Human Microtumors Generated in 3D: Novel Tools for Integrated In Situ Studies of Cancer Immunotherapies.

Cellular immunotherapy targeting human tumor antigens is a promising strategy to treat solid tumors. Yet clinical results of cellular immunotherapy are disappointing. Moreover, the currently available in vitro human tumor models are not designed to study the optimization of T-cell therapies of solid tumors. Here, we describe a novel assay for multiparametric in situ analysis of therapeutic effects on individual human three-dimensional (3D) tumors. In this assay, tumors of several millimeter diameter are generated from human cancer cell lines of different tumor entities in a collagen type I microenvironment. A newly developed approach for efficient morphological analysis reveals that these in vitro tumors resemble many characteristics of the corresponding clinical cancers such as histological features, immunohistochemical staining patterns, distinct tumor growth compartments and heterogeneous protein expression. To assess the response to therapy with tumor antigen specific T-cells, standardized protocols are described to determine T-cell infiltration and tumor destruction by monitoring soluble factors and tumor growth. Human tumors engineered in 3D collagen scaffolds are excellent in vitro surrogates for avascular tumor stages allowing integrated analyses of the antitumor efficacy of cancer specific immunotherapy in situ.

Possible role of minor h antigens in the persistence of donor chimerism after stem cell transplantation; relevance for sustained leukemia remission.

Persistent complete donor chimerism is an important clinical indicator for remissions of hematological malignancies after HLA-matched allogeneic stem cell transplantation (SCT). However, the mechanisms mediating the persistence of complete donor chimerism are poorly understood. The frequent coincidence of complete donor chimerism with graft-versus-leukemia effects and graft-versus-host disease suggests that immune responses against minor histocompatibility antigens (mHags) are playing an important role in suppressing the host hematopoiesis after allogeneic SCT. Here, we investigated a possible relationship between donor immune responses against the hematopoiesis-restricted mHag HA-1 and the long-term kinetics of host hematopoietic chimerism in a cohort of 10 patients after allogeneic HLA-matched, HA-1 mismatched SCT. Functional HA-1 specific CTLs (HA-1 CTLs) were detectable in 6/10 patients lysing host-type hematopoietic cells in vitro. Presence of HA-1 CTLs in the peripheral blood coincided with low host hematopoiesis levels quantified by highly sensitive mHag specific PCR. Additionally, co-incubation of host type CD34+ cells with HA-1 CTLs isolated after allogeneic SCT prevented progenitor and cobblestone area forming cell growth in vitro and human hematopoietic engraftment in immunodeficient mice. Conversely, absence or loss of HA-1 CTLs mostly coincided with high host hematopoiesis levels and/or relapse. In summary, in this first study, presence of HA-1 CTLs paralleled low host hematopoiesis levels. This coincidence might be supported by the capacity of HA-1 CTLs isolated after allogeneic SCT to specifically eliminate host type hematopoietic stem/progenitor cells. Additional studies involving multiple mismatched mHags in more patients are required to confirm this novel characteristic of mHag CTLs as factor for the persistence of complete donor chimerism and leukemia remission after allogeneic SCT.

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.

Multicenter analyses demonstrate significant clinical effects of minor histocompatibility antigens on GvHD and GvL after HLA-matched related and unrelated hematopoietic stem cell transplantation.

The effect of minor H antigen mismatching on the occurrence of graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) after HLA-matched hematopoietic stem cell transplantation (HSCT) has mainly been demonstrated in single-center studies. Yet, the International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 20 laboratories of the IHIW, the roles of 10 autosomal and 10 Y chromosome-encoded minor H antigens were investigated on GvHD and relapse incidence in 639 HLA-identical related donor (IRD) and 210 HLA-matched unrelated donor (MUD) HSCT recipients. Donor and recipient DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, and HY. The correlations with the primary outcomes GvHD (acute or chronic GvHD), survival, and relapse were statistically analyzed. The results of these multicenter analyses show that none of the HLA class I-restricted HY antigens were found to be associated with any of the primary outcomes. Interestingly, of the HLA class II-restricted HY antigens analyzed, HLA-DQ5 positive recipients showed a significantly increased GvHD-free survival in female-to-male HSCT compared with male-to-female HSCT (P = .013). Yet, analysis of the overall gender effect, thus independent of the known HY antigens, between the gender groups demonstrated an increased GvHD incidence in the female-to-male transplantations (P < .005) and a decreased GvHD-free survival in the female-to-male transplantations (P < .001). Of all autosomally encoded minor H antigens, only mismatching for the broadly expressed minor H antigen HA-8 increased the GvHD incidence in IRD HSCT (Hazard ratio [HR] = 5.28, P < .005), but not in MUD HSCT. Most striking was the influence of hematopoietic restricted minor H antigens on GvL as mismatching for hematopoietic minor H antigens correlated with lower relapse rates (P = .078), higher relapse-free survival (P = .029), and higher overall survival (P = .032) in recipients with GvHD, but not in those without GvHD. In conclusion, the significant GvHD effect of the broadly expressed minor H antigen HA-8 favors matching for HA-8 in IRD, but not in MUD, patient/donor pairs. The GvHD-GvL association demonstrating a significant lower relapse in hematopoietic minor H antigen mismatched patient/donor pairs underlines their clinical applicability for adoptive immunotherapy, enhancing the GvL effect in a GvHD controllable manner.

We are all born as microchimera.

It is well accepted that pregnancy establishes microchimerism. Lately, transmaternal passage of cells from elder siblings has been suggested as possible source of non-fetal microchimerism in nulliparous women. Recently, we identified both non-maternal microchimerism and minor H antigen specific cellular immune responses against these microchimeric cells in umbilical cord blood. The identification of the latter microchimeric cells from birth onwards explains the high incidence of microchimerism in healthy people and in people with different diseases. The circulating microchimeric cells of different origin can induce a wealth of antigen specific responses. Thus, nobody is born naïve: we are all microchimera's.

Minor histocompatibility antigen typing by DNA sequencing for clinical practice in hematopoietic stem-cell transplantation.

In HLA-matched stem-cell transplantation (SCT), minor H antigens are key molecules driving allo-immune responses in both graft-versus-host disease (GvHD) and in graft-versus-leukemia (GvL) reactivity. Dissection of the dual function of minor H antigens became evident through their different modes of tissue and cell expression, i.e., hematopoietic system restricted or broad. Broadly expressed minor H antigens are the targets of immune responses in both arms of graft-versus-host (GvH) responses, i.e., both GvHD and GvL, whereas the immune responses against the hematopoietic system-specific minor H antigens are restricted to the GvL arm of SCT. Evidently, it is this latter group of minor H antigens that can function as curative tools for stem-cell (SC)-based immunotherapy of hematological malignancies and disorders. The HLA-matched patient/donor combinations, incompatible for one of the hematopoietic-specific minor H antigens, are suitable for minor H antigen immunotherapy (Goulmy, Immunol Rev 157:125-140, 1997). Information on the minor H antigen phenotype is therefore needed. Hereto, genomic typing for minor H antigens has been implemented in many HLA laboratories. Here, we firstly summarize the relevance of minor H antigens particularly in hematopoietic SCT. Secondly, we describe a method for typing the various polymorphic minor H antigens molecularly identified to date by DNA sequencing.

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.

In situ detection of HY-specific T cells in acute graft-versus-host disease-affected male skin after sex-mismatched stem cell transplantation.

HY-specific T cells are presumed to play a role in acute graft-versus-host disease (aGVHD) after female-to-male stem cell transplantation (SCT). However, infiltrates of these T cells in aGVHD-affected tissues have not yet been reported. We evaluated the application of HLA-A2/HY dextramers for the in situ detection of HY-specific T cells in cryopreserved skin biopsy specimens. We applied the HLA-A2/HY dextramers on cryopreserved skin biopsy specimens from seven male HLA-A2(+) pediatric patients who underwent stem cell transplantation with confirmed aGVHD involving the skin. The dextramers demonstrated the presence of HY-specific T cells. In skin biopsy specimens of three male recipients of female grafts, 68% to 78% of all skin-infiltrating CD8(+) T cells were HY-specific, whereas these cells were absent in biopsy specimens collected from sex-matched patient-donor pairs. Although this study involved a small and heterogeneous patient group, our results strongly support the hypothesis that HY-specific T cells are actively involved in the pathophysiology of aGVHD after sex-mismatched stem cell transplantation.

IL-7 receptor expression identifies suicide gene-modified allospecific CD8+ T cells capable of self-renewal and differentiation into antileukemia effectors.

In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy-that is, the genetic induction of a conditional suicide phenotype into donor T cells-allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk(+) T cells contained HA-1- and H-Y-specific CD8(+) cytotoxic T cells (CTL) precursors. Thymidine kinase-positive HA-1- and H-Y-specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.

Exogenous Addition of Minor H Antigen HA-1+ Dendritic Cells to Skin Tissues Ex Vivo Causes Infiltration and Activation of HA-1-Specific Cytotoxic T Cells.

T cells specific for hematopoietic system restricted minor Histocompatibility (H) antigens target normal and malignant hematopoietic cells. Thus, cellular immune responses against the latter miHAS eradicate the recipient's hematopoiesis including residual leukemic cells after HLA-matched minor H antigen-mismatched stem-cell transplantation (SCT). However, there are controversial reports on the role of HA-1 in the development of graft-versus-host-disease (GVHD) as well. Here, we address the behavior of HA-1-specific cytotoxic T cells (CTLs) in an ex vivo in situ skin explant model wherein HA-1-expressing dendritic cells (DCs) were added as antigen-presenting cells (APCs). Infiltration and activation of HA-1 CTLs occurred only in those cases where both HLA-A2 and HA-1 were expressed, either by the skin or by the DCs, or by the combination of HLA-A2(+) skin and HA-1(+) DCs. These results point toward the role of recipient's HA-1(+) DCs in the chimeric patient suffering from GVHD after HA-1-mismatched SCT. Although in our model the infiltrated and activated CTLs did not cause skin tissue destruction, our results provide a first step in understanding the reported association of HA-1 mismatching with clinical GVHD.

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.

The presence of HLA-antibodies in recurrent miscarriage patients is associated with a reduced chance of a live birth.

Anti-paternal HLA-antibodies are considered a harmless phenomenon during most pregnancies, whereas their role in recurrent miscarriage (RM) patients is disputed. In contrast to primary RM, patients with secondary RM have carried a fetus to term pregnancy prior to a series of miscarriages, which increases the chance that allogeneic fetal cells appear in the maternal circulation. This study investigates the frequency of HLA-antibodies in secondary RM, primary RM patients and parous controls and analyzes whether the presence of HLA-antibodies in early pregnancy is associated with pregnancy outcome. Sera from women with secondary RM (n=56), primary RM (n=13) and parous controls (n=24) were tested for HLA-antibodies using an ELISA assay and complement dependent cytotoxicity. Samples were taken at gestational week 4-5 in 62 (90%) of the patients. HLA-antibodies were significantly more frequent in secondary RM patients with a boy prior to the miscarriages (62%) compared to secondary RM patients with a firstborn girl (29%, p=0.03), primary RM patients (23%, p=0.02) and parous controls (25%, p=0.005). Forty-one percent of HLA-antibody positive pregnant RM patients had a live birth compared to 76% of HLA-antibody negative RM patients, p=0.006 (adjusted OR: 0.22 (0.07-0.68), p=0.008). In conclusion, HLA-antibodies are significantly more frequent in secondary RM patients with a firstborn boy than in other RM patients and controls. The presence of these antibodies in early pregnancy is associated with a reduced chance of a live birth. Further exploring this association may increase our understanding of maternal acceptance of the fetal allograft.

Impact of genomic risk factors on outcome after hematopoietic stem cell transplantation for patients with chronic myeloid leukemia.

BACKGROUND: Non-HLA gene polymorphisms have been shown to influence outcome after allogeneic hematopoietic stem cell transplantation. Results were derived from heterogeneous, small populations and their value remains a matter of debate. DESIGN AND METHODS: In this study, we assessed the effect of single nucleotide polymorphisms in genes for interleukin 1 receptor antagonist (IL1RN), interleukin 4 (IL4), interleukin 6 (IL6), interleukin 10 (IL10), interferon (IFNG), tumor necrosis factor (TNF) and the cell surface receptors tumor necrosis factor receptor II (TNFRSFIB), vitamin D receptor (VDR) and estrogen receptor alpha (ESR1) in a homogeneous cohort of 228 HLA identical sibling transplants for chronic myeloid leukemia. Three good predictors of overall survival, identified via statistical methods including Cox regression analysis, were investigated for their effects on transplant-related mortality and relapse. Predictive power was assessed after integration into the established European Group for Blood and Marrow Transplantation (EBMT) risk score. RESULTS: Absence of patient TNFRSFIB 196R, absence of donor IL10 ATA/ACC and presence of donor IL1RN allele 2 genotypes were associated with increased transplantation-related mortality and decreased survival. Application of prediction error and concordance index statistics gave evidence that integration improved the EBMT risk score. CONCLUSIONS: Non-HLA genotypes were associated with survival after allogeneic hematopoietic stem cell transplantation. When three genetic polymorphisms were added into the EBMT risk model they improved the goodness of fit. Non-HLA genotyping could, therefore, be used to improve donor selection algorithms and risk assessment prior to allogeneic hematopoietic stem cell transplantation.

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.

Effects of mismatching for minor histocompatibility antigens on clinical outcomes in HLA-matched, unrelated hematopoietic stem cell transplants.

Several studies in HLA-matched sibling hematopoietic stem cell transplantation (HSCT) have reported an association between mismatches in minor histocompatibility antigens (mHAg) and outcomes. We assessed whether single and multiple minor mHAg mismatches are associated with outcomes in 730 unrelated donor, HLA-A, B, C, DRB1, and DQB1 allele-matched hematopoietic stem cell transplants (HSCT) facilitated by the National Marrow Donor Program (NMDP) between 1996 and 2003. Patients had acute and chronic leukemia or myelodysplastic syndrome (MDS), received myeloablative conditioning regimens and calcineurin inhibitor-based graft-versus-host-disease (GVHD) prophylaxis, and most received bone marrow (BM; 85%). Donor and recipient DNA samples were genotyped for mHAg including: HA-1, HA-2, HA-3, HA-8, HB-1 and CD31(125/563). Primary outcomes included grades III-IV acute GVHD (aGVHD) and survival; secondary outcomes included chronic GVHD (cGVHD), engraftment, and relapse. Single disparities at HA-1, HA-2, HA-3, HA-8, and HB-1 were not significantly associated with any of the outcomes analyzed. In HLA-A2-positive individuals, single CD31(563) or multiple mHAg mismatches in the HVG vector were associated with lower risk of grades III-IV aGVHD. Based on these data, we conclude that mHAg incompatibility at HA-1, HA-2, HA-3, HA-8, HB-1, and CD31 has no detectable effect on the outcome of HLA matched unrelated donor HSCT.

The relevance of minor histocompatibility antigens in solid organ transplantation.

PURPOSE OF REVIEW: Disparities in minor histocompatibility antigens between HLA-matched organ and hematopoietic stem cell donors and recipients create the risks of graft failure and graft-versus-host disease (GvHD) respectively. A decade ago, technical advances combined with genomic information resulted in the identification of the chemical nature of the first series of minor histocompatibility antigens, facilitating their molecular typing. A new era of research had begun in exploring the role of minor histocompatibility antigens in physiological and nonphysiological settings. Here we summarize, to the best of our knowledge, human studies on the relevance of minor histocompatibility antigens in solid organ transplantation with a main focus on renal allografting. RECENT FINDINGS: The minor histocompatibility antigen HY is associated with acute rejection, and male grafts in female recipients have reduced graft survival; both cellular and humoral responses are observed. Studies on autosomal minor histocompatibility antigens on graft rejection are less conclusive; their role in transplant tolerance, however, offers perspective. SUMMARY: Information on the clinical relevance of minor histocompatibility antigen allo-immune responses in solid organ allografting is still scarce. The possible implications of the minor histocompatibility allo-immune responses for future clinical practice in solid organ transplantation are discussed in relation to their possible detrimental or beneficial effects on the host.

Steric hindrance and fast dissociation explain the lack of immunogenicity of the minor histocompatibility HA-1Arg Null allele.

The di-allelic HLA-A2 restricted minor histocompatibility Ag HA-1 locus codes for the highly immunogenic HA-1(His) and the nonimmunogenic HA-1(Arg) nonapeptides, differing in one amino acid. The HA-1(His) peptide is currently used for boosting the graft-vs-tumor responses after HLA matched HA-1 mismatched stem cell transplantation; usage of the HA-1(Arg) peptide would significantly enlarge the applicability for this therapy. Our studies on mechanisms causing the HA-1 unidirectional immunogenicity revealed marginal differences in proteasomal digestion, TAP translocation, and binding affinity, whereas both dissociation rates and structural analyses clearly showed marked differences in the stability of these two HLA-A2 bound alleles. These data provide a rationale for the lack of HA-1(Arg) peptide immunogenicity essential for the choice of tumor peptides for stem cell-based immunotherapeutic application.

Association of HY-restricting HLA class II alleles with pregnancy outcome in patients with recurrent miscarriage subsequent to a firstborn boy.

Healthy females, pregnant with a boy, generate immune responses against male-specific minor histocompatibility (HY) antigens. The clinical importance of these responses is evident in stem cell transplantation. Birth of a boy prior to a series of miscarriages reduces the chance of a subsequent live birth. This study explores the putative impact of known HY-presenting HLA alleles on future pregnancy outcome in women with at least three consecutive miscarriages following a birth [secondary recurrent miscarriage (SRM)]. HLA-A, -B, -DRB1, DRB3-5 and DQB1 genotyping was performed in 358 SRM patients and in 203 of their children born prior to the miscarriages. The subsequent live birth in women with boys prior to the miscarriages compared with girls is lower in women with HY-restricting HLA class II alleles [odds ratio (OR): 0.17 (0.1-0.4), P = 0.0001]. One HY-restricting HLA class II allele in women with firstborn boys significantly reduces the chances of a live birth [OR: 0.46 (0.2-0.9), P = 0.02]. Two HY-restricting HLA class II alleles further reduced this chance [OR: 0.21 (0.1-0.7), P = 0.02]. HY-restricting HLA class II did not reduce the chances of a live birth in SRM women with firstborn girls. HY-restricting HLA class II alleles are associated with a decreased chance of a live birth in SRM women with firstborn boys. These findings strongly indicate an aberrant maternal immune reaction against fetal HY-antigens in SRM. The results may shed light on the as-yet unknown immunological causes of SRM and may help understand the successful maternal acceptance of the fetal semi-allograft.