J.H. Frederik Falkenburg

Mismatches of Minor Histocompatibility Antigens between HLA-Identical Donors and Recipients and the Development of Graft-Versus-Host Disease after Bone Marrow Transplantation

BACKGROUND Graft-versus-host disease (GVHD) can be a major complication of allogeneic bone marrow transplantation even when the donor and recipient are siblings and share identical major histocompatibility antigens. The explanation may be a mismatch of minor histocompatibility antigens. We previously characterized five minor histocompatibility antigens, HA-1, 2, 3, 4, and 5, that are recognized by T cells in association with the major histocompatibility antigens HLA-A1 an A2. METHODS We collected peripheral-blood leukocytes from 148 bone marrow recipients and their sibling donors, who were genotypically HLA identical. Fifty pairs were positive for HLA-A1, 117 were positive for HLA-A2, and 19 were positive for both. The pairs were typed with cytotoxic-T-cell clones specific for minor histocompatibility antigens HA-1, 2, 3, 4, and 5. RESULTS Mismatches of HA-3 were equally distributed among recipients in whom GVHD developed and those in whom it did not. By contrast, a mismatch of only HA-1 was significantly correlated with GVHD of grade II or higher (odds ratio, infinity; P = 0.02) in adults. One or more mismatches of HA-1, 2, 4, and 5 were also significantly associated with GVHD (odds ratio, infinity; P = 0.006) in adults. These associations were not observed in children. CONCLUSIONS A mismatch of minor histocompatibility antigen HA-1 can cause GVHD in adult recipients of allogeneic bone marrow from HLA-identical donors. Prospective HA-1 typing may improve donor selection and identify recipients who are at high risk for GVHD.

Mesenchymal stem cells promote engraftment of human umbilical cord blood–derived CD34+ cells in NOD/SCID mice

OBJECTIVE Mesenchymal stem cells (MSC) have been implicated as playing an important role in hematopoietic stem cell engraftment. We identified and characterized a new population of MSC derived from human fetal lung. In cotransplantation experiments, we examined the homing of MSC as well as the effect on engraftment of human umbilical cord blood (UCB)-derived CD34(+) cells in NOD/SCID mice. MATERIALS AND METHODS Culture-expanded fetal lung-derived CD34(+) cells were characterized by immune phenotyping and cultured under conditions promoting differentiation to osteoblasts or adipocytes. Irradiated (3.5 Gy) NOD/SCID mice (n = 51) were transplanted intravenously with 0.03 to 1.0 x 10(6) UCB CD34(+) cells in the presence or absence of 1 x 10(6) culture-expanded fetal lung-derived MSC, irradiated CD34(-) cells, B cells, or with cultured MSC only. RESULTS Culture-expanded fetal lung CD34(+) cells were identified as MSC based on phenotype (CD105(+), SH3(+), SH4(+), CD160(+)) and their multilineage potential. Cotransplantation of low doses of UCB CD34(+) cells and MSC resulted in a three-fold to four-fold increase in bone marrow engraftment after 6 weeks, whereas no such effect was observed after cotransplantation of irradiated CD34(-) or B cells. Homing experiments indicated the presence of MSC in the lung, but not in the bone marrow, of NOD/SCID mice. CONCLUSIONS We identified a population of MSC derived from human fetal lung. Upon cotransplantation, MSC, but not irradiated CD34(-) or B cells, promote engraftment of UCB CD34(+) cells in bone marrow, spleen, and blood by mechanisms that may not require homing of MSC to the bone marrow.

Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia

Donor lymphocyte infusion (DLI) into patients with a relapse of their leukemia or multiple myeloma after allogeneic stem cell transplantation (alloSCT) has been shown to be a successful treatment approach. The hematopoiesis-restricted minor histocompatibility antigens (mHAgs) HA-1 or HA-2 expressed on malignant cells of the recipient may serve as target antigens for alloreactive donor T cells. Recently we treated three mHAg HA-1- and/or HA-2-positive patients with a relapse of their disease after alloSCT with DLI from their mHAg HA-1- and/or HA-2-negative donors. Using HLA-A2/HA-1 and HA-2 peptide tetrameric complexes we showed the emergence of HA-1- and HA-2-specific CD8+ T cells in the blood of the recipients 5–7 weeks after DLI. The appearance of these tetramer-positive cells was followed immediately by a complete remission of the disease and restoration of 100% donor chimerism in each of the patients. Furthermore, cloned tetramer-positive T cells isolated during the clinical response specifically recognized HA-1 and HA-2 expressing malignant progenitor cells of the recipient and inhibited the growth of leukemic precursor cells in vitro. Thus, HA-1- and HA-2-specific cytotoxic T lymphocytes emerging in the blood of patients after DLI demonstrate graft-versus-leukemia or myeloma reactivity resulting in a durable remission. This finding implies that in vitro generated HA-1- and HA-2-specific cytotoxic T lymphocytes could be used as adoptive immunotherapy to treat hematological malignances relapsing after alloSCT.

Allo-HLA reactivity of virus-specific memory T-cells is common

Graft-versus-host disease and graft rejection are major complications of allogeneic HLA-mismatched stem cell transplantation or organ transplantation that are caused by alloreactive T cells. Because a range of acute viral infections have been linked to initiating these complications, we hypothesized that the cross-reactive potential of virus-specific memory T cells to allogeneic (allo) HLA molecules may be able to mediate these complications. To analyze the allo-HLA reactivity, T cells specific for Epstein-Barr virus, cytomegalovirus, varicella zoster virus, and influenza virus were tested against a panel of HLA-typed target cells, and target cells transduced with single HLA molecules. Eighty percent of T-cell lines and 45% of virus-specific T-cell clones were shown to cross-react against allo-HLA molecules. The cross-reactivity of the CD8 and CD4 T-cell clones was directed primarily against HLA class I and II, respectively. However, a restricted number of CD8 T cells exhibited cross-reactivity to HLA class II. T-cell receptor (TCR) gene transfer confirmed that allo-HLA reactivity and virus specificity were mediated via the same TCR. These results demonstrate that a substantial proportion of virus-specific T cells exert allo-HLA reactivity, which may have important clinical implications in transplantation settings as well as adoptive transfer of third-party virus-specific T cells.

Reprogramming of virus-specific T cells into leukemia-reactive T cells using T cell receptor gene transfer.

T cells directed against minor histocompatibility antigens (mHags) might be responsible for eradication of hematological malignancies after allogeneic stem cell transplantation. We investigated whether transfer of T cell receptors (TCRs) directed against mHags, exclusively expressed on hematopoietic cells, could redirect virus-specific T cells toward antileukemic reactivity, without the loss of their original specificity. Generation of T cells with dual specificity may lead to survival of these TCR-transferred T cells for prolonged periods of time in vivo due to transactivation of the endogenous TCR of the tumor-reactive T cells by the latent presence of viral antigens. Furthermore, TCR transfer into restricted T cell populations, which are nonself reactive, will minimize the risk of autoimmunity. We demonstrate that cytomegalovirus (CMV)-specific T cells can be efficiently reprogrammed into leukemia-reactive T cells by transfer of TCRs directed against the mHag HA-2. HA-2-TCR–transferred CMV-specific T cells derived from human histocompatibility leukocyte antigen (HLA)-A2+ or HLA-A2− individuals exerted potent antileukemic as well as CMV reactivity, without signs of anti–HLA-A2 alloreactivity. The dual specificity of these mHag-specific, TCR-redirected virus-specific T cells opens new possibilities for the treatment of hematological malignancies of HLA-A2+ HA-2–expressing patients transplanted with HLA-A2–matched or –mismatched donors.

Minor histocompatibility antigens in human stem cell transplantation

Minor histocompatibility antigens (mHags) play a major role in graft rejection, the induction of detrimental graft-vs-host disease (GVHD), and the development of the beneficial graft-vs-leukemia (GVL) effect after allogeneic stem cell transplantation (SCT). mHags can be defined as amino acid polymorphisms in cellular proteins that can lead to differential presentation of antigenic peptides in HLA molecules and therefore to differential recognition by T cells. The tissue distribution of the mHags and the HLA molecules by which they can be presented play a significant role in the clinical outcome of T-cell responses against these antigens. In part, differential recognition by T cells of mHags specifically expressed in hematopoietic cells, including the malignant cells from the recipient may result in GVL reactivity without concurrent GVHD. Furthermore, T-cell responses against proteins solely expressed in hematopoietic cell lineages from which the malignancy is derived may be appropriate mediators of GVL reactivity without GVHD induction. Characterization of clinical immune responses in patients treated for relapsed leukemia after allogeneic SCT with donor lymphocyte infusion in the absence of GVHD may lead to the characterization of new mHags that can be exploited to generate tumor-specific immune responses. By in vitro generation of T-cell responses against defined mHags, the efficacy and specificity of cellular immunotherapy against hematologic malignancies in the context of allogeneic transplantation may be improved.

Identification of HLA class II-restricted H-Y-specific T-helper epitope evoking CD4+ T-helper cells in H-Y-mismatched transplantation

BACKGROUND Stem-cell grafts between HLA-identical siblings are less likely to succeed when there is a sex mismatch. This lack of success can be interpreted as enhanced activity directed against minor histocompatibility antigens encoded by the Y chromosome (H-Y). So far, in man, only cytotoxic T lymphocytes (CTLs) specific for several minor histocompatibility antigens have been reported. We aimed to identify and clarify the role of MHC class II-restricted H-Y-specific T-helper cells in these transplant settings. METHODS H-Y-specific MHC class II-restricted CD4+ T cells were isolated from blood of a female patient who rejected an HLA-identical male stem-cell transplant. By molecular cloning of H-Y genes and functional T-helper experiments, we elucidated antigen specificity and the functional properties of these H-Y-specific T-helper cells. FINDINGS CD4+ T-helper cells recognise the Y gene-encoded peptide VIKVNDTVQI presented by HLA-DRbeta3*0301. These T-helper cells mature dendritic cells and enhance expansion of minor histocompatibility antigen-specific MHC class I-restricted CD8+ CTLs. INTERPRETATION Characterisation of an MHC class II-restricted H-Y epitope that evoked CD4+ T-helper responses adds a novel cellular component to the alloimmune response against Y chromosome-encoded minor histocompatibility antigens. This component completes the H-Y-directed alloimmune response and aids understanding of the poorer outcome of sex-mismatched transplants.

Mixed T cell receptor dimers harbor potentially harmful neoreactivity

Adoptive transfer of T cell receptor (TCR)-transduced T cells may be an attractive strategy to target both hematological malignancies and solid tumors. By introducing a TCR, large numbers of T cells with defined antigen (Ag) specificity can be obtained. However, by introduction of a TCR, mixed TCR dimers can be formed. Besides the decrease in TCR expression of the introduced and endogenous TCR, these mixed TCR dimers could harbor potentially harmful specificities. In this study, we demonstrate that introduction of TCRs resulted in formation of neoreactive mixed TCR dimers, composed of the introduced TCR chains pairing with either the endogenous TCR α or β chain. Neoreactivities observed were HLA class I or class II restricted. Most neoreactive mixed TCR dimers were allo-HLA reactive; however, neoreactive mixed TCR dimers with autoreactive activity were also observed. We demonstrate that inclusion of an extra disulfide bond between the constant domains of the introduced TCR markedly reduced neoreactivity, whereas enhanced effectiveness of the introduced TCR was observed. In conclusion, TCR transfer results in the formation of neoreactive mixed TCR dimers with the potential to generate off-target effects, underlining the importance of searching for techniques to facilitate preferential pairing.

NCI First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation: Report from the Committee on Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation

Relapse is a major cause of treatment failure after allogeneic hematopoietic stem cell transplantation (alloHSCT). Treatment options for relapse have been inadequate, and the majority of patients ultimately die of their disease. There is no standard approach to treating relapse after alloHSCT. Withdrawal of immune suppression and donor lymphocyte infusions are commonly used for all diseases; although these interventions are remarkably effective for relapsed chronic myelogenous leukemia, they have limited efficacy in other hematologic malignancies. Conventional and novel chemotherapy, monoclonal antibody therapy, targeted therapies, and second transplants have been utilized in a variety of relapsed diseases, but reports on these therapies are generally anecdotal and retrospective. As such, there is an immediate need for well-designed, disease-specific trials for treatment of relapse after alloHSCT. This report summarizes current treatment options under investigation for relapse after alloHSCT in a disease-specific manner. In addition, recommendations are provided for specific areas of research necessary in the treatment of relapse after alloHSCT.

GENERATION OF DENDRITIC CELLS EXPRESSING BCR-ABL FROM CD34-POSITIVE CHRONIC MYELOID LEUKEMIA PRECURSOR CELLS

Patients with a relapse of chronic myeloid leukemia (CML) after allogeneic bone marrow transplantation can be successfully treated with blood mononuclear cells from the original bone marrow donor. However, the antileukemic effect of this treatment is often accompanied by graft-versus-host disease (GVHD). Treatment with cytotoxic T-lymphocyte (CTL) lines or clones that are specifically generated against leukemic antigen-presenting cells from the patient, may separate antileukemic effects from GVHD. In this report we demonstrate that after culturing CD34-positive cells purified from bone marrow of patients with chronic phase CML in medium containing human serum, GM-CSF, TNF alpha, and IL-4 up to 28% of the cultured cells were dendritic cells, characterized by morphology, phenotypic analysis, and their efficient capacity to stimulate allogeneic T lymphocytes. The expression of HLA and costimulatory molecules and the stimulatory capacity of the dendritic cell-enriched cell suspensions were optimal between days 7 and 10 after onset of the cultures. Fluorescence in situ hybridization revealed that all cultured dendritic cells contained the CML specific t(9;22) translocation. PCR analysis showed expression of the translocation specific bcr-abl mRNA. These leukemic dendritic cells may enhance the induction and proliferation of CTL lines and clones with more specificity for the leukemic cells.