Skip to main content
. Author manuscript; available in PMC: 2018 Aug 15.
Published in final edited form as: Cell Rep. 2018 Jul 10;24(2):379–390.e6. doi: 10.1016/j.celrep.2018.06.054

Figure 7. Blockade of APC-Intrinsic PD-1 Enhances the Synaptic Enrichment of T Cell-Intrinsic PD-1 and Inhibits T Cell-Mediated Cytotoxicity.

Figure 7

(A) Left: cartoon showing a Jurkat cell expressing PD-1–mGFP (shown as PD-1 in green) conjugated with a Raji cell co-transduced with PD-L1–mCherry and unlabeled PD-1. Right: representative confocal images of the conjugate at the indicated channel acquired 2 min after cell-cell contact. Scale bars, 10 µm.

(B–D) Same as (A), except that Raji (PD-L1+/PD-1+) cells (B), Jurkat (PD-1–mGFP) cells (C), or both (D) were preincubated with pembrolizumab and washed extensively prior to conjugation. Scale bars, 10 µm.

(E) Bar graph comparing the interface enrichment indices (calculated as described in STAR Methods) of the four conditions shown in (A)–(D). Data are expressed as mean ± SEM; n = 40 cells from three independent experiments.

(F) Cartoon illustrating the cytotoxicity assay, in which EL4 cells were used as the target for OT-I cytotoxic T cells.

(G) Bar graph summarizing the OT-I cytotoxicity under indicated conditions, with PD-1 blockade antibody J43 or isotype antibody (Iso) preincubated with neither cell, only EL4, only OT-I, or both cell types. See STAR Methods for details. Cytotoxicity was normalized to the no blockade antibody condition (i.e., EL4 and OT-I preincubated with the isotype control). n.s., not significantly different. Data are presented as mean ± SEM from three independent replicates.

(H) Left: FACS histograms showing PD-1 and PD-L1 surface expression in parental EL4 (EL4 WT), PD-1 knockout EL4 (EL4 PD-1 KO). Right: bar graph summarizing the OT-I cytotoxicity under indicated conditions. OT-I cells preincubated with either PD-1 blockade antibody J43 or its isotype was co-cultured with parental EL4 (EL4 WT) and PD-1 knockout EL4 (EL4 KO), as described in STAR Methods. Cytotoxicity was normalized to the condition containing isotype-treated OT-I and WT EL4 (i.e., OT-I [Iso] + EL4 [WT]. n.s., not significant. Data are presented as mean ± SEM from three independent replicates.