Recognition Of Minor Histocompatibility Antigens Research Articles

T cell optimization for graft-versus-leukemia responses.

Protection from relapse after allogeneic hematopoietic cell transplantation (HCT) is partly due to donor T cell-mediated graft-versus-leukemia (GVL) immune responses. Relapse remains common in HCT recipients, but strategies to augment GVL could significantly improve outcomes after HCT. Donor T cells with αβ T cell receptors (TCRs) mediate GVL through recognition of minor histocompatibility antigens and alloantigens in HLA-matched and -mismatched HCT, respectively. αβ T cells specific for other leukemia-associated antigens, including nonpolymorphic antigens and neoantigens, may also deliver an antileukemic effect. γδ T cells may contribute to GVL, although their biology and specificity are less well understood. Vaccination or adoptive transfer of donor-derived T cells with natural or transgenic receptors are strategies with potential to selectively enhance αβ and γδ T cell GVL effects.

Identification of Biological Relevant Minor Histocompatibility Antigens within the B-lymphocyte-Derived HLA-Ligandome Using a Reverse Immunology Approach.

T-cell recognition of minor histocompatibility antigens (MiHA) not only plays an important role in the beneficial graft-versus-leukemia (GVL) effect of allogeneic stem cell transplantation (allo-SCT) but also mediates serious GVH complications associated with allo-SCT. Using a reverse immunology approach, we aim to develop a method enabling the identification of T-cell responses directed against predefined antigens, with the goal to select those MiHAs that can be used clinically in combination with allo-SCT. In this study, we used a recently developed MiHA selection algorithm to select candidate MiHAs within the HLA-presented ligandome of transformed B cells. From the HLA-presented ligandome that predominantly consisted of monomorphic peptides, 25 polymorphic peptides with a clinically relevant allele frequency were selected. By high-throughput screening, the availability of high-avidity T cells specific for these MiHA candidates in different healthy donors was analyzed. With the use of MHC multimer enrichment, analyses of expanded T cells by combinatorial coding MHC multimer flow cytometry, and subsequent single-cell cloning, positive T-cell clones directed to two new MiHA: LB-CLYBL-1Y and LB-TEP1-1S could be demonstrated, indicating the immunogenicity of these two MiHAs. The biologic relevance of MiHA LB-CLYBL-1Y was demonstrated by the detection of LB-CLYBL-1Y-specific T cells in a patient suffering from acute myeloid leukemia (AML) that experienced an anti-leukemic response after treatment with allo-SCT.

LB-ARHGDIB-1R As Novel Minor Histocompatibility Antigen For Therapeutic Application

Patients with hematological malignancies can be successfully treated with allogeneic hematopoietic stem cell transplantation (alloSCT). Beneficial Graft-versus-Leukemia (GvL) reactivity, however, is often accompanied with undesired Graft-versus-Host Disease (GvHD). In HLA-matched alloSCT, donor T-cells can mediate GvL reactivity by recognition of minor histocompatibility antigens (MiHA) on malignant cells, but also GvHD when MiHA are recognized on non-hematopoietic tissues. To decrease the incidence and severity of GvHD, T-cells can be (partially) depleted from the graft and re-administered later as donor lymphocyte infusion (DLI). MiHA are polymorphic peptides that are presented in HLA-molecules and can be recognized as non-self by donor-derived T-cells. Only a minority of MiHA have therapeutic relevance based on hematopoietic-restricted expression and only 25% and 40% of recipients transplanted with sibling and unrelated donors, respectively, are eligible for T-cell therapies in which one of the known hematopoietic-restricted MiHA is targeted. Therefore, to increase the efficacy and limit the toxicity of T-cell therapy, more therapeutic MiHA are needed. Recently, we identified a MiHA encoded by ARHGDIB, a gene that has been described to be highly expressed on hematopoietic cells, and we here studied the therapeutic relevance of LB-ARHGDIB-1R in detail. First, we confirmed hematopoietic-restricted expression of ARHGDIB by microarray gene expression analysis and demonstrated >10-fold overexpression in the majority of malignant and healthy hematopoietic versus non-hematopoietic cells. In line with its hematopoietic-restricted gene expression profile, LB-ARHGDIB-1R in the context of HLA-B* 07:02 was specifically recognized on different hematological malignancies, but not on non-hematopoietic fibroblasts and keratinocytes cultured in the absence or presence of IFN-γ, which was added to mimick the pro-inflammatory cytokine milieu of the early post-transplantation period. Next, we investigated the cytolytic capacity of LB-ARHGDIB-1R specific T-cells, and demonstrated specific lysis of patient, but not donor, EBV-B cells and specific lysis of an ALL sample in a 10 hrs 51Cr-release assay. Specific lysis of additional ALL and AML samples could be measured after 48 hrs of co-incubation in a flowcytometry-based cytotoxicity assay. To determine the in vivo immunogenicity of LB-ARHGDIB-1R, 11 MiHA-disparate HLA-B* 07:02 positive patient-donor pairs were screened for specific CD8+ T-cells by dual color tetramer analysis. All patients received partial T-cell depleted alloSCT and sampling was done at different time points after alloSCT (and DLI). In 4 out of 11 patients, LB-ARHGDIB-1R-specific T-cells (>0.01%) could be detected directly ex vivo and in 4 additional patients after 7 days of in vitro peptide stimulation, indicating that LB-ARHGDIB-1R is highly immunogenic. High frequencies of LB-ARHGDIB-1R specific T-cells were measured ex vivo in a patient whose hematological relapse was successfully treated with DLI, without development of GvHD, further supporting the therapeutic relevance of LB-ARHGDIB-1R. In summary, we confirmed hematopoietic-restricted expression of LB-ARHGDIB-1R and demonstrated T-cell mediated lysis of primary leukemic cells in long-term co-incubation assays. Furthermore, we showed that LB-ARHGDIB-1R is highly immunogenic and that specific T-cells could be detected in a patient who responded to DLI in the absence of GvHD. Altogether, our data support the clinical relevance of LB-ARHGDIB-1R as therapeutic MiHA with the potential to shift the delicate balance between GvL and GvHD in favor of a desired anti-tumor effect. Disclosures: No relevant conflicts of interest to declare.

Durable Remission of Renal Cell Carcinoma After Donor Lymphocyte Infusion Is Unavoidably Linked with Graft Versus Host Disease As Illustrated by the Detection of Allo Reactive T Cells Recognizing a Novel Minor Histocompatibility Antigen Encoded by the FUCA2 Gene

AbstractAbstract 4467Allogeneic stem cell transplantation (allo SCT) followed by donor lymphocyte infusion (DLI) can be applied as immunotherapeutic intervention to treat malignant diseases. In hematological malignancies, remissions can be observed both in the presence and absence of graft versus host disease (GvHD), illustrating that graft versus leukemia and GvHD can be clinically separated. For efficient induction of an allo immune response, professional (hematopoietic) antigen presenting cells (APC) are required, which may result in skewing towards recognition of minor histocompatibility antigens (MiHA) selectively expressed by hematopoietic cells. However, for treatment of solid tumors including renal cell carcinoma (RCC), recognition of MiHA on non hematopoietic cells is essential. Therefore in these malignancies APC have to present broadly expressed MiHA to be targeted on non hematopoietic cells, making it inevitable that the graft versus tumor (GvT) effect is accompanied by GvHD.Here we describe the treatment of a patient who presented with progressive metastatic clear cell RCC with T cell depleted non myeloablative allo SCT. At 7 months after allo SCT the patient received DLI at a single dose of 0.5*107 T cells/kg. Acute skin GvHD occurred within 30 days after DLI and developed soon thereafter into persistent extensive chronic GvHD for several years that eventually resolved after prolonged topical and systemic immune suppression. Whereas the patient was mixed chimeric after allo SCT (84%, 80% and 95% donor after 2, 3 and 5 months, respectively), treatment with DLI resulted in conversion to full donor chimerism, which remained complete during the following years. The strong immune response was accompanied by an approximately 50% reduction in size of the measurable lung metastasis (according to RECIST criteria) and slight regression of the smaller lung lesions. To characterize the specificity of the immune response, we measured known MiHA, and detected a disparity for the previously identified MiHA LRH-1. T cells specific for this MiHA were detectable in peripheral blood at the onset of GvHD (0.14% of CD8+ T cells). Isolated LRH-1 specific T cells strongly recognized patient derived EBV-LCL, but were irresponsive to monocyte derived dendritic cells (monoDC), fibroblasts, keratinocytes, and a panel of LRH-1+ RCC cell lines and are therefore considered irrelevant for the GvT effect. In addition, we directly cloned activated CD8+ T cells and isolated an HLA-B*07 restricted T cell clone with unknown specificity. Whole genome association scanning unraveled a patient specific single nucleotide polymorphism in the FUCA2 gene encoding a novel MiHA which we designated LB-FUCA2-1V. High frequencies of circulating LB-FUCA2-1V specific T cells were detected after DLI (1.9% of CD8+ cells). Moreover, in contrast to LRH-1 specific T cells, LB-FUCA2-1V specific T cells recognized monoDC, and skin derived keratinocytes and fibroblasts after IFN-γ pretreatment. Our data also show recognition of 4 RCC cell lines, and therefore support a dominant role for LB-FUCA2-1V specific T cells in mediating tumor regression. Unfortunately, coinciding with the gradual resolvement of chronic GvHD, the GvT effect declined and eventually the metastases became progressive almost 3 years after allo SCT. In an attempt to re-initiate the GvT effect, escalating DLI doses of 0.2*107, 0.5*107, 1*107 and 5*107 T cells/kg were given. No GvHD developed but also no GvT effect could be achieved and the patient died of progressive disease 8.5 years after allo SCT.In conclusion, we observed a strong donor derived immune response targeting the broadly expressed LB-FUCA2-1V MiHA that durably controlled metastatic clear cell RCC. Initiation of this response likely depended on the presence of patient hematopoiesis, including APC, since all attempts to re-induce the MiHA specific response to treat disease progression with escalating doses of DLI under full donor chimerism were ineffective. Our data suggest that successful treatment of solid tumors may require induction of an effective immune response against broadly expressed MiHA, inevitably inducing damage to normal tissue cells resulting in GvHD. 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.

High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations

T-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, the number of MiHA identified to date remains limited, making clinical application of MiHA reactive T-cell infusion difficult. This study represents the first attempt of genome-wide prediction of MiHA, coupled to the isolation of T-cell populations that react with these antigens. In this unbiased high-throughput MiHA screen, both the possibilities and pitfalls of this approach were investigated. First, 973 polymorphic peptides expressed by hematopoietic stem cells were predicted and screened for HLA-A2 binding. Subsequently a set of 333 high affinity HLA-A2 ligands was identified and post transplantation samples from allo-SCT patients were screened for T-cell reactivity by a combination of pMHC-tetramer-based enrichment and multi-color flow cytometry. Using this approach, 71 peptide-reactive T-cell populations were generated. The isolation of a T-cell line specifically recognizing target cells expressing the MAP4K1IMA antigen demonstrates that identification of MiHA through this approach is in principle feasible. However, with the exception of the known MiHA HMHA1, none of the other T-cell populations that were generated demonstrated recognition of endogenously MiHA expressing target cells, even though recognition of peptide-loaded targets was often apparent.Collectively these results demonstrate the technical feasibility of high-throughput analysis of antigen-specific T-cell responses in small patient samples. However, the high-sensitivity of this approach requires the use of potential epitope sets that are not solely based on MHC binding, to prevent the frequent detection of T-cell responses that lack biological relevance.

Concurrent Detection of Circulating Minor Histocompatibility Antigen-Specific CD8+ T Cells in SCT Recipients by Combinatorial Encoding MHC Multimers

Allogeneic stem cell transplantation (SCT) is a potentially curative treatment for patients with hematologic malignancies. Its therapeutic effect is largely dependent on recognition of minor histocompatibility antigens (MiHA) by donor-derived CD8+ T cells. Therefore, monitoring of multiple MiHA-specific CD8+ T cell responses may prove to be valuable for evaluating the efficacy of allogeneic SCT. In this study, we investigated the use of the combinatorial encoding MHC multimer technique to simultaneously detect MiHA-specific CD8+ T cells in peripheral blood of SCT recipients. Feasibility of this approach was demonstrated by applying dual-color encoding MHC multimers for a set of 10 known MiHA. Interestingly, single staining using a fluorochrome- and Qdot-based five-color combination showed comparable results to dual-color staining for most MiHA-specific CD8+ T cell responses. In addition, we determined the potential value of combinatorial encoding MHC multimers in MiHA identification. Therefore, a set of 75 candidate MiHA peptides was predicted from polymorphic genes with a hematopoietic expression profile and further selected for high and intermediate binding affinity for HLA-A2. Screening of a large cohort of SCT recipients resulted in the detection of dual-color encoded CD8+ T cells following MHC multimer-based T cell enrichment and short ex vivo expansion. Interestingly, candidate MiHA-specific CD8+ T cell responses for LAG3 and TLR10 derived polymorphic peptides could be confirmed by genotyping of the respective SNPs. These findings demonstrate the potency of the combinatorial MHC multimer approach in the monitoring of CD8+ T cell responses to known and potential MiHA in limited amounts of peripheral blood from allogeneic SCT recipients.

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition.

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1(H) versus HA-1(R)) in the HMHA1 gene. The HA-1(H) peptide is restricted by HLA-A2 and is immunogenic in HA-1(R/R) into HA-1(H) transplants, while HA-1(R) has been suggested to be a "null allele" in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1(R) variant. To understand these findings, we determined the structure of an HLA-A2-HA-1(H) complex to 1.3A resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1(H)-specific T-cells bound HA-1(H) peptide with moderate affinity but failed to bind HA-1(R), indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition.

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.

Aberrant Expression in Human Epithelial Cancers of the P2X5-Encoded Minor Histocompatibility Antigen LRH-1: Implications for Graft-Versus-Tumor Immunity Against Solid Tumors.

Allogeneic stem cell transplantation (SCT) in combination with donor lymphocyte infusion (DLI) is an experimental treatment for patients with metastatic solid tumors. The therapeutic efficacy is attributed to the graft-versus-tumor (GVT) response during which donor-derived T cells eliminate malignant cells via recognition of minor histocompatibility antigens (MiHA). To reduce accompanying GVHD, it is crucial to identify MiHA which are selectively expressed on hematopoietic cells and solid tumor cells. Previously, we identified a hematopoietic cell-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene (J. Clin. Invest. 2005: 115:3506–3516). Here, we report that LRH-1, in addition to its hematopoietic cell-restricted expression, is aberrantly expressed on epithelial tumor cell lines. We observed that P2X5 mRNA is significantly expressed in 14 out of 42 (33%) solid tumor cell lines tested by real-time quantitative RT-PCR. We detected P2X5 transcripts in 3 out of 11 renal cell carcinoma cell lines, 2 out of 4 melanoma cell lines, 3 out of 7 colorectal carcinoma cell lines, 4 out of 10 brain tumor cell lines and 2 out of 10 breast cancer cell lines. To determine whether P2X5 mRNA expression in solid tumor cell lines results in susceptibility to lysis by LRH-1-specific CTL, we performed flow cytometry-based cytotoxicity assays using P2X5-expressing tumor cell lines. Based on LRH-1 genotyping analysis, we selected six solid tumor cell lines for the cytotoxicity studies. Remarkably, LRH-1-specific CTL efficiently lysed and inhibited the growth of DAOY brain tumor cells up to 3 days of co-culture. The renal cell carcinoma cell lines SKRC-33 and SKRC-18 and the melanoma cell line BLM were also susceptible to LRH-1 CTL-mediated lysis, although less effectively. However, pre-incubation of these tumor cell lines with IFNγ and TNFα significantly increased the susceptibility to LRH-1-specific CTL and resulted in complete target cell lysis. Furthermore, these cytokine-stimulated cell lines induced higher levels of CTL degranulation as determined by CD107a staining. No cytotoxicity was observed against LRH-1-negative FM3 melanoma and SKRC-24 renal cell carcinoma cell lines. These findings illustrate that the GVT reactivity observed in solid tumors after allogeneic SCT may be selectively enhanced by LRH-1-specific immunotherapy.

DNA Fusion Vaccines Induce Epitope-Specific Cytotoxic CD8+ T Cells against Human Leukemia-Associated Minor Histocompatibility Antigens

The graft-versus-leukemia effect of allogeneic stem-cell transplantation is believed to be mediated by T-cell recognition of minor histocompatibility antigens on recipient cells. For minor histocompatibility antigens HA-1 and HA-2, normal cell expression is restricted to hemopoietic cells, and boosting the immune response to these antigens may potentiate graft-versus-leukemia effect without accompanying graft-versus-host disease. To increase efficacy, expansion of HA-1- or HA-2-specific CTL before transplantation is desirable. However, primary HA-1- or HA-2-specific CTL expanded in vitro are often of low avidity. An alternative approach is to prime specific CTL responses in vivo by vaccination. Clearly, donor vaccination must be safe and specific. We have developed DNA fusion vaccines able to induce high levels of epitope-specific CTL using linked CD4(+) T-cell help. The vaccines incorporate a domain of tetanus toxin (DOM) fused to a sequence encoding a candidate MHC class I binding peptide. This design generates antitumor CD8(+) T-cell responses and protective immunity in preclinical models. For clinical application, we constructed vaccines encoding HLA-A*0201-restricted peptides from human HA-1 and HA-2, which were fused to DOM, and tested their performance in HLA-A*0201-transgenic mice. Priming induced epitope-specific, IFNgamma-producing CD8(+) T cells with cytotoxic function boosted to high levels with electroporation. Strikingly, these mouse T cells efficiently killed human lymphoblastoid cell lines expressing endogenous HA-1 or HA-2. High avidity is indicated by the independence of cytolysis from CD8/MHC class I interaction. These safe epitope-specific vaccines offer a potential strategy to prime HA-1- or HA-2-specific CTL in transplant donors before adoptive transfer.

Molecular persistence of chronic myeloid leukemia caused by donor T cells specific for lineage-restricted maturation antigens not recognizing immature progenitor-cells

Although donor lymphocyte infusion (DLI) induces complete remissions in 70% of patients with relapsed chronic myeloid leukemia (CML) after allogeneic stem-cell transplantation (SCT), some patients are refractory to DLI by showing disease persistence. In a patient who received DLI for relapsed CML, we observed persisting molecular disease despite a hematological and cytogenetic remission in the absence of graft-versus-host disease (GVHD). To determine the nature of this immune response, we isolated leukemia-reactive donor T-cell clones from the bone marrow (BM) of the patient at the time of clinical response. Four different types of CD8+ HLA class I restricted T-cell clones were obtained that were cytotoxic against Ebstein-Barr virus-transformed B-cell lines (EBV-LCL) of the patient, but not the donor, indicating recognition of minor histocompatibility antigens (mHags). By using survival studies with CFSE labelled BM cells populations, a hematopoietic progenitor cell inhibition assay and direct morphological examination we showed that the T-cell clones recognized mature monocytic and myeloid cells, whereas immature BM progenitor cells were insufficiently lysed. This patient's refractoriness for DLI appears to be caused by inadequate lysis of progenitor cells by these cytotoxic T cells. These findings support the hypothesis that for eradication of CML a cytotoxic T-cell response against leukemic progenitor cells is essential.

Sharing of Class I MHC Molecules Between Donor and Host Promotes the Infiltration of Allografts by mHAg-Reactive CD8+ T cells

Indirect recognition of minor histocompatibility antigens (mHAg) and/or MHC-encoded allopeptides is an important barrier to long-term allograft acceptance following solid organ transplantation. Efficient priming of CD8+ T cells can occur after allotransplantation as a consequence of cross-presentation of donor-derived proteins by the graft recipient's APC. Consistent with this, draining lymph node clonal expansion of OVA-reactive OT-I CD8+ T cells following placement of OVA-transgenic skin grafts did not depend on graft expression of K(b). However, OT-I T cells did accumulate in OVA-transgenic skin grafts most efficiently only when both the donor and host expressed K(b). OT-I infiltration of (B6-OVA x BALB/c)F1 grafts in B6 recipients was not suppressed by graft expression of H-2d. Furthermore, B6 animals transplanted with both B6-OVA and BALB/c-OVA skin had more OT-I T cells infiltrating their B6-OVA MHC-matched graft. Therefore, class I MHC matching between donor and host may not always favor an avoidance of alloreactivity within the graft tissue.

Separation of CD4+ T cell-mediated graft-versus-host responses from the graft-versus-leukemia effect

Graft-versus-host disease (GVHD) can result from transplantation of blood and bone marrow cells into major histocompatibility complex (MHC)-matched recipient mice due to donor T cell recognition of minor histocompatibility antigens (miHA). Graft-versus-leukemia (GVL) responses can be directed to distinct or over expressed leukemia-restricted antigens presented by MHC class I or class II molecules. In addition, donor T cells which are responsible for the induction of GVHD may also mount a GVL effect directed to shared host miHA on tumor cells.

The MBP-reactive repertoire is shaped by recognition of minor histocompatibility antigens

While it is known that the degeneracy of T-cell antigen recognition is involved in many aspects of T cell-immunology, its importance in the selection of the T cell repertoire remains an aspect to be better investigated. Here we examined if an intrathymic degenerate T cell recognition mechanism shapes the myelin basic protein (MBP)-reactive repertoire inducing resistance to experimental autoimmune encephalomyelitis (EAE) in some MHC and/or minor histocompatibility antigens (MiHAs) heterozygous F1 mice bearing the H-2 s susceptibility allele. We found a considerable degree of cross-reactivity between MBP and MiHAs encoded in various EAE resistant mouse strains: (1) MBP-specific T cells can be re-stimulated in vitro by cells expressing these MiHAs and maintain their encephalitogenic activity, and (2) lymphoid cells from parental strains that generate EAE resistant F1 hybrids can induce disease relapse when injected into EAE-susceptible hosts. The results suggest that heterozygosity, through the degeneracy of T cell antigen recognition mechanism, may provide further means to constrain the potential autoreactive repertoire.

The DBY gene codes for an HLA-DQ5–restricted human male-specific minor histocompatibility antigen involved in graft-versus-host disease

AbstractGraft rejection or graft-versus-host (GVH) disease after HLA-identical stem cell transplantation is the result of recognition of minor histocompatibility antigens (mHags) by immunocompetent T lymphocytes from recipient or donor origin, respectively. Cytolytic T lymphocyte (CTL) clones can be isolated during graft rejection and GVH disease to identify mHags and their corresponding genes. Thus far, all human mHags identified appeared to be HLA class I–restricted. Here, we report the characterization of the first human HLA class II–restricted sex-linked mHag involved in GVH disease. Previously, we isolated an HLA-DQ5–restricted CD4+ CTL clone from a male patient with chronic myeloid leukemia who developed acute GVH disease grade III-IV after transplantation of HLA genotypically identical female stem cells. Using a panel of female HLA-DQ5+ EBV cells that we stably transfected with Y chromosome–specific genes, we determined that the HLA class II male-specific mHag (H-Y) was encoded by the Y chromosome–specific gene DBY. The H-Y epitope was localized in the DBY protein using female HLA-DQ5+peripheral blood mononuclear cells loaded with DBY protein fragments. The minimal peptide sequence leading to maximal recognition by the specific HLA-DQ5–restricted CTL clone was characterized as the 12–amino acid sequence HIENFSDIDMGE. Although the epitope differed by 3 amino acids from its X-homolog DBX, only 2 polymorphisms were shown to be essential for recognition by the CTL clone.

DFFRY codes for a new human male-specific minor transplantation antigen involved in bone marrow graft rejection

Graft rejection after histocompatibility locus antigen (HLA)-identical stem cell transplantation results from the recognition of minor histocompatibility antigens on donor stem cells by immunocompetent T lymphocytes of recipient origin. T-lymphocyte clones that specifically recognize H-Y epitopes on male target cells have been generated during graft rejection after sex-mismatched transplantation. Previously, 2 human H-Y epitopes derived from the same SMCY gene have been identified that were involved in bone marrow graft rejection. We report the identification of a new male-specific transplantation antigen encoded by the Y-chromosome-specific gene DFFRY. The DFFRY-derived peptide was recognized by an HLA-A1 restricted CTL clone, generated during graft rejection from a female patient with acute myeloid leukemia who rejected HLA-phenotypically identical bone marrow from her father. The identification of this gene demonstrates that at least 2 genes present on the human Y-chromosome code for male-specific transplantation antigens.

Recognition of minor histocompatibility antigens on lymphocytic and myeloid leukemic cells by cytotoxic T-cell clones

Clinical studies indicated an enhanced antileukemic effect of allogeneic bone marrow transplantation (BMT), as compared with autologous BMT. After allogeneic HLA-identical BMT, donor-derived cytotoxic T lymphocytes (CTLs) directed at minor histocompatibility (mH) antigens on the recipients, tissues can be shown. To evaluate the antileukemic reactivity of mH antigen-specific CTLs, we analyzed the expression of mH antigens on circulating lymphocytic and myeloid leukemic cells. We show that the defined mH specificities HA-1 through HA-5 and H-Y are present on leukemic cells, indicating that mH antigen- specific CTLs are capable of HLA class I-restricted antigen-specific lysis of leukemic cells. Compared with interleukin-2-stimulated normal lymphocytes, leukemic cells of lymphocytic origin are less susceptible to T-cell-mediated cytotoxicity by the HA-2 mH antigen-specific CTL and the anti-HLA-A2 CTL clone. A possible explanation for this phenomenon is impaired expression of the LFA-1 adhesion molecule. Our study suggests that mH antigen-specific HLA class I-restricted CD8+ CTLs may be involved in the graft-versus-leukemia reactivity after allogeneic BMT.

Recognition of Minor Histocompatibility Antigens on Lymphocytic and Myeloid Leukemic Cells by Cytotoxic T-Cell Clones

AbstractClinical studies indicated an enhanced antileukemic effect of allogeneic bone marrow transplantation (BMT), as compared with autologous BMT. After allogeneic HLA-identi-cal BMT, donor-derived cytotoxic T lymphocytes (CTLs) directed at minor histocompatibility (mH) antigens on the recipients, tissues can be shown. To evaluate the antileukemic reactivity of mH antigen-specific CTLs, we analyzed the expression of mH antigens on circulating lymphocytic and myeloid leukemic cells. We show that the defined mH specificities HA-1 through HA-5 and H-Y are present on leukemic cells, indicating that mH antigen-specific CTLs are capable of HLA class I-restricted antigen-specific lysis of leukemic cells. Compared with interleukin-2-stimulated normal lymphocytes, leukemic cells of lymphocytic origin are less susceptible to T-cell-mediated cytotoxicity by the HA-2 mH antigen-specific CTL and the anti-HLA-A2 CTL clone. A possible explanation for this phenomenon is impaired expression of the LFA-1 adhesion molecule. Our study suggests that mH antigen-specific HLA class l-restricted CD8+ CTLs may be involved in the graft-versus-leukemia reactivity after allogeneic BMT.

Inhibitory effect of herpes simplex virus infection to target cells on recognition of minor histocompatibility antigens by cytotoxic T lymphocytes.

Target cell lysis by CTL specific for minor histocompatibility Ag (minor HA), which were generated in (C3H/He x BALB/c)F1 mice immunized with A/J mouse spleen cells, was dramatically reduced by infection of HSV to Neuro-2a (A/J mouse origin) cells as target. The reduction was apparent at 5 h after infection of HSV to target cells, when many viral proteins were produced in the cells. Conversely, MHC-restricted HSV-specific CTL-mediated cell lysis increased time dependently. Using an RNA virus, vesicular stomatitis virus, significant reduction of minor specific CTL-mediated target cell lysis was also found. During the time when this reduction of target cell lysis by HSV occurred, the surface expression of class I H-2Dd molecules was maintained, and anti-H-2a allo-MHC-specific CTL lysed HSV-infected Neuro-2a cells as strongly as uninfected Neuro-2a cells. When HSV-infected or uninfected Neuro-2a cells were treated with Brefeldin A that selectively blocks transportation of newly synthesized proteins out of endoplasmic reticulum, both HSV- and minor HA-specific CTL-mediated cell lyses were blocked. These observations demonstrated that minor HA are continuously synthesized and associated with class I molecules at pre-Golgi and transported via trans Golgi system with quick turnover, and that newly synthesized HSV Ag, which are also associated with class I molecules and transported via the same system, should take the place of intrinsic minor HA and be presented on the surface of the cells to be recognized by MHC-restricted CTL.