Dendritic Cell Vaccination in Acute Leukemia Patients Induces Reduction of Myeloid-Derived Suppressor Cells : Immunological Analysis of a Pilot Study

Background and Purpose: Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population that have an immunesuppresive function and play an important role in tumor immunology and self tolerance. Elevated levels of MDSCs in peripheral blood (PB) are reported in various cancers and hematological malignancies such as MDS, NHL and MM. We reported that vaccination with Wilms’ tumor 1 (WT1) peptide-pulsed dendritic cells (DC) in NHL patients induced not only WT1 specific immune responses but also innate immunity which correlated with clinical efficacy (ASH 2013). In the present study, we focused on MDSCs and investigated the effects of DC vaccination on the alteration of MDSCs which have negative influence on disease progression.

Patients and Methods: 8 patients with acute leukemia (6 AML and 2ALL patients; aged 16-77) who were treated with WT1 peptide-loaded DC vaccination were enrolled in the present study. MDSCs were analyzed by FACS. Linage negative, CD11b⁺CD33⁺HLA-DR^- cells were defined as MDSC. Monocytic and granulocytic MDSC were defined as CD14⁺HLA-DR^low/- and CD15⁺CD11b⁺CD33⁺HLA-DR^-, respectively. For proliferation assay, CD14⁺DR^- cells and CD3⁺ T cells were isolated by magnetic beads method. CFSE-labeled CD3⁺ T cells were stimulated with anti-CD3 and anti-CD28 moAb together with IL-2 in the presence or absence of CD14⁺DR^-cells for 4 days and cell division was analyzed by FACS. Cytoplasmic staining of indoleamine 2,3-deoxygenase (IDO) and arginase 1 were performed by permeabilization using saponin followed by moAb staining. Arginase 1 protein levels in plasma was assessed using a commercially available ELISA kit.

Results: 3 patients were in complete remission (responder) and 5 others were in disease progression (non-responder) following one course of vaccination. WT1 specific T cell responses evaluated by a HLA-tetramer assay and a delayed type hypersensitivity (DTH) test were observed in all the responders and 2 non-responders. Mean percentage of MDSCs, monocytic and granulocytic MDSCs in PB was higher in acute leukemia patients than in healthy donors (1.60±0.72%, 16.56±6.07%, 1.34±0.69%, respectively in patients; 0.60±0.31%, 13.1±4.1%, 0.37±0.1%, respectively in healthy donors). Absolute number of circulating monocytic and granulocytic MDSCs before and after vaccination in responders was 359.4±117.8/μl and 35.2±14.8/μl, 282.8±119.2 /μl and 14.4±12.0/μl, respectively. On the other hand, those in non-responders were 126.3±60.2/μl and 36.3±21.7/μl, 181.3±124.0/μl and 65.1±12.0/μl, respectively. While DC vaccination resulted in reduction of both monocytic and granulocytic MDSCs by 21.3% and 59.1%, respectively in responders, it induced augmentation of these MDSCs by 43.5% and 79.3%, respectively in non-responders. A portion of MDSCs expressed IDO and arginase 1 in cytoplasm. While the percentage of IDO positive cells in monocytic MDSCs increased by 2.2 and 2.3 times in responders and non-responders, respectively following vaccination, the positivity of arginase 1 decreased by 59% in responders. On the other hand, the positivity of IDO and arginase 1 in granulocytic MDSCs decreased at the same level in both responders and non-responders following vaccination. CD14⁺DR^-cells inhibited the proliferation of CD3⁺ T cells in a dose dependent manner, indicating that these cells share characteristics of MDSCs. The inhibition of the proliferation of CD3⁺T cells was partially restored by the addition of 1-methyl-DL-tryptophan (IDO inhibitor) and N^ω-hydroxy-nor-arginine (arginase 1 inhibitor), suggesting that the inhibition was mediated at least in part by IDO and arginase 1. Amount of arginase 1 in the plasma before and after vaccination decreased by 25% in responders, not in non-responders.

Conclusions: These results demonstrated that DC vaccination resulted in the reduction of the absolute number of MDSCs which seems to be correlated with clinical efficacy. DC vaccination may have inhibitory effects on several functions of MDSCs that can suppress immune surveillance against leukemia, which is favorable for regression of leukemia cells.