Immune-Activated B Cells Are Dominant in Prostate Cancer

Abstract

B cells are multifaceted immune cells responding robustly during immune surveillance against tumor antigens by presentation to T cells and switched immunoglobulin production. However, B cells are unstudied in prostate cancer (PCa). We used flow cytometry to analyze B-cell subpopulations in peripheral blood and lymph nodes from intermediate-high risk PCa patients. B-cell subpopulations were related to clinicopathological factors. B-cell-receptor single-cell sequencing and VDJ analysis identified clonal B-cell expansion in blood and lymph nodes. Pathological staging was pT2 in 16%, pT3a in 48%, and pT3b in 36%. Lymph node metastases occurred in 5/25 patients (20%). Compared to healthy donors, the peripheral blood CD19⁺ B-cell compartment was significantly decreased in PCa patients and dominated by naïve B cells. The nodal B-cell compartment had significantly increased fractions of CD19⁺ B cells and switched memory B cells. Plasmablasts were observed in tumor-draining sentinel lymph nodes (SNs). VDJ analysis revealed clonal expansion in lymph nodes. Thus, activated B cells are increased in SNs from PCa patients. The increased fraction of switched memory cells and plasmablasts together with the presence of clonally expanded B cells indicate tumor-specific T-cell-dependent responses from B cells, supporting an important role for B cells in the protection against tumors.

Keywords: B cells; T cells; prostatic neoplasms; sentinel lymph node biopsy.

Figure 1

Representative gating strategy using the Freiburg panel. The B cells can be divided into five subpopulations based on expression of surface markers (a). Gating strategy revealing the five subpopulations. The numbers correspond to different subpopulations (b). All cells are gated on live CD19⁺ cells.

Figure 2

CD19+ B cell distribution in different cell compartments. Cells isolated from blood and lymph nodes were stained with surface markers and analyzed on a flow cytometer. (a–f) Mean percentages of CD19⁺ B cells in total lymphocytes and of the B-cell subpopulations in CD19⁺ lymphocytes are shown. (g) Igλ/Igκ isotype distribution was determined by flow cytometry. The proportion is calculated by dividing Igλ with Igκ percentages of CD19⁺ cells. (n blood = 13; n LN = 74). All error bars indicate SEM. Percentages of lymphocyte subpopulations were compared with Student’s two-tailed t-test. p: * < 0.05, **** < 0.0001.

Figure 3

Peripheral B-cell compartment in prostate cancer patients and healthy donors. Buffy coat cells isolated from blood were stained and analyzed on a flow cytometer. Mean percentages of CD19⁺ B cells in total lymphocytes and of the B-cell subpopulations in CD19⁺ lymphocytes are shown. Igλ/Igκ isotype distribution was determined by flow cytometry. The proportion is calculated by dividing Igλ with Igκ percentages of CD19⁺ cells. All error bars indicate SEM. Percentages of lymphocyte subpopulations were compared with the Student two-tailed t-test. p: * ≤ 0.05, ** ≤ 0.01 *** ≤ 0.001.

Figure 4

Correlation of clinical parameters and CD19 B cells in peripheral blood and lymph nodes of patients. Patients were stratified based on clinical parameters and analyzed for B-cell populations. Mean percentages of CD19⁺ B cells in total lymphocytes and of the B-cell subpopulations in CD19⁺ lymphocytes are shown. Dotted line marks the expected normal IgL/IgK ratio of 0.7. All error bars indicate SEM. Percentages of lymphocyte subpopulations were compared with the Student two-tailed t-test. p: * ≤ 0.05, ** ≤ 0.01 *** ≤ 0.001.

Figure 5

B-cell distribution in SN and N-SN. (a) Correlation of clinical parameters and CD19 B cells in lymph nodes of patients. Patients were stratified based on clinical parameters and analyzed for B-cell populations. (b,c) B cells stained for different isotypes. Mean percentages of CD19⁺ B cells in total lymphocytes and of the B-cell subpopulations in CD19⁺ lymphocytes are shown. All error bars indicate SEM. Percentages of lymphocyte subpopulations were compared with the Student two-tailed t-test. p: * ≤ 0.05, ** ≤ 0.01 *** ≤ 0.001 **** ≤ 0.0001.

Figure 6

V(D)J sequencing and analysis. B cells were purified from N-SN (N), peripheral blood (P), and SN (S) from three patients (patient 1–N1, P1, S1–PSA: 9.9, Gleason Score: 7a, Stage: T3a, Age: 71; patient 3–N3, P3, S3- PSA: 8.6, Gleason Score: 9, Stage: T3a, Age: 78; patient 4–N4, P4, S4–PSA: 8.6, Gleason Score: 6, Stage: T2, Age: 67). Total RNA was isolated and subsequently used for cDNA synthesis and library preparation of CDR3 region of V(D)J segments using SMARTer Human BCR IgG IgM H/K/L Profiling Kit (Takara). The PCR product was sequenced (MiSeq Illumina platform) and analyzed using the Immunarch package. (a) Relative abundance for clonotypes identified in N-SN, peripheral blood, and SN. (b) Heatmap representing hierarchical clustering according to the number of public clonotypes across light (left) and heavy (right) chains of IgG and IgM isotypes. (c) Visualization of tracking the top 5 Igκ clonotypes identified in SN across all samples. (d) Visualization of tracking the top 5 Igλ clonotypes identified in SN across all samples.