Fig 1: CD8+T cell predominance is governed by NF1/RAS regulation of Ccr4 expression. A, CD8+ T cells content in Nf1-OPG optic nerves (n = 10) is similar to controls (n = 8) at 3, 4.5, 6, and 9 WOA, but increases by 12 WOA (P < 0.0001). Nf1+/– mice (n = 6) have similar numbers of CD8+ T cells as controls from 3 to 12 WOA. B, CD4+ T cell content does not change in Nf1-OPG optic nerves relative to controls at 3, 4.5, 6, 9, and 12 WOA. C, Ccl2 did not increase CD4+ T cell migration in WT or Nf1+/– mice. Ccl12 or medium (control) does not increase CD4+ T cell migration. Ccl2 increased Nf1+/– CD8+ T cell migration relative to WT controls (P < 0.0001). Nf1+/- and WT CD8+ T cells treated with medium (Control) or Ccl12 exhibit similar migration. D, Nf1+/- and WT CD4+ T cells had similar levels of Ccr4 expression. Nf1+/– CD8+ T cells have increased Ccr4 expression relative to their WT counterparts (P = 0.0474). E, Ccr4 inhibition (AZD2098; 15µM) in CD8+ T cells decreases Ccl2-induced, but not Ccl12-induced, migration (P = 0.0063). F, Nf1+/– and WT CD4+ T cells had similar levels of Ras activity. Nf1+/– CD8+ T cells have increased Ras activity relative to their WT counterparts (P = 0.0363) G, Inhibition of Ras activity (10µM IN-1) reduces Ccl2-induced,but not Ccl12-induced, CD8+ T cell migration (P = 0.0104). H, RAS activity inhibition (10µM IN-1) decreases Ccr4 expression in Nf1+/- CD8+ T cells (P = 0.0069). I, Proposed model of Ccl2 signaling through Ccr4 in Nf1+/– CD8+ T cells.
Fig 2: Anti-PD1+/TIGIT+ ICI treatment reduces CD8+ T cell infiltration by decreasing TAM-mediated Ccl12 and Cxcl13 chemoattraction.A Schematic representation of the anti-PD1 (α-PD1), anti-TIGIT (α-TIGIT), and combined (α-PD1 + TIGIT) antibody treatments. Nf1-OPG mice were treated (200 µg/dose/ i.p., twice per week) from 12 to 16 weeks of age, and tissues were analyzed at 16 weeks. The control group was injected with anti-IgG isotype control antibodies. Created in BioRender. Chatterjee, J. (2024) BioRender.com/r98n914. B Immunohistochemistry and quantification CD8+ T cells in the entire optic nerve (IgG, n = 10 mice; α-TIGIT, n = 10 mice; α-PD1, n = 6 mice; α-PD1 + TIGIT, n = 5 mice). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated (α-TIGIT, P = 0.0054; α-PD1, P = 0.0003; α-PD1 + TIGIT, P = 0.0032). Scale bar, 200 µm. C Ccl2 RNA expression in the optic nerves of Nf1-OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 5, 2 pooled optic nerves per sample; α-TIGIT n = 5, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. D Volcano plot showing fold change and P value comparing Nf1-OPG TAM to Nf1+/ and WT monocytes in the optic nerves of 12-week-old mice. Upregulated genes in red, downregulated genes in blue. Differential analyses were performed using gene specific analysis (GSA). E Ccr2, Cxcr3, and Cxcr5 expression in T cell populations from 12-week-old Nf1-OPG mouse optic nerves, color-coded by T cell type. F Cxcl9, Ccl12, and Cxcl13 RNA expression in the optic nerves of 12-week-old WT and Nf1-OPG mice. Data are represented relative to the WT group (Cxcl9; WT n = 3, 2 pooled optic nerves per sample; Nf1-OPG n = 4, 2 pooled optic nerves per sample; Ccl12; WT n = 4, 2 pooled optic nerves per sample; Nf1-OPG n = 4, 2 pooled optic nerves per sample; Cxcl13; WT n = 4, 2 pooled optic nerves per sample; Nf1-OPG n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. To evaluate statistical differences, a two-tailed non-parametric Mann–Whitney test was performed. Exact P values are indicated. ns, not significant. G Graph showing the percentage of migrated Nf1+/- CD8+ T cells treated with medium (Control) (n = 5), Ccl12 (n = 6), or Cxcl13 (n = 5). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. Created in BioRender. Chatterjee, J. (2024) BioRender.com/r98n914. H Ccl12 RNA expression in the optic nerves of Nf1-OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 5, 2 pooled optic nerves per sample; α-TIGIT n = 5, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 3, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Tukey’s post-test correction. Exact P values are indicated (α-TIGIT, P = 0.0012; α-PD1, P = 0.0187; α-PD1 + TIGIT, P = 0.0013). I Cxcl13 RNA expression in the optic nerves of Nf1-OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 4, 2 pooled optic nerves per sample; α-TIGIT n = 4, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Tukey’s post test correction. Exact P values are indicated (α-TIGIT, P = 0.0003; α-PD1, P = 0.0007; α-PD1 + TIGIT, P = 0.0001).
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