Fig. 3: Immune and tumor heterogeneity as a possible cause of therapy resistance to anti-PD-1.

a–d Heterogeneous distribution of leukocytes and immune cells in tumors after PD-1 treatment. a. Tumor (A375) bearing Hu-mice that received anti-PD-1 as in Fig. 2h, showed dense leukocyte infiltration of leukocytes (bottom panel; scale bar: 500 μm) when compared to mice that received control mouse IgG (top panel; scale bar: 100 μm) as determined by H&E staining. b Tumor (A375) bearing Hu-mice that received anti-PD-1 showed a heterogeneous distribution of CD4⁺ and CD8⁺ T cells (stitched image right panel; scale bar: 500 μm) when compared control Ig treated Hu-mice that had a sparse distribution of T cells (right panel). Please see Supplementary Fig. 9 for digital quantification of CD4⁺ and CD8⁺ T cells. c Tumor (A375) bearing Hu-mice that received anti-PD-1 showed either low to moderate (left panel; scale bar: 50 μm) or robust (right panel; scale bar: 50 μm) tumor-infiltration of CD4⁺ (brown) and CD8⁺ (blue) T cells within the same tumor. d MassCyTOF staining shows heterogeneous and higher distribution of CD8⁺ T cells (magenta) within the nestin⁺ tumor (A375) cells (dark blue) in anti-PD-1 treated tumor-bearing mice (lower panels) as compared to low infiltration of CD8⁺ T cells (upper panels) in untreated Hu-mice (see f for quantification). Distribution of GrB⁺ T cells (yellow arrows; 2nd to right bottom panel) was heterogeneous as they were higher on the bottom half (digital quantification:131 counts) of the tumor section when compared to the remainder of other nestin⁺ tumor cell areas (digital quantification: 37 counts). e CD8⁺ T cells are of memory phenotype as they stain for CD45RO (light blue; top panel) and areas not infiltrated by CD8⁺ T cells reveal the presence of CD4⁺/FOXP3⁺ cells (magenta arrows; bottom panel). f Quantification of MassCyTOF images by ImageJ software indicates a significant increase (p = 0.0049) in CD8⁺ T cells and Granzyme B⁺ T cells; CD45RO⁺ memory T cells compared to untreated mice and increase in FOXP3⁺ Treg cells in the indicated top or bottom half panels. All histology and MassCyTOF staining were consistent and confirmed in replicates of 2. g, h Downmodulation of HLA class I (white arrows) was observed in tumor areas that were associated with higher FOXP3⁺ cells (p = 0.0351; see e bottom panel; a representative image). Confirmation by quantitative analysis of mean intensity (MI) of HLA-class I expression (h). A one-sided paired t-test was used for analysis when p-values are provided. Representative scale bars in d, e, g represent 50 μm. All observations (a–e, g) were consistent, were observed in technical replicates (CyTOF), confirmed in histology staining, and in replicate experiments.