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. Author manuscript; available in PMC: 2022 Dec 12.
Published in final edited form as: Sci Immunol. 2021 Jul 9;6(61):eabf9792. doi: 10.1126/sciimmunol.abf9792

Fig. 3. KIR3DL3 hampered both TCR dependent and independent function of CD8+ T cells.

Fig. 3

(A) KIR3DL3 and HHLA2 colocalized on T cell immune synapses. Left: Representative confocal images of cell conjugates acquired after Jurkat-Raji contact. Scale bars, 10 𝜇m. Right: The bar graph show the frequencies of KIR3DL3 clusters and HHLA2-YFP or control-YFP clusters in all cell conjugates (HHLA2-YFP+ Raji, n=76; Control-YFP+ Raji, n=43). Mouse Tim-3-YFP/Raji was used as the control.

(B-D) KIR3DL3+ CD8+ T redirected cytotoxicity assay against P815. (B) The lysis of P815 cells (n=5). (C) The degranulation (CD107a) and cytokine production (IFN-γ and TNF-α) of KIR3DL3+ CD8+ T cells (n=5). CD28 and KIR3DL1 served as a negative and positive control, respectively. (D) Cytokine production in the co-culture supernatant of KIR3DL3+ CD8+ T cells with indicated antibody-coated P815. The differentially secreted cytokines are shown in the heatmap (n=5). Data are represented as mean ± SD.

(E-F) RNA sequencing of KIR3DL3+ and KIR3DL3− CD8+ TEMRA (n=3). (E) The volcano plot of RNA-seq showing downregulated (blue) and upregulated (red) differentially expressed genes (DEGs). (F) Gene set enrichment analysis (GESA) in KIR3DL3+ and KIR3DL3− subset. Data shown are GESA plots and the heatmaps for the leading-edge genes.

(G-H) The T (G) and NK (H) cell surface receptor expression on CD8+ TN, TCM, TEM, KIR3DL3− and KIR3DL3+ TEMRA subsets as accessed by spectral flow cytometry (n=12). Data are represented as mean ± SD.

(I) Lysis of HHLA2/K562 or control K562 by KIR3DL3+ CD8+ T cells at indicated E:T ratios. Data are mean for duplicate measurements. Results are representative of three independent experiments.

(J) Lysis of scrambled control or HHLA2KO HCC827 by KIR3DL3+ CD8+ T cells at E:T =10:1 (n=4).

(K) The cytotoxicity, degranulation (CD107a) and cytokine production (IFN-γ and TNF-α) of KIR3DL3+ CD8+ T cell against HCC827 in the presence of anti-KIR3DL3 (26E10) or mIgG1 at E: T=10:1 (lysis) or 2:1 (CD107a, IFN-γ, and TNF-α) (n=4).

P values by a one-way ANOVA (B, C, G, H), and a two-tailed paired t-test (D, J, K). *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001.