Therapy-induced antibodies to MHC class I chain-related protein A antagonize immune suppression and stimulate antitumor cytotoxicity

Abstract

The activation of NKG2D on innate and adaptive cytotoxic lymphocytes contributes to immune-mediated tumor destruction. Nonetheless, tumor cell shedding of NKG2D ligands, such as MHC class I chain-related protein A (MICA), results in immune suppression through down-regulation of NKG2D surface expression. Here we show that some patients who respond to antibody-blockade of cytotoxic T lymphocyte-associated antigen 4 or vaccination with lethally irradiated, autologous tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor generate high titer antibodies against MICA. These humoral reactions are associated with a reduction of circulating soluble MICA (sMICA) and an augmentation of natural killer (NK) cell and CD8(+) T lymphocyte cytotoxicity. The immunotherapy-induced anti-MICA antibodies efficiently opsonize cancer cells for dendritic cell cross-presentation, which is correlated with a diversification of tumor antigen recognition. The anti-MICA antibodies also accomplish tumor cell lysis through complement fixation. Together, these findings establish a key role for the NKG2D pathway in the clinical activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade and granulocyte-macrophage colony-stimulating factor secreting tumor cell vaccines. Moreover, these results highlight the therapeutic potential of anti-MICA antibodies to overcome immune suppression and effectuate tumor destruction in patients.

Fig. 1.

CTLA-4 blockade after autologous tumor vaccination elicited a potent humoral reaction to MICA that was temporally associated with a reduction in sMICA. (Upper) Longitudinal sera samples from MEL15 were diluted 1:500 and analyzed by ELISA with recombinant MICA protein. Reactivity was determined with a pan-IgG secondary. Downward arrows denote tumor cell vaccinations and upward arrows depict infusions of MDX-010. (Lower) Serial sMICA levels were measured with a sandwich ELISA.

Fig. 2.

Therapy-induced anti-MICA antibodies antagonized sMICA suppression of innate immune responses. (A) Normal donor PBMCs were incubated for 48 h in sera, and NKG2D expression on gated NK cells (CD56⁺ and CD3⁻) was determined with flow cytometry. Sera samples were obtained from a healthy donor, vaccinated melanoma patients without sMICA (M2 and M9), and MEL15 during immunization or after CTLA-4 antibody blockade. Anti-MICA monoclonal antibodies or isotype controls were added where indicated. (B) Healthy donor PBMCs were incubated in normal or MEL15 sera for 48 h and washed; NK cells were then purified with magnetic bead selection and tested for lytic activity against ⁵¹Cr-labeled K562 targets. The NKG2D-dependent lysis was determined with the addition of the mAb 1D11 (anti-NKG2D) or isotype control (IgG).

Fig. 3.

Immunotherapy restored protective antitumor innate responses in MEL15. (A) PBMCs were obtained from MEL15 during vaccination or after CTLA-4 blockade, and NKG2D expression on gated NK cells was determined with flow cytometry. (B) Magnetic bead-purified healthy donor or MEL15 NK cells obtained at different times were tested for lytic activity against ⁵¹Cr-labeled K562 targets.

Fig. 4.

Therapy-induced anti-MICA antibodies antagonized sMICA suppression of adaptive immune responses and enhanced MICA-dependent cross-presentation. (A) Normal donor PBMCs were incubated for 48 h in sera, and NKG2D expression on gated CD8⁺ T cells (CD8⁺ and CD3⁺) was determined with flow cytometry. Sera samples were obtained from a healthy donor, vaccinated melanoma patients without sMICA (M2 and M9), and MEL15 during immunization or after CTLA-4 antibody blockade. Anti-MICA monoclonal antibodies or isotype controls were added where indicated. (B) Dendritic cells were generated from adherent PBMCs of HLA-A2.1⁺ donors, loaded with MEL15 early or late sera-coated MEL15-T or MEL15-T-MICA tumor cells, matured with LPS, and used to stimulate autologous purified CD8⁺ T cells for 7 days. IFN-γ production was measured by ELISPOT against the indicated targets. No reactivity against unpulsed dendritic cells or dendritic cells loaded with unopsonized tumors was observed (data not shown). (C) HLA-A2.1⁺ dendritic cells were loaded with MEL15 late sera-coated K008-T melanoma cells (MICA+), LPS matured, and used to stimulate autologous CD8⁺ T cells in the presence of early or late MEL15 sera. IFN-γ production was measured by ELISPOT against the indicated targets.

Fig. 5.

Immunotherapy restored protective antitumor innate responses and enhanced cross-presentation in MEL15. (A) PBMCs were obtained from MEL15 during vaccination or after CTLA-4 blockade, and NKG2D expression on gated CD8⁺ T cells was determined with flow cytometry. (B) PBMCs were obtained from MEL15 during vaccination or after CTLA-4 blockade and used to generate dendritic cells. These cells were loaded with K008-T melanoma cells coated in early or late MEL15 sera and then matured with LPS. Purified CD8⁺ T cells from the same time points were then stimulated in vitro with the respective tumor-loaded dendritic cells for 7 days. IFN-γ production was measured by ELISPOT against the indicated targets. (C) MEL15 CD8⁺ T cells were purified from PBMCs collected during vaccination or after CTLA-4 blockade and tested for IFN-γ production against the indicated targets without prior in vitro stimulation. Melanoma inhibitor of apoptosis protein (ML-IAP) is a previously characterized tumor rejection antigen (13).

Fig. 6.

Immunotherapy-induced anti-MICA antibodies mediate complement-dependent lysis. 293T and 293T-MICA embryonic kidney cells were incubated in patient sera and complement, and cell viability was determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay after 6 h. (Upper) Prevaccination sera. (Lower) Postvaccination sera. M35 was a vaccinated, long-term surviving metastatic melanoma patient (7 years) without anti-MICA antibodies. The differences were abrogated by heat inactivation of the complement.