Caroline E. Rutten

High-Throughput Characterization of 10 New Minor Histocompatibility Antigens by Whole Genome Association Scanning

Patients with malignant diseases can be effectively treated with allogeneic hematopoietic stem cell transplantation (allo-SCT). Polymorphic peptides presented in HLA molecules, the so-called minor histocompatibility antigens (MiHA), play a crucial role in antitumor immunity as targets for alloreactive donor T cells. Identification of multiple MiHAs is essential to understand and manipulate the development of clinical responses after allo-SCT. In this study, CD8+ T-cell clones were isolated from leukemia patients who entered complete remission after allo-SCT, and MiHA-specific T-cell clones were efficiently selected for analysis of recognition of a panel of EBV-transformed B cells positive for the HLA restriction elements of the selected T-cell clones. One million single nucleotide polymorphisms (SNP) were determined in the panel cell lines and investigated for matching with the T-cell recognition data by whole genome association scanning (WGAs). Significant association with 12 genomic regions was found, and detailed analysis of genes located within these genomic regions revealed SNP disparities encoding polymorphic peptides in 10 cases. Differential recognition of patient-type, but not donor-type, peptides validated the identification of these MiHAs. Using tetramers, distinct populations of MiHA-specific CD8+ T cells were detected, demonstrating that our WGAs strategy allows high-throughput discovery of relevant targets in antitumor immunity after allo-SCT.

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.

HLA-DP as specific target for cellular immunotherapy in HLA class II-expressing B-cell leukemia

Mismatching for human leukocyte antigen (HLA)-DPB1 in unrelated donor hematopoietic stem cell transplantation (URD-SCT) has been associated with a decreased risk of disease relapse, indicating that HLA-DP may represent a target for graft-versus-leukemia (GVL) reactivity in HLA class II-expressing hematological malignancies. To investigate whether HLA-DP-specific T cells could mediate GVL reactivity following HLA-DPB1-mismatched URD-SCT and donor lymphocyte infusion (DLI), we analyzed the immune response in a patient with leukemic lymphoplasmacytic lymphoma responding to DLI without graft-versus-host disease. The emergence of leukemia-reactive CD4+ T cells during the clinical immune response was demonstrated by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot(ELISPOT)analysis. Following clonal isolation of these leukemia-reactive CD4+ T cells, blocking studies, panel studies and retroviral transduction experiments of both mismatched HLA-DPB1 alleles identified HLA-DPB1*0201 and HLA-DPB1*0301 as the targets of this immune response. The HLA-DPB1-specific CD4+ T-cell clones were capable of recognizing and lysing several HLA-DP-expressing myeloid and lymphoid hematological malignant cells. Since HLA-DP expression is mainly restricted to hematopoietic cells, HLA-DP may be used as a specific target for immunotherapy following T-cell-depleted URD-SCT. Therefore, in patients with HLA class II-expressing hematological malignancies HLA-DP-mismatched SCT may be preferable over fully matched SCT allowing DLI to induce a GVL effect.

Patient HLA-DP-specific CD4+ T cells from HLA-DPB1-mismatched donor lymphocyte infusion can induce graft-versus-leukemia reactivity in the presence or absence of graft-versus-host disease.

Clinical studies have demonstrated that HLA-DPB1-mismatched allogeneic stem cell transplantation (allo-SCT) is associated with a decreased risk of disease relapse and an increased risk of graft-versus-host disease (GVHD) compared with HLA-DPB1-matched SCT. In T cell-depleted allo-SCT, mismatching of HLA-DPB1 was not associated with an increased risk of severe GVHD, but a significant decreased risk of disease relapse was still observed. To investigate whether patient HLA-DP-specific CD4(+) T cell responses were frequently induced after T cell-depleted HLA-DPB1-mismatched allo-SCT and donor lymphocyte infusion (DLI), we developed a method to screen for the presence of HLA-DP-specific CD4(+) T cells using CD137 as an activation marker and analyzed 24 patient-donor combinations. The patients suffered from various B cell malignancies, multiple myeloma, and myeloid leukemias. Patient HLA-DP-specific CD4(+) T cells were detected after DLI in 13 of 18 patients who exhibited a clinical response to DLI, compared with only 1 of 6 patients without a clinical response to DLI. Eight patients developed significant GVHD. These data show that patient HLA-DP-specific CD4(+) T cells frequently occur after HLA-DPB1-mismatched T cell-depleted allo-SCT and DLI, and are associated with graft-versus-leukemia reactivity both in the presence and absence of GVHD.

Both permissive and nonpermissive HLA-DPB1 mismatches can induce polyclonal HLA-DPB1 specific immune responses in vivo and in vitro.

To the editor: Clinical studies have indicated that human leukocyte antigen (HLA)–DPB1 functions as a classical transplantation antigen in allogeneic stem cell transplantation (SCT). Mismatching for HLA-DPB1 was associated with an increased risk of graft-versus-host disease (GVHD) but also a

HLA-DPB1 mismatching results in the generation of a full repertoire of HLA-DPB1-specific CD4+ T cell responses showing immunogenicity of all HLA-DPB1 alleles.

Clinical studies have indicated that HLA-DPB1 functions as a classical transplantation antigen in allogeneic stem cell transplantation. Mismatching for HLA-DPB1 was associated with an increased risk of graft-versus-host disease (GVHD), but also a decreased risk of disease relapse. However, specific HLA-DPB1 mismatches were associated with poor clinical outcome. It was suggested that this unfavorable effect was caused by a difference in immunogenicity between HLA-DPB1 alleles. To analyze whether immunogenicity of HLA-DPB1 mismatches could be predicted based on the presence or absence of specific amino acid sequences we developed a model to generate allo-HLA-DPB1 responses in vitro. We tested in total 48 different stimulator/responder combinations by stimulating CD4(+) T cells from 5 HLA-DPB1 homozygous individuals with the same antigen-presenting cells transduced with different allo-HLA-DPB1 molecules. HLA-DPB1 molecules used for stimulation comprised 76% to 99% of HLA-DPB1 molecules present in different ethnic populations. We show that all HLA-DPB1 mismatches as defined by allele typing resulted in high-frequency immune responses. Furthermore, we show that crossrecognition of different HLA-DPB1 molecules is a broadly observed phenomenon. We confirm previously described patterns in crossrecognition, and demonstrate that a high degree in similarity between HLA-DPB1 molecules is predictive for crossrecognition, but not for immunogenicity.

Selective graft-versus-leukemia depends on magnitude and diversity of the alloreactive T cell response

Patients with leukemia who receive a T cell–depleted allogeneic stem cell graft followed by postponed donor lymphocyte infusion (DLI) can experience graft-versus-leukemia (GVL) reactivity, with a lower risk of graft-versus-host disease (GVHD). Here, we have investigated the magnitude, diversity, and specificity of alloreactive CD8 T cells in patients who developed GVL reactivity after DLI in the absence or presence of GVHD. We observed a lower magnitude and diversity of CD8 T cells for minor histocompatibility antigens (MiHAs) in patients with selective GVL reactivity without GVHD. Furthermore, we demonstrated that MiHA-specific T cell clones from patients with selective GVL reactivity showed lower reactivity against nonhematopoietic cells, even when pretreated with inflammatory cytokines. Expression analysis of MiHA-encoding genes showed that similar types of antigens were recognized in both patient groups, but in patients who developed GVHD, T cell reactivity was skewed to target broadly expressed MiHAs. As an inflammatory environment can render nonhematopoietic cells susceptible to T cell recognition, prevention of such circumstances favors induction of selective GVL reactivity without development of GVHD.