T regulatory type 1 cells in squamous cell carcinoma of the head and neck: mechanisms of suppression and expansion in advanced disease

Abstract

Purpose: Regulatory T cells play a major role in tumor escape from immunosurveillance. T regulatory cells type 1 (Tr1), a subset of regulatory T cells present in the tumor and peripheral circulation of patients with head and neck squamous cell carcinoma (HNSCC), mediate immune suppression and might contribute to tumor progression.

Experimental design: CD4+CD25-T cells were isolated from peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) of 26 HNSCC patients and 10 normal controls. The Tr1 cell phenotype was determined before and after culture in the presence of interleukin (IL)-2, IL-10, and IL-15, each at 10 to 20 IU/mL. Suppression was measured in carboxyfluorescein diacetate succinimidyl ester-based proliferation assays with or without neutralizing anti-IL-10 or anti-transforming growth factor-beta1 (TGF-beta1) monoclonal antibodies in Transwell systems. ELISA was used to define the Tr1 cytokine profile.

Results: Tr1 cells originate from CD4(+)CD25(-) precursors present in TIL and PBMC of HNSCC patients. Cytokine-driven ex vivo expansion of Tr1 precursors yielded CD4+CD25-Foxp3lowCD132+IL-10+TGF-beta1+ populations that mediated higher suppression than Tr1 cells of normal controls (P < 0.0001). Tr1 cells suppressed proliferation of autologous responders via IL-10 and TGF-beta1 secretion. Expression of these cytokines was higher in TIL-derived than PBMC-derived Tr1 cells (P < 0.0001). The Tr1 cell frequency and suppressor function were significantly higher in patients presenting with advanced than early disease stages and in patients "cured" by oncologic therapies than in those with active disease.

Conclusions: In HNSCC, Tr1 cell generation is promoted at the tumor site. Tr1 cells use TGF-beta and IL-10 to mediate suppression. They expand during disease progression and also following cancer therapy in patients with no evident disease.

Fig. 1

Phenotypic analysis of Tr1 cells obtained from PBMC or TIL of HNSCC patients. A, phenotypic characteristics of single-cell sorted CD4⁺CD25⁻ lymphocytes obtained from circulating PMBC ( ) or from tumor tissue of HNSCC patients (■). B, flow cytometry dot blots showing relative expression of selected markers on sorted fresh PBMC (top row) or tumor-derived cells (TIL; bottom row). Data are representative for CD4⁺CD25⁻ cells sorted from lymphocytes of different HNSCC patients. C, phenotypic analysis of single-cell sorted CD4⁺CD25⁻ T cells obtained from fresh PBMC versus TIL from HNSCC patients and cultured in the presence of “Tr1 cytokines” for 10 d. A and C, are mean ± SD percent positive cells from experiments done with cells of 26 different HNSCC patients. *, P < 0.0006; **, P < 0.0001. Multicolor flow cytometry was done with gates set on CD3⁺CD4⁺ T cells. D, box plots show differences in MFI in IL-10 or TGF-β₁ expression in PBMC versus TIL. E, representative flow cytometry dot blots showing relative expression of selected markers on CD4⁺CD25⁻ lymphocytes after culture in the presence of “Tr1 cytokines” for 10 d.

Fig. 2

Functional analysis of Tr1 cells obtained from PBMC or TIL of HNSCC patients. A, histograms generated using the ModFit software show proliferation of CFSE-labeled responder cells stimulated with anti-CD3/CD28 and cocultured with autologous suppressor cells for 5 d as described in Materials and Methods. Responder cells are CD4⁺CD25⁻ freshly separated T cells obtained from PBMC of normal controls or HNSCC patients or from TIL. Suppressor cells are autologous CD4⁺CD25⁻ freshly separated T cells from PBMC of normal controls or from TIL (top row). Bottom row, responder cells are CD4⁺CD25⁻ freshly separated T cells, whereas suppressor cells are autologous CD4⁺CD25⁻ cells after culture in the presence of “Tr1 cytokines” for 10 d to generate Tr1 cells. Suppressor cells were added to responder cells at the start of the cocultures at the1:1 ratio. Percent inhibition of proliferation relative to proliferation of responder cells alone is indicated in every panel. The results are from one representative experiment out of10 done with cells obtained from 10 different normal controls and 26 HNSCC patients. B, percentages of suppression of proliferation in CFSE-labeled CD4⁺CD25⁻ responder cells coincubated at the 1:1 ratio with suppressor cells derived as described in A from autologous fresh PBMC ( ) or TIL (■). C, titration of Tr1 cells (suppressor cells) derived from PBMC or TIL into CFSE-labeled autologous proliferating responder cell cultures. Suppression by Tr1 cells expanded from TIL (■) or from PBMC (◆) of proliferating responder cells is shown at various ratios. D, percentages of suppression of proliferation in single-cell sorted CFSE-labeled CD4⁺CD25⁻ responder cells by autologous suppressor cells cultured with “Tr1 cytokines” for 10 d. Suppressor cell/responder cell ratio was 1:1. Mean ± SD percent suppression for cocultures done with cells of 10 normal controls and 26 HNSCC patients.

Fig. 3

Mechanism of suppression mediated by Tr1 cells. A, responder cells are (CFSE-labeled autologous CD4⁺CD25⁻ T cells) stimulated with anti-CD3/CD28 mAb and cocultured for 5 d as described in Materials and Methods. Tr1 cells (suppressor cells) are autologous cultured CD4⁺CD25⁻ cells derived from TIL of HNSCC patients titrated into responder cells at the 1:1 ratio. Suppressor cells mediated strong suppression in the cocultures. Addition of anti-IL-10 and/or anti-TGF-β₁, but not isotype control IgG, reduced the level of suppression (P < 0.001). B, suppression of responder cells mediated by Tr1 cells was not decreased when the same cells were cocultured in presence of Transwell inserts. Addition of anti-IL-10 and anti-TGF-β₁ mAb in the presence of Transwell inserts reduced levels of suppression (P < 0.0001). A and B, mean ± SD percent suppression of proliferation of CFSE-labeled autologous responder cells observed in experiments done with cells of 10 different donors.

Fig. 4

Tr1 and Tr1 precursors in the tumor. Tissue sections of a HNSCC were stained as described in Materials and Methods and examined using immunofluorescence and confocal microscopy. A, a representative tumor section was stained with (a) anti-CD4-FITC (green), (b) anti-CD25-PE (red), and (c) anti-Foxp3-Cy5 (pseudocolored blue). d, the overlap shows the presence of CD4⁺CD25⁻Foxp3^low Tr1 cells (arrows), CD4⁺CD25⁻ Foxp3⁻ Tr1 precursor cells, and CD4⁺CD25⁺Foxp3⁺ nTreg. B, a representative tumor section stained with (a) anti-CD4-FITC (green) and anti-CD132-PE (red), with a cluster of CD4⁺CD132⁺ T cells (orange); (b) anti-IL-10-Cy5 (pseudocolored blue) and 4′,6-diamidino-2-phenylindole (white) to show cell nuclei; and (c) an overlay shows that the same cluster of cells is CD4⁺CD132⁺ and IL-10⁺. Magnification, ×400.

Fig. 5

Levels of Tr1-associated cytokines in supernatants of lymphocyte cultures. Supernatants from different Tr1 cocultures were collected on day 10 as described in Materials and Methods. Control cultures included activated T cells cultured alone and immature dendritic cells cocultured with irradiated tumor cells (data not shown). A, IL-10 was not detectable in control T-cell or immature dendritic cell cultures, whereas all Tr1 cell cultures were positive for IL-10. B, TGF-β₁ was undetectable in acidified supernatants from control cultures, whereas cultures with Tr1 cells contained TGF-β₁. Mean ± SD measured in supernatants of different cocultures done with cells obtained from 36 different donors.

Fig. 6

Correlations between Tr1 cell frequency or function and disease stage. A, IL-10, TGF-β₁, or CD132 expression and suppressor function of freshly isolated CD4⁺CD25⁻ T cells or Tr1 cells expanded in culture were measured. T cells were obtained from PBMC of HNSCC patients and were analyzed relative to the early disease stage (International Union Against Cancer I/II) versus late disease stage (International Union Against Cancer III/IV) at the time of surgery. B, IL-10, TGF-β₁, and suppressor function of Tr1 cells obtained from PBMC of HNSCC patients were analyzed relative to disease activity (active disease versus no evident disease) defined at the time of phlebotomy. The data are displayed as box-whisker plots that present mean, spread, and distribution of the data set. Suppressor function of Tr1 was measured using responder T cells in CFSE assays as described in Materials and Methods. A and B, mean ± SD percent positive cells or suppression from experiments done with PBMC of 16 different HNSCC patients. Wilcoxon rank-sum tests were used for comparisons. Significance level of 0.0028 was used to adjust for multiple comparisons.