Hematopoiesis-restricted Minor Histocompatibility Antigens Research Articles

Collateral Damage of Nonhematopoietic Tissue by Hematopoiesis-Specific T Cells Results in Graft-versus-Host Disease During an Ongoing Profound Graft-versus-Leukemia Reaction

After allogeneic stem cell transplantation (allo-SCT), donor T cells may recognize minor histocompatibility antigens (MiHA) specifically expressed on cells of the recipient. It has been hypothesized that T cells recognizing hematopoiesis-restricted MiHA mediate specific graft-versus-leukemia (GVL) activity without inducing graft-versus-host disease (GVHD), whereas T cells recognizing ubiquitously expressed MiHA induce both GVL and GVHD reactivity. It also has been hypothesized that alloreactive CD4 T cells are capable of mediating specific GVL reactivity due to the hematopoiesis-restricted expression of HLA class II. However, clinical observations suggest that an overt GVL response, associated with expansion of T cells specific for hematopoiesis-restricted antigens, is often associated with GVHD reactivity. Therefore, we developed in vitro models to investigate whether alloreactive T cells recognizing hematopoiesis-restricted antigens induce collateral damage to surrounding nonhematopoietic tissues. We found that collateral damage to MiHA-negative fibroblasts was induced by misdirection of cytotoxic granules released from MiHA-specific T cells activated by MiHA-positive hematopoietic cells, resulting in granzyme-B–mediated activation of apoptosis in the surrounding fibroblasts. We demonstrated that direct contact between the activated T cell and the fibroblast is a prerequisite for this collateral damage to occur. Our data suggest that hematopoiesis-restricted T cells actively participate in an overt GVL response and may contribute to GVHD via induction of collateral damage to nonhematopoietic targets.

Collateral Damage of Non-Hematopoietic Tissue by Hematopoiesis-Specific T Cells Results in GvHD During An Ongoing Profound Gvl Reaction,

Abstract 4031Following HLA-matched allogeneic stem cell transplantation (SCT) donor T cells may recognize polymorphic antigens specifically expressed on cells of the recipient, designated as minor histocompatibility antigens (MiHA). It has been hypothesized that hematopoiesis-restricted MiHA may give rise to a specific graft versus leukemia (GvL) effect without graft versus host disease (GvHD), whereas ubiquitously expressed MiHA can be targets for both GvL and GvHD. We and others have observed that during an overt GvL response, associated with the occurrence of T cells specific for hematopoiesis-restricted MiHA, may coincide with limited GvHD. Therefore, we investigated whether allo-reactive T cells recognizing hematopoiesis-restricted MiHA on leukemic cells may induce collateral damage to surrounding non-hematopoietic tissues. To test this, we stimulated MiHA-specific cytotoxic T lymphocytes (CTLs) in vitro with MiHA positive hematopoietic targets and analyzed whether this resulted in lysis of surrounding MiHA negative primary human fibroblasts seeded in the same well. Using chromium release assays, quantitative FACS analysis and fluorescence microscopy, we demonstrated that MiHA-specific CTLs (CD8 n=3, CD4 n=2) and allo-reactive CTLs upon proper activation by MiHA-positive hematopoietic stimulator cells (EBV-LCL/ DCs), induced 80% (60–100%) bystander cytotoxicity to the surrounding fibroblasts at a 5/1 T cell/fibroblast-ratio within 24 hrs. T cell receptor (TCR)-specific T cell activation by the hematopoietic cells was pivotal for the induction of this collateral damage since no bystander kill was observed when MiHA-specific CTLs were exposed to MiHA negative stimulators. Next, we investigated the influence of the strength of T cell activation by stimulating the CTLs with MiHA-negative stimulator cells loaded with increasing concentrations of the relevant peptide resulting in increasing T cell activation measured by IFNg ELISA and expression of the T cell activation marker CD137. We demonstrated that this increase in the strength of T cell activation is correlated with an increased severity of collateral damage. Moreover, we showed also that T cell activation in a cell free system using PHA (0,5ug/ml) or antiCD3/CD28 beads resulted in induction of collateral damage of the surrounding non-hematopoietic cells illustrating that the MiHA-negative fibroblasts are not attacked due to cross-presentation of HLA/peptide complexes from apoptotic hematopoietic cells. Next, we further investigated the prerequisites for collateral damage induction to occur. Using a transwell system, we demonstrated that direct T cell-fibroblast interaction was required. Furthermore, ICAM-1 expression on the non-hematopoietic targets, induced by pro-inflammatory cytokines which were secreted by the activated CTLs, was demonstrated to be crucial for strong T cell-fibroblast interaction. To investigate the contribution of the different T cell effector mechanisms, we retrovirally overexpressed the anti-apoptotic proteins c-FLIP, Bcl-2 or PI-9 in the fibroblasts, thereby inhibiting the death receptor-, mitochondrial-, or granzyme-B (GzB)-mediated pathways, respectively. High expression of c-FLIP or Bcl-2 did not inhibit induction of collateral damage. In contrast, increased PI-9 expression in the fibroblasts partially inhibited induction of collateral damage. Moreover, blockade of Ca2+-dependent secretion of perforin by EGTA, could also inhibit collateral damage suggesting that the main killing pathway of collateral damage is via release of GzB- and perforin-containing granules by the T cells upon TCR-triggering. Misdirection of the release of cytotoxic granules towards the MiHA negative non-hematopoietic targets may induce collateral damage. In conclusion, these data suggest that hematopoiesis-restricted T cells actively participating in an overt GvL response can induce GvHD by collateral damage to MiHA-negative non-hematopoietic targets by misdirection of their cytotoxic execution machinery when they are in close proximity of the non-hematopoietic tissue. Disclosures:No relevant conflicts of interest to declare.

HLA-Peptidomics and the Identification of Clinical Relevant Minor Histocompatibility Antigens,

Abstract 4038T-cell recognition of minor histocompatibility antigens (MiHA) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, MiHA recognition is also associated with graft-versus-host disease (GVHD). It is assumed that the selective infusion of T-cells reactive with hematopoiesis-restricted MiHA may help to separate the GVT and GVHD effects of allo-SCT. However, the number of attractive MiHA identified to date remains limited.In this study we aimed to determine whether it is feasible to identify MiHA using HLA-peptidomics in a reverse-immunology approach, based on bona fide eluted MiHA epitopes. Successful development of such a technology could allow the rapid identification of new MiHA, required to make antigen-selective adoptive T-cell therapy a realistic option. In addition, when compared to classical forward approaches, this strategy may provide tools to efficiently identify favorable GVT-involved MiHA, rather than random identifying targets of activated T-cells isolated during a GVT-response.To identify biological relevant MiHA candidates, HLA class I peptides were isolated from lysed EBV-transformed B-cells (EBV-LCL), analyzed by mass spectrometry (MS) and matched with a human protein database (IPI). This effort resulted in a set of fifteen thousand peptides, encoded in the normal reading frame with high probability MS scores. To identify potential MiHA candidates, the total set was matched with our newly developed public available Human Short Peptide Variation Database (http://srs.bioinformatics.nl/hspv), dedicated to polymorphic peptides. The quality of this peptide set was demonstrated by a detection efficiency of fifty percent of known MiHA including various length variants and eluted MiHA counterparts. Subsequently the combined use of gene expression databases, validated single nucleotide polymorphism (SNP) arrays and HLA-peptide binding assays resulted in a further selection of 27 high potential HLA-A*0201 and B*0701 MiHA candidates. This set was used for the generation of pMHC tetramers by UV-mediated exchange technology. Next, pMHC tetramer positive specific T-cell lines were generated from eighteen healthy SNP-typed PBMC donors following MACS isolation. To decrease the incidence of isolating low affinity T-cells, due to self-tolerance induction, pMHC tetramer isolations were only performed using donors homozygous negative for the specific SNP. After repeated pMHC tetramer pull down, in vitro expanded cell samples were analyzed on a multi-color FACS LSRII flow cytometer and clonally expanded following FACS cell sorting.Using this approach we were able to detect 16 unique pMHC tetramer positive T-cell populations corresponding with 70% of eluted MiHA candidates. Most of these pMHC tetramer positive T-cell populations were detected in multiple individuals, and appeared to be oligoclonal. Although most T-cell clones produced IFN-γ when co-cultured with peptide-pulsed target cells, there appeared to be a wide variety of peptide affinity among the pMHC tetramer positive T-cell clones. High throughput screening of all clones for MiHA specific recognition patterns of SNP-typed EBV-LCL panels revealed a clear correlation between the peptide-affinity of the T-cell clone and its capacity to recognize endogenously processed and presented peptide. Collectively these efforts resulted in the validation of two previously described MiHA and the identification of three new biological relevant MiHA.In summary, this study resulted in the establishment of an algorithm for the high-throughput identification of MiHA based on the combined use of HLA-peptidomics and reverse-immunology by pMHC tetramers. Our data indicate that the technology developed within this project can be of great value to the efficient identification of novel MiHA with potential clinical value especially when epitope selection criteria are supplemented with gene expression data, allowing pre-selection for those MiHA candidates with a hematopoiesis restricted gene expression patterns that may direct reactivity towards GVT. Disclosures:No relevant conflicts of interest to declare.

Myeloid leukemic progenitor cells can be specifically targeted by minor histocompatibility antigen LRH-1–reactive cytotoxic T cells

AbstractCD8+ T cells recognizing minor histocompatibility antigens (MiHAs) on leukemic stem and progenitor cells play a pivotal role in effective graft-versus-leukemia reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified a hematopoiesis-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene. We found that P2X5 is significantly expressed in CD34+ leukemic subpopulations from chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) patients. Here, we demonstrate that LRH-1–specific CD8+ T-cell responses are frequently induced in myeloid leukemia patients following donor lymphocyte infusions. Patients with high percentages of circulating LRH-1–specific CD8+ T cells had no or only mild graft-versus-host disease. Functional analysis showed that LRH-1–specific cytotoxic T lymphocytes (CTLs) isolated from 2 different patients efficiently target LRH-1–positive leukemic CD34+ progenitor cells from both CML and AML patients, whereas mature CML cells are only marginally lysed due to down-regulation of P2X5. Furthermore, we observed that relative resistance to LRH-1 CTL-mediated cell death due to elevated levels of antiapoptotic XIAP could be overcome by IFN-γ prestimulation and increased CTL-target ratios. These findings provide a rationale for use of LRH-1 as immunotherapeutic target antigen to treat residual or persisting myeloid malignancies after allogeneic SCT.

SNP-Based Genome-Wide Identification of Hematopoiesis-Restricted Minor Histocompatibility Antigens

T-cell recognition of minor histocompatibility antigens (mHag) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, mHag recognition is also associated with graft-versus-host disease (GVHD). It is assumed that the selective infusion of T cells reactive with hematopoiesis-restricted mHag may help to separate the GVT and GVHD effects of allo-SCT. However, the number of attractive mHag identified to date remains limited. In this study we aimed to determine whether it is feasible to perform genome-wide identification of hematopoiesis-restricted minor histocompatibility antigens. Successful development of such a technology could allow the rapid identification of sets of hematopoiesis-restricted minor histocompatibility antigens required to make antigen-selective adoptive T cell therapy a realistic option. To this purpose we performed microarray analyses of hematopoietic progenitor and non-hematopoietic restricted cell types and merged our data with a microarray database to select approximately 80 genes with a hematopoiesis-restricted gene expression pattern. Subsequently, 322 single nucleotide polymorphism (SNPs) were identified in this set of genes and HLA-A2 binding peptides were predicted in both the normal and alternative reading frames using four different HLA-binding algorithms. The resulting set of 1300 putative HLA-A2 ligands was synthesized and tested for binding affinity using a MHC-peptide exchange technology based on UV-sensitive conditional ligands. Binding affinity was determined based on the ability of the peptides to induce MHC-complex recovery after photo cleavage of the conditional ligand. Based on these affinity measurements 400 high- to medium affinity HLA-A2 binding peptides were selected and used to generate tetramers by MHC-ligand exchange. To enable screening with this vast number of tetramers we developed a combinatorial coding method that allows the detection of up to 25 different T-cell populations in one sample. Furthermore, to overcome the problem of limited availability of patient PBMC and low-frequent T cell responses we performed an in vitro T-cell enrichment and expansion step. In this method tetramer positive lymphocytes are isolated with magnetic cell separation columns and cultured in presence of cytokines, feeders and anti-CD3/28 coated beads. Using this technological platform, we have begun to analyze hematopoiesis-restricted mHag responses in transplant recipients with different hematologic malignancies. This screen confirmed the existence of a known hematopoiesis-restricted mHag response and suggested the presence of a number of novel hematopoiesis-restricted mHag responses. Our data indicate that the technology developed within this project is likely to be of value to the identification of collections of novel minor histocompatibility antigens with potential clinical value.

Genetic engineering of virus-specific T cells with T-cell receptors recognizing minor histocompatibility antigens for clinical application

Donor lymphocyte infusion is an effective form of adoptive immunotherapy for hematologic malignancies after allogeneic stem cell transplantation. Graft-versus-host disease, however, often develops due to recognition of ubiquitously-expressed minor histocompatibility antigens. Transfer of T-cell receptors recognizing hematopoiesis-restricted minor histocompatibility antigens to virus-specific T cells may be a powerful anti-tumor therapy with a low risk of graft-versus-host disease. The purpose of this study was to develop an optimal T-cell receptors-encoding multi-cistronic retroviral vector and an efficient method for generating T-cell receptors-engineered virus-specific T cells. Retroviral vectors encoding the T-cell receptors for the hematopoiesis-restricted minor histocompatibility antigen HA-2 with and without selection markers were compared for T-cell receptors surface expression and HA-2-specific lysis. In addition, two different methods, i.e. peptide stimulation of CD8(+) cells and Pro5 MHC pentamer-based isolation of antigen-specific T cells, were investigated for their efficiency to generate T-cell receptors-transduced virus-specific T cells. Bi-cistronic vectors without selection markers most efficiently mediated T-cell receptors surface expression and HA-2-specific lysis. Furthermore, both methods were useful for generating gene-modified cells, but the purity of virus-specific T cells was higher after pentamer isolation. Finally, the capacity of gene-modified cells to express the transgenic T-cell receptors at the cell surface markedly differed between virus-specific T cells and was correlated with lysis of relevant target cells. Our data support T-cell receptors gene transfer to pentamer-isolated virus-specific T cells using bi-cistronic retroviral vectors and illustrate the relevance of selection of gene-modified T cells with appropriate transgenic T-cell receptors surface expression for clinical gene therapy.

Primary Allogeneic T-Cell Responses Against In Vitro Modified Mantle Cell Lymphoma (MCL) Cells: Implications for Adoptive Immunotherapy after HLA-Matched Stem Cell Transplantation (SCT).

Allogeneic SCT is being explored as treatment modality for patients with advanced MCL. Complete sustained remissions have been observed after allogeneic SCT illustrating susceptibility of MCL cells to graft-versus-lymphoma (GVL) effect.To potentiate this GVL effect and to reduce graft-versus-host disease (GVHD) reactivity, adoptive transfer of in vitro-selected cytotoxic T cells (CTLs) with specificity for MCL or for hematopoiesis-restricted minor histocompatibility antigens could be an attractive approach. The lack of expression of costimulatory molecules on MCL cells hampers the generation of MCL-reactive T cell-responses. To transform MCL cells into efficient antigen-presenting cells (APCs) we tested the B-lineage specific activating cytokines (IL-4), the unique MCL proliferating cytokine (IL-10) and the ligand of toll like receptor 9, CpG.Furthermore, CD40 triggering using irradiated CD40-L transfected murine fibroblasts (tCD40L) in combination with the cytokines and CpG was examined. The expression of the costimulatory and adhesion molecules CD80, CD86, CD83, CD54 and CD58 of MCL cells of 7 patients, all carrying the t(11;14) translocation, was analyzed by flowcytometry. No upregulation of any of these molecules was observed using the cytokines or CpG. Ligation of CD40 on MCL cells caused a significant upregulation of CD54,CD58, CD80 and CD86 (p<0.01) with maximal expression after 4 days of stimulation. No additional upregulation was induced from IL- 4, IL -10 or CpG. The cumulative production of IL-12 and IL-10 by the MCL cells in response to the various stimuli after 4 days was measured. High amounts of IL-12 (median 1640 pg/mL, range 67–8800 pg/mL) in the absence of IL-10(<100 pg/mL) were synthesized by MCL cells after CD40 activation. Additional stimulation with CpG enhanced the production of IL-12 (1870 pg/mL, range 77–30000 pg/mL) but also the production of IL-10(299 pg/mL, range 0–418 pg/mL). MCL cells were unable to produce IL-12 without CD40 triggering (<5 pg/mL). To analyze the antigen-presenting capacity of primary MCL cells as well as CD40-activated MCL cells (MCL-APC), CD8+ T cells from an unrelated HLA-A and B matched and from a HLA-class I matched donor were stimulated with MCL or MCL-APC cells. Primary MCL cells were not capable of generating T-cell lines. Using a newly developed flowcytometry-based cytotoxicity assay in which the target cells were labeled with CFSE (Jedema, Blood 2004; 103:2677) we investigate whether the CTL lines, generated against MCL-APC were cytotoxic against MCL-specific targets. The CD8+ CTL lines from both donors effectively killed at an E/T ratio of 10:1 primary MCL (53%) and MCL-APC (83%) and not PHA blasts from the donor. Using limiting dilution assay, in both donor/patient pairs MCL-reactive CTL clones could be generated. 60 out of 89 proliferating CD8+ T cell clones from the first patient/donor pair and 29 out of 74 proliferating CD8+ T cell clones from the second combination showed specific lysis of primary MCL, MCL-APC and PHA blasts from the patient and not of PHA blasts of the donor. Blocking studies using anti-HLA class I antibodies of both CTL lines and clones confirmed class I restricted recognition of the target cells. In conclusion, CD40 activation transforms MCL cells into malignant APC, capable of producing high levels of IL-12 and capable of inducing vigorous MCL-reactive T-cell responses.

Direct cloning of leukemia-reactive T cells from patients treated with donor lymphocyte infusion shows a relative dominance of hematopoiesis-restricted minor histocompatibility antigen HA-1 and HA-2 specific T cells.

Donor T cells recognizing hematopoiesis-restricted minor histocompatibility antigens (mHags) HA-1 and HA-2 on malignant cells play a role in the antileukemia effect of donor lymphocyte infusion (DLI) in patients with relapsed leukemia after allogeneic stem cell transplantation. We quantified the contribution of HA-1 and HA-2 specific T cells to the total number of leukemia-reactive T cells in three HA-2 and/or HA-1 positive patients responding to DLI from their mHag negative donors. Clinical responses occurring 5-7 weeks after DLI were accompanied by an increase in percentages HLA-DR expressing T cells within the CD8+ T cell population. To clonally analyze the leukemia-reactive immune response, T cells responding to the malignancy by secreting IFNgamma were isolated from peripheral blood, directly cloned, and expanded. Tetramer analysis and specific lysis of peptide-pulsed target cells showed that 3-35% of cytotoxic T lymphocyte (CTL) clones isolated were specific for HA-1 or HA-2. TCR VB analysis showed oligoclonal origin of the HA-1 and HA-2 specific CTL clones. The HA-1 and HA-2 specific CTL clones inhibited leukemic progenitor cell growth in vitro. The relatively high frequency of HA-1 and HA-2 specific T cells within the total number of tumor-reactive T cells illustrates relative immunodominance of mHags HA-1 and HA-2.