CyTOF analyses of CD3⁺cells. (A) FDLs generated from X-shift within VorteX visualization environment of CD3⁺ cells from 7 primary cHLs and 10 RLNTs. A total of 7350 events was collected from each sample, and the resulting 124 950 events were pooled together prior to clustering. The X-shift algorithm clusters events according to similarities in expression of CyTOF panel proteins, grouping events with shared lineage, differentiation, and polarization within the pool. Every identified unique population is labeled with a specific color based on the hex color code. (B) CD4⁺, CD8⁺, and CD3⁺/CD4⁻/CD8⁻ T-cell clusters defined by the relative expression of CD4⁺, CD8⁺, or neither marker. (C) Heat map of relative expression of the indicated CyTOF panel proteins in clusters denoted by hex color code (y-axis). Relative expression defined by a z-score. Indicated clusters contain >5% of sampled events per case. Clusters are defined by the expression of lineage, differentiation, polarization, and function markers (top, x-axis; bottom, phenotype key). (D) Graphical representation of relative PD-1 levels in CD4⁺ cell subsets. The 3 TFH subsets express the highest levels of PD-1, and naive cells do not express PD-1. By comparison, Tregs are negative for PD-1, and Th1-polarized effector cells (EM cells and TEMRA cells) have increased PD-1 expression.