(B) Kaplan-Meier curves for disease-free survival. (GZMB+CD8+ and GZMB+CD68+ TIL plus Th1 gene expression) immune responses. Analysis of active versus inactive TLS in untreated patients revealed that the former are associated with positive clinical outcomes. TLS also contain functional T follicular regulatory (Tfr) TIL, which are characterized by a CD25+CXCR5+GARP+FOXP3+ phenotype and a demethylated gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-Cdependent mechanism. The activity of tumor-associated TLS was dictated by the relative balance between functional Tfh TIL and functional Tfr TIL. These data provide mechanistic insight into TLS processes orchestrated by functional Th1-oriented Tfh TIL, including TIL-B and CD8+ TIL activation and immunological memory generation. Tfh TIL, regulated by functional Tfr TIL, are an expected key target of PD-1/PD-L1 blockade. = 168; clinicopathological parameters in Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI139905DS1) scored for TIL and TLS (as in ref. 3). In line with previous studies, infiltrating lymphocytes increased in BC tissues, with higher TIL densities (TIL/mg of tissue) more frequently observed in HER2+ and TN BC (Supplemental Figure 1A) (3, 24). Examination of CXCR5+ TIL subpopulations revealed significantly lower frequencies of CD4+CXCR5+ TIL and higher frequencies of CD8+CXCR5+ TIL and CD19+CXCR5+ TIL-B compared with what was found in human tonsils (mean  values in Figure 1A; gating strategies in Supplemental Figure 1B and Supplemental Table 2). Open in a separate window Figure 1 CXCR5+ TIL are primarily localized in human BC-associated TLS.(A) Lymphocytes from fresh tissue homogenates (ref. 29) of human tonsils (= 10) or BC (= 168; luminal A [LumA; = 82], luminal B [LumB; = 36], HER2+ [= LH 846 24], TN [= 26]) were immunophenotyped (flow cytometry). The mean frequency () of CXCR5+ cells within the CD4, CD8, and CD20 subpopulations is shown. Upper LH 846 plots: CD3+CD4+CD45+ T cells; middle plots: CD3+CD8+CD45+ T cells; lower plots: CD19+CD45+ B cells. (B) TIL densities in human BC tumors (= 168; number of TIL/mg of tumor; flow cytometry) for CXCR5+ Tfh TIL, CXCR5+ TIL-B, and CD8+CXCR5+ TIL correlated with one another (linear regression analysis). (C) TIL densities (CXCR5+Tfh TIL + CD8+CXCR5+ TIL + CXCR5+ TIL-B/mg of tumor; flow cytometry) were correlated with TIL aggregates or TLS scored on dual CD3/CD20 (brown/red) cIHC-stained FFPE tissue sections (as described in refs. 3, 39) from the same tumor (= 79; linear regression analysis). (D) Upper panel: representative dual CD3/CD20 cIHC (TN BC 0989); lower panel: zoom images of 3 TLS areas: 1, inside a TLS showing the T cell zone and B cell follicle; 2 and 3, focused on areas outside the TLS. Upper panel: magnification 3.5; lower panel magnification: 35. DFNB39 (E) IF staining of a consecutive tissue section showing the 3 areas in D. Left panels: colocalization of CD20+CXCR5+ TIL-B (yellow) and CD4+CXCR5+ Tfh TIL (purple) or CD8+CXCR5+ TIL (purple) inside the TLS. Magnification 60. Right panels: CXCR5C CD4+/CD8+ (blue) or CD20+ (green) TIL outside the TLS. The T:B border is the junction between the T cell zone and the B cell follicle. Upper right magnification 100; lower right magnification 150. CD4+ TIL contain a mixture of functionally and phenotypically LH 846 diverse subpopulations, with CD4+CXCR5+ T cells classically designated Tfh, now known to include both Tfh and T follicular regulatory (Tfr) cells. A summary of the CD4+ T cell subpopulation phenotypes used in this study is provided in Table 1. Infiltration by Tfh TIL, CD8+CXCR5+ TIL, and CXCR5+ TIL-B was highly correlated (Figure 1B) and independent of the BC subtype (Supplemental Figure 1C). The global CXCR5+ TIL densities in fresh tissues (determined by flow cytometry) were well correlated with TLS scored on dual CD3/CD20 chromogenic IHCCstained (cIHC-stained) tissues from the same patient (Figure 1C and Supplemental Table 3). In contrast, TIL aggregates containing T cell TIL (CD4+and CD8+) and TIL-B (CD20+) (23) were not correlated with CXCR5+ TIL. These data suggest an important association exists between the coinfiltration of CXCR5+ TIL subpopulations and the LH 846 formation and/or presence of a TLS. Table 1 Phenotypes of CD4+ helper T cell subpopulations in human breast tumors and tonsils Open in a separate window TLS were next identified and analyzed on sequential tissue sections from TIL-positive BC (= 7) using CD3/CD20 cIHC (Figure 1D) and CXCR5, CD4 or CD8, and CD20 immunofluorescent (IF) labeling (Figure.