Figure 3. Functionality of AIM2-specific T-cell cultures. Four FACS-sorted, expanded AIM2-specific T-cell cultures were established from patients MM5, MM7, MM8, and MM9. (A) Three of these cultures were stimulated with BM36.1 cells pulsed with the AIM2 decapeptide or HIV-A1 (control peptide) at a 10:1 effector:target ratio, and cytokine production was measured by intracellular cytokine staining. Background was <0.4% and was subtracted from positive samples. The frequency of AIM2-specific multimer⁺CD8⁺ T lymphocytes (among total CD8⁺ T cells) in each culture was: 19.6%, 14%, and 61.6% for MM7, MM8, and MM9, respectively. (B) T-cell cultures from patients MM7, MM8, and MM9 were stimulated with BM36.1 cells pulsed with the AIM2 decapeptide or HIV-A1 (control peptide) at the indicated effector:target ratio and cytotoxicity was measured by ⁵¹Cr-release assays. The frequency of AIM2 specific multimer⁺ T cells (among total live cells) was: 17.2%, 2.7%, and 31.6% for MM7, MM8, and MM9, respectively. (C) The T-cell culture established from patient MM5 was stimulated with the indicated melanoma cell lines at the indicated effector:target ratio and cytotoxicity was measured by ⁵¹Cr-release assays. FM28 cells are HLA-A1⁺ while FM48 and FM74 cells are HLA-A1⁻, yet all express AIM2 (as determined by PCR; data not shown). To test peptide-specificity and HLA-restriction, FM28 cells were pulsed with the AIM2-derived decapeptide or incubated with an anti-MHC Class I antibody (W6/32). Furthermore, cold target inhibition was performed using unlabeled BM36.1 cells pulsed with either HIV-A1 or the AIM2-derived decapeptide at an inhibitor:target ratio of 20:1. There were 68% AIM2 specific T cells in the culture. Data are reported as mean ± SD of 2 replicate measurements.