Fig 2: Immunization with GX-19 elicits Th1-biased T cell responses in mice. BALB/c mice (n = 3–7/group) were immunized at weeks 0 and 2 with indicated doses of GX-19 or pGX27 (empty control vector) as described in the Methods (a–c). Sera were collected at 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG1 and IgG2a/b. Endpoint titers (a), and endpoint tier ratios of IgG2a/b to IgG1 (b) were calculated. At 2 weeks post-boost, mouse splenocytes were isolated and re-stimulated with peptide pools spanning the SARS-CoV-2 S protein ex vivo. Indicated cytokines in the supernatants of culture were quantified using a Th1/Th2 cytometric bead array kit. Mean value of the medium alone background (mean ± s.d., pg ml−1) was 19.17 ± 8.61 for IFN-γ, 57.12 ± 6.53 for TNF-α, 33.10 ± 6.72 for IL-2, 7.83 ± 0.45 for IL-4, and 4.66 ± 0.13 for IL-5 (d). T cell responses were measured by IFN-γ ELISPOT in splenocytes stimulated with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 106 splenocytes (c). Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2. Shown are the frequency of S-specific CD4+ or CD8+ T cells after subtraction of background (DMSO vehicle, Sigma-Aldrich, St. Louis, MO, USA) (e). Data representative of two independent experiments. All data are represented as individual values. * p < 0.05, ** p < 0.01 as determined by the Mann–Whitney test.

Fig 3: Viral Infection Induces Increased Number of miR-181a-Deficient Memory CD4 T Cells(A) Equal numbers of congenically marked WT and miR-181a−/−YFP+ SMARTA CD4 T cells were co-transferred into B6 mice subsequently infected with LCMV 1 day later. Representative flow plots of SLAM and CXCR5 expression by WT and miR-181a−/− SMARTA CD4 T cells (left) and dot plots of the total numbers of SLAM+ Th1 and CXCR5+ Tfh SMARTA CD4 T cells in the spleen (right) on day 8 after infection.(B) Representative flow plots of SLAM and CXCR5 expression by IAb GP66 tetramer+ YFP+ CD4 T cells from LCMV-infected WT and miR-181a−/− mice (left) and dot plots of the total numbers of Th1 and Tfh tetramer+ CD4 T cells in the spleen (right) on day 8.(C–E) Numbers of IAb GP66 tetramer+ YFP+ CD4 T cells in the spleen at indicated time points (C) and in mesenteric and inguinal lymph nodes on day 85 (D). Representative flow plots of IAb GP66 tetramer+ cells gated on YFP+ CD4 T cells (E, left), representative histogram of tetramer binding intensity (middle), and dot plot of tetramer MFI (right). Filled gray in the histogram indicates naive CD4 T cells.(F–H) Dot plots show MFI of indicated cytokines (F), the proportion of cells co-producing IFNγ, IFNα, and IL-2 (G) and the effective GP61 peptide concentration required to elicit a half-maximal IFNγ production (H) by GP61-specific YFP+ CD4 T cells in the spleen on day 85 after LCMV.Unless stated otherwise, data are representative of two independent experiments with 4–5 mice per group (A and B) or pooled from two independent experiments with 6–9 mice per group (C–H). Data are presented as means. Statistical significance by two-tailed paired (A) or unpaired (B–H) t test. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant.

Fig 4: 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) IFNγ+ CD8 T cells, showing gating for IFNγ, TNF-α, and IL-2. (C) The percentage of CD8 T cells positive for one or more of the cytokines IFNγ, TNF-α, 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 IFNγ (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 5: Antitumor activity of Cal/ICG@MPs in H22 tumor-bearing mice upon 808 nm laser irradiation.a Schematic schedule for the antitumor experiment in stroma-rich H22 tumor-bearing mice after intravenous injection of PBS, ICG, Cal/ICG, ICG@MPs, Cal@MPs or Cal/ICG@MPs derived from H22 cells at the ICG dosage of 8 mg kg-1 and Cal dosage of 120 µg kg-1, followed with or without 808 nm laser irradiation (1.5 W cm-2, 10 min) at 2 h after the last injection. b, c Average tumor growth curves (b) and tumor weights (c) of stroma-rich H22 tumor-bearing mice after treatment indicated in a. Data are presented as means ± s.d. (n = 8 mice per group; two-way ANOVA followed by Tukey’s multiple comparisons post-test for b; n = 6 mice per group; one-way ANOVA followed by Tukey’s HSD post hoc test for c). d Kaplan–Meier survival plot of stroma-rich H22 tumor-bearing mice after treatment indicated in a. (n = 8 mice per group). e, f Percentages of CD8+ Tcm (e) and CD8+ Tem cells (f) in spleens of stroma-rich H22 tumor-bearing mice after treatment indicated in a. Data are presented as means ± s.d (n = 6 mice per group; one-way ANOVA followed by Tukey’s HSD post hoc test). g, h Tumor growth curves after rechallenge with H22 cells (2 × 106 cells, g) and 4T1 cells (2 × 106 cells, h) in naive mice or Cal/ICG@MPs with 808 nm laser irradiation-cured mice indicated in a. Data are presented as means ± s.d. (n = 3 mice per group). i, j Percentages of CD8+ Tcm cells (i) and CD8+ Tem cells (j) in spleens of the above tumor-rechallenged mice. Data are presented as means ± s.d. (n = 3 mice per group; two-tailed unpaired t test). k Proliferation ratios of splenocytes after the splenocytes from the above tumor-rechallenged mice were treated with or without H22 cell lysates (antigen) for 72 h. Data are presented as means ± s.d. (n = 3 mice per group; two-way ANOVA followed by Tukey’s multiple comparisons post test). l–o Percentages of IFN-?+ (l), IL-2+ (m), TNF-a+ (n), and GzmB+CD8+ T cells (o) after the splenocytes from the above tumor-rechallenged mice were treated with or without H22 cell lysates for 72 h. Data are presented as means ± s.d. (n = 3 mice per group; two-way ANOVA followed by Tukey’s multiple comparisons post test). p In vitro cytotoxicity of splenocytes against H22 cells after the splenocytes (effector cells) from the above tumor-rechallenged mice were pretreated with or without H22 cell lysates for 72 h and then incubated with H22 cells (target cells) at the effector/target ratio of 20:1 for 6 h. Data are presented as means ± s.d. (n = 3 mice per group; two-way ANOVA followed by Tukey’s multiple comparisons post-test). Source data and exact P values for e are provided as a Source Data file.