Fig 2: Cytokine and granzyme production by effector cells demonstrates greater terminal differentiation in mice with a fixed B cell repertoire(A–G) Seven days after immunization with the combined-adjuvant vaccine, splenocytes were incubated with SIINFEKL peptide ex vivo and assessed for cytokine production. (A) Representative flow cytometry plots of total CD8 T cells and (B) IFN?+ CD8 T cells, showing gating for IFN?, TNF-a, and IL-2. (C) The percentage of CD8 T cells positive for one or more of the cytokines IFN?, TNF-a, and IL-2. (D) The distribution of single-, double-, and triple-positive cells within all cytokine-producing CD8 T cells. (E) Multi-functional CD8 T cells, defined as producing more than one cytokine, as a percentage of all cytokine-producing CD8 T cells. (F) The percentage of cytokine-producing cells making IL-2. (G) The gMFI for IFN? (left) and IL-2 (right) of the respective cytokine+ cells.(H and I) Ag-specific CD8 T cells were identified by tetramer-staining splenocytes 7 days after mice received combined-adjuvant vaccine. (H) Representative plots displaying CD127 and granzyme B staining of tetramer+ cells. (I) Granzyme-B-positive cells as a percentage of tetramer+ CD8 T cells.(J) The gMFI of granzyme-B-positive tetramer+ cells.Data shown are means ± SEM; n = 3 mice per group, representative of 2 experiments. Significance was defined by two-tailed, unpaired Student’s t tests, where *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 3: Suppressive effect of TPP on early activation and proliferation of stimulated lymphocytes. (A) The percentage of IL-2 producing cells in TPP group, mixture group and PBS group was detected by FCM. (B) The proliferation of lymphocytes in TPP group, mixture group and PBS group was detected by FCM using CelltraceViolet. (*p<0.05, **p<0.01,***p<0.001, ****p<0.0001).

Fig 4: Assessment of RBMRNA-176-induced T cell responses and effects on proliferation of immune cell populations in the spleen. BALB/c mice were vaccinated with two shots of 20 µg RBMRNA-176 at an interval of 21 days. 7 days post-boost, splenocytes were isolated from 3 mice per group, and lymphocyte subset percentages (CD3+, CD4+, CD8+ and Tem) were measured (A–D). Splenocytes were re-stimulated with 1 µg SARS-CoV-2 Spike trimer protein. After 6 h, CD4+ T cells (E) or CD8+ T cells (F) expressing IFN-?, IL-2, IL-4, and IL-5 in response to the S Protein were detected by intracellular cytokine staining (ICS). * p < 0.05, ** p < 0.01.

Fig 5: SIRT5 deficiency has no evident impacts on CD8+ T cell activation in vitro. (A) SIRT5 protein expression in purified effector or memory CD8+ T cells and peritoneal macrophages (PM). The activated CD8+ T cells were further induced by IL-2/7 or IL-7/15 for another 4 days, to induce the effector and memory fate, and the phenotypes were confirmed by flow cytometry analysis. PMs were obtained from mice which were injected ip with 2 ml of sodium thioglycollate 3 days before. The adherent macrophages were thus stimulated with LPS for 12 h. (B) Quantification of Western blot images for SIRT5 from five biological replicates. Mean ± SD (n = 5), Student’s t-test, *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns, not significant. (C) Western blot analysis of SIRT5 expression in CD8+ T cells from Sirt5+/+ and Sirt5-/- OT-1 mice. (D) Surface activation marker expression of Sirt5+/+ and Sirt5-/- OT-1 cells at the time points of 6 and 72 h assessed by flow cytometry. (E) Representative histograms of MTG and TMRE of Sirt5+/+ and Sirt5-/- OT-1 cells on the sixth day after activation. (F) Statistical analysis of mean fluorescence intensity (MFI) of MTG and TMRE. Mean ± SD (n = 12), Student’s t-test, ***: p < 0.001.