Functional Th1-oriented T follicular helper cells that infiltrate human breast cancer promote effective adaptive immunity
Grégory Noël, … , Denis Larsimont, Karen Willard-Gallo
Grégory Noël, … , Denis Larsimont, Karen Willard-Gallo
Published August 19, 2021
Citation Information: J Clin Invest. 2021;131(19):e139905. https://doi.org/10.1172/JCI139905.
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Research Article Immunology Oncology

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.

Authors

Grégory Noël, Mireille Langouo Fontsa, Soizic Garaud, Pushpamali De Silva, Alexandre de Wind, Gert G. Van den Eynden, Roberto Salgado, Anaïs Boisson, Hanne Locy, Noémie Thomas, Cinzia Solinas, Edoardo Migliori, Céline Naveaux, Hugues Duvillier, Sophie Lucas, Ligia Craciun, Kris Thielemans, Denis Larsimont, Karen Willard-Gallo

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Figure 5

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

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The balance between functional Tfh and Tfr TIL mediates CD8+ immune resp...
(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-1hiICOSint (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-1lo and PD-1int 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.