Journal: bioRxiv

Article Title: Anti-PD-1/PD-L1 Therapy Triggers Cognitive Deficits and Anxiety-Like Behaviors Through Tumor-Initiated Neuroinflammatory Niches in Male Mice

doi: 10.1101/2025.09.03.673981

Figure Lengend Snippet: a. Histograms representing statistical comparison of the number (n/ml) of myeloid (left) and lymphocyte (right) immune cells populations from whole blood collected from B16 or MC38-bearing mice treated with anti-PD1, anti-PD-L1or IgG. Data are represented as bars with symbols for individual data points and they are expressed by mean ± SEM, (n=4-5), * p<0.05, ** p <0.01. Statistical analyses were performed by one-way ANOVA or Kruskal-Wallis test with Bonferroni or Dunn’s correction for multiple comparisons (B16F10-IgG vs B16F10-PD-1 or B16F10-PD-L1 and MC38-IgG vs MC38-PD-1 or MC38-PD-L1 and PBS vs B16F10-IgG or MC38-IgG). b. Schematic timeline of immuno-neutralization of γδ T lymphocytes in B16- and MC38-bearing mice treated with anti-PD-L1 and IgG. B16F10 mice were injected (i.p.) with anti-PD-L1 or IgG on day 2-4-7 and with an anti-TCRγδ on days 4-7. MC38 mice were injected (i.p.) with anti-PD-L1 or IgG on days 6-10-13 and with anti-TCRγδ on days 6-10-13. Depending on the kinetics of tumor growth (B16: D5-D8, MC38: D13-D16), mice behavior was evaluated by means of OFT, NOR, EPM and TST tests, then sacrificed the day after the end of the behavioral session (B16: D9, MC38: D17) and brains, blood and tumors were collected for further analysis. c . Curves of tumoral volumes represented as mean ± SEM (n = 10) of B16-IgG/B16-PD-L1 and MC38-IgG/MC38-PD-L1 mice receiving or not anti-TCRγδ at indicated time points. Statistical comparison was performed by two-way ANOVA with Bonferroni test for multiple comparisons (MC38 IgG vs. MC38 IgG-TCRγδ, MC38 IgG vs. MC38 PD-L1, MC38 IgG vs. MC38 PD-L1-TCRγδ, MC38 IgG-TCRγδ vs. MC38 PD-L1, MC38 IgG-TCRγδ vs. MC38 PD-L1-TCRγδ, MC38 PD-L1 vs. MC38 PD-L1-TCRγδ, *** p<0.001 (IgG vs PD-L1), $ p<0.05, $$$ p<0.001 (IgG vs IgG-TCRγδ). d. Impact of anti-TCRγδ treatment on the proportion of circulating CD3 + TCRγδ + lymphocytes in blood of B16- and MC38-bearing mice treated with anti-PD-L1 and IgG. Statistical comparison was performed by one-way ANOVA test with Bonferroni correction for multiple comparisons (B16F10 IgG vs. B16F10 PD-L1, B16F10 IgG-TCRγδ vs. B16F10 PD-L1-TCRγδ, B16F10 PD-L1 vs B16F10 PD-L1-TCRγδ, B16F10 IgG vs B16F10 IgG-TCRγδ, MC38 IgG vs. MC38 PD-L1, MC38 IgG-TCRγδ vs. MC38 PDL1-TCRγδ, MC38 PD-L1 vs. MC38 PD-L1-TCRγδ, MC38 IgG vs. MC38 IgG-TCRγδ). Data are represented as bars with symbols for individual data points and they are expressed by mean ± SEM, * p<0.05, ** p<0.01. e. Impact of anti-TCRγδ treatment on anxiety-like behaviors in EPM and on short-term memory in NORT in B16- and MC38-bearing mice treated with anti-PD-L1 and IgG. Statistical analyses were performed (n=10) using one-way ANOVA or Kruskal-Wallis with Bonferroni or Dunn’s correction for multiple comparisons (B16F10 IgG vs. B16F10 PD-L1, B16F10 IgG-TCRγδ vs. B16F10 PD-L1-TCRγδ, B16F10 PD-L1 vs B16F10 PD-L1-TCRγδ, B16F10 IgG vs B16F10 IgG-TCRγδ, MC38 IgG vs. MC38 PD-L1, MC38 IgG-TCRγδ vs. MC38 PDL1-TCRγδ, MC38 PD-L1 vs. MC38 PD-L1-TCRγδ, MC38 IgG vs. MC38 IgG-TCRγδ). Data are represented as violin plot with symbols for individual data points and they are expressed by mean ± SEM, * p<0.05, ** p <0.01. CD: cluster of differentiation, CD11b: integrin alpha M subunit, CD11c: complement component 3 receptor 4 subunit, CD206: cluster of differentiation 206, CD45: leukocyte common antigen, D: day, DAPI: 4’,6-diamidino-2-phenylindol, EPM: elevated plus maze, FoxP3: forkhead box P3, IgG: immunoglobulin G, i.p.: intraperitoneal injection, Ly6C: lymphocyte antigen 6 family member C, Ly6G: lymphocyte antigen 6 family member G, MHCII: major histocompatibility complex 2, M-MDSC: monocytic-derived myeloid derived suppressor cells, NeuN: neuronal nuclei antigen, NK: natural killer, NOR: novel object recognition test, OFT: open field test, PD-1: programmed cell death 1, PD-L1: programmed cell death ligand 1, PMN-MDSC: polymorphonuclear myeloid derived suppressor cells, ROR: Retinoic acid-related Orphan Receptors, SSC: side scattering gating, TCR: T cell receptor, TST: tail suspension test.

Article Snippet: The following anti-mouse antibodies were used, with clone, fluorophore, and reference indicated in parenthesis: CD45 (REA737, VioGreen, 130-110-803, Miltenyi Biotec), CD11b (M1/70, FITC, 101206, Biolegend), CD11c (N418, PerCP, 117328, Biolegend), MHCII (M5/114.15.2, BV605, 107639, Biolegend), CD206 (APC, FAB2535A, R&D systems), Ly6G (1A8, BV421, 1238135, Sony Biotechnology), Ly6C (REA796, Vioblue, 130-111-921, Miltenyi Biotec), CD3 (17A2, BV421, 100228, Biolegend), CD4 (REA1211, PE, 130-123-206, Miltenyi Biotec), CD8 (53-6.7, FITC, 11-0081-82, Invitrogen), NK1.1 (PK136, PerCP, 1143630, Sony Biotechnology), CD19 (6D5, AF700, 115527, Biolegend), FoxP3 (MF-14, AF647, 1232040, Sony Biotechnology), RORγt (Q31-378, BV786, BD Biosciences) and TCRγδ (GL3, BV605, 118129, Biolegend).

Techniques: Comparison, Neutralization, Injection, Immunopeptidomics, Derivative Assay, Suspension