Class II-associated invariant chain peptide down-modulation enhances the immunogenicity of myeloid leukemic blasts resulting in increased CD4+ T-cell responses

Abstract

Background: Disease recurrence in patients with acute myeloid leukemia may be partially explained by the escape of leukemic blasts from CD4(+) T-cell recognition. The current study investigates the role of aberrant HLA class II antigen presentation on leukemic blasts by determining both the clinical and functional impact of the class II-associated invariant chain peptide (CLIP).

Design and methods: The levels of expression of CLIP and HLA-DR on blood and bone marrow samples from 207 patients with acute myeloid leukemia were correlated with clinical outcome. Irradiated CLIP(-) and CLIP(+) leukemic blasts were compared for their ability to induce CD4(+) T cells during mixed leukocyte reactions. To discriminate between these blasts, we down-modulated CLIP expression on myeloid leukemic cell lines by RNA interference of the invariant chain, a chaperone protein critically involved in HLA-DR processing, and performed flow cytometric sorting for their isolation from primary acute myeloid leukemia samples.

Results: We found that patients with leukemic blasts characterized by a high amount of HLA-DR occupied by CLIP (relative amount of CLIP) had a significantly shortened disease-free survival. The clear reductions in amount of HLA-DR occupied by CLIP on blasts of the THP-1 and Kasumi-1 myeloid leukemic cell lines after treatment with invariant chain short interfering RNA resulted in enhanced rates of allogeneic CD4(+) T-cell proliferation. Similar findings were obtained in an autologous setting, in which there were strong increases in proliferation of remission CD4(+) T cells stimulated with CLIP(-)-sorted leukemic blasts from HLA-DR(+) acute myeloid leukemia patients, in contrast to CLIP(+)-sorted leukemic blasts from the same patients.

Conclusions: These data highlight the relevance of CLIP expression on leukemic blasts and the potential of CLIP as a target for immunomodulatory strategies to enhance HLA class II antigen presentation and CD4(+) T-cell reactivity in acute myeloid leukemia.

Figure 1.

Kaplan-Meier curves for disease-free survival of AML patients. A significant difference was observed between patients with HLA-DR⁺CLIP⁻ leukemic blasts and patients with HLA-DR⁺CLIP⁺ leukemic blasts, both in the total (panel A; P=0.013, log-rank) and intermediate cytogenetic risk (panel B; P=0.025, log-rank) groups of patients. Cut-off levels of 45% of blasts positive for HLA-DR and 35% of blasts positive for CLIP were used.

Figure 2.

Screening of several HLA-DR⁺ myeloid leukemic cell lines for expression of proteins involved in HLA class II antigen presentation. (A) Fluorescence histograms displaying HLA-DR (DR), CLIP and invariant chain (Ii) as well as HLA-DM (DM) and HLA-DO (DO) expression intensities (unfilled peaks) of KG-1, ME-1, THP-1 and Kasumi-1 blasts with their appropriate isotype controls (gray filled peaks). Flow cytometric analysis was performed on 7AAD⁻ blasts. (B) Western blot analysis of DMα (35 kD) expression in blasts of the THP-1 and Kasumi-1, compared to the KG-1 cell line. As a loading control, β-actin (42 kD) was used.

Figure 3.

The effect of invariant chain (Ii) silencing in myeloid leukemic cell lines on the relative amount of CLIP. THP-1 and Kasumi-1 blasts were analyzed for the expression of intracellular Ii (panel A and C, respectively) and extracellular HLA-DR (DR) and CLIP (panel B and D, respectively) by flow cytometry. Due to variable sensitivity for the retroviral transduction and selection procedure of THP-1 and Kasumi-1 blasts, measurements on viable blasts could not be performed until day 27 and 35, respectively, after transduction. Absolute MFI values and relative CLIP amounts were defined as described in the Design and Methods section. As an example, DR and CLIP levels of one of two independent Ii siRNA transductants are depicted. The M1 and M2 regions indicate the percentages of CLIP⁺ and DR⁺ blasts, respectively. Ii expression and relative CLIP amounts represent the means (± SD) of two independent Ii siRNA transductants; N/A, not applicable.

Figure 4.

Proliferation assays of allogeneic CD4⁺ T cells stimulated with CLIP down-modulated blasts of myeloid leukemic cell lines. The ability of CLIP⁺ (wild type) and CLIP⁻ (Ii-silenced) THP-1 blasts (panel A) and Kasumi-1 blasts (panel B) to induce allogeneic CD4⁺ T-cell proliferation was compared in different MLR. CD4⁺ T cells were obtained from three independent healthy donors. Stimulator-to-responder ratios of 1:5, 1:10, 1:20, 1:40 and 1:80 were used together with a negative control (only stimulator cells; ‘Neg’). MLR were carried out in triplicate at each stimulator-to-responder ratio. MLR with CD4⁺ T cells from donor 1 were performed twice (donor 1.1 and 1.2) for both cell lines to confirm reproducibility. In each graph, the days after Ii-siRNA transduction is noted at which Ii-silenced blasts were tested in the MLR. Results show the means (± SEM) of [³H]-thymidine incorporation in counts per minute (cpm), as an indicator of CD4⁺ T-cell proliferation.