In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination

Abstract

The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic alpha-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the alphaDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell-mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models.

Figure 1.

Characterization of monoclonal IgG:OVA conjugates. (A) IgG:OVA conjugates at various stages of conjugation. Nonreduced gel (left) of the 80-kD monovalent IgG after MESNA treatment, and reduced and boiled (right) to show heavy and light chains. (B) Western analysis of antibody (DEC-205 and III/10 isotype control) OVA conjugates. (C) C57BL/6 or DEC-205^−/− mice were injected i.v. with 10⁶ CFSE-labeled OT-I or OT-II T cells and 24 h later with either antibody conjugates (DEC-205 and III/10 isotype control at the same doses) containing 50 ng of OVA or 25 μg soluble OVA s.c. 3 d later, proliferation in lymph nodes was evaluated by flow cytometry. (D) As in C, but graded doses of OVA conjugated to IgG- or endotoxin-free OVA were used. For the III/10 isotype control, the highest dose of conjugate with 250 ng of OVA was used. Representative of two or more experiments.

Figure 2.

αDEC-205:OVA with αCD40 primes both CD4⁺ and CD8⁺ T cells in vivo. (A) αDEC-205:OVA containing 500 ng of OVA was administered to naive C57BL/6 mice s.c. with 25 μg of αCD40. 7 d later, spleen cell suspensions were CFSE labeled and restimulated in vitro for 5 d with LPS-free OVA (500 μg/ml) to evaluate proliferation by flow cytometry. (B) As in A, but the cells were restimulated with either SIINFEKL (1.0 μM) or LSQAVHAAHAEINEAGR (2.0 μM) peptides for 2 d and IFN-γ secretion evaluated by ELISPOT. (C) Mice were immunized with grade doses of OVA as a soluble protein or conjugated to αDEC-205. IFNγ secretion was evaluated after 7 d in the lymph nodes and spleen as in B. Representative of at least two experiments.

Figure 3.

αDEC-205:OVA in combination with αCD40 induces durable and strong OVA-specific responses by CD8⁺ T cells. (A) αDEC-205:OVA containing 50 ng of OVA was administered to naive C57BL/6 mice s.c. with 25 μg of αCD40. 14, 21, 60, and 90 d later, intracellular IFN-γ staining was evaluated by flow cytometry without or with OVA peptide restimulation. Indicated percentages are percent IFN-γ⁺ CD8⁺ cells. (B) Wild-type, DEC-205^−/−, CD8^−/−, and CD4^−/− mice were treated as in A. 14 d later, 7 × 10⁶ of each, CFSE-labeled syngeneic splenocytes pulsed with peptide (CFSE^hi) or not (CFSE^lo), were injected i.v. to detect active killer cells in the lymph nodes. (C) As in B, but mice were evaluated after 60 d. Data are representative of two or more experiments.

Figure 4.

Enhanced efficacy of αDEC-205:OVA plus αCD40 relative to other immunization approaches. (A) C57BL/6 mice were immunized s.c. with several methods: Spleen DC pulsed ex vivo with 10 μg/ml each of αDEC-205:OVA and αCD40; 500 μg OVA in CFA; 50 μg OVA with 25 μg αCD40; 50 μg of SIINFEKL peptide with 25 μg αCD40; or 50 ng of OVA in αDEC-205:OVA with 25 μg of αCD40. 7 or 30 d later, lymph nodes were harvested and T cell expansion evaluated by K^b-SIINFEKL–PE tetramer and CD62L staining. The gate for the y-axis was placed relative to the CD62L-negative tetramer binding cells in the right panel. Indicated percentages are percent of CD8⁺ lymphocytes. (B) As in A, but IFN-γ secretion evaluated by intracellular cytokine staining. Data are means of three experiments.

Figure 5.

Systemic antigen presentation after DEC-205 targeting in situ. (A) C57BL/6 mice were given 10 μg of Alexa₄₈₈-conjugated antibodies s.c. At the indicated time points, CD11c⁺ cells were enriched from the draining or distal lymph nodes or spleen for evaluation by flow cytometry. The frequencies of DCs capturing the injected Igs are shown, and the DEC-205 and CD8 high subset of splenic DCs arrowed. (B) C57BL/6 mice were given 10 μg of αDEC-205:OVA, isotype:OVA, or PBS s.c. and, after 18 h, CD11c⁺ cells were enriched from draining or distal lymph nodes or spleen. The presence of OVA was evaluated by intracellular staining with Alexa₄₈₈-conjugated αOVA and flow cytometry. (C) 15 h after s.c. treatment with 5 μg of αDEC-205:OVA or the isotype conjugate ± αCD40, CD11c⁺ lymph node or spleen DCs were selected and used to stimulate OT-I T cells without further addition of OVA. (D) As in C, but mice were treated with αCD40 and either αDEC-205:OVA (5 μg), OVA (500 μg), or PBS. Data are representative of at least two experiments.

Figure 6.

Prolonged MHC class I, but not MHC class II, presentation after DEC-205 targeting in situ. (A) C57BL/6 mice were immunized to OVA under the conditions listed above each panel for 15, 7, 3, or 1 d before transferring 10⁶ CFSE-labeled OT-I T cells. Proliferation in the lymph nodes was monitored by flow cytometry 3 d later. (B) As in A, but CFSE-labeled OT-I or OT-II T cells were transferred. (C) C57BL/6 mice were treated with 50 ug MHC class I binding peptide (SIINFEKL) in CFA, 50 μg MHC class II binding peptide (LSQAVHAAHAEINEAGR) in CFA, CFA alone, or PBS. IFN-γ secretion was evaluated after 12 d in the lymph nodes as in Fig. 2 B. Data are representative of at least two experiments.

Figure 7.

Immunization with a single low dose of αDEC-205:OVA and αCD40 elicits resistance to OVA-modified pathogens. (A) C57BL/6 mice were vaccinated as described in Fig. 3 A. 60 d later, mice were challenged with 5 × 10⁶ MO4 cells s.c. and tumor growth evaluated. (B) C57BL/6 mice were inoculated with MO4 tumor cells as in (A). 7 d later, mice were treated as in Fig. 4 A and tumor growth evaluated. (C) C57BL/6 mice were treated as in Fig. 3 A. 30 d after vaccination, mice were challenged with 10⁵ PFU of vaccinia–OVA intranasally. 7 d later, lungs were harvested and virus titer evaluated by a plaque-forming assay. (D) As in C, but mice were weighed daily after viral challenge. Data are representative of at least two experiments.