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 2

Functional Tfh TIL infiltrate tumors with active immune responses.

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Functional Tfh TIL infiltrate tumors with active immune responses. (A) C...
(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-1lo/intICOSlo), TfhX13 TIL (CXCR5PD-1hiICOSint), 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-1hiICOSint; 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-1lo/intICOSlo) and functional (PD-1hiICOSint) 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.