Analysis of β2M and MHC class I expression on HRS cells by CyTOF. (A) Rosetted HRS cells separated from bare HRS cells by sequential gates. Events from the nonsinglet population are split according to CD3⁺ expression. Events within the CD3⁻ gate are further gated to identify the CD4⁻/CD8⁻ population. The CD3⁻/CD4⁻/CD8⁻ subset was positively gated to select cells that coexpressed CD30 and CD15, representing bare HRS cells. Within the CD3⁺ population, CD15⁺/CD30⁺ coexpression identifies rosetted HRS cells. (B) CD3⁺ expression on rosetted HRS cells (upper panels) and bare HRS cells (lower panels). β2M (C) and MHC class I (D) expression on bare HRS cells (cyan) and CD3⁺ rosetted HRS cells (dark blue) in each of the primary cHLs (1-7). Significant differences were identified using the Wilcoxon rank-sum test. (E) Dual immunohistochemical analysis (PAX5 [red] and MHC class I [brown]) of primary cHLs (cases 2, 5, and 7) and a cHL with known HRS cell expression of MHC class I (positive control [Pos.]); arrows denote HRS cell membranes.³