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Immune system aberrations are suggested to be an important factor in the pathophysiology of unexplained secondary recurrent pregnancy loss (sRPL). The objective was to investigate if the sex ratio of the firstborn child in sRPL patients differs from the background population and whether the sex of the firstborn child has a negative impact on the pregnancy prognosis alone and/or in combination with carriage of male-specific minor histocompatibility (H-Y) restricting HLA class II alleles. From January 2016 to October 2022, 582 patients with unexplained RPL were admitted to the RPL Center of Western Denmark and continuously followed-up. HLA-DRB1 and -DQB1 typing was performed as part of the routine diagnostic work-up. In sRPL patients, a history of a firstborn boy was significantly more frequent than in the Danish background population and was associated with significantly lower odds of a successful reproductive outcome in the first pregnancy after admission compared to a firstborn girl (OR=0.41, 95% CI: 0.20-0.83, p = 0.014). The odds of a successful reproductive outcome were enhanced in patients carrying ≥ 1 H-Y-restricting HLA class II alleles with a first-born girl compared to a firstborn boy (OR=3.33, 95% CI: 1.40-7.88, p = 0.005), while no difference in successful reproductive outcome was seen in sRPL patients not carrying these alleles (OR=1.20, 95% CI: 0.33-4.43, p = 0.781). The sex ratio of children born after RPL was similar to the Danish background population. These findings confirm previous findings and suggests that a harmful immune response triggered by H-Y-antigen exposure during a previous pregnancy in preconditioned women may cause sRPL., Competing Interests: Declaration of interest None, (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Polled intersex syndrome (PIS), a physiological defect associates polledness and intersexuality, will directly affect the reproduction and other phenotypic traits of goats, thus, it is a hazard to the development of the goat industry. Recent studies have revealed that goat PIS was caused by an 11.7-kb deletion located in chromosome 1q43, but its DNA detection method was little reported. Herein, a total of 503 goats from three goat breeds were used to successfully establish a simple, rapid, accurate and effective method for the detection of goat PIS mutation. Furthermore, based on this assay, the associations between the PIS mutation and growth traits were analyzed in Guanzhong dairy goats. These findings would provide the potential practical application for molecular breeding to accelerate the development of the goat industry.

Naturally derived regulatory T cells (Tregs) may prevent graft-versus-host disease (GVHD) while preserving graft-versus-leukemia (GVL) activity. However, clinical application of naturally derived regulatory T cells has been severely hampered by their scarce availability and nonselectivity. To overcome these limitations, we took alternative approaches to generate Ag-specific induced Tregs (iTregs) and tested their efficacy and selectivity in the prevention of GVHD in preclinical models of bone marrow transplantation. We selected HY as a target Ag because it is a naturally processed, ubiquitously expressed minor histocompatibility Ag (miHAg) with a proven role in GVHD and GVL effect. We generated HY-specific iTregs (HY-iTregs) from resting CD4 T cells derived from TCR transgenic mice, in which CD4 cells specifically recognize HY peptide. We found that HY-iTregs were highly effective in preventing GVHD in male (HY(+)) but not female (HY(-)) recipients using MHC II-mismatched, parent→F1, and miHAg-mismatched murine bone marrow transplantation models. Interestingly, the expression of target Ag (HY) on the hematopoietic or nonhematopoietic compartment alone was sufficient for iTregs to prevent GVHD. Furthermore, treatment with HY-iTregs still preserved the GVL effect even against pre-established leukemia. We found that HY-iTregs were more stable in male than in female recipients. Furthermore, HY-iTregs expanded extensively in male but not female recipients, which in turn significantly reduced donor effector T cell expansion, activation, and migration into GVHD target organs, resulting in effective prevention of GVHD. This study demonstrates that iTregs specific for HY miHAgs are highly effective in controlling GVHD in an Ag-dependent manner while sparing the GVL effect., (Copyright © 2015 by The American Association of Immunologists, Inc.)

A role for complement, particularly the classical pathway, in the regulation of immune responses is well documented. Deficiencies in C1q or C4 predispose to autoimmunity, while deficiency in C3 affects the suppression of contact sensitization and generation of oral tolerance. Complement components including C3 have been shown to be required for both B-cell and T-cell priming. The mechanisms whereby complement can mediate these diverse regulatory effects are poorly understood. Our previous work, using the mouse minor histocompatibility (HY) model of skin graft rejection, showed that both C1q and C3 were required for the induction of tolerance following intranasal peptide administration. By comparing tolerance induction in wild-type C57BL/6 and C1q-, C3-, C4- and C5-deficient C57BL/6 female mice, we show here that the classical pathway components including C3 are required for tolerance induction, whereas C5 plays no role. C3-deficient mice failed to generate a functional regulatory T (Treg) -dendritic cell (DC) tolerogenic loop required for tolerance induction. This was related to the inability of C3-deficient DC to up-regulate the arginine-consuming enzyme, inducible nitric oxide synthase (Nos-2), in the presence of antigen-specific Treg cells and peptide, leading to reduced Treg cell generation. Our findings demonstrate that the classical pathway and C3 play a critical role in the peptide-mediated induction of tolerance to HY by modulating DC function., (© 2014 The Authors. Immunology published by John Wiley & Sons Ltd.)

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 clinical success of allogeneic T cell therapy for cancer relies on the selection of antigens that can effectively elicit antitumor responses with minimal toxicity toward nonmalignant tissues. Although minor histocompatibility antigens (MiHA) represent promising targets, broad expression of these antigens has been associated with poor responses and T cell dysfunction that may not be prevented by targeting MiHA with limited expression. In this study, we hypothesized that antitumor activity of MiHA-specific CD8 T cells after allogeneic bone marrow transplantation (BMT) is determined by the distribution of antigen relative to the site of tumor growth. To test this hypothesis, we utilized the clinically relevant male-specific antigen HY and studied the fate of adoptively transferred, HY-CD8(+) T cells (HY-CD8) against a HY-expressing epithelial tumor (MB49) and pre-B cell leukemia (HY-E2APBX ALL) in BMT recipients. Transplants were designed to produce broad HY expression in nonhematopoietic tissues (female → male BMT, [F → M]), restricted HY expression in hematopoietic tissues (male → female BMT, [M → F]) tissues, and no HY tissue expression (female → female BMT, [F → F]). Broad HY expression induced poor responses to MB49 despite sublethal graft-versus-host disease and accumulation of HY-CD8 in secondary lymphoid tissues. Antileukemia responses, however, were preserved. In contrast, restriction of HY expression to hematopoietic tissues restored MB49 responses but resulted in a loss of antileukemia responses. We concluded that target alloantigen expression in the same compartment of tumor growth impairs CD8 responses to both solid and hematologic tumors., (Copyright © 2014 American Society for Blood and Marrow Transplantation. All rights reserved.)

Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-D(b)-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that D(b)-Uty(+) and D(b)-Smcy(+) T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8(+) T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation., (© 2013.)

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.

Recombinant antigen arrays represent a new frontier in parallel analysis of multiple immune response profiles requiring only minute blood samples. In this article, we review the benefits and pitfalls of recombinant antigen microarrays developed for multiplexed antibody quantification. In particular, we describe the development of antigen arrays presenting a set of Y chromosome-encoded antigens, called H-Y antigens. These H-Y antigens are immunologically recognized as minor histocompatibility antigens (mHA) following allogeneic blood and organ transplantation. Clinically relevant B-cell responses against H-Y antigens have been demonstrated in male patients receiving female hematopoietic stem cell grafts and are associated with chronic graft versus host development. This chapter discusses our recombinant antigen microarray methods to measure these clinically relevant allo-antibodies.

Graft-versus-host disease is still a major complication in stem-cell transplantation. In HLA-identical stem-cell transplantation, mismatches in minor histocompatibility antigens contribute to the development of graft-versus-host disease. As treatment of graft-versus-host disease with immunosuppressive drugs potentially also reduces the graft-versus-leukemia responses, antigen-specific tolerance induction may be used to reduce graft-versus-host disease while leaving the graft-versus-leukemia responses intact. In mouse models, induction of antigen-specific tolerance using IgG-antigen-transduced B cells can prevent and ameliorate autoimmunity. In this study, the principle of the in vivo animal model has been applied to a human in vitro model wherein induction of tolerance against the HLA class II-restricted male-specific minor histocompatibility antigen RPS4Y1 has been analyzed. T cells precultured in the presence of B cells expressing the IgG-coupled HY antigen showed reduced responsiveness selectively against the minor HY antigen RPS4Y1 in different in vitro protocols. These first observations serve as a basis for further studying and understanding the tolerizing potential of IgG constructs with human T cells.

Nonvirus-specific bystander CD8 T cells bathe in an inflammatory environment during viral infections. To determine whether bystander CD8 T cells are affected by these environments, we examined P14, HY, and OT-I TCR transgenic CD8 T cells sensitized in vivo by IFN-alphabeta-inducing viral infections or by polyinosinic:polycytidylic acid. These sensitized cells rapidly exerted effector functions, such as IFN-gamma production and degranulation, on contact with their high-affinity cognate Ag. Sensitization required self-MHC I and indirect effects of IFN-alphabeta, which together upregulated the T-box transcription factor Eomesodermin, potentially enabling the T cells to rapidly transcribe CTL effector genes and behave like memory cells rather than naive T cells. IL-12, IL-15, IL-18, and IFN-gamma were not individually required for sensitization to produce IFN-gamma, but IL-15 was required for upregulation of granzyme B. These experiments indicate that naive CD8 T cells receive signals from self-MHC and IFN-alphabeta and that, by this process, CD8 T cell responses to viral infection can undergo distinct differentiation pathways, depending on the timing of Ag encounter during the virus-induced IFN response.

T-cell immunotherapy that targets minor histocompatibility (H) antigens presented selectively by recipient hematopoietic cells, including leukemia, could prevent and treat leukemic relapse after hematopoietic cell transplantation without causing graft-versus-host disease. To provide immunotherapy that can be applied to a majority of transplantation recipients, it is necessary to identify leukemia-associated minor H antigens that result from gene polymorphisms that are balanced in the population and presented by common human leukocyte antigen alleles. Current approaches for deriving minor H antigen-specific T cells, which provide essential reagents for the molecular identification and characterization of the polymorphic genes that encode the antigens, rely on in vivo priming and are often unsuccessful. We show that minor H antigen-specific cytotoxic T lymphocyte precursors are found predominantly in the naive CD8(+) T-cell subset and provide an efficient strategy for in vitro priming of native T cells to generate T cells to a broad diversity of minor H antigens presented with common human leukocyte antigen alleles. We used this approach to derive a panel of stable cytotoxic T lymphocyte clones for discovery of genes that encode minor H antigens and identify a novel antigen expressed on acute myeloid leukemia stem cells and minimally in graft-versus-host disease target tissues.

Background: The chance of a live birth after a diagnosis of secondary recurrent miscarriage (SRM) is reduced in patients who, prior to the miscarriages, gave birth to a boy and carry HLA class II alleles that efficiently present male-specific (H-Y) antigens to the immune system. Information about obstetric complications in births prior and subsequent to the SRM diagnosis is limited. The relations between maternal carriage of H-Y-restricting HLA, fetal sex, obstetric complications and prognosis are unknown., Methods: Women with unexplained SRM referred to the Danish Recurrent Miscarriage Clinic between 1986 and 2006 (n = 358) were included; 213 gave birth after the diagnosis. Controls, retrieved from the Danish National Birth Registry, were all women with singleton birth of parity 0, 1982-2005 (n = 608,068) and parity 1, 1986-2008 (n = 510,264). Cross-linkage to the National Discharge Registry identified birth complication diagnoses related to the relevant births among patients and controls., Results: The sex ratio was 1.49 in births prior to SRM and 0.76 in birth after SRM (P < 0.0001). For SRM patients with only late miscarriages (>10 weeks gestation), the corresponding sex ratios were 2.31 and 0.21. Compared with the control groups, obstetric complications were more frequent both before (39% versus 24% P

Background: The Dombrock (Do) blood group system consists of five distinct antigens: Do(a), Do(b), Gy(a), Hy, and Jo(a). Our finding of a patient whose plasma contained a Do-related alloantibody suggested the presence of a sixth antigen., Study Design and Methods: Standard hemagglutination, flow cytometry, and polymerase chain reaction (PCR)-based methods were used throughout. Protein homology modeling was used to map the amino acid change on the protein structure., Results: The patient's red blood cells (RBCs) typed as Do(a-b-), Hy+(w), Jo(a+(w)), and Gy(a+(w)). The patient's plasma agglutinated RBCs with common Dombrock phenotypes. Reactivity with Hy- and Jo(a-) RBC samples was weak, and Gy(a-) RBC samples were nonreactive. DNA analysis showed the patient to be DO*793A (DO*A/DO*A), DO*323G, and DO*350C, which predicts the Do(a+b-), Hy+, and Jo(a+) phenotype, and revealed a homozygous single-nucleotide change of 547T>G in Exon 2 that is predicted to change tyrosine at Amino Acid Position 183 to aspartic acid. This missense substitution introduced a BtgZI restriction enzyme site. The sequence data were confirmed with a PCR-restriction fragment length polymorphism assay and revealed that the patient's parents and children were heterozygous DO*547T/G. Homology modeling predicted that the 183Tyr substitution by Asp altered the Cys182 environment and influenced the formation and/or stability of the Cys182-Cys231 disulfide bond., Conclusion: The patient's DO genes have a single-nucleotide change, which leads to the absence of the high-prevalence antigen DOYA. The absence of this antigen is associated with 183Asp and silencing of Do(a) and weakening of Gy(a), Hy, and Jo(a) antigens.

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.

During thymocyte development, the T-cell receptor (TCR) can discriminate major histocompatibility complex (MHC)/peptide ligands over a narrow range of affinities and translate subtle differences into functional fate decisions. How small differences in TCR input are translated into absolute differences in functional output is unclear. We examined the effects of galectin-1 ablation in the context of class-I-restricted thymocyte development. Galectin-1 expression opposed TCR partial agonist-driven positive selection, but promoted TCR agonist-driven negative selection of conventional CD8(+) T cells. Galectin-1 expression also promoted TCR agonist-driven CD8alphaalpha intestinal intraepithelial lymphocytes (IEL) development. Recombinant galectin-1 enhanced TCR binding to agonist/MHC complexes and promoted a negative-selection-signaling signature, reflected in intensified rapid and transient extracellular signal-regulated kinase (ERK) activation. In contrast, galectin-1 expression antagonized ERK activity in thymocytes undergoing positive selection. We propose that galectin-1 aids in discriminating TCR-directed fate decisions by promoting TCR binding to agonist/MHC complexes and enforcing agonist-driven signals, while opposing partial-agonist signals. In this way, galectin-1 widens the distinction between TCR-directed functional fate cues.

A developmental block is imposed on CD25(+)CD44(-) thymocytes at the beta-selection checkpoint in the absence of the pre T cell receptor (preTCR) alpha-chain, pTalpha. Early surface expression of a transgenic alphabeta TCR has been shown to partially circumvent this block, such that thymocytes progress to the CD4(+)CD8(+) double-positive stage. We wanted to analyze whether a restricting MHC element is required for alphabeta TCR-expressing double-negative (DN) thymocytes to overcome the developmental block in pTalpha-deficient animals. We used the HY-I knock-in model that endows thymocytes with alphabeta TCR expression in the DN compartment but has the advantage of physiological expression levels, in contrast to conventional TCR transgenes. On a pTalpha-deficient background, this HY-I TCR transgene 'rescued' CD25(+)CD44(-) thymocytes from apoptosis and enabled progression to later differentiation stages. On a non-selecting MHC background, however, pTalpha-deficient HY-I mice presented a pronounced reduction in numbers of splenocytes and thymocytes when compared to animals of selecting MHC genotype, showing that MHC restriction is necessary to drive HY-TCR-mediated rescue of pTalpha-deficient thymocytes.

Adaptors play a critical role in regulating signaling pathways that control lymphocyte development and activation. Adaptor in lymphocytes of unknown function X (ALX) and Rlk/Itk-binding protein (RIBP) are adaptors related by structure and sequence, coexpressed in T cells. Mice deficient for each adaptor demonstrated that ALX and RIBP, respectively, negatively and positively regulate T cell activation in response to TCR/CD28 stimulation. However, these results did not preclude that they may function redundantly in other cell populations, or in response to other stimuli. Therefore, to understand the relationship between these related adaptors, ALX/RIBP-deficient mice were generated. We demonstrate that although ALX and RIBP are expressed throughout T cell development, T cell development occurs normally in these mice. Using the H-Y TCR transgenic model, positive and negative selection were found to proceed unimpeded in the absence of ALX and RIBP. We demonstrate that RIBP is also expressed in B cells; however, RIBP- and ALX/RIBP-deficient mice had normal B cell development, and responded equivalently to wild type in response to IgM, CD40, B cell-activating factor/B lymphocyte stimulator, CpG, and LPS. Interestingly, T cells deficient in both ALX and RIBP behaved similarly to those deficient in ALX alone during T cell activation in response to TCR/CD28, exhibiting increased IL-2 production, CD25 expression, and proliferation, thus showing that ALX deficiency masked the effect of RIBP deficiency. ALX/RIBP-deficient T cells did not have any alterations in either activation-induced cell death or Th1/2 polarization. Therefore, we did not find any functional redundancy or synergy during lymphocyte development, selection, activation, or survival in ALX/RIBP-deficient mice, demonstrating that these molecules function independently.

This study examines thymic nurse cell (TNC) function during T-cell development. It has been suggested that TNCs function in the removal of nonfunctional and/or apoptotic thymocytes and do not participate in major histocompatibility complex restriction. We analyzed TNCs isolated from both normal C57BL/6 mice and C57BL/6 TgN (TCRHY) mice (HY-TCR transgenic mice). Using confocal microscopic analyses of TNCs isolated from C57BL/6 animals, we showed that 75%-78% of the enclosed thymocyte subset was viable, and 87%-90% of these cells expressed both CD4 and CD8. CD4 and CD8 also were expressed on TNC thymocytes isolated from both male and female HY-TCR transgenic mice. The transgenic female thymus was shown to have 17 times more TNCs per milligram of thymus than the transgenic male thymus. TNCs from HY-TCR transgenic females were 8-10 microm larger than transgenic male TNCs, and the female TNCs contained five times more thymocytes within intracytoplasmic vacuoles, with less than 4% apoptosis. However, more than 42% of the thymocytes within transgenic male TNCs were apoptotic. The large number and size of TNCs containing viable thymocytes in the female transgenic thymus suggest that TNC function is not limited to the removal of apoptotic thymocytes. We believe that the selective uptake of viable double-positive thymocytes by TNCs in C57BL/6 and HY-TCR transgenic female mice provides evidence that this interaction occurs during the process of major histocompatibility complex restriction.

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.

In vitro stimulation of human female T cells with male HLA-identical dendritic cells resulted in the generation of HLA-DQB1*0501/0502-restricted minor histocompatibility H-Y antigen-specific CD4(+) T cell clones. Two clones generated from different HLA-identical pairs were analyzed. Use of HLA-DQ5-expressing female Epstein-Barr virus transformed B lymphoblastoid cell lines transfected with various H-Y genes and loaded with overlapping peptides demonstrated that both T cell clones are specific for a peptide encoded by DDX3Y. Previously, an HLA-DQ5-restricted T cell clone specific for the same peptide was isolated from a patient with graft-versus-host disease. Thus, we compared the T cell receptor (TCR) rearrangements of the 2 in vitro generated T cell clones and the ex vivo isolated T cell clone. All 3 clones shared the same TCRBV5-4* gene segment and 2 of 3 clones also used similar TCR-Valpha segments. Our results suggest that T cells recognizing the HLA-DQ5/DDX3Y T cell epitope might be characterized by a relatively limited TCR-beta repertoire. The differences in the junctional TCR-beta region had no effect on the antigen specificity, but altered the capacity of the TCR to distinguish the HLA-DQ5/DDX3Y complex from its allelic counterpart. The results also demonstrate that in vitro stimulation of T cells with allogeneic HLA-identical dendritic cells may facilitate the characterization of in vivo, potentially relevant HLA class II-restricted minor H epitopes.

Background: Minor histocompatibility (H) antigens are peptides of allelic intracellular proteins that play an important role in human leukocyte antigen (HLA) matched transplantations. In an animal model of keratoplasty, minor H antigens have even been reported to exceed the immunogenicity of major H antigens (MHC). This investigation is to assess any benefit of matching the broadly expressed gender (H-Y) and HA-3 antigens in HLA-A1 donor positive human keratoplasty., Methods: A total of 229 HLA-A1 donor positive keratoplasties were analyzed. A Cox proportional hazards model and Kaplan-Meier analysis were applied to estimate the effect of H-Y or HA-3 mismatches on rejection-free graft survival., Results: Eighty-one cases were mismatched for H-Y (male donor to female recipient). A mean follow up of two years showed graft survival as high as 88% in the H-Y compatible group compared to only 77% in the H-Y mismatched group (P = 0.02). Eight out of 62 cases were mismatched for HA-3. No statistically significant influence of HA-3 matching on rejection-free graft survival was observed (85% vs. 73%, P=0.52)., Conclusion: HLA-A1/H-Y matching and matching for other broadly expressed minor H antigens may further improve prognosis in keratoplasty.

The Ets family of transcription factors function as key regulators of multiple aspects of immune cell development and function. To date, Ets-1 has been implicated in regulating early stages of thymic maturation and lymphocyte function and homeostasis. This report describes a novel role for Ets-1 in supporting later stages of thymic selection, in that positive selection of MHC class I-restricted CD4+CD8+ double-positive thymocytes is markedly inhibited in mice expressing a hypomorphic allele of Ets-1. This effect is thymocyte intrinsic, as Ets-1 mutant thymocytes fail to efficiently generate CD8+ single-positive thymocytes in mixed bone marrow chimeric backgrounds. Although peripheral CD8+ T cells are present in Ets-1 mutant mice, both CD4+ and CD8+ subsets contain an elevated proportion of cells with an effector memory (CD62L-CD44+) phenotype. In addition, while thymic expression of Thy1 is relatively normal, peripheral T cells isolated from Ets-1 mutant mice display a striking loss of Thy1 expression. These data identify Ets-1 as a key transcription factor regulating thymocyte positive selection and lineage commitment of MHC class I-restricted thymocytes.

The evidence that proteasomes are involved in the processing of cross-presented proteins is indirect and based on the in vitro use of proteasome inhibitors. It remains, therefore, unclear whether cross-presentation of MHC class I peptide epitopes can occur entirely within phagolysosomes or whether it requires proteasome degradation. To address this question, we studied in vivo cross-presentation of an immunoproteasome-dependent epitope. First, we demonstrated that generation of the immunodominant HY Uty(246-254) epitope is LMP7 dependent, resulting in the lack of rejection of male LMP7-deficient (LMP7(-/-)) skin grafts by female LMP7(-/-) mice. Second, we ruled out an altered Uty(246-254)-specific T cell repertoire in LMP7(-/-) female mice and demonstrated efficient Uty(246-254) presentation by re-expressing LMP7 in male LMP7(-/-) cells. Finally, we observed that LMP7 expression significantly enhanced cross-priming of Uty(246-254)-specific T cells in vivo. The observations that male skin grafts are not rejected by LMP7(-/-) female mice and that presentation of a proteasome-dependent peptide is not efficiently rescued by alternative cross-presentation pathways provide strong evidence that proteasomes play an important role in cross-priming events.

Adaptation of the T cell activation threshold may be one mechanism to control autoreactivity. To investigate its occurrence in vivo, we engineered a transgenic mouse model with increased TCR-dependent excitability by expressing a Zap70 gain-of-function mutant (ZAP-YEEI) in postselection CD8 thymocytes and T cells. Increased basal phosphorylation of the Zap70 substrate linker for activation of T cells was detected in ZAP-YEEI-bearing CD8 T cells. However, these cells were not activated, but had reduced levels of TCR and CD5. Moreover, they produced lower cytokine amounts and showed faster dephosphorylation of linker for activation of T cells and ERK upon activation. Normal TCR levels and cytokine production were restored by culturing cells in the absence of TCR/spMHC interaction, demonstrating dynamic tuning of peripheral T cell responses. The effect of avidity for self-ligand(s) on this sensory adaptation was studied by expressing ZAP-YEEI in P14 or HY TCR transgenic backgrounds. Unexpectedly, double-transgenic animals expressed ZAP-YEEI prematurely in double-positive thymocytes, but no overt alteration of selection processes was observed. Instead, modifications of TCR and CD5 expression due to ZAP-YEEI suggested that signal tuning occurred during thymic maturation. Importantly, although P14 x ZAP-YEEI peripheral CD8 T cells were reduced in number and showed lower Ag-induced cytokine production and limited lymphopenia-driven proliferation, the peripheral survival/expansion and Ag responsiveness of HY x ZAP-YEEI cells were enhanced. Our data provide support for central and peripheral sensory T cell adaptation induced as a function of TCR avidity for self-ligands and signaling level. This may contribute to buffer excessive autoreactivity while optimizing TCR repertoire usage.

Minor histocompatibility Ags derive from self-proteins and provoke allograft rejection and graft-vs-host disease in MHC-matched donor-recipient combinations. In this study, we define the HYD(k) epitope of the HY minor histocompatibility Ag as the 8mer peptide RRLRKTLL derived from the Smcy gene. Using HY tetramers, the response to this peptide was found to be immunodominant among the four characterized MHC class I-restricted HY epitopes (HYD(k)Smcy (defined here), HYK(k)Smcy, HYD(b)Uty, and HYD(b)Smcy). Indirect presentation stimulated a robust primary HYD(k)Smcy response. Indirect presentation and priming of HY-specific CD8+ T cells is also operative in the presence of a full MHC mismatch. To determine whether the indirect route of Ag presentation is required for HY priming, female parent into F1 (H2bxk) female recipient bone marrow chimeras were immunized with male cells of the other parental haplotype, limiting presentation to the direct pathway. The dominant H2b HY response (HYD(b)Uty) was dependent on indirect presentation. However, the dominant H2k HY response (HYD(k)Smcy) could be stimulated efficiently by the direct pathway. In contrast, secondary expansion of both HYD(k)Smcy and HYD(b)Uty-specific CD8+ T cells was effective only when Ag was presented by the direct route. Transgenic overproduction of Smcy mRNA within the immunizing cells resulted in a corresponding increase in the HYD(k)Smcy, HYD(b)Smcy, and HYK(k)Smcy-specific CD8+ T cell responses when presented via the direct pathway but did not enhance indirect presentation demonstrating the independent regulation of MHC class I-peptide occupancy in the two Ag-processing pathways.

Bladder cancer is regarded as a promising candidate for innovative therapies in the field of immune and gene therapy. In this paper, we present the subcutaneous, metastatic and a novel orthotopic model of murine MB49 bladder cancer in C57BL/6 mice. We further show the potential of using adenoviral vectors together with different transduction enhancers to augment in vivo gene delivery. Finally, we present candidate genes for tumour detection, therapy or targeting. The MB49 tumour grew rapidly in mice. The subcutaneous model allowed for tumour detection within a week and the possibility to monitor growth rate on a day-by-day basis. Injection of MB49 cells intravenously into the tail vein gave rise to lung metastases within 16 days, while instillation of tumour cells into pretreated bladders led to a survival time of 20-40 days. Adenoviral vectors can be used as a vehicle for gene transfer to the bladder. By far, the most potent transduction enhancer was Clorpactin, also known as oxychlorosene. Last, we show that MB49 cells express tumour-associated antigens like bladder cancer-4, prostate stem cell antigen and six-transmembrane epithelial antigen of the prostate. Given the possibility for efficient genetic modification of the bladder and the presence of known tumour antigens, the MB49 models can be used in innovative ways to explore immunogene therapy.

In vivo studies suggest that vascular endothelial cells (ECs) can acquire and cross-present exogenous Ag on MHC-I but the cellular mechanisms underlying this observation remain unknown. We tested whether primary female mouse aortic ECs could cross-present exogenous male Ag to the T cell hybridoma, MHH, specific for HYUty plus D(b). MHC-I-deficient male spleen cells provided a source of male Ag that could not directly stimulate the MHH cells. Addition of male but not female MHC-I-deficient spleen cells to wild-type syngeneic female EC induced MHH stimulation, demonstrating EC cross-presentation. Lactacystin treatment of the donor male MHC-I-deficient spleen cells, to inhibit proteasome function, markedly enhanced EC cross-presentation showing that the process is most efficient for intact proteins rather than degraded peptide fragments. Additional experiments revealed that this EC Ag-processing pathway is both proteasome and TAP1 dependent. These studies demonstrate that cultured murine aortic ECs can process and present MHC-I-restricted Ag derived from a separate, live cell, and they offer insight into the molecular requirements involved in this EC Ag presentation process. Through this pathway, ECs expressing cross-presented peptides can participate in the effector phase of T cell-mediated inflammatory responses such as autoimmunity, anti-tumor immunity, and transplant rejection.

T cells undergo negative selection in the thymus to eliminate potentially autoreactive cells. The signals generated through the alphabeta TCR following receptor interactions with peptide/MHC complexes in the thymus control these selection processes. Following receptor ligation, a fraction of the TCR zeta subunit appears as two distinct tyrosine-phosphorylated forms of 21 and 23 kDa (p21 and p23). Previous data have reported elevated levels of p21 in some murine models of autoimmunity. We have examined the contributions of both the p21 and p23 to T cell negative selection in the HY TCR-transgenic system using ITAM-substituted TCR zeta and CD3 epsilon transgenic mice. Expression of just p21, in the absence of p23, partially impairs negative selection of self-reactive HY-specific T cells. This results in the emergence of potentially autoreactive peripheral T cells and an elevated population of CD11b(+)B220(+) B cells in the spleen. These data clearly identify a specific and unique role for p21 during negative selection.

We have designed DNA fusion vaccines able to induce high levels of epitope-specific CD8(+) T cells, using linked CD4(+) T cell help. Such vaccines can activate effective immunity against tumor Ags. To model performance against minor histocompatibility (H) Ags important in allogeneic hemopoietic stem cell transplantation, responses against the H2D(b)-restricted Uty and Smcy male HY epitopes have been investigated. Vaccination of females induced high levels of tetramer-specific, IFN-gamma-producing CD8(+) T cells against each epitope. Vaccines incorporating a single epitope primed effector CTL able to kill male splenocytes in vitro and in vivo, and HY(Db)Uty-specific vaccination accelerated rejection of syngeneic male skin grafts. Priming against either epitope established long-term memory, expandable by injection of male cells. Expanded CD8(+) T cells remained specific for the priming HY epitope, with responses to the second suppressed. To investigate vaccine performance in a tolerized repertoire, male mice were vaccinated with the fusion constructs. Strikingly, this also generated epitope-specific IFN-gamma-producing CD8(+) T cells with cytotoxic function. However, numbers and avidity were lower than in vaccinated females, and vaccinated males failed to reject CFSE-labeled male splenocytes in vivo. Nevertheless, these findings indicate that DNA fusion vaccines can mobilize CD8(+) T cells against endogenous minor H Ags, even from a profoundly tolerized repertoire. In the transplantation setting, vaccination of donors could prime and expand specific T cells for in vivo transfer. For patients, vaccination could activate a potentially less tolerized repertoire against similar Ags that may be overexpressed by tumor cells, for focused immune attack.

Previous evidence suggested that the hemopoietic-specific nuclear factor Ikaros regulates TCR signaling thresholds in mature T cells. In this study, we test the hypothesis that Ikaros also sets TCR signaling thresholds to regulate selection events and CD4 vs CD8 lineage determination in developing thymocytes. Ikaros null mice were crossed to three lines of TCR-transgenic mice, and positive selection, negative selection, and CD4 vs CD8 lineage decisions were analyzed. Mice expressing a polyclonal repertoire or a MHC class II-restricted TCR transgene exhibited enhanced positive selection toward the CD4 lineage. Moreover, in the absence of Ikaros, CD4 development can occur with decreased thresholds of TCR signaling. In addition, CD4 single-positive thymocytes were detected in MHC class I-restricted TCR-transgenic Ikaros null mice. To assess the role of Ikaros in negative selection, we analyzed deletion of T cells induced by conventional Ag or by endogenous superantigen. Surprisingly, negative selection was impaired in Ikaros null thymocytes despite evidence of high levels of TCR signal and no intrinsic defect in apoptosis ex vivo. To our knowledge, these data identify Ikaros as the first nuclear factor that plays a critical role in regulating negative selection as well as CD4 vs CD8 lineage decisions during positive selection.

Background: RBCs of the Hy- phenotype have, in the past, been typed as Gy(a+w), Hy-, Jo(a-), and RBCs with the Jo(a-) phenotype type Gy(a+), Hy+w, and Jo(a-). Anti-Hy and anti-Joa are difficult to identify mainly because appropriate reagent RBCs are poorly characterized. Historically, anti-Joa has not reacted with RBCs with either phenotype. This report describes a case of an anti-Joa that shows Hy- RBCs express some Joa antigen, albeit weakly., Case Report: Anti-Joa was identified in a serum sample of a 71-year-old woman. The antibody reacted 1+ to 2+ by the IAT with all untreated and ficin-treated panel RBCs and did not react with Gy(a-) RBCs and Jo(a-) RBCs. Unexpectedly, the serum sample reacted weakly with six of eight RBC samples with the Hy- phenotype. The anti-Joa was adsorbed onto and eluted from Hy- RBCs, indicating the presence of weak Joa antigen. The patient's RBCs typed Gy(a+), Hy+, Jo(a-). DNA studies using PCR-RFLP analysis showed the patient to be homozygous for the JO allele, which is consistent with the serologically determined Jo(a-) status., Conclusion: The DNA and serologic evidence of this case show that Hy- RBCs may express low levels of Joa antigen, which contradicts previously published data concerning the Joa type of Hy- RBCs.

Minor histocompatibility antigens (mHAs) recognized by donor T cells play a central role as immunologic targets of graft-versus-host disease (GVHD) and graft versus leukemia after allogeneic hematopoietic stem cell transplantation (HSCT). Men who have undergone sex-mismatched allogeneic HSCT are at high risk for GVHD because of immune responses directed against mHAs encoded by genes on the Y chromosome (termed H-Y antigens). We hypothesized that the immunogenicity of mHAs results in a coordinated response involving B cells as well as T cells. To test this, we measured antibody responses to a well-characterized H-Y antigen, dead box RNA helicase Y (DBY), and its homolog, DBX, in 150 HSCT patients. Using Western blot and enzyme-linked immunosorbent assay (ELISA), we found that 50% of male patients who received stem cell grafts from female donors developed antibody responses to recombinant DBY protein. Antibodies to DBY were also detected in 17% of healthy women, but not in healthy men. Antibody responses were directed primarily against areas of amino acid disparity between DBY and DBX. These studies demonstrate that the immune response to mHA includes the generation of specific antibodies and suggests that the serologic response to these antigens may also be useful in the identification of new mHAs.

Homeostatic signals that control the overall size and composition of the naive T cell pool have recently been identified to arise from contact with self-MHC/peptide ligands and a cytokine, IL-7. IL-7 presumably serves as a survival factor to keep a finite number of naive cells alive by preventing the onset of apoptosis, but how TCR signaling from contact with self-MHC/peptide ligands regulates homeostasis is unknown. To address this issue, murine polyclonal and TCR-transgenic CD8+ cells expressing TCR with different affinities for self-MHC/peptide ligands, as depicted by the CD5 expression level, were analyzed for their ability to respond to and compete for homeostatic factors under normal and lymphopenic conditions. The results suggest that the strength of the TCR affinity determines the relative "fitness" of naive T cells to compete for factors that support cell survival and homeostatic proliferation.

To evaluate the priming and trafficking of male Ag-reactive CD4(+) T cells in vivo, we developed an adoptive transfer model, using Marilyn (Mar) TCR transgenic T cells that are specific for the H-Y minor transplantation Ag plus I-A(b). By manipulating donor and recipient strain combinations, we permitted the Mar CD4(+) T cells to respond to the H-Y Ag after processing and presentation by recipient APCs (indirect pathway), or to the male Ag as expressed on donor APCs (direct pathway). Mar CD4(+) T cells responding through the indirect pathway specifically proliferated and expressed activation markers between days 2 and 4 posttransplant, migrated to the graft 2-3 days before cessation of graft heartbeat, and were detected in close proximity to transplant-infiltrating recipient APCs. Intriguingly, adoptively transferred Mar T cells did not respond to male heart or skin grafts placed onto syngeneic MHC class II-deficient female recipients, demonstrating that activation of Mar T cell preferentially occurs through cognate interactions with processed male Ag expressed on recipient APCs. The data highlight the potency of indirect processing and presentation pathways in vivo and underscore the importance of indirectly primed CD4(+) T cells as relevant participants in both the priming and effector phases of acute graft rejection.

It is widely accepted that developing T cells can undergo clonal deletion in the thymus in response to a high affinity self-Ag. This is largely based on studies of TCR transgenics. However, encounter with high affinity self-Ag can also result in receptor editing in TCR transgenic models. Because all TCR transgenics display ectopic receptor expression, the tolerance mechanism that predominates in normal mice remains an open question. When self-Ag drives receptor editing during T cell development, one expects to find in-frame, self-reactive TCRalpha joins on TCR excision circles (TRECs), which are the products of secondary V/J recombination in the TCRalpha locus. Such joins are not expected if clonal deletion occurs, because the progenitor cell would be eliminated by apoptosis. To test the relative utilization of receptor editing vs clonal deletion, we determined the frequency of in-frame, male-specific joins on TRECs in male and female HYbeta transgenic mice. In comparison with female HYbeta transgenic mice, our analysis showed a lower frequency of TRECs with male-reactive V17J57 joins in male mice. Thus, it would appear that receptor editing is not a predominant tolerance mechanism for this self-Ag.

Although T cell receptor (TCR)gammadelta+ and TCRalphabeta+ cells are commonly viewed as functionally independent, their relatedness and potential interdependence remain enigmatic. Here we have identified a gene profile that distinguishes mouse gammadelta cell populations from conventional alphabeta T cells. However, this profile was also expressed by sets of unconventional alphabeta T cells. Therefore, whereas TCR specificity determines the involvement of a T cell in an immune response, the cell's functional potential, as assessed by gene expression, does not segregate with the TCR. By monitoring the described gene profile, we show that gammadelta T cell development and function in TCRbeta-deficient mice was impaired because of the absence of alphabeta T cell progenitors. Thus, normal gammadelta cell development is dependent on the development of conventional alphabeta T cells.

Negative selection serves as a major mechanism to maintain self-tolerance. We previously reported that LIGHT (a cellular ligand for herpes virus entry mediator and lymphotoxin receptor), a TNF family member, plays an important role in thymocyte development via promoting apoptosis of double-positive thymocytes. Here, we demonstrated that LIGHT-mediated deletion of thymocyte requires the strong interaction of TCR with MHC/self-peptide. Transgenic mice overexpressing LIGHT in thymocytes were bred with a transgenic mouse line expressing a TCR recognizing the H-Y male Ag in the context of H-2b class I MHC molecules. In male H-Y/LIGHT double-transgenic mice, more efficient negative selection of H-Y T cells occurred, and total thymocyte number was further reduced compared with H-Y/negative littermates. In contrast, the presence of LIGHT transgene had no evident impact on the thymocyte development of female H-Y/LIGHT double-transgenic mice. Taken together, LIGHT plays a role in negative selection of thymocytes via inducing the apoptosis of thymocytes bearing high affinity TCR during negative selection.

T cell differentiation in the thymus is dependent upon signaling through the TCR and is characterized by the resulting changes in expression patterns of CD4 and CD8 surface coreceptor molecules. Although recent studies have characterized the effects of proximal TCR signaling on T cell differentiation, the downstream integration of these signals remains largely unknown. The growth factor independence-1 (GFI1) and GFI1B transcriptional repressors may regulate cytokine signaling pathways to affect lymphocyte growth and survival. In this study, we show that Gfi1 expression is induced upon induction of the T cell program. Gfi1B expression is low and dynamic during T cell development, but is terminated in mature thymocytes. Transgenic expression of GFI1 and GFI1B in T cells allowed us to determine the functional consequences of constitutive expression. GFI1 potentiates response to TCR stimulation and IL-2, whereas GFI1B-transgenic T cells are defective in T cell activation. Moreover, GFI1B-transgenic thymocytes display reduced expression of the late-activation marker IL-7R alpha, and a decrease in CD4(-)8(+) single-positive T cells that can be mitigated by transgenic expression of BCL2 or GFI1. These data show that GFI1 and GFI1B are functionally unique, and implicate a role for GFI1 in the integration of activation and survival signals.

Minor histocompatibility (H) antigens are a diverse assemblage of major histocompatibility complex (MHC)-bound peptides with the unifying property of acting as alloantigens that induce allogeneic tissue rejection. They are a consequence of any form of accumulated genetic variation that translates to differential MHC-presented peptide epitopes, the most common form of which is simple sequence polymorphisms. The universe of potential minor H antigens is large when transplantation is performed between genetically unrelated, MHC-matched individuals, especially considering the remarkable discriminative sensitivity of T cells. However, the phenomenon of immunodominance greatly simplifies immune responses that ensue. One mouse minor H antigen, H60, stands out in that the preponderance of the CD8 T cell response elicited in a complex alloantigenic setting is directed against this single minor H antigen epitope. Its immunodominance is because mice lacking H60 develop an unusually robust T cell repertoire dedicated to this single minor H antigen. The now well-characterized mouse minor H antigen system should provide a vehicle to assess the degree to which immunodominant alloantigens contribute to transplant rejection.

Background: In HLA-identical sibling bone marrow transplantation, certain recipient cytokine gene polymorphism genotypes and minor histocompatibility differences influence the occurrence and severity of acute graft-versus-host disease (aGvHD). The present study investigated the role of cytokine tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 gene polymorphisms HY, HA-1, and CD31 minor histocompatibility antigen (mHag) mismatch in the development of aGvHD after unrelated cord blood (CB) transplant (CBT)., Methods: DNA samples of 115 CB recipients and their unrelated CB grafts were analyzed for genotype associated with TNF-alpha (TNFd3/d3) and IL-10 (IL-10(-1064), 11-16) and for disparities in major and three minor histocompatibility antigens, HY, HA-1, and CD31 codon 125. Results were correlated with the incidence of aGvHD grades II to IV., Results: Neither the donor nor the recipient GvHD risk alleles TNFd3/d3 and IL-10(-1064) (11-16) were associated with the development of aGvHD grades II to IV and I to IV. Because of the heterogeneity of CBTs, the data were reanalyzed separately for patients with malignancies (n=83) or with inborn errors (n=24). No significant association was observed between the severity of aGvHD and the possession of either TNFd3/d3 or IL-10 (11-16) genotypes. Mismatches for the mHags HY, HA-1, and CD31 exon 125 between donor and recipient did not associate with aGvHD grades II to IV., Conclusions: In contrast to HLA-identical sibling bone marrow transplantation, in mismatched unrelated CBT, neither the cytokine genotypes TNFd3/d3 alone or in combination with IL-10(-1064) alleles nor the minor histocompatibility antigens HY, HA-1, and CD31 exon 125 were associated with aGvHD grades II to IV. Further determination of the cytokine gene polymorphism genotypes in CBTs compared with bone marrow transplants may identify those polymorphisms that could be potential predictive markers for the occurrence of aGvHD.

Allelic exclusion is inefficient at the TCRalpha locus, allowing a sizeable portion of T cells to carry two functional TCRs. The potential danger of dual TCR expression is a rescue of autoreactive TCRs during selection in the thymus and subsequent development of autoimmunity. In this study, we examine the reason(s) for replacing an autoreactive TCR and for allowing the survival of cells carrying two TCRs. We compared development of TCR transgenic CD4(+)CD8(-) thymocytes in the presence or absence of MHC class II autoantigen that does not induce deletion of thymocytes. Contrary to the expected negative effect of the presence of autoantigen, approximately 100% more CD4(+)CD8(-) thymocytes were found in the presence of MHC class II autoantigen than in the neutral background. A further increase in the strength of autoantigenic signal via expression of a human CD4 transgene led to an additional increase in the numbers of CD4(+)CD8(-) thymocytes. Thus, editing autoreactive TCR results in more efficient positive selection, and this may be both a reason and a reward for risking autoimmunity.

Separation between primary and secondary lymphoid organs is a universal feature in jawed vertebrates. Strikingly, oncostatin M (OM)-transgenic mice present massive extrathymic T cell development, localized exclusively in the lymph nodes (LN). According to the prevailing paradigm, the thymus is the main source of T lymphocytes in gnathostomes mainly because thymic epithelial cells have a unique ability to support early steps in T cell development. It is therefore remarkable that productive T cell development occurs in the OM(+) LN, despite the absence of epithelial cells. The present study shows that in the OM(+) LN: 1) MHC class I expression strictly on hemopoietic cells is sufficient to support the development of a diversified repertoire of CD8 T cells; 2) the efficiency of positive selection of specific TCR-transgenic T cells is not the same as in the thymus; 3) negative selection is very effective, despite the lack of an organized thymic-like medulla. Furthermore, our data suggest that extrathymic T lymphocytes developing in the OM(+) LN undergo extensive postselection expansion because they live in the microenvironment in which they were positively selected. This work illustrates how the division of labor between primary and secondary lymphoid organs influences the repertoire and homeostasis of T lymphocytes.

We derived Rag2-deficient mice bearing two rearranged alphabeta TCR transgenes, one specific for the HY male Ag and the second specific for the gp33-41 peptide of lymphocytic choriomeningitis virus, both restricted to the MHC H-2D(b) class I molecule. We found that, in female double transgenic (DTg) mice, most CD8 T cells express only the TCRbeta chain from the aHY transgene. By comparing the mRNA species for both beta-chains, we observed that in T cells from DTg mice the aHY TCRbeta chain transcripts are abundant, whereas the anti-lymphocytic choriomeningitis virus TCRbeta chain transcripts are rare. In contrast to TCRbeta chain expression, most of the T cells from DTg mice express two TCRalpha chains. We examined the thymus selection of the dual-receptor CD8 T cells in the presence of self-Ag. We found that the presence of a second TCRalpha chain allows a significant number of CD8 T cells expressing a self-reactive receptor to escape central deletion and migrate to the peripheral pools of male mice. Differences in TCR and coreceptor expression between female and male MoaHY and DTg mice suggest that peripheral T cell survival requires an optimal level of signaling, which implies a process of "adaptation" of lymphocyte populations to the host environment.

The outcome of thymocyte selection is influenced by the nature of Ca2+ signals transduced by the TCR. Robust Ca2+ responses characterize high-affinity, negatively selecting peptide/TCR interactions, while modest responses typify lower-affinity, positively selecting interactions. To elucidate mechanisms by which thymocytes decode distinct Ca2+ signals, we examined selection events in mice lacking Ca2+/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr), which is enriched in thymocytes. CaMKIV/Gr-deficient thymocytes exhibited impaired positive selection and defective Ca2+-dependent gene transcription. Significantly, CaMKIV/Gr deficiency raised the selection threshold of peptide/TCR interactions such that a peptide that normally induced weak negative selection instead promoted positive selection. These results demonstrate an important role for CaMKIV/Gr in sensitizing thymocytes to selection by low-affinity peptides.

We studied Rag2-deficient mice bearing two rearranged alphabeta TCR transgenes, both restricted to the MHC H-2D(b) class I molecule. We have previously shown that, in these DTg mice, most peripheral CD8 T cells express one TCRbeta chain associated with two TCRalpha chains, as in one-third of the mature T cells from normal mice. We examined the functional behavior of the dual-receptor CD8 T cells developing either in the absence or in the presence of self-Ag. The dual-receptor CD8 T cells, which develop in absence of self-Ag, show efficient responses to immunization and remain sensitive to induction of peripheral tolerance. In contrast to single TCR T cells, the dual-TCR cells, when tolerized upon exposure to high levels of self-Ag, are not deleted and therefore may exert important regulatory functions. When developing in the presence of self-Ag, the dual-receptor-expressing CD8 T cells escape central deletion, but are not fully competent to respond to cognate stimuli. Overall, we found that the dual-TCR CD8 T cells show a poor competitive value and can be out-competed by single-TCR cells, both in the course of immune responses and in reconstitution experiments. The decreased fitness of the dual-receptor cells may contribute to diminishing the autoimmune hazard that they could represent.

E2Fs are important regulators of proliferation, differentiation, and apoptosis. Here we characterize the phenotype of mice deficient in E2F2. We show that E2F2 is required for immunologic self-tolerance. E2F2(-/-) mice develop late-onset autoimmune features, characterized by widespread inflammatory infiltrates, glomerular immunocomplex deposition, and anti-nuclear antibodies. E2F2-deficient T lymphocytes exhibit enhanced TCR-stimulated proliferation and a lower activation threshold, leading to the accumulation of a population of autoreactive effector/memory T lymphocytes, which appear to be responsible for causing autoimmunity in E2F2-deficient mice. Finally, we provide support for a model to explain E2F2's unexpected role as a suppressor of T lymphocyte proliferation. Rather than functioning as a transcriptional activator, E2F2 appears to function as a transcriptional repressor of genes required for normal S phase entry, particularly E2F1.

The development of TCR alphabeta(+), CD8alphabeta(+) intestinal intraepithelial lymphocytes (IEL) is dependent on MHC class I molecules expressed in the thymus, while some CD8alphaalpha(+) IEL may arise independently of MHC class I. We examined the influence of MHC I allele dosage on the development CD8(+) T cells in RAG 2(-/-) mice expressing the H-2D(b)-restricted transgenic TCR specific for the male, Smcy-derived H-Y Ag (H-Y TCR). IEL in male mice heterozygous for the restricting (H-2D(b)) and nonrestricting (H-2D(d)) MHC class I alleles (MHC F(1)) were composed of a mixture of CD8alphabeta(+) and CD8alphaalpha(+) T cells, while T cells in the spleen were mostly CD8alphabeta(+). This was unlike IEL in male mice homozygous for H-2D(b), which had predominantly CD8alphaalpha(+) IEL and few mostly CD8(-) T cells in the spleen. Our results demonstrate that deletion of CD8alphabeta(+) cells in H-Y TCR male mice is dependent on two copies of H-2D(b), whereas the generation of CD8alphaalpha(+) IEL requires only one copy. The existence of CD8alphabeta(+) and CD8alphaalpha(+) IEL in MHC F(1) mice suggests that their generation is not mutually exclusive in cells with identical TCR. Furthermore, our data imply that the level of the restricting MHC class I allele determines a threshold for conventional CD8alphabeta(+) T cell selection in the thymus of H-Y TCR-transgenic mice, whereas the development of CD8alphaalpha(+) IEL is dependent on, but less sensitive to, this MHC class I allele.

The response of H-Y-specific TCR-transgenic CD8(+) T cells to Ag is characterized by poor proliferation, cytolytic activity, and IFN-gamma secretion. IFN-gamma secretion, but not cytotoxic function, can be rescued by the B7.1 molecule, suggesting that costimulation can selectively enhance some, but not all, effector CD8(+) T cell responses. Although the H-Y epitope binds H-2D(b) relatively less well than some other epitopes, it can induce potent CTL responses in nontransgenic mice, suggesting that the observed poor responsiveness of transgenic CD8(+) T cells cannot be ascribed to the epitope itself. Previously reported reactivity of this TCR to H-2A(b) is also not the cause of the poor responsiveness of the H-Y-specific CD8(+) T cells, as H-Y-specific CD8(+) T cells obtained from genetic backgrounds lacking H-2A(b) also responded poorly. Rather, reducing the levels of H-2(b) class I molecules by breeding the mice to (C57BL/6 x B10.D2)F(1) or TAP1(+/-) backgrounds partially restored cytotoxic activity and enhanced proliferative responses. These findings demonstrate that the self MHC class I gene dosage may regulate the extent of CD8(+) T cell responsiveness to Ag.

Genes controlling both testis determining and expression of the male-specific transplantation antigen, HY, are located on the short arm of the mouse Y chromosome, and on the X and Y-linked translocation, Sxr(a). A mutation of Sxr(a) was discovered in a cross between an Sxr carrier male and a T16H/X female. This was designated Sxr(b) and found to affect both the expression of HY and spermatogenesis, but not testis differentiation, thereby disproving Ohno's hypothesis that HY controlled testis determination. Molecular genetic analysis showed the mutation to be caused by fusion of two duplicated genes, Zfy1 and Zfy2, deleting the intervening DNA. This deletion interval, deltaSxr(b), contained a number of genes, each a candidate HY gene. Expression cloning with HY-specific T cell clones identified Smcy, Uty and Dby as encoding peptide epitopes of this transplantation antigen. The human homologues SMCY and UTY likewise express HY antigens and these are targets of damaging graft-versus-host (GVH) responses and potentially therapeutic graft-versus-leukaemia (GVL) responses following bone marrow transplantation (BMT). Knowledge of the peptide identity of HY epitopes allows monitoring of immune responses following BMT, using fluorescent tetramers, and also offers the possibility of inducing immunological tolerance.