Fig. 4.

Differential expression of MHC-I and MHC-II exist in the ONB TIME. (A) Representative photomicrographs at 20x magnification from two ONBs of merged and single-color immunofluorescence images assessing the presence of MHC-I⁺ cells with a validated panel of six biomarkers, CD8 (yellow, Opal 570), HLA-DR (red, Opal 690), Ki-67 (turquoise, Opal 480), MHC-II (orange, Opal 620), CD4 (green, Opal 520), and synaptophysin (white, Opal 780). Multispectral immunofluorescence images are counterstained with DAPI. Expression of synaptophysin identified tumor cells and MHC-I identified MHC-I⁺ cells. Right column is representative of a high Hyams Grade tumor (Hyams grade III) and the left column is representative of a low Hyams Grade Tumor (Hyams Grade I). (B) Quantification of percentage of MHC-I⁺ tumor cells by Hyams Grade and (C) MHC-I expression by density of CD8⁺ T cells. (D) Representative photomicrographs at 20x magnification from two ONBs of merged and single-color immunofluorescence images assessing the presence of MHC-II⁺ cells and CD4⁺ T cells with a validated panel of six biomarkers, CD8 (yellow, Opal 570), HLA-DR (red, Opal 690), Ki-67 (turquoise, Opal 480), MHC-II (orange, Opal 620), CD4 (green, Opal 520), and synaptophysin (white, Opal 780). Multispectral immunofluorescence images are counterstained with DAPI. Expression of synaptophysin identified tumor cells, HLA-DR identified MHC-II⁺ cells, and CD4 identified CD4⁺ T cells. (E) Left column is representative of a high CD4⁺ infiltrated tumor and the right column is representative of a low CD4⁺ infiltrated tumor. (F) Quantification of percentage of MHC-II⁺ tumor cells by Hyams Grade and MHC-II expression by density of CD4⁺ T cells. All lines are graphed to indicate the median value. Mann-Whitney U tests were used to test for statistical significance. **p ≤ 0.01, ns, non-significant