Radiation Therapy Activates Interferon-Responsive Genes in Follicular B-Cells in the Tumor-Draining Lymph Node.

Purpose/Objective(s) Radiation therapy (RT) constitutes a fundamental treatment modality for head and neck squamous cell carcinoma. As the use of checkpoint blockade immunotherapy becomes more prevalent and combinatorial therapies are explored, it is imperative to understand how RT alters specific immune cell subsets. Prior studies have investigated the effect of RT on T-cells and myeloid cells; more recent studies have begun to study B-cells and tertiary lymphoid structures, yet much remains to be uncovered as B-cells are critical in facilitating an antigen-specific adaptive anti-tumor response. Materials/Methods Wild type C3H mice were injected subcutaneously with 5 × 105 cells of AT-84-E7-OVA into the right flank. Mice were then treated with 12 Gy of focused RT direct at the tumor. Tumor-draining lymph nodes were dissected and FACS was used to isolate B-cells. Cells were then counted and loaded onto a 10x-Genomics Chromium controller. 10x-Genomics v3 libraries were then prepared per manufacturer recommendations and then sequenced with a target of a minimum of 20000 reads per cell using an Illumina Hiseq 4000. Single-cell RNA sequencing data were then initially processed using Cell Ranger, converted into matrix files, and subsequently analyzed using Seurat v3. Pathway analysis was performed using Metascape. Results Single-cell RNA-sequencing data of mice that were untreated and treated with RT alone revealed 7 distinct clusters of B-cells in the tumor-draining lymph node that could be broadly distinguished as being follicular vs germinal center B cells. Amongst the follicular B-cells, there was one cluster that constituted 1.7% of B-cells in untreated mice and 5.1% in mice treated with RT. Compared to naive follicular B-cells, this cluster had 88 upregulated genes; pathway analysis demonstrated that these genes were involved in: “response to interferon-alpha” (7 genes, log(P) = -11.3), and “positive regulation of interferon-beta production” (5 genes, log(P) = -6.5). Upon closer analysis, cells in this cluster derived from mice treated with RT had 77 genes upregulated compared to those from untreated mice. Successive pathway analysis exhibited that these genes were related to: “generation of precursor metabolites and energy” (10 genes, log(P) = -5.9), “positive regulation of cell cycle” (6 genes, log(P) = -2.7), amongst others. Conclusion RT significantly increased the proportion of B-cells that expressed genes involved in interferon-related pathways and additionally altered the intrinsic phenotype of this B-cell subset. Continued investigation into how B-cells react to conventional therapies may reveal further therapeutic targets to successively polarize B-cell activation and exploit B-cell-mediated anti-tumor responses.