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Supporting Table 1
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10

Fig. 7. Therapy-induced anti-MHC class I chain-related protein A (anti-MICA) antibodies do not block natural killer (NK) cell lysis of K562 cells. Magnetic bead-purified healthy donor or MEL15 NK cells obtained at different times were tested for lytic activity against ⁵¹Cr-labeled K562 targets. Autologous sera were added to the lytic assay where indicated.

Fig. 8. Vaccination with irradiated, autologous granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting tumor cells alone elicited a potent humoral reaction to MHC class I chain-related protein A (MICA) that was temporally associated with a reduction in sMICA in some patients. (Upper) Longitudinal sera samples from vaccinated NSCLC patient L1 and vaccinated melanoma patients M37 and M34 were diluted 1:500 and analyzed by ELISA with recombinant MICA protein. Reactivity was determined with a pan-IgG secondary. (Lower) Serial soluble MICA (sMICA) levels were measured with a sandwich ELISA. Upward arrows depict tumor cell vaccinations. L1 exhibited disseminated, progressive nonsmall cell lung carcinoma upon study enrollment, but vaccination stimulated dense T and B cell infiltrates in metastatic lesions and disease stabilization for nearly 2 years (survival of 31 months). M37 entered study with visceral melanoma metastases, which were completely resected for vaccine manufacture. He was immunized for 2 years and remains disease-free 6 years after enrollment without further therapy. M34 presented with visceral melanoma metastases and achieved a partial clinical response to an initial course of immunization. Subsequent isolated recurrences were processed into additional vaccines, whereas the patient succumbed to progressive disease 4.5 years after study entry.

Fig. 9. Vaccine-induced anti-MHC class I chain-related protein A (anti-MICA) antibodies antagonized soluble MICA (sMICA) suppression of innate immune responses. (A) Normal donor peripheral blood mononuclear cells (PBMCs) were incubated for 48 h in sera, and NKG2D expression on gated natural killer (NK) cells was determined with flow cytometry. Sera samples were obtained before vaccination and at the end of therapy. (B) Healthy donor PBMCs were incubated in L1 pre- and postvaccination sera for 48 h and washed; NK cells were then purified with magnetic bead selection and tested for lytic activity. (C) NK cells were evaluated with M37 pre- and postvaccination sera as above.

Fig. 10. Vaccine-induced anti-MHC class I chain-related protein A (anti-MICA) antibodies antagonized soluble MICA (sMICA) suppression of adaptive immune responses and enhanced MICA-dependent cross-presentation. (A) Normal donor peripheral blood mononuclear cells (PBMCs) were incubated for 48 h in L1 and M37 sera, and NKG2D expression on gated CD8⁺ T cells was determined with flow cytometry. (B) The effects of L1 sera on the cross-presentation of M34-T and M34T-MICA cells was determined as in Fig. 3. (C) M37 sera.

Table 1. Targets identified in melanoma cDNA expression libraries using MEL15 sera