Targeted complement inhibition using bispecific antibodies that bind local antigens and endogenous complement regulators.

Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or in development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned a novel class of bispecific antibodies (bsAbs) which are capable of site-directed complement inhibition by bringing endogenous complement regulators in the vicinity of defined cell surface antigens. Here, we analyzed a comprehensive set of obligate bsAbs designed to crosslink a specific target with either complement regulator factor H (FH) or C4b-binding protein (C4BP). The bsAbs were assessed for their capacity to inhibit complement activation and cell lysis in an antigen-targeted manner. We observed that the bsAbs inhibited classical, lectin, and alternative pathway complement activation in which sufficient endogenous serum FH and C4BP could be recruited to achieve local inhibition. Importantly, the bsAbs effectively protected antigen-positive liposomes, erythrocytes, and human leukocytes from complement-mediated lysis. In conclusion, localized complement inhibition by bsAbs capable of recruiting endogenous human complement regulators (such as FH or C4BP) to cell surfaces potentially provides a novel therapeutic approach for the targeted treatment of complement-mediated diseases.

Human anti-C1q autoantibodies bind specifically to solid-phase C1q and enhance phagocytosis but not complement activation.

Autoantibodies directed against complement component C1q are commonly associated with autoimmune diseases, especially systemic lupus erythematosus. Importantly, these anti-C1q autoantibodies are specific for ligand-bound, solid-phase C1q and do not bind to fluid-phase C1q. In patients with anti-C1q, C1q levels are in the normal range, and the autoantibodies are thus not depleting. To study these human anti-C1q autoantibodies at the molecular level, we isolated C1q-reactive B cells and recombinantly produced nine monoclonal antibodies (mAbs) from four different healthy individuals. The isolated mAbs were of the IgG isotype, contained extensively mutated variable domains, and showed high affinity to the collagen-like region of C1q. The anti-C1q mAbs exclusively bound solid-phase C1q in complex with its natural ligands, including immobilized or antigen-bound IgG, IgM or CRP, and necrotic cells. Competition experiments reveal that at least 2 epitopes, also targeted by anti-C1q antibodies in sera from SLE patients, are recognized. Electron microscopy with hexameric IgG-C1q immune complexes demonstrated that multiple mAbs can interact with a single C1q molecule and identified the region of C1q targeted by these mAbs. The opsonization of immune complexes with anti-C1q greatly enhanced Fc-receptor-mediated phagocytosis but did not increase complement activation. We conclude that human anti-C1q autoantibodies specifically bind neo-epitopes on solid-phase C1q, which results in an increase in Fc-receptor-mediated effector functions that may potentially contribute to autoimmune disease immunopathology.

Epitope Stealing as a Mechanism of Dominant Protection by HLA-DQ6 in Type 1 Diabetes.

The heterozygous DQ2/8 (DQA1*05:01-DQB1*02:01/DQA1*03:01-DQB1*03:02) genotype confers the highest risk in type 1 diabetes (T1D), whereas the DQ6/8 (DQA1*02:01-DQB1*06:02/DQA1*03:01-DQB1*03:02) genotype is protective. The mechanism of dominant protection by DQ6 (DQB1*06:02) is unknown. We tested the hypothesis that DQ6 interferes with peptide binding to DQ8 by competition for islet epitope ("epitope stealing") by analysis of the islet ligandome presented by HLA-DQ6/8 and -DQ8/8 on dendritic cells pulsed with islet autoantigens preproinsulin (PPI), GAD65, and IA-2, followed by competition assays using a newly established "epitope-stealing" HLA/peptide-binding assay. HLA-DQ ligandome analysis revealed a distinct DQ6 peptide-binding motif compared with the susceptible DQ2/8 molecules. PPI and IA-2 peptides were identified from DQ6, of DQ6/8 heterozygous dendritic cells, but no DQ8 islet peptides were retrieved. Insulin B6-23, a highly immunogenic CD4 T-cell epitope in patients with T1D, bound to both DQ6 and DQ8. Yet, binding of InsB6-23 to DQ8 was prevented by DQ6. We obtained first functional evidence of a mechanism of dominant protection from disease, in which HLA molecules associated with protection bind islet epitopes in a different, competing, HLA-binding register, leading to "epitope stealing" and conceivably diverting the immune response from islet epitopes presented by disease-susceptible HLA molecules in the absence of protective HLA.

Heterogeneity of circulating CD8 T-cells specific to islet, neo-antigen and virus in patients with type 1 diabetes mellitus.

Auto-reactive CD8 T-cells play an important role in the destruction of pancreatic ß-cells resulting in type 1 diabetes (T1D). However, the phenotype of these auto-reactive cytolytic CD8 T-cells has not yet been extensively described. We used high-dimensional mass cytometry to phenotype autoantigen- (pre-proinsulin), neoantigen- (insulin-DRIP) and virus- (cytomegalovirus) reactive CD8 T-cells in peripheral blood mononuclear cells (PBMCs) of T1D patients. A panel of 33 monoclonal antibodies was designed to further characterise these cells at the single-cell level. HLA-A2 class I tetramers were used for the detection of antigen-specific CD8 T-cells. Using a novel Hierarchical Stochastic Neighbor Embedding (HSNE) tool (implemented in Cytosplore), we identified 42 clusters within the CD8 T-cell compartment of three T1D patients and revealed profound heterogeneity between individuals, as each patient displayed a distinct cluster distribution. Single-cell analysis of pre-proinsulin, insulin-DRIP and cytomegalovirus-specific CD8 T-cells showed that the detected specificities were heterogeneous between and within patients. These findings emphasize the challenge to define the obscure nature of auto-reactive CD8 T-cells.

Dendritic Cells Guide Islet Autoimmunity through a Restricted and Uniquely Processed Peptidome Presented by High-Risk HLA-DR.

Identifying T cell epitopes of islet autoantigens is important for understanding type 1 diabetes (T1D) immunopathogenesis and to design immune monitoring and intervention strategies in relationship to disease progression. Naturally processed T cell epitopes have been discovered by elution from HLA-DR4 of pulsed B lymphocytes. The designated professional APC directing immune responses is the dendritic cell (DC). To identify naturally processed epitopes, monocyte-derived DC were pulsed with preproinsulin (PPI), glutamic acid decarboxylase (65-kDa isoform; GAD65), and insulinoma-associated Ag-2 (IA-2), and peptides were eluted of HLA-DR3 and -DR4, which are associated with highest risk for T1D development. Proteome analysis confirmed uptake and processing of islet Ags by DC. PPI peptides generated by DC differed from those processed by B lymphocytes; PPI signal-sequence peptides were eluted from HLA-DR4 and -DR3/4 that proved completely identical to a primary target epitope of diabetogenic HLA-A2-restricted CD8 T cells. HLA-DR4 binding was confirmed. GAD65 peptides, eluted from HLA-DR3 and -DR4, encompassed two core regions overlapping the two most immunodominant and frequently studied CD4 T cell targets. GAD65 peptides bound to HLA-DR3. Strikingly, the IA-2 ligandome of HLA-DR was exclusively generated from the extracellular part of IA-2, whereas most previous immune studies have focused on intracellular IA-2 epitopes. The newly identified IA-2 peptides bound to HLA-DR3 and -DR4. Differential T cell responses were detected against the newly identified IA-2 epitopes in blood from T1D patients. The core regions to which DC may draw attention from autoreactive T cells are largely distinct and more restricted than are those of B cells. GAD65 peptides presented by DC focus on highly immunogenic T cell targets, whereas HLA-DR-binding peptides derived from IA-2 are distinct from the target regions of IA-2 autoantibodies.

Discovery of a Selective Islet Peptidome Presented by the Highest-Risk HLA-DQ8trans Molecule.

HLA-DQ2/8 heterozygous individuals are at far greater risk for type 1 diabetes (T1D) development by expressing HLA-DQ8trans on antigen-presenting cells compared with HLA-DQ2 or -DQ8 homozygous individuals. Dendritic cells (DC) initiate and shape adaptive immune responses by presenting HLA-epitope complexes to naïve T cells. To dissect the role of HLA-DQ8trans in presenting natural islet epitopes, we analyzed the islet peptidome of HLA-DQ2, -DQ8, and -DQ2/8 by pulsing DC with preproinsulin (PPI), IA-2, and GAD65. Quality and quantity of islet epitopes presented by HLA-DQ2/8 differed from -DQ2 or -DQ8. We identified two PPI epitopes solely processed and presented by HLA-DQ2/8 DC: an HLA-DQ8trans-binding signal-sequence epitope previously identified as CD8 T-cell epitope and a second epitope that we previously identified as CD4 T-cell epitope with increased binding to HLA-DQ8trans upon posttranslational modification. IA-2 epitopes retrieved from HLA-DQ2/8 and -DQ8 DC bound to HLA-DQ8cis/trans. No GAD65 epitopes were eluted from HLA-DQ. T-cell responses were detected against the novel islet epitopes in blood from patients with T1D but scantly detected in healthy donor subjects. We report the first PPI and IA-2 natural epitopes presented by highest-risk HLA-DQ8trans. The selective processing and presentation of HLA-DQ8trans-binding islet epitopes provides insight in the mechanism of excessive genetic risk imposed by HLA-DQ2/8 heterozygosity and may assist immune monitoring of disease progression and therapeutic intervention as well as provide therapeutic targets for immunotherapy in subjects at risk for T1D.

HY immune tolerance is common in women without male offspring.

BACKGROUND: Sex difference is an established risk factor for hematopoietic stem cell transplantation (HSCT)-related complications like graft versus host disease (GVHD). CD8pos cytotoxic T cells specific for Y chromosome-encoded minor Histocompatibility antigens (HY) play an important role therein. Prior to HSC donation, female donors may encounter HY antigens through fetomaternal or transmaternal cell flow, potentially leading to the induction of HY-specific cytotoxic or regulatory immune responses. Whether HY priming occurs independent of parity, and whether HY priming is dependent on the presence of male microchimerism, is as yet unknown. METHODS: We investigated the presence of HY-specific regulatory T cells (Treg) and male microchimerism in 45 healthy women with a fully documented pregnancy and family history. HY peptide-induced linked suppression, a commonly reported functional feature of CD4pos and CD8pos Treg, was measured by trans vivo Delayed Type Hypersensitivity testing. As source of HY antigens, male microchimerism was analyzed by real-time PCR and defined by the presence of male DNA in at least one purified leukocyte cell type. RESULTS: HLA class I or class II restricted HY-specific Treg were detected in 26/42 (62%) women eligible for analysis. The prevalence of HY-specific Treg was significantly higher in women who had never given birth to sons than in women with male offspring (p = 0.004). Male microchimerism could be detected in 24 out of 45 (53%) women but did not correlate with the presence of HY specific Treg. CONCLUSIONS: HY-specific Treg in women with male offspring have been described previously. Here we show for the first time that, in fact, HY specific Treg are more common in nulliparous women and in parous women with female offspring. Their presence is independent of the presence of male microchimerism. Whether HY-specific Treg presence in female stem cell grafts might decrease the GVHD incidence in male HSCT recipients needs to be investigated.

The human minor histocompatibility antigen 1 is a RhoGAP.

The human minor Histocompatibility Antigen HMHA-1 is a major target of immune responses after allogeneic stem cell transplantation applied for the treatment of leukemia and solid tumors. The restriction of its expression to hematopoietic cells and many solid tumors raised questions regarding its cellular functions. Sequence analysis of the HMHA-1 encoding HMHA1 protein revealed the presence of a possible C-terminal RhoGTPase Activating Protein (GAP) domain and an N-terminal BAR domain. Rho-family GTPases, including Rac1, Cdc42, and RhoA are key regulators of the actin cytoskeleton and control cell spreading and migration. RhoGTPase activity is under tight control as aberrant signaling can lead to pathology, including inflammation and cancer. Whereas Guanine nucleotide Exchange Factors (GEFs) mediate the exchange of GDP for GTP resulting in RhoGTPase activation, GAPs catalyze the low intrinsic GTPase activity of active RhoGTPases, resulting in inactivation. Here we identify the HMHA1 protein as a novel RhoGAP. We show that HMHA1 constructs, lacking the N-terminal region, negatively regulate the actin cytoskeleton as well as cell spreading. Furthermore, we show that HMHA1 regulates RhoGTPase activity in vitro and in vivo. Finally, we demonstrate that the HMHA1 N-terminal BAR domain is auto-inhibitory as HMHA1 mutants lacking this region, but not full-length HMHA1, showed GAP activity towards RhoGTPases. In conclusion, this study shows that HMHA1 acts as a RhoGAP to regulate GTPase activity, cytoskeletal remodeling and cell spreading, which are crucial functions in normal hematopoietic and cancer cells.

Transmaternal cell flow leads to antigen-experienced cord blood.

Umbilical cord blood (UCB) is used for HSCT. It is known that UCB can comprise Ag-specific T cells. Here we question whether solely transmaternal cell flow may immunize UCB. Twenty-three female UCB samples were collected from healthy mothers and analyzed for minor histocompatibility Ag HY-specific responses. Forty-two of 104 tetramer(pos) T-cell clones, isolated from 16 of 17 UCB samples, showed male-specific lysis in vitro. Male microchimerism was present in 6 of 12 UCB samples analyzed. In conclusion, female UCB comprises HY-specific cytotoxic T cells. The immunization is presumably caused by transmaternal cell flow of male microchimerism present in the mother. The presence of immune cells in UCB that are not directed against maternal foreign Ags is remarkable and may explain the reported clinical observation of improved HSCT outcome with younger sibling donors.

Peptide length extension skews the minor HA-1 antigen presentation toward activated dendritic cells but reduces its presentation efficiency.

Minor histocompatibility Ags (mHags) are important targets of the graft-versus-leukemia effect after HLA-matched allogeneic stem cell transplantation. mHags are HLA-restricted polymorphic peptides expressed on normal and leukemia cells. Vaccination with hematopoiesis-restricted mHag peptides, such as HA-1, may boost the graft-versus-leukemia effect. However, some animal studies indicate that peptides exactly reflecting immunogenic T cell epitopes (short peptides [SPs]) induce tolerance that is potentially due to systemic Ag spreading. Peptide length extension (long peptides [LPs]) may optimize immune responses by restricting and prolonging Ag presentation on dendritic cells (DCs). In this study, we compared the in vitro characteristics and T cell-stimulatory capacities of a human 30-mer HA-1 LP with the 9-mer HA-1 SP. DCs presented the HA-1 LP and SP and expanded HA-1-specific cytotoxic T cell lines. As hypothesized, HA-1 LP presentation, but not SP presentation, was largely restricted to activated DCs and was nearly absent on other hematopoietic cells. However, DCs presented the HA-1 LP 2-3 log levels less efficiently than the SP. Finally, the decay of HA-1 LP and SP presentation on DCs was comparable. We conclude that HA-1 LP and SP differ in their in vitro characteristics and that only comparative clinical studies after allogeneic stem cell transplantation may reveal the optimal HA-1 vaccine.

Naturally acquired tolerance and sensitization to minor histocompatibility antigens in healthy family members.

Bidirectional cell transfer during pregnancy frequently leads to postpartum persistence of allogeneic cells and alloimmune responses in both the mother and in her offspring. The life-long consequences of naturally acquired alloimmune reactivity are probably of importance for the outcome of allogeneic stem cell transplantation. We investigated the presence of CD8(pos) minor histocompatibility (H) antigen-specific cytotoxic T lymphocytes (T(CTL)) and CD8(pos) minor H antigen-specific T regulator cells (T(REG)) in peripheral blood cells obtained from 17 minor H antigen-disparate mother-offspring pairs. Absence of minor H antigen-specific T(REG), as marked by the feasibility to expand T(CTL) from isolated tetramer(pos) populations, was observed in 6 mothers and 1 son. The presence of minor H alloantigen-specific T(REG) was observed in 4 mothers and 5 sons. These T(REG) were detected within isolated tetramer(dim) staining fractions and functioned in a CTLA-4-dependent fashion. Our study indicates that both T(CTL) and T(REG) mediated alloimmunity against minor H antigens may be present in healthy female and male hematopoietic stem cell donors, potentially influencing graft-versus-host reactivity in different ways.

Hypomethylating drugs convert HA-1-negative solid tumors into targets for stem cell-based immunotherapy.

Clinical responses of solid tumors after allogeneic human leukocyte antigen-matched stem cell transplantation (SCT) often coincide with severe graft-versus-host disease (GVHD). Targeting minor histocompatibility antigens (mHags) with hematopoiesis- and cancer-restricted expression, for example, HA-1, may allow boosting the antitumor effect of allogeneic SCT without risking severe GVHD. The mHag HA-1 is aberrantly expressed in cancers of most entities. However, an estimated 30% to 40% of solid tumors do not express HA-1 (ie, are HA-1(neg)) and cannot be targeted by HA-1-specific immunotherapy. Here, we investigated the transcriptional regulation of HA-1 gene expression in cancer. We found that DNA hypermethylation in the HA-1 promoter region is closely associated with the absence of HA-1 gene expression in solid tumor cell lines. Moreover, we detected HA-1 promoter hypermethylation in primary cancers. The hypomethylating agent 5-aza-2'-deoxycytidine induced HA-1 expression only in HA-1(neg) tumor cells and sensitized them for recognition by HA-1-specific cytotoxic T lymphocytes. Contrarily, the histone deacetylation inhibitor trichostatin A induced HA-1 expression both in some HA-1(neg) tumor cell lines and in normal nonhematopoietic cells. Our data suggest that promoter hypermethylation contributes to the HA-1 gene regulation in tumors. Hypomethylating drugs might extend the safe applicability of HA-1 as an immunotherapeutic target on solid tumors after allogeneic SCT.

A uniform genomic minor histocompatibility antigen typing methodology and database designed to facilitate clinical applications.

BACKGROUND: Minor Histocompatibility (H) antigen mismatches significantly influence the outcome of HLA-matched allogeneic stem cell transplantation. The molecular identification of human H antigens is increasing rapidly. In parallel, clinical application of minor H antigen typing has gained interest. So far, relevant and simple tools to analyze the minor H antigens in a quick and reliable way are lacking. METHODOLOGY AND FINDINGS: We developed a uniform PCR with sequence-specific primers (PCR-SSP) for 10 different autosomal minor H antigens and H-Y. This genomic minor H antigen typing methodology allows easy incorporation in the routine HLA typing procedures. DNA from previously typed EBV-LCL was used to validate the methodology. To facilitate easy interpretation for clinical purposes, a minor H database named dbMinor (http://www.lumc.nl/dbminor) was developed. Input of the minor H antigen typing results subsequently provides all relevant information for a given patient/donor pair and additional information on the putative graft-versus-host, graft-versus-tumor and host-versus-graft reactivities. SIGNIFICANCE: A simple, uniform and rapid methodology was developed enabling determination of minor H antigen genotypes of all currently identified minor H antigens. A dbMinor database was developed to interpret the genomic typing for its potential clinical relevance. The combination of the minor H antigen genomic typing methodology with the online dbMinor database and applications facilitates the clinical application of minor H antigens anti-tumor targets after stem cell transplantation.

Adult and cord blood T cells can acquire HA-1 specificity through HA-1 T-cell receptor gene transfer.

BACKGROUND AND OBJECTIVES: Minor histocompatibility antigen (mHag)-specific graft-versus-leukemia reactivities are observed following unselected donor lymphocyte infusion for the treatment of relapse after HLA-matched mHag-mismatched stem cell transplantation (SCT). Adoptive transfer of donor-derived ex vivo-generated HA-1-specific oligoclonal T cells or HA-1 peptide patient vaccination are currently being explored as curative tools for stem cell based immunotherapy of hematologic malignancies. Another treatment modality to eradicate residual leukemic cells after SCT is the transfer of the HA-1 hematopoietic-specific T-cell receptor (TCR) into cells from the stem cell donor. This strategy would be particularly useful in case of relapse after cord blood transplantation (CBT) and is explored in this study. DESIGN AND METHODS: HLA-A2(neg) adult peripheral blood and cord blood mononuclear cells were transduced with the genes encoding the HA-1 alpha and beta TCR chains derived from established HA-1 specific cytotoxic T lymphocyte clones. RESULTS: The T cells transduced with HA-1 TCR alpha beta showed consistent marker gene expression, but low staining with HLA-A2/HA-1 tetrameric complexes. They did, however, show hematopoietic-restricted cytolytic activity against HLA-A2(pos)/HA-1(pos) target cells, including leukemic cells. INTERPRETATION AND CONCLUSIONS: The low level of HA-1-specific tetramer staining of HA-1 TCR alpha beta transduced T cells may be caused by hybrid TCR formation of the transferred TCRalpha and beta chains with endogenous TCR alpha and beta chains. This may cause unwanted alloreactivity and requires attention. The HA-1 TCR alpha beta transduced T cells show that the HA-1 TCR can be functionally transferred into donor mononuclear cells, which can be exploited in immunotherapeutic settings of SCT and CBT for hematologic malignancies.

Detection of microchimerism by minor histocompatibility antigen HA-1 allele-specific nested polymerase chain reaction.

Minor histocompatibility antigens (mHags) can induce T-cell reactivities with important consequences for the graft-versus-leukemia effect and the development of graft-versus-host disease in HLA-matched stem cell transplantation settings. Recently, mHag-specific T cells were also demonstrated in multiparous woman and in solid organ transplant recipients. Microchimeric cells have been detected in the latter settings. To study whether microchimerism is instrumental in the induction and/or maintenance of mHag T cells, we developed an HA-1 allele-specific nested polymerase chain reaction. To optimize and validate the reliability of this method at different levels of microchimerism, serial dilutions of HA-1(H) cells titrated into HA-1(R) cells were tested. We demonstrated that the HA-1(H) allele can be reliably and consistently detected at concentrations as low as 1:10(5) without losing specificity. The developed HA-1-specific nested polymerase chain reaction is an important tool that facilitates the detection of HA-1 microchimerism in various clinical specimens and that promotes investigation of the effects of microchimerism on induction of mHag-specific T cells in the various settings of immunization.

Minor H antigen HA-1-specific regulator and effector CD8+ T cells, and HA-1 microchimerism, in allograft tolerance.

The role of the hematopoietic lineage-restricted minor histocompatibility (H) antigen HA-1 in renal allograft tolerance was explored. We obtained peripheral blood samples from three recipients of histocompatibility leukocyte antigen (HLA)-matched, HA-1-mismatched renal transplants, one of which had discontinued immunosuppression >30 yr ago while sustaining normal kidney function. Peripheral blood mononuclear cells (PBMCs) were injected into the footpads of severe combined immunodeficiency mice to measure human delayed type hypersensitivity (DTH) responses. All three patients manifested regulated DTH responses to HA-1H peptide. By differential tetramer staining intensities, we observed two distinct minor H antigen HA-1-specific CD8+ T cell subsets. The one that stained dimly had the characteristics of a T regulatory (TR) cell and produced interleukin (IL) 10 and/or transforming growth factor (TGF) beta. These HA-1-specific TR cells coexisted with bright tetramer-binding CD8+ T effector (TE) cells. The CD8+ TE cells mediated HA-1-specific DTH and produced interferon-gamma. Suppression of these TE functions by TR cells was TGFbeta, IL-10, and cytotoxic T lymphocyte-associated antigen 4 dependent. In addition, HA-1 microchimerism was detected in two recipients, primarily in the dendritic cell fraction of the PBMCs. This is the first demonstration of coexisting CD8+ memory TR and TE cells, both specific for the same HA-1 antigen, in the context of renal allograft tolerance.

Pregnancy induces minor histocompatibility antigen-specific cytotoxic T cells: implications for stem cell transplantation and immunotherapy.

Recipients of HLA-identical stem cell transplants have a poorer transplant outcome if the donor is female rather than male. We analyzed whether pregnancy primes for minor histocompatibility (H) antigens. Peripheral blood mononuclear cells (PBMCs) from healthy multiparous female blood donors were depleted for CD4+, CD14+, CD16+, and CD19+ cells, stained with minor H antigen-specific HLA-A2 tetramers, sorted by fluorescence-activated cell sorting, and tested for cytotoxic activity. Minor H antigens HY-, HA-1-, and HA-2-specific cytotoxic T cells (CD8+, CD45RA-) were present in PBMCs from 4 of 7 female donors up to 22 years after the last delivery. Interestingly, in 2 of the 4 cases microchimerism of the putative immunizing minor H antigen was observed. Thus, pregnancy can lead to alloimmune responses against the infant's paternal minor H antigens. The minor H antigen immunization status of female donors raises important questions for the clinical practice of stem cell transplantation.

Quantification of the HA-1 gene product at the RNA level; relevance for immunotherapy of hematological malignancies.

Minor histocompatibility antigens can induce cytotoxic T cells that play an important role in the graft-versus-leukemia and graft-versus-host-disease (GvHD) activity after stem cell transplantation. Minor histocompatibility antigens (mHags) with expression limited to the hematopoietic system may have a prominent role in the graft-versus-leukemia reaction. Earlier in vitro studies demonstrated that cytotoxic T cells specific for the minor histocompatibility antigen HA-1 only lysed cells of hematopoietic origin. Despite this limited expression, an HA-1 mismatch is associated with GvHD. Yet, the hematopoietic-restricted HA-1 membrane expression motivated us to develop an ex vivo HA-1-specific protocol for cellular immunotherapy of relapsed leukemia. To ensure the feasibility and safety of such cellular therapy, broad HA-1 RNA analysis is indispensable. Here we demonstrate the hematopoietic-restricted expression at the HA-1 gene transcriptional level with high RNA expression in normal and in malignant hematopoietic cells and background expression levels in nonhematopoietic cells. In tissues that showed low HA-1 RNA expression, hematopoietic cells were present as demonstrated by CD45 RNA expression analyzed in parallel. Thus, the mHag HA-1 can function as an excellent target antigen for immunotherapy of hematological malignancies with no or low risk of GvHD.

The minor histocompatibility antigen HA-3 arises from differential proteasome-mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein.

Minor histocompatibility (H) antigens crucially affect the outcome of human leukocyte antigen (HLA)-identical allogeneic stem cell transplantation (SCT). To understand the basis of alloimmune responses against minor H antigens, identification of minor H peptides and their antigenicity-determining mechanisms is essential. Here we report the identification of HA-3 and its encoding gene. The HA-3 peptide, VTEPGTAQY (HA-3T), is encoded by the lymphoid blast crisis (Lbc) oncogene. We thus show for the first time that a leukemia-associated oncogene can give rise to immunogenic T-cell epitopes that may have participated in antihost and antileukemic alloimmune responses. Genotypic analysis of HA-3- individuals revealed the allelic counterpart VMEPGTAQY (HA-3M). Despite the lack of T-cell recognition of HA-3- cells, the Thr-->Met substitution had only a modest effect on peptide binding to HLA-A1 and a minimal impact on recognition by T cells when added exogenously to target cells. This substitution did not influence transporter associated with antigen processing (TAP) transport, but, in contrast to the HA-3T peptide, HA-3M is destroyed by proteasome-mediated digestion. Thus, the immunogenicity of minor H antigens can result from proteasome-mediated destruction of the negative allelic peptide.

The hematopoietic system-specific minor histocompatibility antigen HA-1 shows aberrant expression in epithelial cancer cells.

Allogeneic stem cell transplantation (SCT) can induce curative graft-versus-tumor reactions in patients with hematological malignancies and solid tumors. The graft-versus-tumor reaction after human histocompatibility leukocyte antigen (HLA)-identical SCT is mediated by alloimmune donor T cells specific for polymorphic minor histocompatibility antigens (mHags). Among these, the mHag HA-1 was found to be restricted to the hematopoietic system. Here, we report on the HA-1 ribonucleic acid expression by microdissected carcinoma tissues and by single disseminated tumor cells isolated from patients with various epithelial tumors. The HA-1 peptide is molecularly defined, as it forms an immunogenic peptide ligand with HLA-A2 on the cell membrane of carcinoma cell lines. HA-1-specific cytotoxic T cells lyse epithelial tumor cell lines in vitro, whereas normal epithelial cells are not recognized. Thus, HA-1-specific immunotherapy combined with HLA-identical allogeneic SCT may now be feasible for patients with HA-1(+) carcinomas.