Fig 2: Conjugating peptide antigens to CPPs increases antigen-specific T cell responses in vitro and in vivo. (A) Schematic structure of antigen-CPP conjugates prepared using azide/alkyne click chemistry and sequences of eight tested CPPs. (B and C) Splenocytes from C57BL/6 mice were pulsed for 1 h with the indicated gp100 peptide or CPP conjugate then cocultured with naïve CFSE-labeled pmel-1 CD8+ T cells. Pmel-1 T cell activation was assessed by flow cytometry analysis of CD69 up-regulation at 24 h (B) and proliferation (CFSE dilution) at 72 h (C). Significance relative to the gp100 long peptide was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test: ****P < 0.0001. (D–H) C57BL/6 mice (n = 4 to 5 animals per group) were immunized twice with 5 nmol of the indicated antigen and 25 µg cyclic-di-GMP, followed by restimulation of peripheral blood mononuclear cells (PBMCs) at day 21 with peptide to detect cytokine-producing antigen-specific T cells by flow cytometry. (D) Timeline of immunization experiments. (E) Representative flow cytometry plots (Left) and quantification (Right) of gp100-specific IFN-γ+/TNF-α+ CD8+ T cells. (F) Representative flow cytometry plots (Left) and quantification (Right) of Adpgk-specific IFN-γ+/TNF-α+ CD8+ T cells. (G) Linker placement for the antigen gp100 to pAntp and MPG. The original, a click linkage between the antigen C terminus and the CPP N terminus, is denoted “c-n”; “c-c” denotes a click linker between the antigen C terminus and the CPP C terminus; “–“ denotes a variant with a peptide bond linking the antigen and the CPP (i.e., synthesizing the antigen-CPP as a single long peptide). (H) Percentage of IFN-γ+ of CD8+ T cells after a prime and boost with the CPP conjugates shown in G. Significance relative to the gp100 long peptide (or Adpgk) was determined by two-way ANOVA followed by Dunnett’s multiple comparisons test: ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig 3: In the absence of B7-CD28 co-stimulation, the frequency of cells undergoing clonal deletion at DP and CD4SP stages is decreased.a–d WT and B7 DKO thymocytes were gated on developmental stages as defined by TCR and CD69 expression in Fig. 1. a Frequency of clonally deleting cells (Helios+ PD-1high) was decreased in Stage III (CD4+ CD8low) thymocytes in B7 DKO mice. Each group n = 3. Data are representative of three independent experiments. p = 0.0365. b Frequency of clonally deleting cells (Helios+) was decreased in Stage IV and V SP4 Tconv (CD25/Foxp3nega) in B7 DKO mice. Each group n = 3. Data are representative of three independent experiments. Stage IV p = 0.0004, Stage V p = 0.0003. c Frequency of clonally deleting cells (Active Caspase3+) was decreased in TCR-signaled DP and SP cells in B7 DKO mice. Each group n = 5. Data are pooled results of two independent experiments. Signaled DP p < 0.0001, Signaled SP4 p = 0.0038. d Frequency of clonally deleting cells (Helios+) was decreased in Stage IV and V SP4 Tconv (CD25/Foxp3nega) in Bcl2-Tg/B7 DKO mice. WT n = 9, B7 DKO n = 3, Bcl2-Tg n = 5, Bcl2-Tg/B7 DKO n = 3. Data are pooled results of three independent experiments. Stage IV (WT and B7DKO) p = 0.0003, Stage IV (Bcl2-Tg and Bcl2-Tg/B7DKO) p = 0.0037, Stage V (WT and B7DKO) p = 0.0008, Stage V (Bcl2-Tg and Bcl2-Tg/B7DKO) p = 0.020. e Nur77-GFPhigh SP4 cells were increased in B7 DKO mice. Left panel, representative FACS plot. Right panel, quantification of Nur77-GFPhigh and SP4 Nur77-GFPhigh populations. Each group n = 6. Data are pooled results of three independent experiments. Total p < 0.00001, SP4 p = 0.0010. f CD5 expression level (MFI) of SP4 Nur77-GFPhigh population. Left panel, representative FACS plot. Right panel, Quantification of CD5 MFI. Each group n = 4. Data are combined results of three independent experiments. p = 0.0005. a–f Data are mean ± SEM with dots representing individual values of biologically independent animals. Statistical differences between groups were calculated using (a–d) unpaired, two-tailed Student’s t-test, and (e, f) paired, two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Fig 4: 6PGD-/- CD8+ T cells display a TEFF phenotype(A) CD8+ T cells were isolated from spleen of 6PGD-/- and 6PGDfl/fl mice and stimulated for 48 h with aCD3 + aCD28 mAbs, followed by RNA extraction and RNA-sequencing analysis. Heatmap of gene expression for 6PGD-/- versus 6PGDfl/fl CD8+ T cells is shown.(B) For confirmation, naive (CD62Lhigh CD44low) CD8+ T cells were isolated and stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL). Expression of representative genes was confirmed by real-time PCR on day 0 and day 4.(C–M) Naive CD8+ T cells were stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL) followed by assessment of IFN-? expression by intracellular staining (C) and quantification of IFN-? production by ELISA (D). Expression of granzyme B (E–F), T-bet (G and H), Fas (CD98) (I and J), FasL (CD178) (K and L), and CD69 (M) was assessed by flow cytometry. Results are representative of three independent experiments with n = 6 mice per group. Error bars represent ± SEM. ***p < 0.001; ****p < 0.0001.

Fig 5: In the absence of B7-CD28 co-stimulation, thymocyte numbers increase progressively after positive selection.a The number of single-positive thymocytes was increased in B7 DKO mice. SP4 p = 0.0026, SP8 p = 0.0047. b Thymocyte developmental stages defined by TCR vs. CD69 expression. c CD4 and CD8 profile in each thymocyte developmental stage. d Thymocyte number in each developmental stage. Stage III p = 0.005, Stage IV p = 0.0089, Stage V p = 0.0026. e Thymocyte cell number in subpopulations of stage III. CD4hiCD8lo p = 0.006, CD4loCD8lo p = 0.0012. f Relative thymocyte number in B7 DKO compared to WT mice increased progressively after stage III. Each group n = 3. Data are representative of three independent experiments. a, d, e Data are mean ± SEM with dots representing individual values of biologically independent animals. Statistical differences between groups were calculated using unpaired, two-tailed Student’s t-test. ** p < 0.01. n.s.; not significant. Source data are provided as a Source Data file.