Fig 2: Intermittent 3HB scheduling affects systemic expression of T cell inhibitory receptor and their ligands.(A and B) Experimental setting for intemittent diet and combination immune checkpoint blockers (cICB) in the RET model and Kaplan Meier curves of overall survival for 12 animals/group, according to therapeutic scheme comparing continuous (Cont) versus intermittent (On/off) diet interventions. (C) Flow cytometric analyses of PD-1 and CTLA-4 expression in splenic CD8+ T lymphocytes at day 17, after diet (ketogenic diet [KD] or 3-hydroxybutyrate [3HB] Cont or On/off) and systemic cICB combinatorial regimen in RENCA bearing BALB/c mice. Refer to Supplemental Figure 5, A and B, for Tim-3, Lag-3, and 4-1BB in all splenic T cells. (D and E) Flow cytometry analysis of MFI for the membrane expression of costimulatory (CD86 in D) or inhibitory (PD-L1 in E) on macrophages (D) and other myeloid subsets from the spleens at day 17, after complete diet (3HB On/off) and systemic cICB combinatorial regimen in RENCA-bearing BALB/c mice. The results from 2–3 experiments involving 6 mice/group are depicted; each dot represents 1 spleen. (F and G) In vitro effects of increasing dosing of 3HB onto a DC cell line stimulated or not with rIFN-?. Flow cytometry determination of MHC II/I-Ab (F) and PD-L1 expression (G) as mean fluorescence intensity (MFI) on the surface expression at 48 hours of stimulation. One representative experiment out of 4 is depicted, yielding similar conclusions (n = 12 or each groups). Student’s t test (C), global comparison using Kruskall-Wallis test with post hoc multiple comparisons using Dunn’s test (*P < 0.05) (D and E). Global comparison using ANOVA with post hoc multiple comparisons using Dunnett’s test (*P < 0.05) (F and G). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: 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 4: 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).

Fig 5: 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.