Figure 1.

Frequency of infiltrating and circulating γδ T cells expressing either Vδ1 or Vδ2 TCR δ chains in HD and CRC patients. (A) Cumulative analysis of immune infiltrates of 70 colon cancer specimens. Lymphomonocyte populations were evaluated by the use of cell-surface markers and indicated as percentage of the total number of CD45⁺ cells in each sample. (B) Box plot of percentages of Vδ1 or Vδ2 γδ T cells subsets in healthy tissue, tumor tissue and peripheral blood of CRC patients and peripheral blood of HD subjects. Boxes represent 25th to 75th percentiles; middle bar identifies median; whiskers show minimum and maximum. *p<0.05 performed by nonparametric Mann-Whitney test, unpaired and 2-tailed with confidential interval 95%. (C) Representative dot plots of the gating strategy used to define Vδ1 and Vδ2 T cells from healthy and tumor tissues. The following gating strategy was used to detect γδ T lymphocytes: FSC/SSC, single cells, live cells CD45/CD3, Vδ1 and Vδ2 T cells. (D) Sections from CRC patients were stained with anti-human pan-γδ TCR (red) and anti-CD3 (green) for immunofluorescent (IF) staining. Right panel is a magnified view and the arrows display the colocalization of γδ TCR and CD3. Nuclei were contrasted with DAPI. One of 3 independent experiments is shown. (E) Phenotypical analysis of Vδ1 and Vδ2 T cells among healthy and tumor tissues and PBMC of CRC patients, upon staining with mAbs to CD45RA and CD27, and gating on CD3⁺ Vδ1⁺ or CD3⁺ Vδ2⁺ T cells. Beside, flow cytometry panels of a representative dot plot. Isotype-matched mAbs were used as controls. Viable lymphocytes were gated by forward and side scatter, and analysis was performed on 100,000 acquired events by using FlowJo. PBMC were stained with anti-CD3, anti-Vδ2, anti-CD45RA and CD27 mAbs.