Fig 2: 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.

Fig 3: FOLactis efficiently enhanced the activation of DCs and T cells, and induced tumor cell death in vitro.a Colocalization analysis of FOLactis (DiO; green) in bone marrow-derived DCs (BMDCs) (DiI; red) by confocal microscopy (two-hour incubation) (One representative data was shown from three independently repeated experiments). White scale bars: Up, 50 μm; Down, 10 μm. b The percentage of mature DCs (mDCs, CD11c+CD80+CD86+) after co-incubation with Lactis or FOLactis in vitro for 24 h (n = 6, biologically independent samples). c Western blot analysis of several membrane TLR proteins and NF-κB pathway in BMDCs. The gels were loaded with equal amounts of the proteins (10 μg) (One representative data was shown from three independently repeated experiments). d Pro inflammatory cytokine concentrations in BMDC supernatants (n = 6, biologically independent samples). e Assessment of IFN-γ in coculture supernatants after T cells from the spleen of BALB/c mouse stimulated by Lactis or FOLactis for 24 hours in vitro (n = 6, biologically independent samples). f The quantification of CD69 and CD25 expression on CD8+ and CD4+ T-cell subsets in the T cells (n = 6, biologically independent samples). g Luminescence was detected by the addition of D-luciferin to CT26-GFP-Luc cells after incubated with T cells and/or FOLactis for 24 h (n = 6, biologically independent samples). Cells were examined under the microscope EVOS FL Auto Cell Imaging System (Invitrogen) and photographed. White scale bars, 200 μm. h, i The Flt3L-OX40L-sfGFP fusion protein (FO-sfGFP) was extracted from lysates of FOLactis-sfGFP. Confocal laser scanning microscopy images of mouse BMDCs treated with FO-sfGFP and allophycocyanin (APC)-anti mouse Flt3 for two hours. Confocal laser scanning microscopy images of mouse splenocytes treated with FO-sfGFP and APC anti-mouse OX40 for two hours (One representative data was shown from three independently repeated experiments). FO-sfGFP, green; APC-anti mouse Flt3 (APC anti-mouse OX40), red. White scale bars, Left, 50 μm; Right, 10 μm. For b, d–g, data were mean ± s.e.m. statistical significance was determined by two-tailed unpaired Student’s t-tests. ns represented p > 0.05. Source data are provided as a Source Data file.

Fig 4: Co-inhibitory receptors exert distinct quantitative and qualitative control over T cell re-activation(A) Experimental design for blockade of co-inhibitory receptors.(B) Tg4 Nr4a3-Tocky Il10-GFP mice were immunized s.c. with 80 μg of [4Y] MBP. 24 h later mice were randomized to receive either 0.5 mg isotype pool (1:1 ratio of rat IgG1 and rat IgG2a), anti-Lag3, or anti-PD1 30 min prior to re-challenge with 8 μg [4Y] MBP peptide. Splenic CD4+ T cells were analyzed for Nr4a3-Blue versus Nr4a3-Red analysis 4 h after peptide re-challenge.(C and D) Summary data from (B) detailing the percentage of responders (percent of Nr4a3-Blue+Red+, C) or median Nr4a3-Blue within Nr4a3-Blue+Red+ CD4+ T cells (D) in isotype (n = 5), anti-Lag3 (n = 6), or anti-PD1 (n = 6) treated mice. Bars represent mean ± SEM, dots represent individual mice. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test.(E) Tg4 Nr4a3-Tocky Il10-GFP mice were immunized s.c. with 80 μg of [4Y] MBP. 24 h later, mice were randomized to receive 0.8 mg isotype pool, 0.8 mg anti-Lag3, or 0.8 mg anti-PD1 30 min prior to re-challenge with 8 μg [4Y] MBP peptide. Splenic CD4+ T cells expression of Nr4a3-Blue versus Nr4a3-Red 4 h after peptide re-challenge in pre-sorted (top) and sorted (bottom) populations.(F) RNA was extracted from the sorted populations and 3′ mRNA-seq performed. PCA of the normalized expression data identified 3 clusters, n = 2.(G) KEGG pathway analysis of DEG between isotype and anti-PD1 treated groups.(H) Z score heatmap analysis of log2 transformed and normalized counts displaying the 69 DEG between isotype and anti-PD1 groups, in isotype, anti-PD1, or anti-Lag3 groups.(I) Tg4 Nr4a3-Tocky Il10-GFP mice were immunized s.c. with 80 μg of [4Y] MBP. 24 h later mice received 0.5 mg rat IgG2a or or anti-PD1 30 min prior to re-challenge with 8 μg [4Y] MBP peptide. Splenic CD4+ T cells were analyzed for Nr4a3-Blue versus Nr4a3-Red analysis 4 h after peptide re-challenge. Gates were set on responding T cells.(J) Summary data from (I) for cells responding to dose 2 (n = 4). Bars represent mean ± SEM. ∗p < 0.05 by unpaired t test.(K) Histograms showing expression of Nr4a3, OX40, GITR and ICOS in responding T cells (indicated by gates in I) between isotype (gray)- or anti-PD1 (blue)-treated cells.(L) Summary data of the median of expression of the stated markers in responding T cells, n = 4. Bars represent mean ± SEM. Statistical analysis by unpaired t test. ∗∗p < 0.01, ∗∗∗p < 0.001.(M) Analysis of intracellular IRF8 in CD4+ Tg4 T cells from isotype (gray)- or anti-PD1 (blue)-treated mice, n = 4. Bars represent mean ± SEM. Statistical analysis by unpaired t test. ∗p < 0.05..(N) Analysis of intracellular STAT4 in isotype or anti-PD1 treated mice. Bars represent mean ± SEM, n = 4.Please also see Figures S4 and S5 and Table S3.