Figure 5.

Antibody blockade of LAG3 or PD-L1 boosts ex vivo proliferation and cytokine production of intra-tumoral T cells from MMR-proficient LM-CRC in response to polyclonal stimuli. CFSE-labeled TIL from LM-CRC patients were stimulated with CD3/CD28 beads for four days, in the presence or absence of 10 μg/ml antagonistic antibodies. (A) Representative dot plots of CD3⁺CD8⁺ and CD3⁺CD4⁺ TIL proliferation in response to CD3/CD28 beads (a-CD3/CD28) in the presence or absence of antagonistic antibodies (a-) or isotype controls (iso ctrl). Dotplots indicated by “TIL” show proliferative responses in the absence of CD3/CD28 beads. In all other conditions, CD3/CD28 beads were added. (B) The percentages of proliferating cells (CFSE-low) within CD8⁺ and CD4⁺ T cells derived from the tumor or blood in response to CD3/CD28 beads without addition of any antagonistic antibody. Values of individual patients are presented. (C) Effects of antibody blockade of inhibitory interactions on CD8⁺ and CD4⁺ TIL proliferation (n = 7-9). Because the proliferative responses differed between patients, the results are reported as relative proliferation in the presence of antibodies compared to baseline proliferation, which was calculated by dividing the percentages of proliferating (CFSE-low) T cells in the presence of antagonistic antibody or isotype control antibody by the percentages in the control condition with only CD3/CD28 beads. Values are depicted as means with standard error of the mean. (D) IFN-γ and TNF-α accumulation in culture supernatants was quantified at day four by enzyme-linked immunosorbent assay. Values are depicted as medians with interquartile range (n = 10-11). Differences were analyzed by paired t test or Wilcoxon matched pairs test; *p < 0.05, **p < 0.01.