Figure 6. T_FHX13 tumor-infiltrating lymphocytes (TIL) in human breast cancer (BC) are correlated with specific B-TIL maturation stages.

(A–H) Flow cytometric analyses. (A) CXCR5 expression in specific lymphocyte subpopulations from patient peripheral blood (P-PB) and BC (n = range 16–27 BC). One-way ANOVA followed by Tukey’s test. (A–D) Gating strategies are shown in Supplemental Figure 6A; ^$B cells were analyzed in the total cell gate. (B) Correlation between the number of CXCR5⁺ cells in specific TIL subpopulations and total CXCL13⁺ TIL in fresh BC homogenates. (C) Germinal center (GC) T_FH (PD-1^hiCXCR5^hi) or T_FHX13 (CXCL13⁺CD4⁺; red) cells in tonsils and BC (dot plots); correlation between #GC T_FH and #T_FHX13 BC TIL (graph). (D) Equally high T_FHX13 TIL responses (left) but distinct proliferating (Ki67⁺) B-TIL subpopulations (middle and right) in 2 BC. B cell differentiation markers are shown for total CD19⁺ (green) and Ki67⁺CD19⁺ (blue) B-TIL (GC B cells = CD38⁺IgD^–; plasmablasts/plasma cells [PC] = CD38^hiIgD^– or CD38^hiCD27^int/hi; CD27 = memory B cell marker). (E) Correlation between #GC T_FH and #T_FHX13 TIL defined in C and #B-TIL subpopulations defined in D. (F) Correlation between % memory cells (excluding PC) in Ki67⁺ B-TIL (lymphocyte gate) and PC in Ki67⁺ total B-TIL (total cell gate) from 4 BC with sufficient numbers of Ki67⁺ B-TIL (gating strategies in Supplemental Figure 6B). (G) PD-1/ICOS–defined CD4⁺ T cell subpopulations from tonsils. (H) CD45RA and CXCR5 expression on PD-1^loICOS^loCD4⁺ T cells from tonsils and BC. (I) Expression of PDCD1 (PD-1), CXCL13, and IL21 genes (qPCR) in CD4⁺ T cell subpopulations sorted from 5 tonsils. One-way ANOVA followed by Sidak’s test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.