Results

Thirty three tumor/normal pairs, including 7 MSIhigh samples have been analyzed for their immunologic environment, including lymphocytes subsets, cytokine production, immune checkpoint expression and myeloid populations. Flow cytometry analysis showed Lag-3 or Tim-3 predominant patients in CD4+ and CD8+ cells and these two immune-checkpoints behave in a mutually exclusive pattern. CD4+ cells made higher amounts of IL-17 in MSIlow patients compared to the prognostically better MSIhigh patients group. Conversely, the expression of Tim-3 was higher on CD4+ cells from MSIhigh patients suggesting a more active state of these lymphocytes. In these two genetically distinct CRC populations, immune checkpoints seem to have a different functional significance. In MSIlow samples, there is an increased production of interferon gamma in tumor-infiltrating T cells (CD4+ and CD8+) versus a decreased production of this cytokine in cells expressing LAG-3 or TIM-3. On the contrary, while observing a very strong production of IFNγ in MSIhigh CRC-infiltrating T cells, LAG-3+ or Tim3+ T cells barely decreased IFNγ production. Finally, according to the PD-1 expression of CD4+ cells in MSIhigh patients, we identified two distinct populations of lymphocytes which react differently to Lag-3 or Tim-3 expression. A PD-1 high population, which is a stronger producer of IFNγ and is not inhibited by Lag-3 or Tim-3 expression except in the presence of high levels of Lag-3, and a PD-1 low CD4 population which produces much lower quantities of IFNγ which is suppressed by the presence of the two mentioned immune-checkpoints.