Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity

Abstract

We previously demonstrated that tumor-infiltrating lymphocytes (TIL) in human breast cancer sometimes form organized tertiary lymphoid structures (TLS) characterized by CXCL13-producing T follicular helper (Tfh) cells. The present study found that CD4+ Tfh TIL, CD8+ TIL, and TIL-B, colocalizing in TLS, all express the CXCL13 receptor CXCR5. An ex vivo functional assay determined that only activated, functional Th1-oriented Tfh TIL (PD-1hiICOSint phenotype) provide help for immunoglobulin and IFN-γ production. A functional Tfh TIL presence signals an active TLS, characterized by humoral (immunoglobulins, Ki-67+ TIL-B in active germinal centers) and cytotoxic (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 FOXP3 gene. Functional Tfr inhibited functional Tfh activities via a glycoprotein A repetitions predominant (GARP)-associated TGF-β-dependent 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.

Keywords: Adaptive immunity; Breast cancer; Immunology; Oncology; Th1 response.

Figure 1. CXCR5⁺ TIL are primarily localized in human BC-associated TLS.

(A) Lymphocytes from fresh tissue homogenates (ref. 29) of human tonsils (n = 10) or BC (n = 168; luminal A [LumA; n = 82], luminal B [LumB; n = 36], HER2⁺ [n = 24], TN [n = 26]) were immunophenotyped (flow cytometry). The mean frequency (μ) of CXCR5⁺ cells within the CD4, CD8, and CD20 subpopulations is shown. Upper 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 (n = 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 (n = 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. (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: CXCR5^– 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.

Figure 2. Functional Tfh TIL infiltrate tumors with active immune responses.

(A) CD4⁺ TIL analyzed by flow cytometry for CD25, CXCR5, ICOS, and PD-1 surface expression. (B) Gene expression (qRT-PCR) in sorted CD4⁺CD25^– TIL subpopulations (n = 18; 1-way ANOVA): Th TIL (CXCR5^–PD-1^lo/intICOS^lo), TfhX13 TIL (CXCR5PD-1^hiICOS^int), and Tfh TIL (CXCR5⁺). (C) Representative confocal microscopy images showing CD4⁺ (blue), PD-1⁺ or CXCR5⁺ (red), and CD20⁺ (green) on consecutive sections of the tumors in Figure 1E (BC 0989) and S1F (BC 0906). A zoomed image inside the TLS B cell follicle (BC0989) or at the T:B border (BC 0906) is shown. Upper left panel magnification: ×60; upper right ×170; lower left ×45; lower right ×170. (D) Flow chart for the Tfh functional assay: 3 subpopulations of CD4⁺CD25^– TIL (Th, Tfh and TfhX13 TIL) are sorted from BC homogenates and activated ex vivo with SEB in cocultures with human splenic B cells. Cells are harvested at day 3 for immunophenotyping and culture supernatants at day 7 for immunoglobulin and cytokine quantification. (E) Assay supernatants (n = 9 for Th TIL and Tfh TIL; n = 3 for TfhX13 TIL) analyzed for IgG (left panel) and IgA (right panel) produced in assay cocultures with Th, TfhX13, or Tfh TIL. (F) IgG production in cocultures with Tfh TIL are correlated with the frequency of functional Tfh TIL (PD-1^hiICOS^int; flow cytometry) determined ex vivo (i.e., at the time of sorting for the Tfh functional assay). Left panel: cytokines/chemokines in the assay supernatants containing Th, TfhX13, or Tfh TIL; the 3 subpopulations are subdivided into nonfunctional (n = 5) and functional Tfh TIL (n = 4; Student’s t test). Middle panel: gene-expression analysis (qRT-PCR) of sorted nonfunctional (PD-1^lo/intICOS^lo) and functional (PD-1^hiICOS^int) Tfh TIL (n = 4; Student’s t test). Right panel: intracytoplasmic CXCL13 staining (% positive cells) in the Th, TfhX13, or Tfh TIL subpopulations (flow cytometry; n = 8; 1-way ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3. The proportion of Tfr TIL in human BC is associated with TLS activity.

(A) Four distinct CD4⁺ TIL subpopulations are identified based on CD25 and CXCR5 surface expression (flow cytometry): (a) Tfr (CD25⁺CXCR5⁺), (b) Tregs (CD25⁺CXCR5^–), (c) Tfh (CD25^–CXCR5⁺), and (d) the remaining Th (CD25CXCR5^–). (B) Left: nuclear Foxp3 expression in CD4⁺ TIL was positive in 11% of CD25^–CXCR5^–, 9% of Tfh, 88% of Tregs, and 86% of Tfr (flow cytometry; n = 30). Right: FOXP3 gene demethylation in sorted TIL: CD4⁺CD25^– (10%+), Treg (80%+) and Tfr (76%+) (n = 5). PBMC sorted from healthy donors: CD4⁺CD25 (4%+), Tregs (84%+) (n = 3). (C) Gene-expression analysis (qRT-PCR) of the sorted CD4⁺ TIL subpopulations in A (1-way ANOVA). (D) Left: representative dual CD4/Foxp3 (red/brown) cIHC-staining of human BC. Upper right: zoomed image inside a TLS; lower right: zoomed image just outside a TLS; both showing Tfh TIL (red membrane, blue nuclei) and Tfr/Treg TIL (red membrane, brown nuclei; location dependent for Tfr or Treg). The TLS shown is 600 μm wide. Magnification: ×40 (left panel); ×100 (right panels). (E) Sorted functional Tfr (PD-1^int) and Tfr PD-1^hi from tonsils activated in Tfh functional assay cocultures with tonsillar Tfh cells plus or minus an anti–TGF-β antibody (n = 9; 1-way ANOVA). Upper left: GARP⁺ cell frequencies among CD4⁺ cells (on assay day 3); upper right: IgG production (on assay day 7). Lower left: tonsillar Tfr subpopulations sorted on ICOS and PD-1 (flow cytometry); lower middle: GARP⁺ cell frequencies and lower right: CD25 and Foxp3 MFI for functional Tfr^int and Tfr^hi tonsillar cells (n = 7; Student’s t test). (F) Left: Tfr TIL subpopulations sorted on ICOS and PD-1 (flow cytometry); middle: GARP⁺ cell frequencies; right: CD25 and Foxp3 MFI for functional Tfr^int and Tfr^hi TIL subpopulations (n = 21; Student’s t test). *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 4. The balance of functional Tfh to functional Tfr TIL governs humoral immune responses in human BC.

(A) Correlation between the density of functional Tfh TIL (no./mg tumor; flow cytometry) and IgG (μg/mg tumor in the fresh tissue supernatant; n = 71; LumA [n = 20], LumB [n = 19], HER2⁺ [n = 12], and TN [n = 20]). (B) Representative FFPE sections (TNBC 1068 shown) were cIHC stained for scoring. Left: dual CD3/CD20 (brown/red) for T cell zones and B cell follicles; middle: dual PD-1/Ki-67 (brown/red); consecutive sections; right: zoomed image of the B cell follicle. The TLS is 1000 μm wide. Magnification: ×35 (left panels); ×140 (right panel). (C) Correlation between the frequency of PD-1⁺ cells (functional Tfh TIL surrogate) and Ki-67⁺ cells (GC TIL-B surrogate) in TLS-associated B cell follicles stained and scored as in B. Each point shows the mean score for all TLS in a given tumor. (D) The ratio of functional Tfh to functional Tfr TIL (flow cytometry) correlated with supernatant Igs (A). Upper panels: IgG (μg)/mg tumor; lower panels: IgA (μg)/mg tumor (n = 71; LumA [n = 20], LumB [n = 19], HER2⁺ [n = 12], TN [n = 20]). (E) mIHC for CD20 (green), Ki-67 (orange), CD4 (blue), PD-1 (red), ICOS (yellow), and Foxp3 (cyan) on a representative FFPE section (2 TLS; TNBC); zoomed image of the T cell zone. Contact scores between functional Tfr TIL (CD4⁺Foxp3⁺ICOS⁺) and Tfh TIL (CD4⁺PD-1^–) or functional Tfh TIL (CD4⁺PD-1⁺) or Tfr TIL (CD4⁺Foxp3⁺ICOS^–) or TIL-B (CD20⁺) in the T cell zone. Magnification: ×80 (upper right); ×100 (upper left); ×200 (lower panels). (F) Using the same mIHC-stained sections as in E, the ratios between (left) total Tfh TIL (PD-1^–+PD-1⁺) and total Tfr TIL (ICOS^–+ICOS⁺) and (right) functional Tfh TIL and functional Tfr TIL are shown for the T cell zones of 5 GC⁺ and 3 GC^– TLS (TNBC; Student’s t test). Statistical testing for all correlations used linear regression analysis. *P < 0.05.

Figure 5. The balance between functional Tfh and Tfr TIL mediates CD8⁺ immune responses in human BC.

(A) ICOS and PD-1 on CD8⁺CD25^– TIL (flow cytometry). (B) Gene expression (qRT-PCR) in sorted subpopulations from A (n = 5; 1-way ANOVA). (C) Intracytoplasmic CXCL13 (% positive cells) in subpopulations from A (n = 8; 1-way ANOVA). (D) Correlation between the frequencies of functional Tfh (in total Tfh) and activated PD-1^hiICOS^int (in total CD8⁺CXCR5⁺ TIL) (n = 71; LumA [n = 20], LumB [n = 19], HER2⁺ [n = 12], TN [n = 20]). (E) Upper left: mIHC: CD4 (blue), CD8 (green), CD20 (yellow), Ki-67 (cyan), PD-1 (red), GZMB (red) in a TLS (representative BC); upper right: GC zoom, lower left, zoom 1: CD4⁺PD-1⁺ TIL (surrogate for functional Tfh TIL; pink) and CD8⁺PD-1⁺ TIL (surrogate for activated CD8⁺ TIL; yellow) in the T cell zone and lower right, zoom 2: CD8⁺GZMB⁺ TIL (green membrane/red cytoplasm), CD4CD8^–CD20^–GZMB⁺ TIL (red sub-membrane) and tumor cells (cyan nucleus). Magnification: upper left ×50; upper right ×125; lower left ×200; and lower right ×135. (F) mIHC: CD4 (blue), CD8 (green), PD-1 (red), and Foxp3 (cyan). Left: TLS T cell zone and B cell follicle (representative BC, TLS in Supplemental Figure 5D); right: zoomed image of TLS T:B border showing functional Tfh TIL (pink membrane), activated CD8⁺PD-1⁺ TIL (yellow membrane), and Foxp3⁺CD4⁺ Tfr TIL (cyan nucleus). Magnification: left ×80; right ×250. (G) mIHC: CD68 (green) and GZMB (red) in TLS-associated T cell zone; CD68⁺GZMB⁺ TIL (orange); CD68^–GZMB⁺ TIL (red; considered CD8⁺). Magnification: ×100. (H) PD-1⁺ cells (functional Tfh TIL) and GZMB⁺ cell (CD8⁺ and CD68⁺ TIL) frequencies in TLS-associated T cell zones (n = 76 TLS; 20 tumors) correlated with CD3/CD20, PD-1/Ki-67, and GZMB cIHC scores (consecutive sections; each point = mean TLS/tumor; same tumors as in Figure 4C). (I) Ratio between functional Tfh and Tfr TIL correlated with the ratio between PD-1^lo and PD-1^int T cells (flow cytometry); upper panel: CD8⁺CXCR5^– TIL, lower panel: CD8⁺CXCR5⁺ TIL (TN BC; n = 18). Statistical tests used linear regression analysis for all correlations. *P < 0.05; **P < 0.01.

Figure 6. Active TLS signal adaptive immunity and are associated with a good prognosis.

(A) Gene-expression analysis using the Nanostring Pan Cancer Immune Profiling Panel on total tumor RNA extracted from FFPE sections of highly infiltrated BC tumors (TN and HER2⁺). The tumors are divided into active TLS (PD-1⁺Ki-67^+/–, n = 6), inactive TLS (PD-1^–Ki-67^–, n = 5), and TIL without TLS (no TLS, n = 6; 1-way ANOVA). TLS numbers and activities were scored on dual cIHC-stained (CD3/CD20 and PD-1/Ki-67) consecutive sections that followed those for RNA extraction. (B) Kaplan-Meier curves for disease-free survival. Left: relative to TLS density (n = 48; HER2⁺ and TN BC); right: relative to active TLS density (n = 32; TLS⁺ BC only) in the resected tumor. (C) Upper graph: total TLS densities; lower graph: active TLS densities compared between BC patients with (n = 13) or without (n = 35) disease recurrence (Student’s t test). (D) TIL subpopulations in all TLS scored on mIHC tissue sections (stained as in Figure 4E) from 11 BC (5 with and 6 without recurrence). Each point represents the mean of all TLS in a given tumor (Student’s t test). Disease-free survival based on (upper left) frequency of functional cells (PD-1⁺) in total Tfh TIL, (upper middle) proliferating cells (Ki-67⁺) in TIL-B, and (upper right) functional cells (ICOS⁺) in Tfr TIL. Ratios between (lower left) total Tfh TIL (PD-1^– + PD-1⁺) and total Tfr TIL (ICOS^– + ICOS⁺) and (lower right) functional Tfh TIL and functional Tfr TIL relative to disease-free survival.*P < 0.05; **P < 0.01; ***P < 0.001.