Squamous Cell Tumors Recruit γδ T Cells Producing either IL17 or IFNγ Depending on the Tumor Stage

Memory phenotype of Vδ1 and Vδ2T cells in TILs and PBMCs of SCC patients. Phenotypical analysis of (A) infiltrating and (B) circulating Vδ1 and Vδ2 T cells from SCC patients and HDs upon staining with mAbs to CD45RA and CD27, after gating on CD3⁺Vδ1⁺ or CD3⁺Vδ2⁺T cells. Data are from three independent experiments. C, Flow cytometry panels of a representative experiment, using the gating strategy described in the legend to Fig. 1. Isotype-matched mAbs were used as controls.

Cytokine production of γδ T cells in SCC patients. A, Representative gating strategy to define Vδ1 and Vδ2T cells making IL17 or IFNγ among TILs or in PBMCs of SCC patients. Cells were stimulated in vitro as described in Materials and Methods and were stained with mAbs to IFNγ and IL17 after gating on the Vδ1⁺ and Vδ2⁺ T-cell populations. B, Histograms of cumulative data from 45 SSC patients. Error bars indicate SEM (**, P < 0.01; *, P < 0.05). Each experiment was repeated three times independently. C, CD45⁺ single cells were used to generate the SPADE tree. Infiltrating cells were grouped in two different populations, CD3^– and CD3⁺ (black outer circles). The color patterns indicate that the nodes contained within the dark black boundary of CD3⁺ subset are Vδ1 or Vδ2 cells. The distribution of the major populations is shown for one representative sample. The branching tree is based on the number of cells included in each node and the legend indicates the range of cell per node according to relative median fluorescence intensity.

γδ T-cell recruitment at the tumor microenvironment of SCC patients. A, Analysis of chemokine receptor expression by circulating Vδ1 and Vδ2 T cells. Values are expressed as MFI; error bars indicate SEM. Data are from three independent experiments. B and D, Luminex analysis of chemokines (B) and cytokines/growth factors (D) production by CSCs, CAFs, and SDACs obtained from SCC patients, and cultured in vitro as described in Materials and Methods. The concentrations of each molecule is evaluated according to a color intensity scale. C, γδ T-cell migration in response of SCC conditioned medium was evaluated as described in Materials and Methods, data are expressed as a percentage of migrated cells among input. Error bars indicate SEM (*, P < 0.05). Each experiment was repeated five times independently.

Correlation between cytokine production, clinical stage of SSC, and clinical outcome. A, Cumulative data of the frequencies of Vδ1 and Vδ2 T cells producing IL17 or IFNγ in SCC patients at early or late clinical stage (I–II vs. III–IV) and in HD subjects. B, Box plot of the percentages of infiltrating Vδ1 and Vδ2 T cells in three different groups: HD, SSC patients at stage I/II, and SSC patients at stage III/IV. C, Cumulative data of the frequencies of IL17- or IFNγ-producing SSC infiltrating Vδ1 and Vδ2 T cells in recurrence/nonrecurrence, dead/live, and no lymph node invasion/lymph node invasion patients. In all experiments, data are reported as mean of percentage of positive cells ± SEM (*, P < 0.05).

Frequency of infiltrating and circulating Tregs in HDs and SCC patients. A, Flow cytometry plot of representative primary data to define Tregs in tissue and peripheral blood of HD subjects and SCC patients. Analysis was performed as described in the legend to Fig. 1. The following gating strategy was used to detect Tregs: FSC/SSC, live cells, single cells, CD3/CD4 double-positive T cells, and CD25⁺FoxP3⁺. Isotype-matched mAbs were used as controls. B, Histogram of cumulative data from 47 patients. Error bars indicate SEM (**, P < 0.01; *, P < 0.05). Data are from three independent experiments. C, Cumulative data of the frequency of Tregs in SCC patients at early or late clinical stage (I–II vs. III–IV) and in HD subjects. Error bars indicate SEM (*, P < 0.05). D, Cumulative data of the frequency of Tregs in SCC patients related with relapse rate and overall survival; error bars indicate SEM (*, P < 0.05).