Fig 2: Circulating CD4+CD8+ cells in Tg8 mice are not exported from the thymus but from the spleen. A) Kinetics of CD4+CD8+ cell appearance in thymus, spleen, mesenteric lymph nodes (mLN), inguinal lymph nodes (ILN), bone marrow (BM), and liver of Tg8 mice determined by flow cytometry (data are representative of n = 4 mice for each indicated time point). Cells were positively gated using CD3 and analyzed for the expression of CD4 and CD8. B) Biotin was injected into the thymus of 5-week-old Tg8 or wild type (WT) mice. 24 h later, biotin+ cells recently emigrated from the thymus were identified in spleen and mLN by staining with an anti-biotin antibody. Data are the mean ± SEM (n = 4 mice per genotype). C) Biotin was injected in the spleen of 5-week-old Tg8 or WT mice. 24 h later, biotin+ cells recently emigrated from the spleen were identified in thymus, mesenteric lymph nodes (mLN), inguinal lymph nodes (ILN), bone marrow (BM), and liver. Data are the mean ± SEM (n = 3 mice per genotype). In (B) and (C) cells were gated using CD3 and analyzed for streptavidin and CD4 and CD8 expression. In (B) and (C): * p < 0.05 ** p < 0.01 (Mann-Whitney test). DP, CD4+CD8+ double-positive cells; 4SP, CD4+ single-positive cells; 8SP, CD8+ single-positive cells; w.o., week-old.

Fig 3: Characterization of the immune response elicited by the RBD vaccine candidate(A) Antibody linear epitopes mapped onto the structure of the FL spike protein. Each domain of the FL protein (NTD, RBD, furin cleavage, FP-fusion peptide, and S2) is outlined with a different color (left), and the linear epitopes are shown as gold spheres within the spike domains used for immunization (center and right). (B) Anti-RBD IgG levels measured in bronchoalveolar lavage (BAL) of FL- and RBD-vaccinated BALB/c mice at day 38. (C) Neutralizing antibody titers in sera collected from RBD- or FL-vaccinated BALB/c mice (n = 5) at day 14 (prime) and day 38 (boost), measured through a neutralization assay with infectious SARS-CoV-2-and Vero cells. (D) Neutralizing antibody titers in sera collected at day 38 from C57BL/6 mice (n = 5) vaccinated with increasing doses of RBD vaccine (5–10–20 μg) in a prime-boost regimen. (E) Serum anti-RBD IgG levels measured over time in sera of RBD-vaccinated C57BL/6 mice (prime-boost regimen, n = 5) up to 6 months starting from prime. (F) T cell immune response (IFN-γ+ and TNF-α+) in CD8+ and CD4+ cells measured by intracellular staining of splenocytes collected from FL- and RBD-vaccinated BALB/c mice (n = 5) at day 38 and restimulated with pool S1 peptides. (G) IFN-γ-producing T cells measured by ELISpot assay performed on BALs collected from BALB/c mice (n = 5) vaccinated with FL and RBD, intranasally challenged with 20 μg RBD protein at day 42, and culled the day after. ∗p value < 0.05.

Fig 4: Impact of anti-tumor immunity on tumor microenvironment (TME) and survival. (A) Changes in Melan-A TNFα and IFNγ functionality of CD8+ and CD4+ cells in PBMCs. Isotype IgG2b, Melan-A VLP (open bar, n = 4); αCTLA-4 i.d., Melan-A VLP (blue, n = 9); αCTLA-4 i.v., αPD-1 i.v., Melan-A VLP (black, n = 7); αCTLA-4 i.d., αPD-1 i.v., Melan-A VLP (purple, n = 10); and αCTLA-4 i.d., αPD-1 i.d., Melan-A VLP (orange n = 10). *, # Denotes statistical significance by unpaired, two-tailed and one-tailed Student’s t test (p < 0.05), respectively. Error bars indicate SEM. (B) Multiplex immunofluorescence (mIF) and H&E of tumor tissue harvested at 17 days post implant (d.p.i.) in (i) untreated animals and animals treated with (ii) αCTLA-4 i.v., αPD-1 i.v., Melan-A VLP; (iii) αCTLA-4 i.d., αPD-1 i.d., Melan-A VLP; and (iv) αCTLA-4 i.d., αPD-1 i.v., Melan-A VLP. Scalebars in miF and H&E panels correspond to 200 μm. (C) Multiplex immunofluorescence of PD-L1+ cells (CK+, F40/80+ and CD45+CD3E+) in TME at 17 d.p.i. for animals treated with IgG2b i.d. (X, grey, n = 5); αCTLA-4 i.v., Melan-A VLP (▲, blue, n = 5); αCTLA-4 i.d., Melan-A VLP (■, green, n = 5); αCTLA-4 i.d., Gag-VLP (○, n = 5); αCTLA-4 i.v., αPD-1 i.v., Melan-A VLP (black, n = 5); αCTLA-4 i.d., αPD-1 i.d., Melan-A VLP (⬣, orange n = 6); and αCTLA-4 i.d., αPD-1 i.v., Melan-A VLP (◆, purple n = 5). * Denotes statistical significance by unpaired, two-tailed Student’s t test (p < 0.05). Error bars indicate SEM. (D) Kaplan-Meier survival curve shows lymphatic delivery of combinational therapy with Melan-A VLP significantly improves survival up to 30 d.p.i. * Indicates significant difference between αCTLA-4 i.d., αPD-1 i.d., Melan-A VLP, and no treatment. i.d., intradermal; i.v., intravenous. Scale bar: 200 μm.

Fig 5: Hypoxia and HIF-1α, but Not HIF-2α, Support CD8+ T Cell Acquisition of Effector Phenotype(A) CD8+ T cells were isolated from spleens of HIF-1αfl/fldlckCRE (maroon) or HIF-2αfl/fldlckCRE (green) and littermate control (black) mice, and activated with αCD3/CD28 for 48 hr, then expanded for 5 days in IL-2 and subjected to 21% or 1% O2 for 24 hr. Expression of CD44 and CD62L by flow cytometry is shown.(B) Intracellular expression of IFNγ, TNFα, and granzyme B in HIF-1αfl/fldlckCRE (maroon) and HIF-1αfl/fl control littermates (black) by flow cytometry after restimulation (n = 3, error bars represent SD).(C) Intracellular expression of IFNγ, TNFα, and granzyme B in HIF-2αfl/fldlckCRE (green) and HIF-2αfl/fl control littermates (gray) by flow cytometry after restimulation (n = 3, error bars represent SD).(D) Surface expression of costimulatory molecules/checkpoint receptors CD137, OX40, GITR, PD-1, TIM-3, and LAG3 on CD8+ T cells isolated from HIF-1αfl/fldlckCRE (maroon) HIF-1αfl/fl control littermates (black), activated and expanded as in (A) (n = 3, error bars represent SD).(E) Same as in (D) on CD8+ T cells isolated from HIF-2αfl/fldlckCRE (green) or HIF-2αfl/fl control littermate mice (gray) (n = 3, error bars represent SD).(F) Relative mRNA levels of Vegfa (left, n = 3) and amount of VEGF-A in media (right, n = 4, error bars represent SD) on CD8+ T cells for the indicated time points after αCD3/CD28 activation.(G) Amount of VEGF-A in media from CTLs expanded as in (A) and cultured under 21% O2 or 1% O2 for 24 hr (n = 4, error bars represent SD).(H) Relative expression of Vegfa in total LLC tumor lysate and immune populations isolated from subcutaneous Lewis lung carcinoma (LLC) tumors 11 days after implantation (n = 10, error bars represent SD).Grouped data were assessed by two-way ANOVA for multiple comparisons with Bonferroni correction. ∗∗∗∗p < 0.00005, ∗∗∗p < 0.0005, ∗∗p < 0.005, ∗p < 0.05. MFI, mean fluorescence intensity. See also Figure S2.