell subset in patients. Analysis of natural cytotoxicity receptors (NCRs) expression identified four distinct clusters of patients with distinct NK cell subset profiles as compared to one major cluster in HD. Notably the cluster characterized by a low circulating level of NKp46+ NK cell subsets was absent in HD. We showed that the rate of circulating NKp46+ CD56dim CD16+ NK cells influenced the patients’ survival. Indeed, the median overall survival in patients exhibiting high versus low level of this NK cell subset was 16 and 27 months respectively (P = 0.02). Finally, we demonstrated that blocking NKp46 receptor in vitro was able to restore spontaneous tumor specific T cell responses in NSCLC patients. In conclusion, this study showed a distinct distribution and phenotype of circulating NK cell subsets in NSCLC. It also supports the regulatory role of NKp46+ NK cell subset in NSCLC patients.

3044 Background: Increasing evidence highlights the crucial roles played by CD4+ Th1 cells in cancer immunity and immunotherapy (Spitzer et al., Cell 2017, Borst et al., Nat rev Immunol 2018). Here, we investigate the relevance of circulating CD4 Th1 response against shared tumor-associated antigens (TAA) in cancer patients treated by anti-PD-1 immunotherapy. Methods: A total of 46 advanced cancer patients (pts) including 32 pts with non-small cell lung cancer (NSCLC), 14 pts with melanoma, were enrolled (ITHER trial NCT02840058). Patients were treated with anti-PD-1 therapy as standard of care (26 pts with nivolumab and 20 pts with pembrolizumab). Peripheral blood mononuclear cells were collected before and after treatment at 1 and 3 months. The presence of circulating TAA-specific Th1 response was measured by IFNy ELISPOT assay using a mixture of 15mer peptides derived from telomerase (TERT) (Laheurte et al., Oncoimmunology 2016 and Br J C 2019). Results: At the baseline, the anti-TERT Th1 response was observed in 37% of pts. After anti-PD-1 therapy, de novo induction and/or amplification of pre-existing anti-TERT Th1 response was found in 26 % of pts (12/46). Whereas, a decrease of this response was documented in 15% of pts (7/46).The presence of anti-TERT Th1 response in peripheral blood during anti-PD-1 treatment was associated with a prolonged progression free-survival (PFS) as compared to the immune non responder pts (14.4 vs 2.6 months respectively, p = 0.006, HR 0.39 [0.2;0.76]). Similar observation was made for the overall survival (OS) (22.3 vs 12.3 months respectively, p = 0.04 HR 0.45 [0.21;0.96]). Notably, de novo reinvigoration of peripheral anti-TERT Th1 response after anti-PD-1 therapy was associated with a better clinical outcome as compared to the group of pts with decreased immune response after treatment (Median OS not reached vs 5.8 months). In contrast, no association with anti-PD-1 response was observed neither with circulating anti-NY-ESO-1 or with anti-viral Th1 response, concurrently measured in these patients. Conclusions: The reinvigoration of circulating CD4 Th1 against telomerase in patients treated by anti-PD-1 is associated with a better clinical outcome. These results underline the potential interest of monitoring circulating antitumor CD4 Th1 response for immune checkpoint inhibitors management.

Purpose: To investigate the immunoprevalence of SALL4-derived peptides in healthy volunteers and cancer patients. Experimental Design: A multistep approach including prediction algorithms was used to design in silico SALL4-derived peptides theoretically able to bind on common HLA-DR and HLA-A/B molecules. The presence of T-cell responses after a long term T-cell assay (28 days) against SALL4 was monitored in 14 healthy donors and the presence of T-cell responses after a short term T-cell assay (10 days) was monitored in 67 cancer patients using IFN-γ ELISPOT assay. A T-cell clone specific for the immunoprevalent A18 K-derived peptide was isolated, characterized and used as a tool to characterize the natural processing of A18 K. Results: A SALL4 specific T-cell repertoire was present in healthy donors (8/14) and cancer patients (29/67) after short term T-cell assay. We further identified two immunoprevalant SALL4-derived peptides, R18 A and A18 K, which bind MHC-class II. In parallel, an A18 K specific Th1 clone recognized monocyte derived Dendritic Cell (moDC) loaded with SALL4 containing cell lysate. The level of IFN-γ secreted by specific T-cell clone was greater in presence of moDC loaded with SALL4 containing cell lysate (49.23 ± 14.02%) than with moDC alone (18.03 ± 3.072%) (p = 0.0477) Conclusion: These results show for the first time immunogenicity of SALL4 oncogenic protein-derived peptides, especially A18 K and R18 A peptides and make them potential targets for personalized medicine. Thus, SALL4 possess major characteristics of a tumor antigen.

The rapalogs everolimus and temsirolimus that inhibit mTOR signaling are used as antiproliferative drugs in several cancers. Here we investigated the influence of rapalogs-mediated immune modulation on their antitumor efficacy. Studies in metastatic renal cell carcinoma patients showed that everolimus promoted high expansion of FoxP3+Helios+Ki67+ regulatory CD4 T cells (Tregs). In these patients, rapalogs strongly enhanced the suppressive functions of Tregs, mainly in a contact-dependent manner. Paradoxically, a concurrent activation of spontaneous tumor-specific Th1 immunity also occurred. Furthermore, a high rate of Eomes+CD8+ T cells was detected in patients after a long-term mTOR inhibition. We found that early changes in the Tregs/antitumor Th1 balance can differentially shape the treatment efficacy. Patients presenting a shift toward decreased Tregs levels and high expansion of antitumor Th1 cells showed better clinical responses. Studies conducted in tumor-bearing mice confirmed the deleterious effect of rapalogs-induced Tregs via a mechanism involving the inhibition of antitumor T-cell immunity. Consequently, the combination of temsirolimus plus CCR4 antagonist, a receptor highly expressed on rapalogs-exposed Tregs, was more effective than monotherapy. Altogether, our results describe for the first time a dual impact of host adaptive antitumor T-cell immunity on the clinical effectiveness of rapalogs and prompt their association with immunotherapies. Cancer Res; 76(14); 4100–12. ©2016 AACR.

Telomerase is a prototype-shared tumor Ag and represents an attractive target for anticancer immunotherapy. We have previously described promiscuous and immunogenic HLA-DR–restricted peptides derived from human telomerase reverse transcriptase (hTERT) and referred as universal cancer peptide (UCP). In nonsmall cell lung cancer, the presence of spontaneous UCP-specific CD4 T cell responses increases the survival of chemotherapy-responding patients. However, the precise mechanisms of hTERT’s uptake, processing, and presentation on MHC-II molecules to stimulate CD4 T cells are poorly understood. In this work, by using well-characterized UCP-specific CD4 T cell clones, we showed that hTERT processing and presentation on MHC-II involve both classical endolysosomal and nonclassical cytosolic pathways. Furthermore, to our knowledge, we demonstrated for the first time that hTERT’s internalization by dendritic cells requires its interaction with surface heparan sulfate proteoglycans. Altogether, our findings provide a novel mechanism of tumor-specific CD4 T cell activation and will be useful for the development of novel cancer immunotherapies that harness CD4 T cells.