LAUSR

Background Donor lymphocyte infusion is an effective treatment for AML relapse post-transplant, but it has the risk of inducing graft versus host disease (GVHD). Isolating and expanding a subset of lymphocytes that contains antigen-specific T cells against AML antigens may increase the potential for a graft-versus-leukemia immune response while reducing the risk of GVHD. We hypothesized that irradiating AML blasts would diminish their immune suppressive capacity while maintaining antigen presentation, resulting in higher activation of antigen-specific CD8+ T cells among peripheral blood mononuclear cells (PBMC) in co-culture. Methods PBMC were isolated from healthy donors’ whole blood. PBMC were co-cultured with live AML K-1 (CRL-2724) cells and irradiated K-1 cells (40 Gy) separately at a 1:2 ratio. Cells were cultured in RPMI complete media supplemented with 10% FBS and IL-2 20 IU/ml. On day 3 of co-culture, CD137 positive cells were labeled by anti-CD137 antibody and isolated by positive selection using a magnetic separation column (MiniMACS; Miltenyi Biotech). PBMC Immunophenotype was evaluated on flow cytometer using surface labels CD3, CD4, CD8, CD137, CD25, CD56 and LAG3 in addition to intracellular IFNγ and FOXP3. Both CD137-selected and unselected cells were expanded in complete media supplemented with IL-2 2000 IU/ml, IL-7 50 ng/ml and IL-15 300 ng/ml. CFSE-labeled K-1 cells were incubated with both CD137-selected and unselected PBMCs separately then viability was measured by 7-ADD on flow cytometry. Results Healthy donor PBMC co-cultured with irradiated K-1 showed significantly higher CD137 expression (9.3% ± 1.21 v. 5.7% ± 3.4; n=7, P Figure 1 A) and IFNγ expression (11.8% ± 3.1 v. 7% ± 3.3; n=7, P=0.012; Figure 1 B) on CD8+ T cells when compared to the live K-1-PBMC co-cultures. There were fewer Tregs (CD4+ CD25+ FOXP3+) in the PBMC co-cultured with irradiated K-1 cells compared to the live K-1-PBMC co-cultures (1.96% ± 0.37 v. 3.39% ± 0.58; n=4, P=0.03; Figure 1 C). CD137-selected cells were isolated and expanded in culture for 2 weeks, and unselected cells were expanded in the same culture conditions. CD137-selected cells expressed more CD3 (60.65% vs 22.8%; n=4, P=0.012) and CD56 (1.6% vs 0.25; n=4, P=0.01) than unselected cells. In comparison to unselected cells. CD137-selected cells showed higher killing of CSFE-labeled AML cells across AML: PBMC ratios (1:5,1:10 and 1:15) at both 24 and 48 hours compared to unselected cells ( Figure 2 ). Conclusion Irradiation of AML cells prior to co-culture increases the expression of activation markers CD137, CD154, and IFNγ. The activation marker CD137 can be used to select a population of cells from healthy donor PBMC. CD137-selected cells can be expanded in vitro and induce efficient killing of AML cells compared to the subset of PBMCs with no CD137 expression.