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. 2022 Jul 6;13:931630. doi: 10.3389/fimmu.2022.931630

Figure 6.

Figure 6

Cytokine hypersecretion in the absence of NKG7 compensates for inefficient synapse-mediated cytotoxicity. (A) Schematic of CRISPR/Cas9-mediated knock out of Nkg7 in CD8+ T cells from OT-I transgenic mice. Naïve CD8+ T cells were isolated and electroporated with Cas9 complexed to sgRNA targeting Nkg7. Cells were then activated with anti-CD3/28 antibodies and used for analyses 72 hours post-activation. (B) Immunofluorescence microscopy showing NKG7 staining in CD8+ T cells electroporated with a non-targeting sgRNA (sgNT) or Nkg7-targeting sgRNA (sgNkg7), quantified in right panel, unpaired t-test, n = 3. (C) NKG7 protein expression detected by flow cytometry in sgNT and sgNkg7 OT-I T cells, and CD8+ T cells from C57BL/6J Nkg7-/- and Nkg7+/+ littermate mice, quantified in right panel; percent of NKG7+ OT-I T cells normalized for sgNT OT-I NKG7 expression equal to 100%, unpaired t-test, n = 3. (D) Specific lysis of 51Cr-labelled MC38-OVA tumor cells (targets) by sgNT or sgNkg7 OT-I T cells (effectors) in a 4-hour co-culture as measured by chromium release at increasing effector to target ratios. Relative killing (right panel) calculated as the relative efficiency of T cells to achieve 50% specific lysis of target cells, unpaired t test, n = 3. (E) Degranulation of sgNT or sgNkg7 OT-I T cells co-cultured with MC38-OVA target cells for 4 hours, measured by OT-I T cell surface exposure of CD107a during the co-culture, detected by flow cytometry, unpaired t test, pooled data from n = 3 independent experiments. (F) Cytokines secreted by sgNT or sgNkg7 OT-I T cells co-cultured with MC38-OVA tumor cells for 4 hours, measured by cytokine bead array on supernatants from co-cultures, unpaired t test, pooled data from n = 3 independent experiments. (G) TNFR1 protein expression detected by flow cytometry in wild-type (WT) MC38-OVA cells or MC38-OVA cells electroporated with Cas9 complexed to sgRNA targeting Tnfrsf1a (gene encoding TNFR1; MC38-OVA-Tnfrsf1a-/-). (H) Specific lysis of 51Cr-labelled MC38-OVA or MC38-OVA-Tnfrsf1a-/- tumor cells (targets) by sgNT or sgNkg7 OT-I T cells (effectors) in 4-hour or 18-hour co-cultures as measured by chromium release at increasing effector to target ratios. (I) Relative killing from (H), calculated as the relative efficiency of T cells to achieve 50% specific lysis of target cells, 2-way ANOVA, Tukey’s multiple comparisons test, pooled data from n = 3 independent experiments. (J) Tumor growth of MC38-OVA-Tnfrsf1a-/- cells implanted subcutaneously in Nkg7+/+ or Nkg7-/- littermates with or without CD8 depletion antibodies administered starting the day prior to tumor inoculation, n = 4-12. (K) Tumor size from (J) on day 22 post tumor inoculation, One-way ANOVA (n = 4-12). (L, M) Data pooled from Figures 1E, F and panels (J, K). All error bars show +/- SEM. ns – not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.