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 3

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

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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-1int) and Tfr PD-1hi 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 Tfrint and Tfrhi 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 Tfrint and Tfrhi TIL subpopulations (n = 21; Student’s t test). *P < 0.05; **P < 0.01; ****P < 0.0001.