Fig 2: Correlation between multiple autologous HLA allele-binding epitopes and PolyPEPI-SCoV-2-specific IFN-?-producing CD8+ T cell responses in COVID-19 convalescent individuals. (A) Correlation between multiple autologous HLA allele-binding epitopes and magnitude of T cell responses. Rs, Spearman coefficient (confirmed by Pearson correlation analysis, too) (B) Magnitude of CD8+ T cell responses detected for PEPIs (binding = 3 autologous HLA class I alleles) and for non-PEPIs (binding <3 autologous HLA class I alleles) by enriched FluoroSpot assay, (p = 0.008, t-test). Median and individual data for each subject are presented, n = 15 (C) Variable dependency analysis using 2 × 2 contingency table and Fisher Exact test. (D) Confirmation of Personal Epitopes (PEPIs) by IFN-? producing CD8+ T cells for each subject [Positive Predictive Value (PPV) = True positive/Total predicted = 37/44 (84%)]. dSFU, delta spot forming units calculated as non-stimulated background corrected spot counts per 106 PBMC; PBMC, peripheral blood mononuclear cells.

Fig 3: T cell–dependent effects of ketogenic diet and synergy with immune checkpoint blockade.(A) Effects of T cell depletion on tumor sizes at day 12 of diet interventions. Tumor sizes in all diet-fed (normal diet [ND], ketogenic diet [KD], or 3-hydroxybutyrate per os [3HBpo]) groups in the presence of i.p. injections of anti-CD4 and anti-CD8 depleting Abs prior to tumor inoculation. A representative experiment out of 2 is depicted, both yielding similar conclusions; each dot represents 1 mouse, and each group contained 5–6 mice. Mann Whitney U statistical analyses was used. *P < 0.05. (B) Experimental setting for combinatorial regimen. (C and D) Effects of diet interventions on tumor size (C) at day 9 after RET inoculation and diet intervention, after only 1 i.p. administration of cICB (anti–CTLA-4 plus anti–PD-1 mAbs) and overall survival (D). Results of 4 concatenated experiments are depicted; each dot represents 1 mouse (n = 30 for ND and KD groups; n = 12 for 3HBpo and 3HBip). (C) Fold increase of tumor sizes in cICB groups fed with ND, KD, or 3HBpo or 3-hydroxybutyrate i.p. (3HBip) (normalized to ND groups). (D) Overall survival appreciated with Kaplan Meier curves for 12 animals/group after a complete treatment combining continuous diet plus cICB, according to B. (E) Flow cytometry analyses of Tc1 (defined as CXCR3+CD8+ T splenocytes) at day 9 of the combinatorial regimen; each dot represents 1 spleen. Results from 2 pooled experiments are shown. (F) Effects of sucrose supplementation on the synergy between diets and cICB against RET tumor progression. Individual tumor sizes at day 19 of 3 concatenated experiments involving 6 mice/group are shown for each diet intervention; each dot represents 1 tumor/mouse (n = 27 for groups without sucrose, n = 28 for groups on sucrose). Student’s t test (A); global comparison using Kruskall-Wallis test and post-hoc multiple comparisons using Dunn’s test (C and E–F). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Flt3–/–mice show more prominent CD8+T-cell accumulation upon Helicobacter infection. (A) Percentage of CD8+ T cells, CD4+ T cells, neutrophils, and macrophages in live single cells and of Tregs in live CD4+ T cells from the stomach of Flt3–/– and WT mice with H felis infection (n = 13–17 for each H felis infection group, n = 9–11 for the noninfection group). Representative flow cytometry plots of T cells (left). (B) As in panel A, but with H pylori infection (WT, n = 7; Flt3–/–, n = 5). Note that the number of CD8+ T cells is higher in Flt3–/– mice. (C) Representative H&E images of the gastric corpus regions of H pylori–infected WT (left) and H pylori–infected Flt3–/– (right) mice were aligned. Scale bar: 200 µm. (D) More severe mucosal infiltration of T cells in the stomach of H felis–infected Flt3–/– mice. Representative images of CD3 IHC (3,3'-diaminobenzidine substrate). Scale bar: 200 µm. (E) The T-cell fraction in the spleens of WT and Flt3–/– mice 8 weeks after H felis infection. Note that DC depletion did not affect CD8+ T cells, CD4+ T cells, or the Treg population in the spleen (WT, n = 10; Flt3–/–, n = 9). (F) Relative IFN-?, Granzyme A, and Perforin mRNA expression levels in gastric corpus tissues from Flt3–/– and WT mice after H felis infection for 8 weeks. Results are shown as fold change compared with the noninfected WT group (n = 6–7 for the H felis infection group, n = 2–3 for the noninfection group). (G) Chemokine levels in gastric corpus tissues from Flt3–/– and WT mice using a magnetic Luminex immunoassay. Measured protein concentrations (pg/mL) were corrected for total protein content (mg/mL) (n = 7 and 9 for groups with 4 and 8 weeks of H felis infection, respectively; n = 5 for noninfection group). Data are presented as means ± SEM. *P < .05 and **P < .01. Weeks, duration of infection.

Fig 5: 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-?-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-a, IFN-?, 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).