Figure 2.

Expansion of effector cells in the blood during pembrolizumab (Pembro) therapy. A, A schematic of lesion occurrences and therapies over time. B-G, PBMC samples were collected at multiple times as depicted and stained for multiple markers, including T-cell subset markers CD8, CD4, and FoxP3, as well as markers for T-cell differentiation (CD45RA, CD45RO, CCR7, and CD62L), activation and inhibition (41BB, PD-1, CTLA-4, ICOS, and OX40), and proliferation (Ki67) by flow cytometry. Live, single cells were gated first by excluding populations expressing CD14, CD16, CD19, CD20, CD56, CD303, and TCRγδ followed by gating on CD3⁺CD4⁺CD8⁻ and CD3⁺CD4⁻CD8⁺ subsets. B, The percentage of proliferating CD4⁺ and CD8⁺ T cells, by Ki67 expression. CD4⁺ (C) and CD8⁺ (D) T cells were assessed for levels of activation and inhibitory markers 41BB, PD-1, CTLA-4, ICOS, and OX40. The percent expression of these markers within the CD4⁺ or CD8⁺ T-cell subset was tracked over time in the blood during therapy. E, The differentiation status of the CD8⁺ T-cell pool tracked over time as a percentage within the CD8⁺ subset with gating as follows (T_N, CD62L⁺CCR7⁺CD45RA⁺CD45RO⁻; T_CM, CD62L⁺CCR7⁺CD45RA⁻CD45RO⁺; T_EM, CD62L⁻CCR7⁻CD45RA⁻CD45RO⁺; T_EMRA, CD62L⁻CCR7⁻CD45RA⁺CD45RO⁻). F, The CD8:Treg ratio was determined by dividing the number CD8⁺ T cells by the number of CD4⁺FoxP3⁺ T cells at the same time point. G, The CD8:CD4 ratio was determined by dividing the number CD8⁺ T cells by the number of CD4⁺FoxP3⁻ T cells at the same time point.