Figure 1.

Tumor-infiltrating CD39⁺ γδT cells are abundant and express higher immunosuppression-related molecules in human colorectal cancer. (A) Representative flow cytometric analysis of CD39 expression in γδT cells in the tumor tissues. Flow plots were gated on CD45⁺ CD3⁺ TCRγδ⁺ cells. (B) Representative flow cytometric analysis of CD39⁺ γδT cells in the tumor and paired normal tissues. Plots were gated on CD45⁺ CD3⁺ T cells. Bar diagram summarizes the percentages of CD39⁺ γδT cells in the CD45⁺ CD3⁺ cells. N: normal tissue; T: tumor tissue. Data are shown as mean ± SEM; n = 109; ***p < 0.001. (C, D) Single-cell suspensions from tumors were stained with a panel of antibodies and analyzed by FCM. Flow plots were gated on CD45⁺ CD3⁺ TCRγδ⁺ CD39⁺, CD45⁺ CD3⁺ CD4⁺ CD39⁺, or CD45⁺ CD3⁺ CD8⁺ CD39⁺ T cells (C). Representative histograms are shown. Bar diagram summarizes the percentages of FOXP3⁺ cells, CTLA-4⁺ cells, and PD-1⁺ cells in CD39⁺ γδT, CD39⁺ CD4⁺ T, and CD39⁺ CD8⁺ T cells, respectively (D). Data are shown as mean ± SEM; n = 5; ns: no significance; *p < 0.05; **p < 0.01. (E) Representative flow cytometric analysis of CD39⁺ γδT and CD4⁺ Treg cells in tumor tissue. Flow plots of CD39⁺ γδT cells were gated on CD45⁺ CD3⁺ TCRγδ⁺ cells, and CD4⁺ Tregs was identified as CD45⁺ CD3⁺ CD4⁺ CD25⁺ CD127^low cells (left panel). Bar diagram summarizes the absolute numbers of CD39⁺ γδT and CD4⁺ Treg cells in the CD45⁺ CD3⁺ cells (10⁵) (right panel). Data are shown as mean ± SEM; n = 8; **p < 0.01. (F) Sorted CD39⁺ γδT, CD4⁺ Treg cells from tumors were in vitro co-cultured with CFSE-labeled allogeneic CD3⁺ T cells in the presence of CD3 and CD28 mAbs. CD3⁺ T-cell proliferation was evaluated on day 6 by FCM (left panel). Bar diagram summarizes the percentages of proliferated cells (CFSE^low) in CD3⁺ T cells (right panel). T: tumor tissue. Data are shown as mean ± SEM; n = 5; *p < 0.05; **p < 0.01.