Activation of STING signaling plays an important role in anti-tumor immunity, and we previously reported the anti-tumor effects of STING through accumulation of M1-like macrophages in tumor tissue treated with a STING agonist. However, myeloid cells express SIRPα, an inhibitory receptor for phagocytosis, and its receptor, CD47, is overexpressed in various cancer types. Based on our findings that breast cancer patients with highly expressed CD47 have poor survival, we evaluated the therapeutic efficacy and underlying mechanisms of combination therapy with the STING ligand cGAMP and an antagonistic anti-CD47 mAb using E0771 mouse breast cancer cells. Anti-CD47 mAb monotherapy did not suppress tumor growth in our setting, whereas cGAMP and anti-CD47 mAb combination therapy inhibited tumor growth. The combination therapy enhanced phagocytosis of tumor cells and induced systemic anti-tumor immune responses, which rely on STING and type I IFN signaling. Taken together, our findings indicate that coadministration of cGAMP and an antagonistic anti-CD47 mAb may be promising for effective cancer immunotherapy.

The current coronavirus disease 2019 (COVID-19) pandemic that is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a significant threat to public health. Although vaccines based on the mRNA of the SARS-CoV-2 spike protein have been developed to induce both cellular and humoral immunity against SARS-CoV-2, there have been some concerns raised about their high cost, particularly in developing countries. In the present study, we aim to identify an immunogenic peptide in the SARS-CoV-2 spike protein to activate cellular immunity, particularly CD4⁺ helper T lymphocytes (Th cells), which are a commander of immune system. SARS-CoV-2 spike protein-derived peptides Spike_448-477 and Spike_{489-513(N501Y)}-specific CD4⁺ Th cell lines were generated by repetitive stimulation of healthy donor-derived CD4⁺T-cells with each peptide. Their HLA-restrictions were addressed by using blocking antibodies against HLA and HLA-transfected L-cells. The epitopes of Spike_448-477-specific CD4⁺ Th cell lines were defined using a series of 7–14-mer overlapping truncated peptides and alanine-substituted epitope peptides. To address responsiveness of these CD4⁺ Th cell lines to several SARS-CoV-2 variants, we stimulated the CD4⁺ Th cell lines with mutated peptides. We addressed whether these identified peptides were useful for monitoring T-cell-based immune responses in vaccinated donors using the IFN-γ ELISpot assay. The Spike_448-477 peptide was found to be a promiscuous peptide presented by HLA- DRB1*08:02, DR53, and DPB1*02:02. Although HLA-DPB1*02:02-restricted CD4⁺ Th cells did not response to some peptides with the L452R and L452Q mutations, the other CD4⁺ Th cells were not affected by any mutant peptides. We developed two tetramers to detect HLA-DRB1*08:02/Spike_449-463- and Spike_449-463(L452R/Y453F)-recognizing CD4⁺ Th cells. Spike_{489-513(N501Y)} peptide was also a promiscuously presented to HLA-DRB1*09:01 and DRB1*15:02. The T-cell responses specific to both peptides Spike_448-477 and Spike_489-513 were detected in PBMCs after vaccinations. In addition, we observed that the Spike_448-477 peptide activated both CD8⁺ T-cells and CD4⁺ Th cells in individuals receiving mRNA vaccines. SARS-CoV-2 spike protein-derived peptides, Spike_448-477 and Spike_489-513, include several epitopes that are presented by multiple HLA-class II alleles to activate CD4⁺ Th cells, which are considered useful for monitoring the establishment of acquired immunity after vaccination.

CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily and activates antigen-presenting cells (APCs) to promote immune responses through producing inflammatory cytokines such as interleukin (IL)-12 and TNFα. Although cancer immunotherapies using agonistic anti-CD40 antibody have been conducted in clinical settings, many clinical trials have been discontinued owing to the occurrence of severe adverse effects. Stimulator of interferon genes (STING) also promotes activating APCs to prime tumor antigens to effector T cells. Recent our studies have shown that intratumoral administration of the STING ligand cyclic GMP-AMP (cGAMP) effectively activated tumor-specific T cells and suppressed tumor growth in mouse models. Based on these findings, we hypothesized that co-activation of both STING and CD40 within the tumor microenvironment could effectively suppress tumor growth and prolong survival in tumor-bearing hosts. In the present study, CD40 expression was upregulated in mouse bone marrow-derived dendritic cells (BMDCs) upon stimulation with cGAMP, and this effect was dependent on TNFα induced by type I IFNs produced in an autocrine and paracrine manner. Furthermore, in mouse tumor models, CD40 was found to be critical for the antitumor effects of STING-targeted therapy, and intratumoral co-activation of STING and CD40 synergistically suppressed tumor growth and prolonged survival in a CD8⁺ T-cell-dependent manner. These findings suggest that intratumoral immunotherapy co-targeting STING and CD40 represents a promising therapeutic strategy with potentially reduced systemic side effects.