Falha na sexagem por inibição do desenvolvimento de embriões bovinos produzidos in vitro com anticorpos anti H-Y / Failure of sexing by developmental arrest of bovine embryos in vitro produced with H-Y antisera

Embriões bovinos produzidos in vitro, em estádio de mórula, foram cultivados em meio contendo anticorpos anti H-Y de alto título proveniente de ratos por 24h e, após este tempo, classificados em dois grupos: 1) embriões inibidos em estádio de mórula (classificados como machos) e 2) embriões que se desenvolveram e formaram a blastocele (classificados como fêmeas). O sexo de 311 embriões, distribuídos em três grupos de concentração dos anticorpos, 3 por cento, 5 por cento ou 7 por cento, foi identificado pela reação em cadeia da polimerase. Não houve desvio da proporção entre machos e fêmeas (P>0,05) nos grupos em que se utilizaram os anticorpos anti H-Y, quando comparadas ao grupo-controle, sem adição de anticorpos anti H-Y. Diferentemente dos resultados obtidos utilizando-se embriões bovinos produzidos in vivo, a sexagem com anticorpos anti H-Y de alto título em embriões produzidos in vitro não propiciou sucesso.

In vitro produced bovine embryos at morula stage were cultured in medium containing high titer of rat H-Y antisera for 24h. The embryos were classified in two groups: 1) embryos arrested at morula stage (classified as males); and 2) embryos that developed and formed a blastocoele (classified as female). The sex of 311 embryos, divided in three groups of concentration of H-Y antisera, 3 percent, 5 percent or 7 percent, was identified by polimerase chain reaction. The results showed no difference (P>0.05) on sexual deviation in groups in which the H-Y antisera was added, in relation to control group, in which no H-Y antisera was added. In contrast with results obtained with in vivo produced bovine embryos, the sexing of in vitro produced bovine embryos with high H-Y antisera titer did not succed.

DDX3Y encodes a class I MHC-restricted H-Y antigen that is expressed in leukemic stem cells.

The Y chromosome encodes male-specific minor histocompatibility (H-Y) antigens that stimulate T- and B-lymphocyte responses after sex-mismatched allogeneic hematopoietic cell transplantation (HCT). A CD8(+) cytotoxic T lymphocyte (CTL) clone that recognizes a novel HLA-B*2705-restricted H-Y antigen encoded by the DDX3Y gene was isolated from a male who had received a hematopoietic cell graft from his human leukocyte antigen (HLA)-identical sister. The antigenic peptide is a decamer that differs from the homologous DDX3X-encoded peptide at 4 positions. Expression of DDX3Y and of the H-Y epitope that it encodes was examined by quantitative polymerase chain reaction (PCR) and by CTL recognition assays. Expression of DDX3Y is detected in all myeloid and lymphoid leukemic cells that carry an intact Y chromosome. Moreover, the DDX3Y-encoded H-Y epitope is presented on the surface of both myeloid and lymphoid leukemic cells from male HLA-B*2705(+) patients. DDX3Y-specific CTLs prevent engraftment of human acute leukemia in nonobese diabetic/severe combined immune deficient mice, demonstrating that the DDX3Y-encoded H-Y antigen is also expressed in leukemic stem cells. These results demonstrate that CD8(+) T-cell responses against DDX3Y have the potential to contribute to graft-versus-leukemia (GVL) activity after female into male allogeneic HCT. This study is registered at http://clinicaltrials.gov as NCT00107354.

In vitro expansion improves in vivo regulation by CD4+CD25+ regulatory T cells.

CD4+CD25+ T regulatory cells (Tregs) can actively suppress immune responses and thus have substantial therapeutical potential. Clinical application is, however, frustrated by their scarcity, anergic status, and lack of defined specificity. We found that a single injection of a small number of expanded but not fresh HY-specific Tregs protected syngeneic male skin grafts from rejection by immune-competent recipients. The expanded Tregs were predominantly located in the grafts and graft-draining lymph nodes. In vitro expanded Tregs displayed a phenotype of CD25highCD4lowFoxp3+CTLA4+, and also up-regulated IL10 and TGFbeta while down-regulating IFN-gamma, GM-CSF, IL5, and TNF-alpha production. Furthermore, expanded Tregs appeared to express a reduced level of Foxp3, which could be prevented by adding TGFbeta to the culture, and they also tended to lose Foxp3 following the repeated stimulation. Finally, a proportion of expanded HY-specific Tregs secreted IL2 in response to their cognate peptide, and this finding could be confirmed using Tregs from Foxp3GFP reporter mice. We not only demonstrated that expanded Tregs are superior to fresh Tregs in suppressing T cell responses against alloantigens, but also revealed some novel immunobiological properties of expended Tregs which are very instructive for modifying current Treg expansion procedures.

Functional HY-specific CD8+ T cells are found in a high proportion of women following pregnancy with a male fetus.

Recent studies have demonstrated that fetal cells can be detected in the maternal circulation during virtually all human pregnancies. These fetal cells can engraft and may be isolated for many decades after pregnancy, leading to a state that may be maintained by the passage of pregnancy-associated progenitor cells. The clinical consequences of fetal cell microchimerism are unclear but may be potentially detrimental or valuable to the mother. One possibility is the generation of an alloreactive immune response by the mother to antigens expressed by the fetus; for example, the HY protein encoded by the Y chromosome. To test this we have screened a cohort of women with a range of parity histories within 8 yr of their last pregnancy for the presence of an HY-specific CD8+ T-cell response. Fluorescent HLA-peptide (HY) tetramers were used to stain short-term T-cell cultures from these women for analysis by flow cytometry. Responses were detected in 37% of women with a history of pregnancies that produced males, and this value rose to 50% in women with two or more pregnancies that produced males. HY-specific CD8+ T cells also could be detected directly in the peripheral blood of women with a history of at least two pregnancies that produced males. These HY-specific CD8+ T cells produced interferon gamma (IFNG) following peptide stimulation, demonstrating their functional capacity. In conclusion, our data indicate that alloreactive CD8+ T cells are generated frequently following normal pregnancy and retain functional capability for years following pregnancy.

[Cloning and analysis of phage Fab antibodies of mouse male specific antigen].

To clone mouse phage antibodies against H-Y antigen from a phage antibody library, three cycles of affinity enrichment of the mouse phage antibody library with male spleen cells and two cycles of nonspecific absorption with female spleen cells were performed. The presence of mouse Fab on the phage surface was determined by ELISA and sequence analysis. 9 of 15 strains can bind to male spleen cells with the specific activity. Recombination rate of the phage antibody library clones is 60%. Sequence analysis of the PCR products of plasmid DNA of E5 clones show VH and Vkappa had common characteristics shared by other known variable region of antibodies. The mouse phage Fab antibody could be used for identifying H-Y antigen, and for the development of sex determination of early embryos in mammals.

In situ visualization of antigen-specific T cells in cryopreserved human tissues.

Tetrameric MHC/peptide complexes are important tools for analyzing antigen-specific T cells. The in situ use of tetrameric MHC/peptide complexes in viable tissue sections has several shortcomings: it does not allow the execution of multiple analyses on one single biopsy, the storage of the biopsies, and the co-staining of the tetramer-positive cells for various intracellular molecules. We have developed a novel approach using overnight pre-labeling of viable human tissues with MHC/peptide tetramers, followed by cryopreservation and labeling of the cryosections. The visualization of antigen-specific T cells, combined with detection of other membrane, cytoplasmic, or nuclear markers is now feasible.

Prolonged Hya-disparate skin graft survival in ethanol-consuming mice: correlation with impaired delayed hypersensitivity.

BACKGROUND: Ethanol consumption impairs cell-mediated immunity and enhances humoral immunity. Among cell-mediated immune reactions, little is known of the effect of ethanol on chronic graft rejection. Allograft responses against the male-specific minor histocompatibility antigen, Hya, are widely used to study chronic graft rejection. METHODS: Female C57BL/6 (B6) mice were fed ethanol-containing liquid diets, were pair-fed an isocaloric liquid control diet, or were fed solid diet and water ad libitum. One week after diet initiation, the mice were grafted with split thickness, orthotopic male tail skin grafts, and the integrity of the grafts was monitored as the diet continued. Delayed hypersensitivity (DTH) was also determined in these same mice. In addition, Hya-cytolytic T-cell-deficient syngeneic major histocompatibility complex mutant B6.C-H2bm13 (bm13) and B6.C-H2bm14 (bm14) mice were assessed for skin graft rejection, DTH, and cytotoxic T-lymphocyte (CTL) activity. RESULTS: Ethanol-consuming female B6 mice are impaired in their ability to reject syngeneic male skin grafts and to develop Hya-specific DTH responses. To address the underlying mechanism, we show that Hya graft rejection correlates with DTH and not with CTL activity. Female B6 mice clearly differ from female bm13 and bm14 mice in their ability to generate CTLs against Hya antigen. Despite their inability to make Hya-specific CTL responses, bm13 and bm14 female mice, nevertheless, make Hya-specific DTH responses and ultimately reject Hya-disparate skin grafts, indicating that Hya-specific graft rejection results from DTH. Ethanol, by impairing Hya-specific DTH, inhibits Hya-specific skin graft rejection. CONCLUSIONS: We demonstrate that ethanol consumption impairs Hya-specific graft rejection. In addition, experiments with mice unable to generate anti-Hya CTLs support previous observations suggesting that DTH responses are sufficient to cause rejection of Hya-incompatible grafts.

Generation of helper and cytotoxic CD4+T cell clones specific for the minor histocompatibility antigen H-Y, after in vitro priming of human T cells by HLA-identical monocyte-derived dendritic cells.

BACKGROUND: There is now convincing evidence that minor histocompatibility antigens (mHag) may play a significant role in the pathogenesis of graft-versus-host disease after HLA-identical bone marrow transplantation. Indeed, in this clinical situation, T cells specific for mHag have been isolated. Here, we addressed whether one can generate mHag-specific T cells in vitro, without any in vivo immunization, among healthy blood donors. METHODS: We used monocyte-derived dendritic cells (Mo-DCs) as antigen presenting cells to induce primary responses between healthy HLA-identical siblings, in mixed lymphocyte dendritic cell reactions (MLDCRs). RESULTS: We show that CD4+ T-cell clones, specific for the mHag H-Y, can be generated in vitro. These clones were derived from a gender-mismatched positive MLDCR pair of HLA-identical siblings and were restricted by the HLA DQB1*0502 molecule. In addition, these CD4+ T clones were also able to lyse allogeneic targets with the same pattern of restriction and specificity than helper function. Finally, acute myeloid leukemia (AML) blast cells were susceptible to lysis by these clones. CONCLUSIONS: Altogether, these results predict that Mo-DCs could help to generate class II-associated, mHag-specific, T-cell lines or clones in vitro, between healthy blood donors, without any need of transplantation-mediated immunization.

Histocompatibility Y antigen compatibility and allograft rejection in corneal transplantation.

PURPOSE: To assess the effects of histocompatibility Y (H-Y) antigen matching on the rate of corneal allograft rejection after penetrating keratoplasty (PKP). METHODS: We retrospectively investigated the graft survival rate and rejection-free graft survival rate after PKP in 396 eyes. The compatible combinations of H-Y antigen included male donors and male recipients (n = 135), female donors and male recipients (n = 107), and female donors and female recipients (n = 60). Incompatible combination was from male donors and female recipients (n = 94). The eyes were classified into two groups--high-risk (168 eyes) and low-risk (228 eyes)--depending on the degree of vascularisation in the recipient corneas or a history of previous allograft rejection. Data were analysed using the Kaplan-Meier life table method, the log-rank test and the Cox proportional hazards model. RESULTS: In both the high-risk and low-risk groups, the graft survival and rejection-free graft survival rates were not affected by the H-Y compatibility. The graft survival (p < 0.001) and rejection-free graft survival (p < 0.001) rates were higher in the low-risk group than in the high-risk group. High-risk PKP was associated with greater risk of graft failure (risk ratio, 2.33) and rejection (risk ratio, 2.05) than low-risk PKP. CONCLUSION: H-Y antigen matching does not influence the rate of allograft rejection after PKP.

Tumor-induced interleukin-10 inhibits type 1 immune responses directed at a tumor antigen as well as a non-tumor antigen present at the tumor site.

Interleukin (IL)-10 is a potent immunosuppressive cytokine that has been found to be present at the tumor site in a wide variety of human cancers, including transitional cell carcinoma of the bladder. Using a murine bladder tumor (MB49), which we show to express the male transplantation antigen (HY), we tested the hypothesis that IL-10 at the tumor site can block the generation of a tumor-specific type 1 immune response. We show that, despite its expression of HY, MB49 fails to prime for an HY-specific type 1 (IFN-gamma) response in normal female mice. Although MB49 does not constitutively produce IL-10, our data support a model whereby MB49 induces infiltrating cells to produce IL-10. This feature rendered the IL-10 knockout (KO) mouse, whose infiltrating cells are incapable of IL-10 production, a suitable model in which to study MB49 in the absence of IL-10. When injected into IL-10 KO mice, MB49 does prime for an HY-specific, type 1 immune response. Furthermore, IL-10 KO mice show prolonged survival and an increased capacity to reject tumors as compared with normal mice. We also tested the ability of tumor-induced IL-10 to inhibit immunization to a non-tumor antigen present at the tumor site. When vaccinia virus encoding beta-galactosidase (beta-gal) is injected into the tumors of normal mice, no beta-gal-specific IFN-gamma response is mounted. However, when this same viral construct is injected into the tumors of IL-10 KO mice, it produces a strong beta-gal-specific, IFN-gamma response. These studies demonstrate that tumor-induced IL-10 can block the generation of a tumor-specific type 1 immune response as well as subvert attempts to elicit a type 1 immune response to a non-tumor antigen at the tumor site.

Do X and Y spermatozoa differ in proteins?

This article reviews the current knowledge about X- and Y-chromosomal gene expression during spermatogenesis and possible differences between X- and Y-chromosome-bearing spermatozoa (X and Y sperm) in relation to whether an immunological method of separation of X and Y spermatozoa might some day be feasible. Recent studies demonstrated that X- and Y-chromosome-bearing spermatids do express X- and Y-chromosomal genes that might theoretically result in protein differences between X and Y sperm. Most, if not all, of these gene products, however, are expected to be shared among X and Y spermatids via intercellular bridges. Studies on aberrant mouse strains indicate that complete sharing might not occur for all gene products. This keeps open the possibility that X and Y sperm may differ in proteins, but until now, this has not been confirmed by comparative studies between flow-cytometrically sorted X and Y sperm for H-Y antigen or other membrane proteins.

HA-1 and the SMCY-derived peptide FIDSYICQV (H-Y) are immunodominant minor histocompatibility antigens after bone marrow transplantation.

BACKGROUND: Allogeneic bone marrow donors can be incompatible at different levels. Even HLA-identical pairs will be still incompatible for numerous minor histocompatibility antigens (mHag). Nevertheless, some incompatibilities are found to be associated with an increased risk of graft-versus-host disease (GVHD), which could be related to the way the immune system recognizes these antigens. METHODS: We determined the specificity of cytotoxic T-cell clones isolated during acute GVHD or during bone marrow graft rejection in patients (n=14) transplanted with marrow from donors who were histoincompatible for different minor and/or major histocompatibility antigens. RESULTS: We found a clear hierarchy among the different types of histoincompatibilities. In three combinations mismatched for a class I allele, all 27 clones isolated during GVHD were specific for the incompatible HLA molecule. In the 11 class I-identical combinations, 14 different mHags were recognized. The mHag HA-1, known to have a significant impact on the development of GVHD, was recognized in the two HA-1-incompatible combinations. In one of these combinations, which was sex mismatched, all 56 clones analyzed were directed against HA-1, demonstrating the dominance of this mHag. In the four HA-1-compatible, sex-mismatched combinations, the anti-H-Y response was directed against one immunodominant epitope rather than against multiple Y-chromosome-encoded epitopes. All male specific cytotoxic T lymphocytes (n=15) recognized the same high-performance liquid chromatography-purified peptide fraction presented by T2 cells. Moreover, all cytotoxic T lymphocytes tested (n=6) were specific for the SMCY-derived peptide FIDSYICQV, originally described as being the H-Y epitope recognized in the context of HLA-A*0201. CONCLUSIONS: Some histocompatibility antigens are recognized in an immunodominant fashion and will therefore be recognized in the majority of mismatched combinations. Only for such antigens, correlations between mismatches and the occurrence of GVHD or graft rejections will be found.

Mismatches of minor histocompatibility antigens between HLA-identical donors and recipients and the development of graft-versus-host disease after bone marrow transplantation.

BACKGROUND: Graft-versus-host disease (GVHD) can be a major complication of allogeneic bone marrow transplantation even when the donor and recipient are siblings and share identical major histocompatibility antigens. The explanation may be a mismatch of minor histocompatibility antigens. We previously characterized five minor histocompatibility antigens, HA-1, 2, 3, 4, and 5, that are recognized by T cells in association with the major histocompatibility antigens HLA-A1 an A2. METHODS: We collected peripheral-blood leukocytes from 148 bone marrow recipients and their sibling donors, who were genotypically HLA identical. Fifty pairs were positive for HLA-A1, 117 were positive for HLA-A2, and 19 were positive for both. The pairs were typed with cytotoxic-T-cell clones specific for minor histocompatibility antigens HA-1, 2, 3, 4, and 5. RESULTS: Mismatches of HA-3 were equally distributed among recipients in whom GVHD developed and those in whom it did not. By contrast, a mismatch of only HA-1 was significantly correlated with GVHD of grade II or higher (odds ratio, infinity; P = 0.02) in adults. One or more mismatches of HA-1, 2, 4, and 5 were also significantly associated with GVHD (odds ratio, infinity; P = 0.006) in adults. These associations were not observed in children. CONCLUSIONS: A mismatch of minor histocompatibility antigen HA-1 can cause GVHD in adult recipients of allogeneic bone marrow from HLA-identical donors. Prospective HA-1 typing may improve donor selection and identify recipients who are at high risk for GVHD.

Papel do cromossomo Y na diferenciaçäo gonadal / Y chromosome role in the gonadal differentiation

Os autores fazem uma revisao sobre a evoluçao dos conceitos de diferenciaçao gonadal, desde a descoberta dos cromossomos X e Y até a localizaçao da seqüência de bases, no braço curto do cromossomo Y, próximo à regiao pseudoautossômica, supostamente responsável pela diferenciaçao da gônada bipotencial a testículo. O assunto desperta grande interesse, pois nem todos os passos deste verdadeiro quebra-cabeças foram desvendados e a questao do virilismo XX ainda se acha aberta à pesquisa científica.

Procedures for Sxs antigen detection by antibody-mediated cytotoxicity tests. A comparative analysis.

Biological reagents used in the serological detection of Sxs antigen by antibody-mediated cytotoxicity tests were compared in order to optimize the method. Our analyses showed that: (a) red cell-free spleen cells are the best target cells, (b) rabbit serum used as the complement source should be obtained from females, and absorbed with female spleen cells before use, (c) antiserum obtained by immunizing females with repeated injections of syngenic male spleen cells provides the highest anti-Sxs antibody titer, and (d) of the different biological fluids investigated, testis supernatant has highest concentration of Sxs antigen.

Daudi supernatant, unlike other H-Y antigen sources, exerts a sex-reversing effect on embryonic chick gonad differentiation.

In vitro cultures of intact chick gonads (organ cultures) and reaggregation cultures of dispersed gonad cells (roller cultures) were made. Gonads or gonad cells from 7-day-old chick embryos, at the stage when sex-specific differentiation begins, were cultured in the presence of presumed H-Y antigen-containing supernatants, or co-cultured in the presence of H-Y antigen-producing cell lines. The H-Y antigen-producing cells tested were of human, mouse, bovine and chicken origin. During organ culture, addition of supernatant of the human lymphoma cell line Daudi, or co-culture with Daudi cells, stimulated a clear proliferation of the germinal epithelium in male gonads, indicating feminization. A similar effect was obtained by treatment with estradiol. In reaggregation culture, the increase in nuclear size of germ cells was chosen as a parameter for feminization. A significant increase of germ cell nuclear size was observed in gonads cultured in the presence of Daudi supernatant. In both organ cultures and reaggregation cultures, other tested H-Y antigen sources and semi-purified H-Y antigen fractions did not exert significant effects on differentiation of the gonads or on the average area of the germ cell nuclei. These findings suggest that it is not H-Y antigen, but another protein produced by Daudi cells, that might be responsible for the sex-reversing effects.

Enhanced chemiluminescence: a high-sensitivity detection system for in situ hybridization and immunohistochemistry.

The breakthrough of chemiluminescence in the field of solution immunoassays and transfer membranes prompted us to explore whether a light-based detection system could provide a gain in sensitivity over chromogenic and FITC markers for nucleic acid and protein detection on histological preparations. A Hamamatsu device and an enhanced chemiluminescence (ECL) luminol substrate of the peroxidase were used to detect epithelial and endothelial components by immunohistochemistry (IHC) and for in situ hybridization (ISH) of papilloma virus DNA. The accuracy of the signal was compared to that obtained with DAB-peroxidase, silver-enhanced DAB-peroxidase, NBT-BCIP-alkaline phosphatase, and FITC. Our results demonstrated the feasibility and high sensitivity of luminescence detection for histological preparations. In part due to the ultrasensitive videocamera and photon-counting imaging, interpretable and reproducible results were obtained within counting times shorter than 5 min, and with dilutions of the primary antibodies 100- to 10,000-fold greater than those used for chromogenic and FITC reactions. As for ISH, with identical concentrations of the HPV 18 DNA probe on HeLa cells, labeling was apparent by luminescence but undetectable with the chromogen. The morphological resolution allowed a discriminatory analysis of the signal. Therefore, at the light microscopic level, enhanced chemiluminescence offers an appealing alternative to FITC and chromogenic markers for detection and quantification of low-concentration molecules.