Fig. 1. FcγRIIB is expressed on CD8⁺ T cells from human PBMCs and is a marker of activated, cytokine-producing cells.

(A) Flow cytometry plots showing gating strategy for excluding dead cells as well as contaminating CD14⁺ and CD19⁺ cells and gating on the FcγRIIB^pos population in human CD8⁺ and CD4⁺ T cell compartments of PBMCs. (B-C) Quantification comparing the frequency of FcγRIIB^pos CD4⁺ and CD8⁺ T cells in (B) human healthy controls and (C) stage IV patients with melanoma. (D) Quantification comparing the frequency of FcγRIIB^pos CD4⁺ T cells between healthy donors and stage IV melanoma patient (n=6). (E) Quantification comparing the frequency of FcγRIIB^pos CD8⁺ T cells between healthy donors and stage IV patients with melanoma (n=6 per group). (F-K) PBMCs from healthy controls were stimulated in vitro for 5 days with anti-CD3/CD28 beads, re-stimulated with PMA/Ionomycin for 4h and stained intracellularly (n=8). Representative flow plots and quantification showing the frequency of (F) Ki-67⁺, of (G) IFNγ⁺, of (H) TNF⁺, of (I) TNF⁺ IFNγ⁺, of (J) CD107a⁺, and of (K) Granzyme B⁺ FcγRIIB^pos and FcγRIIB^neg CD8⁺ T cells. When comparing cell groups within the same donor, P-values were calculated using the Wilcoxon matched-pairs rank test. Mann-Whitney non-parametric, unpaired tests were used when comparing groups between donors. The error bar in summary figures denotes mean ± SEM. *P<0.05 **P<0.01, ns, not significant.