Figure 2.

Tfh cells accumulate in murine spontaneous and transplantable cancer models. (A) Representative flow cytometry analysis of PD-1 and CXCR5 expression on CD4⁺ T cells from 4T1 tumor-bearing mice. (B), Wild-type mice were inoculated with EL4 lymphoma, B16F-10 melanoma, LLC1 lung adenocarcinoma, Tc1 non-small cell lung cancer or MC38 colorectal carcinoma (C57BL/6 mice) or CT26 colorectal carcinoma, 4T1 triple negative breast cancer (BALB/c mice). When tumors reached 100–150 mm², the spleen, non-draining lymph node (ndLN), tumor-dLN and tumor were excised. The percentage of Tfh among CD4⁺ T cells was analyzed by flow cytometry. (C) Representative flow cytometry of CD25, ICOS and BCL6 expression on Tfh cells (CD4⁺ T cells expressing PD-1 and CXCR5) and on CD4⁺ T cells negative for CXCR5 and PD-1 markers in draining lymph nodes from 4T1 tumor bearing mice. (D) Tfh cells (CD4⁺CXCR5⁺PD-1⁺) and other CD4⁺ T cells (CD4⁺CXCR5^-PD-1^-) were sorted by flow cytometry from spleen. BCL6, ASCL2, Pdcd1, CXCR5 and IL21 expression was determined by real-time PCR. The relative gene expression was normalized to Actb. (E) Wild-type C57BL/6 mice were treated by intraperitoneal injection of urethane once a week for 10 weeks. Then, control mice and treated mice were sacrificed 4 months after the first injection. lungs, spleen and draining mediastinal lymph nodes were harvested and the Tfh cell proportion was assessed by flow cytometry. (F) B16 tumor serial sections were stained with anti-CD4, anti-BCL6 and anti-CD8 antibodies and analyzed by immunohistochemistry. Arrow: enlarge image; dotted red line: TLS. Results are shown as mean±SEM of at least three representative and independent experiments; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, versus ‘naïve’ in (B) versus ‘CXCR5^-PD-1‘ in (D) and versus ‘tumor free’ in (E). BCL6, B-cell lymphoma 6; CXCR5, C-X-C motif chemokine Receptor 5; ICOS, Inducible T cell costimulator; IL21, interleukin 21; ns, not significant; PD-1, programmed cell death-1; Tfh, T follicular helper cells.