Cross-dressed CD8α+/CD103+ dendritic cells prime CD8+ T cells following vaccination

Abstract

Activation of naïve cluster of differentiation (CD)8(+) cytotoxic T lymphocytes (CTLs) is a tightly regulated process, and specific dendritic cell (DC) subsets are typically required to activate naive CTLs. Potential pathways for antigen presentation leading to CD8(+) T-cell priming include direct presentation, cross-presentation, and cross-dressing. To distinguish between these pathways, we designed single-chain trimer (SCT) peptide-MHC class I complexes that can be recognized as intact molecules but cannot deliver antigen to MHC through conventional antigen processing. We demonstrate that cross-dressing is a robust pathway of antigen presentation following vaccination, capable of efficiently activating both naïve and memory CD8(+) T cells and requires CD8α(+)/CD103(+) DCs. Significantly, immune responses induced exclusively by cross-dressing were as strong as those induced exclusively through cross-presentation. Thus, cross-dressing is an important pathway of antigen presentation, with important implications for the study of CD8(+) T-cell responses to viral infection, tumors, and vaccines.

Fig. 1.

CD8⁺ T-cell priming is selectively ablated in Batf3^−/− mice following DNA vaccination. (A and B) WT and Batf3^−/- mice (n = 5 per group) were immunized with OVA plasmid DNA. CD8⁺ T-cell responses were measured by IFN-γ enzyme-linked immunosorbent spot assay (ELISPOT) and intracellular cytokine staining. The results are representative of at least three independent experiments. (C) WT and Batf3^−/− mice were vaccinated with SIINFEKL peptide and adjuvant or adjuvant alone. CD8⁺ T-cell responses were measured by IFN-γ ELISPOT. (D) WT and Batf3^−/− mice were vaccinated with OVA plasmid DNA. Sera were collected before and after vaccination. OVA-specific total IgG, IgG₁, and IgG_2a were measured by ELISA, and the levels were equivalent between WT and Batf3^−/− mice. *P < 0.01; ns, not significant; SFC, spot forming cells.

Fig. 2.

Molecular engineering of SCT peptide–MHC complexes precludes cross-presentation. (A) Schematic of the p5Y/K^b SCT, integrating the p5Y peptide, β₂m, and H2-K^b heavy chain (HC). Of note, the disulfide bond (red line) formed between linker 1 and the α1 domain traps the low-affinity p5Y epitope in the peptide binding groove. (B) C57BL/6 mice (n = 4 per group) were vaccinated with the indicated DNA constructs. K^b/SIINFEKL-specific immune responses were measured by ELISPOT. Data are presented as means ± SEM. *P < 0.01; ns, not significant. Similar results were obtained in at least three independent experiments.

Fig. 3.

Engineered SCT vaccines prime CD8⁺ T-cell responses in vivo by cross-dressing. (A) WT or Batf3^−/− mice were vaccinated with K14-p5Y/K^b SCT DNA or control DNA by gene gun. CD8⁺ T-cell responses were measured by IFN-γ ELISPOT. (B) A total of 2 × 10⁶ CFSE-labeled OT-1 cells were adoptively transferred into WT or Batf3^−/− mice. Twenty-four hours later, 5 × 10⁶ necrotic fibroblasts were injected (s.c.). Ninety hours later, draining lymph nodes were harvested, and OT-1 cell proliferation was determined by flow cytometry. The percentage of OT-1 cells (identified as CD8⁺CD45.1⁺) that divided at least once (CFSE^lo) is shown. The results are representative of three independent experiments. Each data point represents an individual animal. (Bars indicate mean values for each experimental group.)

Fig. 4.

Cross-dressing is a robust antigen-presentation pathway in vivo. CFSE-labeled OT-1 cells were adoptively transferred into the C57BL/6→TAP^−/− BM chimeras, which were subsequently vaccinated with K14-OVA or K14-p5Y/K^b SCT. (A) This experimental model system results in CD8⁺ T-cell priming by two distinct, nonoverlapping antigen-presentation pathways. (B) Spleen cells from vaccinated mice were collected and analyzed by flow cytometry. Representative two-color dot plots show expression of CFSE and Vα2 by viable CD8⁺ T cells. Gated regions represent OT-1 cells (expressing CFSE, CD8, and Vα2). Numbers indicate the percentage of CFSE^lo OT-1 cells that had gone through at least one cell division. (C) Summary of data from individual animals (n = 3–5 per group). *P < 0.01; ns, not significant.