Fig. 1.

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.