Fig. 3. FcγRIIB^pos CD8⁺ T cells are resistant to immune checkpoint blockade in an Fc-dependent manner.

(A) Schematic of experimental setup where PBMCs from human healthy controls were cultured with anti-PD-1-mAb or anti-CTLA4 mAb for 5 days and were re-stimulated with PMA/Ionomycin and stained intracellularly. Responses were measured by frequency of Ki-67⁺ of FcγRIIB^pos and FcγRIIB^neg CD8⁺ T cells (n=6). (B) Representative flow cytometry plots showing the frequency of Ki-67⁺ of FcγRIIB^pos and FcγRIIB^neg CD8⁺ T cells in untreated human controls and after incubation with anti-PD-1 or anti-CTLA4 mAbs. Quantification showing the change in frequency of Ki-67⁺ of FcγRIIB^neg and FcγRIIB^pos CD8⁺ T cells after incubation with anti-PD-1 mAb (C) and anti-CTLA-4 mAb (D) in vitro compared to untreated controls. (E) Schematic of experimental setup wherein PBMCs from healthy controls were cultured with anti-PD-1-mAb or anti-PD-1-F(ab) for 5 days and were re-stimulated with PMA/Ionomycin and stained intracellularly. Responses were measured by frequency of Ki-67⁺ of FcγRIIB^pos and FcγRIIB^neg CD8⁺ T cells (n=6).(F) Representative flow plots showing the frequency of Ki-67⁺ of FcγRIIB^pos and FcγRIIB^neg CD8⁺ T cells after incubation with anti-PD-1-mAb or anti-PD-1-F(ab). (G) Quantification comparing the frequency of FcγRIIB^neg CD8⁺ T cells after incubation with anti-PD-1 mAb or antI-PD-1 F(ab). (H) Quantification comparing the change in frequency of Ki-67⁺ of FcγRIIB^pos CD8⁺ T cells after incubation with anti-PD-1-mAb or anti-PD-1-F(ab) compared to untreated controls. *P<0.05, ns, not significant. Wilcoxon matched-pairs rank test.