Fig 2: Anti-PD-1 increased antitumor immunity in the tumor microenvironmentFlow cytometric analysis of tumors from obese mice treated with IgG2a or anti-PD-1 was conducted to quantify frequencies of immune cell infiltrates.(A) Tumor dendritic cells (tDCs).(B) Tumor macrophages (tMacs).(C) M1-like and M2-like tMac subtypes.(D) M1-like/M2-like ratio of tMacs.(E) CD3+ T cells.(F) CD3+ T cell staining by immunofluorescence and quantification in 6 20× fields. Representative 20× images are shown. Scale bar is 200 μM. DAPI nuclear stain shows negative control without primary antibody.(G) CD8+ and CD4+ T cells.Data are shown as mean ± SEM. *p < 0.05 and **p < 0.01 by two-way ANOVA. n = 5–8. See also Figures S1 and S2.

Fig 3: Induction of cellular and humoral immune responses by PolyPEPI-SCoV-2 vaccine in mouse models. Animals received PolyPEPI-SCoV-2 or Vehicle subcutaneously at days 0, 14. IFN-γ-producing T cell responses elicited by PolyPEPI-SCoV-2 expressed as fold change in BALB/c (A) and Hu-mouse (B) models compared to the respective Vehicle cohorts; diversity of vaccine-induced T cell responses after two doses at day 28 in BALB/c (C) and Hu-mouse (D) models by ex vivo ELISpot. Test conditions: stimulation with 30-mer S-pool (three S-peptides), N-pool (four N-peptides), M-peptide, E-peptide, or 9-mer pools (s-pool, n-pool, e1, m1 peptides). For Fold change calculation the average dSFU values of the 30-mer and 9-mer stimulation conditions are pooled. (E,F) PolyPEPI-SCoV-2 induced Th1 response and no significant Th2 cytokine induction shown as average of vaccine-specific CD4+ and CD8+ T cells producing IL-2, TNF-α, IFN-γ, IL-5 or IL-10 in BALB/c (E) and Hu-mouse (F), using Intracellular cytokine staining. Mean +/-SEM are shown. 2 × 105 cells were analyzed, gated for CD45+CD3+CD4+/CD8+. Average percentage was obtained by pooling the background-subtracted values of the 30-mer stimulation conditions for each cytokine for CD4+ and CD8+ splenocytes. IgG production measured from the plasma of BALB/c (G) and Hu-mice (H). N = 6 animals at each time point, *p < 0.05, **p < 0.001 (Mann-Whitney U).

Fig 4: Astrocytic JAM-A increases pro-inflammatory protease and cytokine levels in astrocyte-CD3+ T-cell co-culture. Astrocytes were transfected with JAM-A or non-targeted siRNA (siJAM-A versus siNT), then co-cultured with CD3+ T cells for 24 h and samples processed for human protease and cytokine ELISA immunoassays. (A–C) JAM-A knock-down in astrocytes led to an increase of MMP-1 (relative log2 expression 0.38, P = 0.019) and a decrease of MMP-2 (relative log2 expression −0.13, P = 0.014) in the supernatant and decrease of ADAM9 (relative log2 expression −1.074, P = 0.05) and cathepsin C (relative log2 expression −1.174, P = 0.006) in astrocyte lysates (B). There were no significant changes in protease levels seen in lymphocyte lysates (C). (D–F) Astrocytic JAM-A knock-down led to decreased levels of (D) GM-CSF (relative log2 expression −0.79, P = 0.04) in the supernatant and (E) CCL-2 (relative log2 expression −1.3, P = 0.03), CXCL12 (relative log2 expression −0.13, P = 0.04) and serpin E1 (relative log2 expression −0.3, P = 0.03) in astrocytic lysates. There were no significant changes in cytokine levels seen in lymphocyte lysates (F). Data (A–F) are from three biological replicates; two-tailed paired t-tests were performed on probes demonstrating a visually detectable signal in normalized expression values relative to a reference control.

Fig 5: Astrocytic JAM-A does not change the total number of CD3+ or proportion of CD4+ cells within the CNS or spleen during EAE. (A–H) Flow cytometry was performed on spinal cords and spleens of WT (n = 5) and CKO (n = 5) mice with EAE on Day 5 from the onset of disease. Average EAE score: WT 2.8, CKO 2.6. (A) Representative plots show (B) similar total CD3+ cell counts in the spinal cord of CKO and WT (mean CKO 0.23% versus WT 0.19%, P = 0.42, unpaired two-tailed t-test). (C) Representative plots reflect (D) no difference found in the proportion of CD3+CD4+ cells in the spinal cord of CKOs compared with WTs (mean CKO 84.73% versus WT 87.97%, P = 0.29, unpaired two-tailed t-test). (E–H) In the spleen, total CD3+ and CD3+CD4+ counts were unchanged between CKO and WT groups (CD3+: mean CKO 24.3% versus WT 28.4%, P = 0.25; CD3+CD4+: mean CKO 53.1% versus 55.6%, P = 0.34, unpaired two-tailed t-test).