Fig 2: (A) CD45.2+ mice that had received an adoptive transfer of 1x106 of both OTI (CD45.1+CD3+CD8+) and OTII (CD45.1+CD3+CD4+) T cells via i.v. injection, were treated s.c. in all four hocks on days 1 and 8 with saline, or 20 μg of OVA or OVA-p(GluNAc) (5 μg per hock). On day 38, all mice were sacrificed and the dLNs and spleen were analyzed for OTI and OTII T cell phenotype. (B) OTI CD8+ T cells recovered from dLNs. (C) Central memory OTI CD8+ T cells in dLNs (left) and spleen (right). (D) Ratio of central memory to effector memory OTI CD8+ T cells (left) and representative flow cytometry contour plot of the memory subsets (right) induced in the spleen. (E) PD-1 MFI on OTI CD8+ T cells in dLNs. (F) Lag-3+ OTI CD8+ T cells in dLNs. (G) Foxp3+ OTI CD8+ T cells in dLNs (left) and spleen (right). (H) Splenocytes from the OVA-p(GluNAc) group were restimulated with 100 μg/mL OVA in culture media alone or supplemented with 200 Units/mL (~12 ng/mL) exogenous IL-2, and IFNγ levels were measured in the supernatant 3 days later by ELISA. (I) CD45.2+ mice received a first adoptive transfer of 1x106 OTI CD8+ T cells (groups 1,3) or no cells (group 2), followed by two s.c. OVA-p(GluNAc) treatments on days 1 and 8 for all groups. On day 30, mice from groups 2 and 3 received a second adoptive transfer of 5x105 OTII CD4+ T cells. All mice were administered an OVA+LPS challenge on day 31, and 10 days later, the dLNs and spleen were examined for the OTII CD4+ T cell response. (J) OTII CD4+ T cells recovered from dLNs (top) and spleen (bottom). (K) Lag-3+ OTII CD4+ T cells in dLNs (top) and spleen (bottom). (L) Numbers of IL-2 producing OTII cells in dLNs (top) or spleen (bottom) after a 6-h ex vivo restimulation with OVA323-339 peptide. (M) Numbers of TNFα secreting OTII CD4+ T cells in dLNs (top) or spleen (bottom) after a 6-h ex vivo restimulation with OVA323-339 peptide. Data are pooled from two independent experiments (n= 5-12), and represent the mean ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by one-way ANOVA using Tukey’s post hoc test in (B, C, E–G, J), and unpaired Student’s T test in (D, H, K–M).

Fig 3: Specific T cell responses elicited by the S DNA vaccine at 5 weeks post the 3rd immunization. Splenocytes were collected from each mouse at 5 weeks post the 3rd immunization. Frequencies of S protein specific IFN-γ, IL-2 and TNF-α secreting CD8+ T cells (A) and CD4+ T cells (B) were detected by ICS. iMFI (Specific T cell frequency multiplied by MFI) of specific CD8+ (C) and CD4+ T cells (D) was compared among different inoculation strategies. Data are shown as mean ± SD. Statistical differences among groups was analyzed using t-test.

Fig 4: Metabolic Intervention Modulates Cytokine Production in TB10.4-TET+ Cells(A–C) IFNγ (A), TNFα (Β), and IL-2 (C) production in CD44+CD8+ T cells purified from the lungs of uninfected (UI), Mtb-infected, or BCG-infected mice after re-stimulation with PMA/ionomycin.(D) The proportion of polyfunctional T cells within each CD44+CD8+ T cell subset, on the basis of co-expression of IFNγ, TNFα, and/or IL-2 (single, double, or triple combinations).(E and F) Fold change in IFNγ production following treatment with 2-DG (E) or etomoxir (F) relative to untreated cells re-stimulated with PMA/ionomycin.(G) Cytokine production by TB10.4-TET+ cells in the presence/absence of 2-DG or etomoxir.(H) Glucose uptake (2-NBDG MFI) in cells treated with or without PMA/ionomycin with or without 2-DG or etomoxir and representative histogram. Measurements shown in (G) and (H) represent D35 post-infection.TB10.4-TET+ cells are from Mtb-infected mice. 2-DG, 2-deoxyglucose. Statistics, unless otherwise indicated, are relative to UI. Data are representative of three independent experiments (N = 5 mice per group). “p ≤ 0.05, #p ≤ 0.01, ∞p ≤ 0.005, and ∗p ≤ 0.001 by one-way ANOVA. Error bars are mean ± SD.

Fig 5: Boosting with the S_Omicron DNA vaccine showed no improvement of mono-functional CD4+ T cell responses. Comparisons of IFN-γ (A) TNF-α (B) and IL-2 (C) producing CD4+ T cell response to the spike protein of ancestral Wuhan strain. Comparisons of IFN-γ (D) TNF-α (E) and IL-2 (F) producing CD4+ T cell response to the spike protein of Omicron variant. Data are shown as mean ± SD. Statistical analyses were performed using the method of one-way ANOVA. S_Omicron DNA vaccine, n=8; S_Wuhan DNA vaccine, n=8; PBS control, n=6.