Fig. 4: Ex vivo phenotyping of intratumoral T and myeloid cell subsets by flow cytometry.

A Frequency of intratumoral CD3⁺ T cells gated on CD45⁺ cells, CD4⁺ T cells, CD4⁺ Tconv (CD3⁺CD4⁺CD127^-/+CD25^-), CD4⁺ Tregs (CD3⁺CD4⁺CD127^loCD25^+/high) and of CD8⁺ T cells, expressed as a percentage of CD3⁺ T cells, in PF vs PD. B Spearman correlation between the intratumoral frequency of CD3 and Tregs (both expressed as a percentage of CD45⁺ cells), assessed by FC in PF vs PD. The graphs display non-linear regression curves and 95% confidence intervals. C Distribution of CD4⁺ and CD8⁺ T cell subsets (EMRA = CD45RA⁺CD27⁻; CD45RA⁺CD27⁺ cells; CM = CD45RA^-CD27⁺; EM = CD45RA^-CD27⁻) expressed as a frequency of CD4⁺ and CD8⁺ T cells, respectively, in PF vs PD. D Mean fluorescence intensity (MFI), expressed as Geomean (gMFI) of PD-1 in EMRA CD4⁺ T cell compartment in PF vs PD. E Distribution of the total myeloid compartment, defined as Lineage^- (CD3^-CD19⁻CD56⁻), HLA-DR⁺CD11c⁺, in PF vs PD. F Representative histograms showing the frequency of PD-L1⁺ TAMs (gated on Lineage^-HLA-DR⁺CD11c⁺CD14⁺CD16⁺CD163⁺CD206⁺) in PF vs PD (plain color in orange and gray, respectively) and isotype controls (relative dotted lines) are shown on the left. Cumulative data are shown on the right. G gMFI of ICOS-L in Lineage^-HLA-DR⁺CD11c⁺CD14⁺CD16⁺CD163⁻CD206⁻ TAMs. H Frequency of conventional DC1 (cDC1) cells, expressed as a frequency of CD141⁺ cells, within lineage^-HLA-DR⁺CD11c⁺CD14⁻CD16⁻ cells, in PF vs PD. In all panels, PF: n = 6; PD: n = 4 (biologically independent samples). Data were presented as individual values showing mean ± SEM. Statistical tests: two-tailed unpaired t-test (Mann–Whitney test) (Panels A, C–H). Nonparametric Spearman correlation (Panel B). If not indicated, no statistically significant difference was observed. Tconv T conventional, Treg T regulatory, EM effector memory, CM central memory, EMRA effector memory RA. Source data relative to all panels are provided as Source Data File.