Figure 2.

Identification of 11 CD4⁺ TIL clusters based on the differential expression of ICs. (A) FlowSOM tree of CD4⁺ TILs from cohort 2 (n = 20). The background color represents meta-clustering, and the legends of the star plot and meta-clustering are shown on the right side. (B) Heatmap of the MFI of the PD-1, TIGIT and Tim-3 markers expressed, or not expressed, by the 11 identified clusters of the CD4⁺ TIL population (n = 20, cohort 2). (C) Cell frequency of the 11 generated clusters of CD4⁺ TILs (n = 20, cohort 2). (D) Cell frequency of the 11 generated CD4⁺ TILs clusters (n = 32, cohorts 2 and 3) according to the MSS/MSI status (pale green and dark blue, respectively). The clusters of the two cohorts were pooled and the cluster numbering from cohort 2 was retained; Mann–Whitney test (* p < 0.05). (E) Principal component analysis of tumors from cohorts 2 and 3 based on cell frequencies in each generated CD4⁺ TIL cluster, with MSS tumors in pale green triangles and MSI tumors in dark blue circles. Ellipses were drawn to highlight two new groups of CRC tumors: the IC^high group (in dark blue) and the IC^low group (in pale green).