Figure 1.

The TME of HPV16⁺ OPSCC tumors is highly infiltrated with CD14^‒CD33^‒CD163⁺ myeloid cells. Paraffin-embedded OPSCC tissue of 20 patients was analyzed by triple immunofluorescent confocal microscopy analysis with antibodies directed against CD14 (red), CD33 (blue) and CD163 (green). (A) The left panel depicts an example of an OPSCC sample with dense infiltration of the stromal compartment. the right panel depicts examples of (1) a CD14⁺CD33⁺CD163⁺ cell, (2) a CD14⁺CD33^‒CD163⁺ cell, (3) a CD14^‒CD33⁺CD163^‒ cell and (4) a CD14^‒CD33^‒CD163⁺ cell. (B) Scatter plot depicting (from left to right) CD14⁺CD33^‒CD163^‒, CD14⁺CD33^‒CD163⁺, CD14⁺CD33⁺CD163^‒, CD14⁺CD33⁺CD163⁺, CD14^‒CD33⁺CD163^‒ and CD14^‒CD33^‒CD163⁺ cell infiltrates/mm² for each patient in the epithelium (top) and stroma (bottom). Cells were manually counted and represented as the number of cells per mm² for each slide (average of five images at 250× magnification). (C) Scatter plots and correlation analysis between the number of total CD14^‒CD33^‒CD163⁺ myeloid cells/mm² (epithelium+stroma; Y-axis) and total CD8⁺, CD8⁺Foxp3^‒Tbet⁺ and CD4^‒Foxp3^‒Tbet⁺ (CD4, from left to right; X-axis) cells/mm². Pearson’s correlation with the coefficient of determination (r²) and p value is depicted for each correlation analysis. The dotted lines represent the 95% CI. Data for total CD8⁺, CD8⁺Foxp3^‒Tbet⁺ and CD8^‒Foxp3^‒Tbet⁺ (CD4) have been described before.3 (D) Stacked bar graph depicting (from top to bottom) CD14⁺CD33^‒CD163^‒, CD14⁺CD33^‒CD163⁺, CD14⁺CD33⁺CD163^‒, CD14⁺CD33⁺CD163⁺, CD14^‒CD33⁺CD163^‒ and CD14^‒CD33^‒CD163⁺ cell infiltrates/mm² in eight HPV16⁺ immune response-negative (HPV16⁺IR^‒; left) and 12 HPV16⁺IR⁺ (right) patients with OPSCC. (E) Kaplan-Meier survival curve showing the survival of 19 patients with OPSCC. The patients were grouped into low (blue) or high (red) based on the median numbers of CD14^‒CD33^‒CD163⁺ cells/mm² of the total group. statistical significance of the survival distribution was analyzed by log-rank testing and differences were considered significant when p<0.05. HPV, human papillomavirus; OPSCC, oropharyngeal squamous cell carcinoma; TME, tumor microenvironment.