B Cells Improve Overall Survival in HPV-Associated Squamous Cell Carcinomas and Are Activated by Radiation and PD-1 Blockade

Abstract

Purpose: To characterize the role of B cells on human papilloma virus (HPV)-associated cancer patient outcomes and determine the effects of radiation and PD-1 blockade on B-cell populations.

Experimental design: Tumor RNA-sequencing data from over 800 patients with head and neck squamous cell carcinoma (HNSCC) and cervical cancer, including a prospective validation cohort, was analyzed to study the impact of B-cell gene expression on overall survival (OS). A novel murine model of HPV⁺ HNSCC was used to study the effects of PD-1 blockade and radiotherapy on B-cell activation, differentiation, and clonality including analysis by single-cell RNA-sequencing and B-cell receptor (BCR)-sequencing. Human protein microarray was then used to quantify B-cell-mediated IgG and IgM antibodies to over 16,000 proteins in the serum of patients treated on a clinical trial with PD-1 blockade.

Results: RNA-sequencing identified CD19 and IGJ as novel B-cell prognostic biomarkers for 3-year OS (HR, 0.545; P < 0.001). PD-1 blockade and radiotherapy enhance development of memory B cells, plasma cells, and antigen-specific B cells. BCR-sequencing found that radiotherapy enhances B-cell clonality, decreases CDR3 length, and induces B-cell somatic hypermutation. Single-cell RNA-sequencing identified dramatic increases in B-cell germinal center formation after PD-1 blockade and radiotherapy. Human proteome array revealed enhanced IgG and IgM antibody responses in patients who derived clinical benefit but not those with progressive disease after treatment with PD-1 blockade.

Conclusions: These findings establish a key role for B cells in patient outcomes and responses to PD-1 blockade in HPV-associated squamous cell carcinomas and demonstrate the need for additional diagnostics and therapeutics targeting B cells.

Figure 1:. CD19 and IGJ are B-cell specific prognostic biomarkers for overall survival in HPV-associated squamous cell carcinomas.

(A, E) Overall survival analysis based on CD19 and IGJ expression in all TCGA HNSCC cases. (B, F) Subgroup analysis of HPV+ HNSCC cases. (C, G) Overall survival analysis based on CD19 and IGJ expression in all TCGA cervical cancer cases. (D, H) Overall survival analysis based on CD19 and IGJ expression in all HPV+ HNSCC cases in our validation cohort.

Figure 2:. B-cell depletion promotes tumor growth and anti-PD-L1 immunotherapy and radiation therapy modulate B-cell activation.

(A) Tumor volumes of mice (n = 6–7 per group) inoculated with 5 × 10⁵ cells of AT84-E7-OVA tumor cells. Treatment with radiation (12 Gy x 1) and/or anti-PD-L1 CBI (200 μg every 3 days for 3 total doses) beginning on day 6. This experiment was conducted three times with similar results. (B) Tumor volumes of mice (n = 6–7 per group) inoculated with 1 × 10⁵ cells of B16-OVA tumor cells. (C) Tumor volumes of mice (n=5 per group) inoculated with 5×10⁵ cells of AT-84-E7-OVA tumor cells. Mice were treated with anti-CD20 antibody to deplete B cells. (D, F) Representative contour plots for IgD and IgM expression to define mature, T1, and T2 B-cells in the TDLN. (E, G) MHC II expression within each subpopulation of B-cells in the TDLN. (H) Representative contour plot and gating schematic for regulatory B-cells in the TDLN. (I) Percent regulatory B-cell (CD1dHigh, CD5+) in the TDLN.

Figure 3:. Radiation therapy enhances development of antigen-specific B cells, promotes plasma cell and memory B-cell differentiation, and stimulates antibody production.

(A, C) Representative contour plot of antigen-specific B cells in AT-84-E7-OVA and B16-OVA models. (B, D) Percent IgG+OVA-Multimer+ B-cells in the TDLN. (E, F) Percent plasma cells (CD138+, IgG+) in the TDLN (E) and spleen (F). (G) Antibody concentrations against OVA in B16-OVA bearing mice. (H) Representative contour plot for SP and DP memory populations, gated on IgG+ cells. (I, J) Percent of each memory population in AT-84-E7-OVA-bearing mice (I) and B16-OVA-bearing mice (J). (K-M) Percent of cells in each group that were live (PI-, AnnV-), in early apoptosis (PI-, AnnV+), and in late apoptosis (PI+, AnnV+). Groups contained 6–8 mice per group.

Figure 4:. Anti-PD-L1 immunotherapy and radiation therapy enhance B-cell clonality and somatic hypermutation.

(A) Left, Representative scatterplot demonstrating BCR sequences found in the two or three distinct samples from TDLN; right, frequency of B cell clones occurring multiple TDLN samples. (B, C) Productive clonality (B) and maximum productive frequency (C) of B-cells in the TDLN. (D) Mean number of somatic hypermutations per productive BCR sequence in the TDLN. (E) Proportion of productive BCR templates in the TDLN that possess a CDR3 length of less than 12 amino acids.

Figure 5:. Single cell RNA sequencing demonstrates that radiation combined with anti-PD-L1 immunotherapy significantly enhances development of germinal center B-cells.

(A) UMAP plot of integrated analysis of sorted B-cells from the tumor-draining lymph node of tumor-bearing mice that were untreated, or treated with RT, anti-PD-L1 CBI, or combination therapy divided with clear clusters corresponding to follicular and germinal center (GC) B-cells. (B) Ridge plots demonstrating expression of select genes between the GC and follicular B-cell clusters. (C) Heat map demonstrating the expression profile of the top 10 most differentially expressed genes in each sub-cluster of follicular B-cells (clusters F1-F4). (D) Ridge plots demonstrating expression of select genes between two sub-clusters of GC B-cells corresponding to light zone (GC 1) and dark zone (GC 2). (E) UMAP plots of GC B-cells from individual treatment groups. (F) Proportion of total B-cells from each sample belonging combined germinal center clusters.

Figure 6:. B-cell mediated IgG and IgM antibody responses correlate with objective responses to checkpoint blockade immunotherapy.

Representative plot of IgG and IgM antibody responses for all matched targets in patients treated with CBI +/− SBRT in patients with objective responses (A, B) and progressive disease (C, D). (E) Fluorescence values for top 25 of fewer IgG antibodies in region 1 for each patient shown. (F) Number of antibody responses with an IgG or IgM post-treatment fold-change greater than 8 in patients with clinical benefit versus progressive disease.