CD4+T regulatory cells type 1 are enriched in tumor infiltrating lymphocytes in head and neck squamous cell carcinoma Free

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Objective: Tumor-infiltrating lymphocytes (TIL) in head and neck squamous cell carcinoma (HNSCC) are known to be impaired in anti-tumor immune responses. We hypothesized that this suppression could be related to accumulation and activation of regulatory T cells Type 1 (Tr1 cells) in the tumor. Therefore, we evaluated TIL for the presence and characteristics of Tr1 cells and compared them to Tr1 cells in the peripheral blood of HNSCC patients.

Methods: To generate Tr1 cells, an in vitro culture system containing MACS-separated CD4⁺CD25^- T cells, which were obtained either from TIL or the peripheral blood of 15 HNSCC patients, was used. Lymphocytes were cultured in AIM V medium supplemented with low doses of IL-2, IL-10, IL-15 and 10% FBS for 10 days. In parallel, CD4⁺CD25^- T cells isolated from the peripheral blood of 10 normal donors were cultured in the presence of irradiated HLA-A2⁺ HNSCC cells, autologous immature dendritic cells and the same cytokines to simulate tumor microenvironment and serve as controls. Multi-color flow cytometry was used to phenotype freshly isolated and outgrowing cells stained for CD25, CD122, CD132, FoxP3, CTLA-4, IL-10 and IL-4. Their suppressor activity was measured in CFSE inhibition assays using autologous OKT3-stimulated CD4⁺CD25^- responders. Culture supernatants (SN) were tested for levels of cytokines in ELISA.

Results: In HNSCC patients, freshly isolated CD4⁺CD25^- TIL contained 67% IL-4⁺, 25% IL-10⁺ and 20% each Foxp3⁺ and CTLA-4⁺ T cells. After culture the phenotype of outgrowing T cells corresponded to that of Tr1 cells: low expression of Foxp3 (2.5% ± 3, MFI: 3.8 ± 1) and CTLA-4 (2.1% ± 1, 2.4 ± 1.4) and high expression of IL-10 (88.4% ± 5.1, MFI: 13.5 ± 2.6) and IL-2Rγ (56.2% ± 12, MFI: 195.9 ± 33.1). Tr1 cells outgrowing from PBMC had lower MFI for IL-10 and IL2R γ (p=0.0001). The suppressor activity of Tr1 cells obtained from TIL was higher than that of Tr1 cells expanded from PBMC (85.1% ± 9.7 vs. 64.8% ± 14.5). Levels of IL-10 and TGF-β₁ in culture SN of TIL was significantly higher compared to levels in SN of PBMC cultures (median: 1429pg/mL vs. 1023 pg/mL and median: 3721pg/mL vs. 3006pg/mL, respectively). In normal control cultures, outgrowing Tr1 cells were fewer and had lower suppressor activity. Conclusion: Phenotypically and functionally, Tr1 cells in TIL are different from circulating Tr1 cells in HNSCC patients. Tr1 cells with high suppressor activity are enriched in the tumor microenvironment and may contribute to the impairment of anti-tumor immune responses.