Fig. 7: Anti-PD1⁺/TIGIT⁺ ICI treatment reduces tumor cell proliferation through two distinct mechanisms.

A Immunohistochemistry and quantification of proliferating (%Ki67⁺) cells in the optic nerves of Nf1-OPG mice (IgG, n = 9 mice; α-TIGIT, n = 10 mice; α-PD1, n = 9 mice; α-PD1 + TIGIT, n = 6 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-PD1, P < 0.0001; α-PD1 + TIGIT, P = 0.001). Scale bar, 200 µm. B, C Ccl4 and Ccl5 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 Immunocytochemistry and quantification of proliferating (%Ki67⁺) optic glioma tumor cells (o-GTCs) treated with TCM from unstimulated (none) and exhausted (α-CD3+α-CD28 antibody stimulated) CD8⁺ T cells (Control, n = 8; TCM none, n = 4; TCM α-CD3+α-CD28, n = 4). 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. Scale bar, 200 µm. E Volcano plot showing fold change and P value comparing CD8⁺ PD1⁺ T cells (corresponds to the dataset shown in Fig. 2; n = 10 pooled mice) and in CD8⁺ PD1^- T cells (corresponds to the dataset shown in Fig. 2; n = 10 pooled mice) the optic nerve of 12-week-old Nf1-OPG mice. Upregulated genes in red, downregulated genes in blue. Differential analyses were performed using gene specific analysis (GSA). F Graph showing levels of secreted TGFβ in the TCM of unstimulated (none) and exhausted (α-CD3+α-CD28 antibody stimulated) CD8⁺ T cells by ELISA (none, n = 4; α-CD3+α-CD28, n = 4). 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. G Tgfb1 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 Holm-Šídák post-test correction. Exact P values are indicated. H Schematic representation of the anti-TGFβ treatment (α-TGFβ). Nf1-OPG mice were treated (200 µg/dose, i.p., twice per week) from 12 to 16 weeks of age, 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/z17k582. I Immunohistochemistry and quantification of proliferating (%Ki67⁺) cells in the optic nerves of Nf1-OPG mice (IgG, n = 5 mice; α-TGFβ, n = 4 mice). 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. Scale bar, 200 µm. J Immunohistochemistry and quantification of CD8⁺ T cells in the entire optic nerve of Nf1-OPG mice (IgG, n = 5 mice; α-TGFβ, n = 4 mice). 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. Scale bar, 200 µm. K Schematic representation of the direct and indirect mechanisms by which CD8⁺ exhausted T cells regulate Nf1-OPG growth. Created in BioRender. Chatterjee, J. (2024) BioRender.com/z17k582.