M. Willy Honders

Identification of phosphatidylinositol 4-kinase type II β as HLA class II-restricted target in graft versus leukemia reactivity

Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLIs). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Because HLA class II molecules are predominantly expressed on hematopoietic cells, mHag-specific CD4+ T cells may selectively mediate graft versus leukemia (GvL) reactivity without graft versus host disease (GvHD). In this study, we used a recombinant bacteria cDNA library for the identification of the first autosomal HLA class II (HLA-DQB1*0603)-restricted mHag LB-PI4K2B-1S encoded by the broadly expressed phosphatidylinositol 4-kinase type II β gene. A polyclonal CD4+ T cell response against LB-PI4K2B-1S was demonstrated in a patient with relapsed chronic myeloid leukemia (CML) who responded to DLI after HLA-matched alloSCT. LB-PI4K2B-1S-specific CD4+ T cells recognized and lysed the CD34+ CML cells of the patient and other leukemic cells as well as high HLA-DQ-expressing normal hematopoietic cells. HLA-DQ expression on normal cells of nonhematopoietic origin was moderately up-regulated by IFN-γ and not sufficient for T cell recognition. We hypothesize that LB-PI4K2B-1S-specific CD4+ T cells contributed to the antitumor response by both directly eliminating malignant cells as effector cells and stimulating CD8+ T cell immunity as helper cells.

Deoxycytidine kinase expression and activity in patients with resistant versus sensitive acute myeloid leukemia

Abstract: Resistance to cytarabine (AraC) is a major problem in treatment of patients with acute myeloid leukemia (AML). In contrast to in vitro AraC resistance, deoxycytidine kinase (dCK) mutations are rarely found in patients with refractory or relapsed AML. Previously we have demonstrated alternatively spliced dCK mRNA predominantly expressed in leukemic blasts from patients with resistant AML. In this study we investigated wild‐type (wt) dCK expression and activity to elucidate the possible role of decreased dCK expression or activity in unresponsiveness to AraC in patients with AML. No alterations in dCK mRNA and protein expression or in dCK activity were detected between patients with clinically resistant vs. sensitive AML. In addition, wt dCK expression and activity were not reduced in leukemic blasts expressing alternatively spliced dCK forms as compared to blasts with only wt dCK. Also, no major differences in wt dCK expression and activity were observed between samples obtained from patients with AML and bone marrow or peripheral blood samples from healthy donors. These data implicate that in our patient group of refractory or relapsed AML cases, alterations in dCK expression and/or activity cannot explain unresponsiveness to chemotherapy including AraC.

Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity

Background Patients with hematologic malignancies can be successfully treated with donor lymphocyte infusion after HLA-matched allogeneic hematopoietic stem cell transplantation. The effect of donor lymphocyte infusion is mediated by donor T cells recognizing minor histocompatibility antigens. T cells recognizing hematopoietic restricted minor histocompatibility antigens may induce selective graft-versus-leukemia reactivity, whereas broadly-expressed antigens may be targeted in graft-versus-host disease. Design and Methods We analyzed in detail CD8+ T-cell immunity in a patient with relapsed chronic myelogenous leukemia who responded to donor lymphocyte infusion with minimal graft-versus-host disease of the skin. CD8+ T-cell clones specific for 4 HLA-B*40:01 restricted minor histocompatibility antigens were isolated which were identified by screening a plasmid cDNA library and whole genome association scanning. Detailed T-cell reactivity and monitoring experiments were performed to estimate the clinical and therapeutic relevance of the novel antigens. Results Three antigens were demonstrated to be expressed on primary leukemic cells of various origins as well as subtypes of non-malignant hematopoietic cells, whereas one antigen was selectively recognized on malignant hematopoietic cells with antigen presenting cell phenotype. Skin derived fibroblasts were only recognized after pre-treatment with IFN-γ by two T-cell clones. Conclusions Our data show evidence for different roles of the HLA-B*40:01 restricted minor histocompatibility antigens in the onset and execution of the anti-tumor response. All antigens may have contributed to a graft-versus-leukemia effect, and one minor histocompatibility antigen (LB-SWAP70-1Q) has specific therapeutic value based on its in vivo immunodominance and strong presentation on leukemic cells of various origins, but absence of expression on cytokine-treated fibroblasts.

Inactivation of deoxycytidine kinase and overexpression of P-glycoprotein in AraC and daunorubicin double resistant leukemic cell lines

AraC resistance in vitro is explained by inactivation of dCK, while resistance to DNR is described by overexpression of multidrug efflux pumps like Pgp or MRP. Thus far, no correlation between resistance mechanisms in vitro and in patients with AML has been documented. We generated AraC and DNR double resistant cell lines to investigate resistance mechanisms of both agents. In these cell lines involvement of dCK was extensively investigated and Pgp expression and activity was determined. Our data implicate that similar resistance mechanisms like inactivation of dCK coincided by alternatively spliced dCK forms and overexpression of Pgp are induced in single-as well as in double resistant leukemic cell lines.

Human Leukocyte Antigen–DO Regulates Surface Presentation of Human Leukocyte Antigen Class II–Restricted Antigens on B Cell Malignancies

Hematological malignancies often express surface HLA class II, making them attractive targets for CD4+ T cell therapy. We previously demonstrated that HLA class II ligands can be divided into DM-resistant and DM-sensitive antigens. In contrast to presentation of DM-resistant antigens, presentation of DM-sensitive antigens is suppressed by HLA-DM but can be rescued by HLA-DO. We also showed that HLA-DO expression remains low in nonhematopoietic cells under inflammatory conditions, suggesting that DM-sensitive antigens may be ideal T cell targets with a low risk for graft-versus-host disease. Here, we demonstrated that B cell malignancies often express HLA-DO and that levels are in particular high in chronic lymphocytic leukemia. Moreover, we showed that surface presentation of DM-sensitive antigens is regulated by HLA-DO, and that DM-sensitive antigens are relevant T cell targets for B cell malignancies and, especially, chronic lymphocytic leukemia. These data open the perspective to target HLA class II ligands with specific processing and presentation behavior for CD4+ T cell therapy of hematological malignancies.