CD40 Agonist Combined with Radiation and PD-1 Blockade Enhances Development Of Systemic Tumor-Specific B-Cells And B-Cell Memory

Abstract

Checkpoint blockade immunotherapy (CBI) is approved for head and neck squamous cell carcinoma; however, the majority of patients do not have objective responses and combinatorial therapies are needed to improve outcomes. Radiation therapy (RT) can enhance presentation of tumor antigens, and multiple studies have investigated the immunological effects of combined RT and CBI, particularly focusing on T-cells and myeloid populations. B-cells, however, have been relatively understudied, but their contributions in mounting a highly antigen-specific adaptive immune response are being further elucidated. Wild type C3H mice were injected subcutaneously with 5x105 cells of AT-84-E7-OVA into the right flank. Mice were then treated intravenously with 2 doses of 100 μg agonistic anti-CD40 antibody, 3 doses of 200 μg anti-PD-L1 antibody, and 12 Gy of focused RT. Tumors and spleens were dissected and analyzed by flow cytometry. An OVA-antigen biotinylated multimer was generated inhouse for tracking of tumor specific B-cells. Data were collected using a BD LSRII and analyzed using FlowJo. Mice treated with the combination of RT, PD-1 blockade, and CD40 activation had the highest frequency of tumor specific B-cells recognizing OVA, (from 3.3 +/-0.8 events per 50K live cells in untreated mice to 8.5 +/- 2.4; P = 0.01). We also observed a significant increase in plasma cells (from 11.9 +/- 8.0 events per 50K live cells in untreated mice to 235.5 +/- 131.2; P < 0.001). Additionally, mice treated with triple combination therapy had the highest proportion of plasma cells in the spleen (from 0.42 +/- 0.12% in untreated mice to 0.76 +/- 0.38% of all B220+ cells; P = 0.01), indicating augmented systemic B-cell immune responses induced by local radiation. Furthermore, mice treated with triple combination therapy demonstrated increased differentiation into PD-L2+ memory B cells (from 0.61 +/- 0.37% to 3.2 +/- 1.2% of all B220+ cells; P < 0.0001). Addition of CD40 agonist to RT and CBI significantly increases the frequency of systemic antigen-specific B-cells and B-cell memory. Continued investigation into the roles of distinct B-cell subpopulations and their responses to novel therapeutic combinations are ongoing and will help detail mechanisms combinatory activity.