Selective blockade of inhibitory Fcgamma receptor enables human dendritic cell maturation with IL-12p70 production and immunity to antibody-coated tumor cells

Abstract

The final differentiation or maturation of dendritic cells (DCs) in response to environmental stimuli influences their ability to both initiate immunity and determine the quality of the response to antigens. Circulating immune complexes and cell-bound immunoglobulins present in normal human sera represent a potential stimulus for inadvertent DC activation in the steady state and during autoimmunity. Here, we show that selective blockade of the inhibitory Fcgamma receptor (FcgammaR) FcgammaRIIb with recently developed monoclonal antibodies leads to maturation of human monocyte-derived DCs, which depends on the presence of IgG in normal human plasma. Plasma, in the presence of an FcgammaRIIb blockade, caused the DCs to up-regulate the expression of costimulatory molecules and to produce the inflammatory mediator IL-12p70. FcgammaRIIb blockade of DCs loaded with tumor cells led to increased tumor-specific T cell immunity without the need for exogenous stimuli other than human plasma. Therefore, the activation status of DCs in the presence of normal human serum depends on the balance between activating and inhibitory FcgammaRs and can be enhanced by new antibodies that react selectively with FcgammaRIIb. These data suggest an approach for modifying this balance to enhance immunity to immune complexes and antibody-coated tumor cells and to silence DC activation by immune complexes in autoimmune states.

Fig. 1.

Expression of FcγRIIa and FcγRIIb on human DCs. (A) Expression of FcγRIIa and FcγRIIb on human monocyte-derived DCs. Purified CD14⁺ monocytes were induced to differentiate into DCs in the presence of GMCSF and IL-4. On day 5 of culture, inflammatory cytokines were added to yield mature DCs. Expression of FcγRIIa and FcγRIIb on immature and mature DCs was determined by flow cytometry by using specific antibodies (IV.3 and 2B6, respectively). Data are representative of three experiments. (B) Expression of FcγRIIa and FcγRIIb on myeloid and plasmacytoid subsets of human blood-derived DCs. Myeloid (Lin-1^–, DR⁺, and CD11c⁺) and plasmacytoid (Lin-1^–, DR⁺, and CD123⁺/BDCA2⁺) DCs were isolated from peripheral blood mononuclear cells as described in Materials and Methods. Expression of FcγRIIa and FcγRIIb on immature and mature DCs was determined by flow cytometry by using specific antibodies (IV.3 and 2B6, respectively). Data are representative of three experiments.

Fig. 2.

Blockade of FcγRIIb in the presence of human serum leads to maturation of human monocyte-derived DCs. (A) Monocyte-derived DCs cultured in RPMI medium 1640 with 1% plasma or serum-free media (AIM-V) were incubated overnight with anti-FcγRIIb antibody (2B6, 1 μg/ml) or isotype control antibody. Expression of HLA-DR, CD80, CD86, and CD83 on CD11c⁺ DCs was monitored by flow cytometry. Data are representative of three similar experiments. (B) Monocyte-derived DCs were cultured either in serum-free medium (AIM-V) or AIM-V supplemented with 1% plasma. DCs were cultured with chimeric (ch-2B6) anti-FcγRIIb antibody or isotype control. DC maturation was monitored by flow cytometry. Data are mean/SD of two similar experiments. (C) Representative FACS plot showing expression of maturation marker CD83/CD80 in DCs cultured under conditions described in B. Percentages of CD83⁺ cells are noted. (D) Monocyte-derived DCs were cultured in either RPMI medium 1640 with 1% plasma or RPMI medium 1640 with 1% Ig-depleted plasma. DCs were cultured with chimeric (ch-2B6) anti-FcγRIIb antibody or isotype control. DC maturation was monitored by flow cytometry. Data shown are mean/SD of two similar experiments. Western blot (Lower) shows depletion of Ig from plasma. Lane 1 is RPMI medium 1640 with 1% plasma, and lane 2 is 50× concentrate of RPMI medium 1640 with 1% Ig-depleted plasma. (E) Representative FACS plot showing expression of maturation marker CD83/CD80 in DCs cultured under conditions described in D, with isotype control or chimeric (ch-2B6) or aglycosylated anti-FcγRIIB (agly-2B6) antibody. Percentages of CD83⁺ cells are noted.

Fig. 3.

FcγRIIb blockade leads to IL-12p70 production. Supernatants of immature monocyte-derived DCs treated overnight with anti-FcγRIIb (2B6, 1 μg/ml), isotype control antibody, or inflammatory cytokines were analyzed for IL-12p70 by ELISA.

Fig. 4.

Effect of FcγRIIb blockade on the uptake of tumor cells by DCs. Myeloma cells were labeled with dye (PKH26), opsonized with anti-syndecan-1 antibody, and cocultured with dye (PKH67)-labeled DCs at 4°C or 37°C. DCs were also pretreated with either isotype control antibody or anti-FcγRIIB antibody (2B6). After 4–8 h of coculture, the percentage of double-positive DCs was evaluated by flow cytometry.

Fig. 5.

Effect of FcγRIIb blockade on the expansion of myeloma-reactive T cells by tumor-loaded DCs. (A) Monocyte-derived DCs alone or loaded with opsonized U266 tumor cells were either left untreated (“no maturation cytokines”) or matured ex vivo by using a cytokine mixture as a maturation stimulus (“with maturation cytokines”). DCs were also pretreated with either isotype control or anti-FcγRIIb antibody (2B6). The tumor-loaded and unpulsed DCs were each used to stimulate autologous T cells. IFN-γ producers against U266 (A2⁺) or cag (A2^–) cells as control were analyzed by ELISPOT assay. Data shown are mean/SD of three separate experiments. (B) Immature monocyte-derived DCs from HLA-A2⁺ donors were loaded with opsonized cag (A2^–) myeloma cells in the presence of isotype control or anti-FcγRIIB antibody (2B6) and used to stimulate autologous T cells. After 14 days of culture, T cells were stimulated overnight in ELISPOT plates with autologous DCs pulsed with 10 μM A2 restricted peptides derived from MAGE-A3, NY-ESO-1, or 2.5 μMof an overlapping 15-mer peptide library derived from survivin. IFN-γ producers were quantified by an ELISPOT assay. Data shown are mean/SD of four replicates in independent experiments on two blood donors. *, P value for comparison with no antigen control. MAGE-A3, P = 0.048; NYESO-1 and survivin, P < 0.01.