B7-DC cross-linking restores antigen uptake and augments antigen-presenting cell function by matured dendritic cells

Abstract

Dendritic cells (DCs) are classified in two states: immature DCs (iDCs), which perform sentinel functions, sampling for antigen and danger signals, and mature DCs (mDCs), which exhibit enhanced antigen-presenting functions but are no longer capable of acquiring antigen. We now describe DCs with a different activation phenotype: cells having the strong antigen-presenting functions of mDCs and the antigen-acquiring functions of iDCs. We have described an antibody that binds the costimulatory molecule B7-DC and activates DCs. The resulting phenotype is distinct from iDCs or mDCs matured by using Toll-like receptor (TLR) agonists. Ability to take up antigen increases, while expression of B71/2 costimulatory and MHC molecules remains unchanged. DCs matured with TLR agonists and then superactivated through B7-DC exhibit a previously unrecognized phenotype. These DCs recover the ability to take up antigen, which is normally lost after treatment with TLR-3 and TLR-9 agonists, yet retain the high expression of costimulatory and MHC molecules and strong antigen-presenting functions of mDCs. Immunization using TLR agonists and B7-DC XAb (cross-linking antibody) together as adjuvant resulted in substantially increased cytolytic T cell responses, particularly when minimal peptide antigens were used. By stimulating DCs with two distinct activation signals, a previously unrecognized phenotype exhibiting augmented antigen-presenting functions was obtained.

Fig. 1.

B7-DC XAb induces uptake of antigen by CD11c⁺ cells in vitro. DCs were prepared from bone marrow-derived precursors after culture with granulocyte–macrophage colony-stimulating factor and IL-4. Day-5 cultures of DCs were either not treated or pulsed with the TLR agonists CpG-ODN or poly I:C. On day 6, cells received either sHIgM12 B7-DC XAb or isotype control antibody sHIgM39 and were pulsed with FITC-labeled OVA. Accumulation of FITC in washed cells was assessed by flow cytometry on day 7. Histograms represent FITC incorporation by CD11c⁺ cells. (A) No treatment on day 5, isotype control antibody (black) or B7-DC XAb (gray). (B) No treatment on day 5, isotype control antibody on day 6 (black); CpG-ODN on day 5 and isotype control antibody on day 6 (gray). (C) No treatment on day 5 and isotype control antibody on day 6 (black); poly I:C on day 5 and isotype control antibody on day 6 (gray). (D) CpG-ODN on day 5 and isotype control antibody (black) or B7-DC XAb (gray) on day 6. (E) poly I:C on day 5 and isotype control antibody (black) or B7-DC XAb (gray) on day 6. (F) CpG-ODN on day 5; IgG isotype control antibody (black), anti-CD40 IgG antibody (light gray), or B7-DC XAb (dark gray) on day 6.

Fig. 2.

B7-DC cross-linking does not induce CD80, CD86, and class II expression by DCs. DCs were evaluated for expression of the costimulatory molecule CD80, CD86, and IA^b expression. Mean fluorescent intensity (MFI) of histograms of cells gated for CD11c expression is displayed above each panel. (A) Day-7 CD80 expression on DCs not treated (dotted line) or treated with CpG-ODN (gray) on day 5, followed by treatment with IgM isotype control antibody on day 6. (B) CD86 expression on cells treated as in A. (C) IA^b expression on cells treated as in A.(D, E, and F) CD80, CD86, and IA^b expression as in A, B, and C, respectively, except that DCs were treated with sHIgM12 B7-DC XAb on day 6 instead of isotype control IgM antibody. (G, H, and I) CD80, CD86, and IA^b expression, respectively, of DCs treated on day 6 only with IgG isotype control antibody (solid line), B7-DC XAb (dotted line), or IgG anti-CD40 antibody (gray). Left MFI value is for isotype control, center MFI value is for B7-DC XAb, and right MFI value is for anti-CD40.

Fig. 3.

Treatment of mDCs with B7-DC XAb at the time of antigen pulsing restores antigen-presenting ability. Day-7 DCs from treatment groups shown in Fig. 1 were cocultured with naïve T cells from OT-I and OT-II transgenic mice to evaluate antigen-presenting function. Incorporation of [³H]thymidine was used to evaluate T cell activation. ³H incorporation was evaluated on day 10 (day 3 of coculture with T cells). (A) DCs that received isotype control antibody (open circles) or B7-DC XAb (filled circles) along with soluble OVA on day 6 of culture coincubated for 3 days with OT-I T cells. (B) DCs that received isotype control antibody (open circles) or B7-DC XAb (filled circles) along with soluble OVA on day 6 of culture coincubated for 3 days with OT-II T cells. (C) DCs treated with poly I:C on day 5, isotype control antibody (open circles) or B7-DC XAb (filled circles) and OVA on day 6, and then cocultured with OT-I T cells on day 7. (D) DCs treated with poly I:C on day 5, isotype control antibody (open circles) or B7-DC XAb (filled circles) and OVA on day 6, and then cocultured with OT-II T cells on day 7. (E) Comparison of FITC-OVA incorporation in 7-day DCs that received no treatment on day 5 and isotype control antibody plus OVA on day 6 (black) with DCs that received poly I:C treatment on day 5 and B7-DC XAb and OVA on day 6 (gray). (F) T cell stimulation by DCs from E cocultured with OT-I cells. (G) T cell stimulation by DCs from E cocultured with OT-II cells.

Fig. 4.

B7-DC cross-linking induces antigen uptake in lymph node DCs. Animals received i.v. treatments with 10 μg of isotype control antibody (sHIgM39), B7-DC XAb (sHIgM12), or CD40-binding antibody (1C10) 1 day before and on the day of intradermal challenge with 100 μg of FITC-OVA. The draining lymph nodes and contralateral lymph nodes were excised and assessed for FITC incorporation by CD11c⁺ cells. (A) Draining lymph node cells from animals treated systemically with B7-DC XAb. (B) Contralateral lymph node cells from animals treated systemically with B7-DC XAb. (C) Draining lymph node cells from animals treated systemically with isotype control antibody. (D) Draining lymph node cells from animals treated systemically with CD40-binding antibody. (E) Draining lymph node cells from animals treated systemically with B7-DC XAb and locally at the time of intradermal challenge with FITC-OVA with 10 μg of a rat anti-B7-DC IgG2a antibody. (F) Draining lymph node cells from animals treated systemically with CD40-binding antibodies and locally at the time of intradermal challenge with FITC-OVA with 10 μg of a rat anti-B7-DC IgG2a antibody.

Fig. 5.

Stimulation through TLR together with B7-DC cross-linking induces enhanced responses to protein and peptide antigens. Animals received either whole OVA (A and B) or a mixture of the peptides SIINFEKL and PADRE (C and D) in IFA s.c. The animals received no further treatment, a series of four daily treatments with CpG-ODN beginning on the day of antigen challenge, a series of three daily i.v. B7-DC XAb or isotype control antibody challenges beginning 1 day before antigen challenge, or a combination of the treatments. CTLs were harvested from the draining lymph nodes 7 days after antigen challenge and evaluated directly in a ⁵¹Cr-release assay. Treatment groups shown are as follows: open circles, CTLs from animals treated with isotype control antibody; filled circles, CTLs from animals treated with B7-DC XAb; squares, CTLs from animals treated with CpG-ODN; triangles, CTLs from animals receiving a combination of B7-DC XAb and CpG-ODN treatments. (A) CTL assay using OVA-immunized T cells and OVA-expressing EG7 cells as targets. (B) CTL assay using T cells in A against EL4 cells that do not express OVA antigens. (C) CTL assay using T cells immunized with a SIINFEKL–PADRE mixture and EG7 targets. (D) CTL assay using T cells described in C against EL4 targets.