Immune complex-mediated antigen presentation induces tumor immunity

Abstract

Antigen uptake receptors on dendritic cells (DCs) provide efficient entry for the initiation of antigen-specific adaptive immunity. Here we show that targeting of antigen to Fc receptors on DCs accomplishes combined activation of Th1 CD4 and CD8 effector responses in vivo, namely delayed-type hypersensitivity and tumor immunity. Tumor immunity specific for ovalbumin-expressing tumors was provided by immunization with wild-type but not FcgammaRgamma(-/-) DCs loaded with ovalbumin-containing immune complexes. Tumor protection was eliminated when immune complex-loaded DCs lacked beta(2) microglobulin, TAP, or MHC class II, demonstrating that Fc receptor-targeted antigenic uptake led to both MHC class I- and class II-restricted responses, which together are required for effector tumor immunity. Thus the cross-presentation pathway accessed by antigens acquired endocytically through Fc receptors links humoral and cellular immunity. These data suggest that administration of antitumor antibodies may enhance tumor-specific T cell responses in vivo and provide the rationale for Fc receptor targeting in vaccine development.

Figure 1

Immunization with BMDCs loaded with tumor antigen containing–ICs provides long-lasting and potent tumor immunity. (a) Prevention. Antigen-pulsed DCs (1 × 10⁶ cells per mouse) were administered intravenously to naive WT mice (n = 25 per group). One week later, 2 × 10⁵ MO-4 cells were injected subcutaneously and mice were monitored for the development of palpable tumor. No protection was provided by immunization with OVA-loaded BMDCs; 24 of 25 mice developed tumors. Near-complete protection was seen after immunization with OVA/IC–loaded BMDCs (only 1 of 25 mice developed tumors). (b) Treatment. 2 × 10⁵ MO-4 cells were injected subcutaneously; 1 week later, antigen-pulsed DCs (1 × 10⁶ cells per mouse) were administered intravenously to naive WT mice (n = 10 per group). (c) Recall response. Six months after intravenous immunization with antigen-pulsed BMDCs, WT mice (n = 5) were challenged subcutaneously with 2 × 10⁵ MO-4 cells. No protection was provided by immunization with OVA-loaded BMDCs, but mice were fully protected 6 months after immunization with OVA/IC–loaded BMDCs. No significant differences in tumor protection were found between mice immunized with BMDCs alone and with BMDCs loaded with soluble OVA; for simplicity, the BMDC-alone group is not presented.

Figure 2

IC-loaded BMDCs induce OVA-specific cellular and humoral immunity in vivo. (a) OVA–peptide MHC class I– and II–restricted cellular immune responses result from OVA/IC–loaded BMDC immunization. Proliferative responses occur after addition of MHC class I– or class II–restricted peptides to lymph node cell populations from mice immunized with BMDCs loaded with OVA/ICs but not OVA alone. (b) Antibody responses are enhanced after immunization with OVA/IC–loaded BMDCs. OVA-specific ELISA reveals higher OVA-specific total IgG, IgG1, and IgG2a titers in diluted sera from mice immunized 15 days earlier with IC-pulsed rather than OVA-pulsed BMDCs (ten mice per group).

Figure 3

Phagocytic but not endocytic uptake of antibody-complexed antigen requires activating FcγRs on DCs. Immature BMDCs were incubated for 30 minutes with either OVA-containing ICs for endocytosis or antibody-opsonized SRBCs for phagocytosis. Uptake of soluble OVA or anti-OVA alone was negligible (data not shown), while uptake of OVA/ICs was dramatically enhanced for both WT and FcγRγ^–/– BMDCs, indicating that inhibitory FcRs are capable of efficiently endocytosing OVA/ICs. In contrast, phagocytic uptake of antibody-opsonized SRBCs was seen in WT but not in FcγRγ^–/– BMDCs.

Figure 4

OVA/IC–mediated antigen presentation in vitro induces Th1 CD4 and CD8 cellular proliferation and effector differentiation of naive transgenic T cells. (a) Anti-OVA T cell responses. IC-mediated antigen presentation efficiently drives OT-I and OT-II proliferative responses in vitro. 2 × 10⁵ CD4 OT-II and 4 × 10⁵ CD8 OT-I cells were cultured with 10⁴ or 2 × 10⁴ antigen-pulsed BMDCs in 96-well culture plates. ³H-thymidine incorporation was measured between 36 and 48 hours later and expressed in cpm. OVA (10 μg/ml), OVA-containing ICs (10 μg/ml OVA and 50 μg/ml anti-OVA), or OVA (10 μg/ml) plus “irrelevant” peroxidase/antiperoxidase (PAP) complexes (50 μg/ml) were added to DCs for 48 hours prior to incubation with T cells. The results are representative of five separate experiments. (b) Cytokine production. IC-loaded BMDCs induce IFN-γ production by naive OT-I and OT-II transgenic cells. CD4 OT-II or CD8 OT-I transgenic T cells were cultured at 10⁶/ml with 5 × 10⁴/ml pulsed DCs in 24-well plates. IL-4 and IFN-γ production was determined by ELISA after 3–5 days of culture. The results are from two independent experiments. The sensitivity of the ELISAs were 1 pg/ml. Culture of OT-I or OT-II transgenic T cells with DCs alone (i.e., without OVA peptides or soluble OVA) did not elaborate detectable IFN-γ or IL-4.

Figure 5

Sensitization with IC-loaded BMDCs induces DTH effector responses. Groups of eight to ten mice were immunized with 1 × 10⁶ cells per mouse of OVA-pulsed DCs or IC-pulsed DCs in the front footpad. One week after primary immunization (sensitization), the mice were challenged with PBS in one footpad and OVA in the other footpad. Change in footpad thickness (OVA-injected footpad vs. PBS-injected control footpad) was measured 9 days after initial immunization. Error bars are the SEM of each group.