Low CLIP Expression On Leukemic Blasts From AML Patients Is Essential for the Generation of Effective CD4⁺ T Cell Responses.

Abstract 996

Poster Board I-18

In patients with AML, the outgrowth of minimal residual disease (MRD) is considered as the major cause of relapse, whereby it is hypothesized that residual leukemic blasts are able to escape from immune surveillance. Since CD4⁺ T cells are critical for inducing effective anti-leukemic immunity, certain leukemic blasts might exhibit aberrant HLA class II antigen presentation that interferes with antigen-specific CD4⁺ T cell recognition. Increased binding of the class II-associated invariant chain self peptide (CLIP) to the HLA class II antigen-binding groove may thereby prevent the presentation of antigenic peptides. This study investigates both the clinical and functional role of CLIP expression on myeloid leukemic blasts. Blood and bone marrow samples from a cohort of 207 de novo AML patients were analyzed by flow cytometry for plasma membrane expression of CLIP and HLA-DR (DR). Significantly shortened disease-free and overall survival rates were found for patients with leukemic blasts characterized by a high amount of DR occupied by CLIP (relative CLIP amount). To explore the functional role of CLIP, we transduced blasts of the human Kasumi-1 and THP-1 myeloid leukemic cell lines with retroviral siRNAs specific for the Invariant Chain, a chaperone molecule that is critically involved in DR processing. Significant reductions in relative CLIP amount were found on blasts of both cell lines. Subsequently, CD4⁺ T cells derived from different healthy donors (n=3) were stimulated with either irradiated DR⁺CLIP^- (Ii siRNA-treated) or DR⁺CLIP⁺ (wild type) THP-1 and Kasumi-1 blasts during mixed leukocyte reactions. In contrast to DR⁺CLIP⁺ blasts, DR⁺CLIP^- blasts of both cell lines induced strong increases in allogeneic CD4⁺ T cell proliferation in a stimulator-to-responder dependent manner. To examine the effect of CLIP on CD4⁺ T cell induction in primary samples, we performed flow cytometric sorting experiments to select for CLIP^- and CLIP⁺ leukemic blasts from different DR⁺ AML patients (n=5). CD4⁺ T cells collected from these same patients after achieving complete remission were isolated and stimulated with sorted CLIP^- or CLIP⁺ leukemic blasts during four weeks of culture. In 2 of the 5 patients, marked proliferation of autologous remission CD4⁺ T cells stimulated with CLIP^- leukemic blasts was observed in contrast to stimulation with CLIP⁺ leukemic blasts. In addition, in 4 of the 5 patients, flow cytometric analysis of CD4⁺ T cells showed that CLIP^- leukemic blasts were able to induce both high CD25 and HLA-DR and low CD45RA and CD27 expression as compared to CLIP⁺ leukemic blasts, indicating increased activation of effector memory CD4⁺ T cells. Moreover, CD4⁺ T cells stimulated with CLIP^- leukemic blasts also revealed strongly increased IFN-g/IL-4 ratios in contrast to CD4⁺ T cells stimulated with CLIP⁺ leukemic blasts, as determined by flow cytometry after PMA/ionomycin stimulation. This might imply skewing towards a more Th1 phenotype. In conclusion, these findings not only emphasize that the relative CLIP amount on leukemic blasts predicts clinical outcome, but also reveal that it is a critical factor for CD4⁺ T cell activation in AML. Hence, CLIP may serve as a target for immunomodulatory strategies to optimize HLA class II antigen presentation on AML whole-cell or DC vaccines and induce leukemia-specific CD4⁺ T cell immunity in patients.