CD40 and CD80/86 signaling in cDC1s mediate effective neoantigen vaccination and generation of antigen-specific CX3CR1⁺ CD8⁺ T cells in mice

Abstract

Running title: CX3CR1 as a circulating biomarker for vaccine therapy 33 34 Word counts (excluding abstract and references): 2,476 35 36 Abstract 38Background The use of tumor mutation-derived neoantigen represents a promising approach for 39 cancer vaccines. Preclinical and early-phase human clinical studies have shown the successful 40 induction of tumor neoepitope-directed responses; however, overall clinical efficacy of 41 neoantigen vaccines has been limited. One major limitation of this strategy is the lack of a 42 reliable blood-based surrogate marker for clinical response. Here, we investigated a role of 43 CX3C chemokine receptor 1 (CX3CR1), a marker of T-cell differentiation in predicting response 44 to neoantigen vaccination in a preclinical model. 45Methods Mice bearing MC38 colon adenocarcinoma cells that harbor a single-epitope mutation 46 within the Adpgk protein were treated with mutant Adpgk peptide (AdpgkMut) in combination 47 with toll-like receptor 3 (TLR3) agonist poly(I:C) and/or anti-CD40 antibody (Ab). Antitumor 48 efficacy of neoantigen vaccination and the frequency of peripheral blood AdpgkMut-specific 49 CX3CR1+CD8+ T cells were assessed. Mechanisms underlying the generation of CX3CR1+CD8+ 50 T cells were examined using knockout and bone marrow chimeric mice. 51 ResultsWe found that CD40 stimulation significantly enhanced antitumor efficacy of 52 neoantigen and TLR3 agonist vaccination, which was associated with an increased frequency of 53 circulating AdpgkMut-specific effector CX3CR1+CD8+ T cells. Generation of neoantigen-specific 54 CX3CR1+CD8+ T cells by AdpgkMut/CD40/TLR3 stimulation was dependent on CD40 and Batf3 55 in bone marrow-derived cells. Further mechanistic studies using mixed bone marrow chimeras 56identified key roles for CD40 and CD80/86, but not CD70 signaling in Batf3-dependent 57 conventional type 1 dendritic cells (cDC1s) for generation of neoantigen-specific cytotoxic 58 CX3CR1+CD8+ T cells and therapeutic efficacy of neoantigen vaccine. 59Conclusions Taken together, our results underscore critical roles of cDC1s and an implication of 60 dual CD40/TLR3 stimulation in neoantigen-based therapeutic vaccines, and demonstrate the 61 potential utility of CX3CR1 as a circulating predictive T-cell biomarker in vaccine clinical trials. 62 63

Type 1 conventional dendritic cells and interferons are required for spontaneous CD4+ and CD8+ T‐cell protective responses to breast cancer

“…Together with our preliminary observation of the simultaneous interactions of cDC1 with CD4 + T cells and CTLs in the tumor bed, this suggested that cell‐intrinsic responses of cDC1 to IFN may be critical to promote their ability to deliver to CTL the help from CD4 + T conv in a manner depending on their interactions via CD40/CD40L. This hypothesis is consistent with the demonstration that simultaneous presentation of viral Ags by cDC1 to CTLs and CD4 + T cells is key for robust antiviral cellular immunity 89,90 and with publications linking CD40 expression on cDC1, their ability to activate CD4 + T conv and the CTL‐dependent rejection of tumors 30,91 …”

“…This hypothesis is consistent with the demonstration that simultaneous presentation of viral Ags by cDC1 to CTLs and CD4 + T cells is key for robust antiviral cellular immunity 89,90 and with publications linking CD40 expression on cDC1, their ability to activate CD4 + T conv and the CTL-dependent rejection of tumors. 30,91 To the best of our knowledge, our preliminary confocal microscopy data are the first direct evidence showing that cDC1 in tumors might act as a unique cellular platform docking simultaneously CD4 + T conv and CTLs, which is likely key to their ability to relay CD4 + T-cell help to CTLs in situ in tumor, akin to what had been previously shown in infectious settings. 89,90 However, further experiments including dynamic data and spatial distribution assessments will be required to robustly determine whether cDC1 simultaneously interact with cognate CD4 + and CD8 + T cells in the tumor bed, and more efficiently than other Ag-presenting cells.…”

“…This hypothesis is consistent with the demonstration that simultaneous presentation of viral Ags by cDC1 to CTLs and CD4 + T cells is key for robust antiviral cellular immunity 89 , 90 and with publications linking CD40 expression on cDC1, their ability to activate CD4 + T conv and the CTL‐dependent rejection of tumors. 30 , 91 …”

cDC1 and interferons promote spontaneous CD4+ and CD8+ T cell protective responses to breast cancer

“…Together with our observation of the simultaneous interactions of cDC1 with CD4 + T cells and CTLs in the tumor bed, this suggested that cell-intrinsic responses of cDC1 to IFN may be critical to promote their ability to deliver to CTL the help from CD4 + Tconv in a manner depending on their interactions via CD40/CD40L. This hypothesis is consistent with the demonstration that simultaneous presentation of viral antigens by cDC1s to CTLs and CD4 + T cells is key for robust antiviral cellular immunity (49,50) and with publications linking CD40 expression on cDC1, their ability to activate CD4 + Tconv and the CTL-dependent rejection of tumors (14,51).…”