Fig. 4: Drug-induced TLS harbor antigen-primed class-switched plasma and memory B cells.

a, Upper bar charts: the abundance of B cells in primary KPC tumors on the day of tumor resection (day 14) determined by flow cytometry. Analyses of total B cells (CD19) as well as CD69 and CD44 expression were conducted to determine early and late activation states of B cells, respectively. The abundance of total and activated B cells is presented as a percentage fraction of the total gated lymphocytes. The fraction of memory B cells was determined by CD73/PD-L2/CD19 triple staining. For each data point, three tumors were pooled and dissociated to isolate lymphocytes for the FACS analysis. A total of 6–9 tumors were examined. Data are presented as the mean ± s.e.m. and analyzed using one-way ANOVA with Tukey’s test for statistical significance. *P < 0.05; **P < 0.01. Lower bar charts: similar analyses of tumor-draining lymph nodes on day 14. For each data point, six draining (inguinal) lymph nodes were pooled for FACS analysis. A total of 18 lymph nodes were examined for each group. A representative of two independent experiments is shown for each analysis. b, CD69 and CD19 immunofluorescence of combination therapy-treated KPC tumors showing that the majority of B cells in TLS are activated. Scale bars, 100 μm (upper images) and 20 μm (lower images). c, Accumulation of IgG-expressing CD138⁺ plasma cells in TLS (arrowheads). CD138 (syndecan-1) is also expressed by tumor cells (yellow arrow). The TLS area indicated by the square is shown in higher magnification in the lower images. Scale bar, 300 μm. d, CD73⁺CD19⁺ B cells are present in TLS. The area of CD73/CD19 doubly stained cells is shown in higher magnification in the lower images. Scale bars, 100 μm (upper images) and 20 μm (lower images). Representatives of at least three independent experiments are shown in b–d.