New antigen in the Dombrock blood group system, DOYA, ablates expression of Do(a) and weakens expression of Hy, Jo(a), and Gy(a) antigens.

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.

Self-antigen maintains the innate antibacterial function of self-specific CD8 T cells in vivo.

Self-specific CD8 T cells, which are selected by high-affinity interactions with self-Ags, develop into a lineage distinct from conventional CD8 T cells. We have previously shown that these self-specific cells acquire phenotypic and functional similarities to cells of the innate immune system including the expression of functional receptors associated with NK cells. In this study, we show that these self-specific cells have the ability to produce large amounts of IFN-gamma in response to infection with Listeria monocytogenes in a bystander fashion. The rapid production of IFN-gamma is associated with a dramatic reduction in the number of viable bacteria at the peak of infection. Self-specific CD8 T cells provide only marginal innate protection in the absence of self-Ag; however, the presence of self-Ag dramatically increases their protective ability. Exposure to self-Ag is necessary for the maintenance of the memory phenotype and responsiveness to inflammatory cytokines such as IL-15. Significantly, self-specific CD8 T cells are also more efficient in the production of IFN-gamma and TNF-alpha, thus providing more cytokine-dependent protection against bacterial infection when compared with NK cells. These findings illustrate that self-reactive CD8 T cells can play an important innate function in the early defense against bacterial infection.

Optimization of the MB49 mouse bladder cancer model for adenoviral gene therapy.

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.

LIGHT (a cellular ligand for herpes virus entry mediator and lymphotoxin receptor)-mediated thymocyte deletion is dependent on the interaction between TCR and MHC/self-peptide.

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.

Growth factor independence-1B expression leads to defects in T cell activation, IL-7 receptor alpha expression, and T cell lineage commitment.

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.

CD8+ T lymphocytes in double alpha beta TCR transgenic mice. II. Competitive fitness of dual alpha beta TCR CD8+ T lymphocytes in the peripheral pools.

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.

Absence of CD43 fails to alter T cell development and responsiveness.

Genetic elimination of CD43 has been associated with increased T cell adhesiveness and T cell hyperresponsiveness to mitogens and alloantigens. Therefore, we investigated whether T cell development was perturbed in CD43-deficient mice by breeding CD43(null) mice with male Ag (Hy)-specific TCR-transgenic mice. Neither positive nor negative thymic selection of male Ag-specific T cells were affected by CD43 status. Furthermore, we did not observe a substantial or consistent hyperresponsive pattern in HY-CD43(null) lymph node cells compared with littermate HY-CD43(+/-) lymph node cells upon analysis of in vitro T cell stimulation with male Ag or mitogen. These observations challenged original conclusions associating absence of CD43 with T cell hyperresponsiveness and led us to re-examine this association. Reported phenotypes of CD43(null) mice have been based on mice with a mixed 129xC57BL/6 genetic background. To exclude a possible influence of genetic background differences among individual mice we analyzed CD43(null) littermates that had been back-bred onto the C57BL/6 background for seven to eight generations. We found that CD43(+) and CD43(null) littermates with the C57BL/6 background exhibited no differences in response to mitogen or alloantigen, thereby establishing that T cell hyperresponsiveness is not a general correlate of CD43 absence.

Dendritic cells permit identification of genes encoding MHC class II-restricted epitopes of transplantation antigens.

Minor or histocompatibility (H) antigens are recognized by CD4+ and CD8+ T lymphocytes as short polymorphic peptides associated with MHC molecules. They are the targets of graft versus host and graft versus leukemia responses following bone marrow transplantation between HLA-identical siblings. Several genes encoding class I-restricted minor H epitopes have been identified, but approaches used for these have proved difficult to adapt for cloning class II-restricted minor H genes. We have combined the unique antigen-presenting properties of dendritic cells and high levels of episomal expression following transfection of COS cells to identify a Y chromosome gene encoding two HY peptide epitopes, HYAb and HYEk.

The human UTY gene encodes a novel HLA-B8-restricted H-Y antigen.

The mammalian Y chromosome encodes male-specific minor histocompatibility (H-Y) Ags that are recognized by female T cells in an MHC-restricted manner. Two human H-Y epitopes presented by HLA-A2 and HLA-B7, respectively, have been identified previously and both are derived from the SMCY gene. We previously isolated CD8+ CTL clones that recognized a male-specific minor histocompatibility Ag presented by HLA-B8. In contrast to the SMCY-encoded H-Y epitopes, the B8/H-Y Ag was not presented by fibroblasts from male donors, suggesting that it was encoded by a novel gene. We now report that the HLA-B8-restricted H-Y epitope is defined by the octameric peptide LPHNHTDL corresponding to aa residues 566-573 of the human UTY protein. Transcription of the UTY gene is detected in a wide range of human tissues, but presentation of the UTY-derived H-Y epitope to CTL by cultured human cells shows significant cell-type specificity. Identification of this CTL-defined H-Y epitope should facilitate analysis of its contribution to graft/host interactions following sex-mismatched organ and bone marrow transplantation.

Dual MHC class I and class II restriction of a single T cell receptor: distinct modes of tolerance induction by two classes of autoantigens.

In the final stages of thymic development, immature T cells undergo three distinct processes (positive selection, negative selection, and lineage commitment) that all depend on interactions of thymocyte TCRs with MHC molecules. It is currently thought that TCRs are preferentially restricted by either MHC class I or class II molecules. In this report, we present direct evidence that the TCR previously described as H-Y/H-2Db specific cross-reacts with H-2IAb if expressed in CD4+ cells. We also demonstrate an increase in thymocyte numbers in H-Y TCR-trangenic mice deficient in MHC class II, suggesting a relatively discrete form of negative selection by MHC class II compared with that induced by H-Y/H-2Db. We propose that inability to generate CD4+ T cells expressing H-Y TCR in different experimental settings may be due to tolerance to self-MHC class II. These results, therefore, support an intriguing possibility that tolerance to self may influence and/or interfere with the outcome of the lineage commitment.

Modulation of thymic selection by expression of an immediate-early gene, early growth response 1 (Egr-1).

The potential involvement of early growth response (Egr)-1, a zinc-finger transcription factor belonging to the immediate-early genes, in positive/negative selection of thymocytes has been implicated by its expression in the population of CD4(+)CD8(+) double positive (DP) cells undergoing selection. To further investigate this possibility, transgenic mice overexpressing Egr-1 in thymocytes were bred with a transgenic mouse line expressing a T cell receptor (TCR) recognizing the H-Y male antigen in the context of H-2(b) class I major histocompatibility complex (MHC) molecules. In Egr-1/TCR H-Y double-transgenic mice, efficient positive selection of H-Y CD8(+) T cells occurred, even in mice on either a nonselecting H-2(d) background or a beta2-microglobulin (beta2m)-deficient background in which the expression of class I MHC heavy chains is extremely low; no positive selection was observed on a Kb-/-Db-/-beta2m-/- background where class I MHC expression is entirely absent. Similarly, when the Egr-1 transgene was introduced into a class II MHC-restricted TCR transgenic mouse line, Egr-1/TCR double-transgenic mice revealed increased numbers of CD4(+) T cells selected by class II MHC, as well as significant numbers of CD8(+) T cells selected by class I MHC (for which the transgenic TCR might have weak affinity). Thus, Egr-1 overexpression allows positive selection of thymocytes via TCR-MHC interactions of unusually low avidity, possibly by lowering the threshold of avidity required for positive selection. Supporting this possibility, increased numbers of alloreactive T cells were positively selected in Egr-1 transgenic mice, resulting in a strikingly enhanced response against allo-MHC. These results suggest that expression of Egr-1 and/or its target gene(s) may directly influence the thresholds required for thymocyte selection.

An H-YDb epitope is encoded by a novel mouse Y chromosome gene.

Rejection of male tissue grafts by genotypically identical female mice has been explained by the existence of a male-specific transplantation antigen, H-Y (ref. 1), but the molecular nature of H-Y antigen has remained obscure. Hya, the murine locus controlling H-Y expression, has been localized to delta Sxrb, a deletion interval of the short arm of the Y chromosome. In mice, H-Y antigen comprises at least four distinct epitopes, each recognized by a specific T lymphocyte clone. It has recently been shown that one of these epitopes, H-YKk, is a peptide encoded by the Y-linked Smcy gene, presented at the cell surface with the H-2Kk major histocompatibility complex (MHC) molecule. However, deletion mapping and the analysis of variable inactivation of H-Y epitopes has suggested that the Hya locus may be genetically complex. Here we describe a novel mouse Y chromosome gene which we call Uty (ubiquitously transcribed tetratricopeptide repeat gene on the Y chromosome). We identify the peptide WMHHNMDLI derived from the UTY protein as an H-Y epitope, H-YDb. Our data formally demonstrate that H-Y antigen is the product of more than one gene on the Y chromosome.

T cell tolerance and activation to a transgene-encoded tumor antigen.

Much has been learned in recent years concerning the nature of tumor antigens recognized by T cells. To apply this knowledge clinically, the nature of the host response to individual and multiple tumor antigens has to be characterized. This will help to define the efficacy of immune surveillance and the immune status of the host following exposure to tumor antigens expressed on pre-neoplastic tissue. To approach these questions, we have developed a transgenic mouse which expresses the tumor-specific antigen P91A. The single amino acid substitution in P91A results in the expression of a new MHC class I (H-2Ld)-binding peptide. In transgenic tissue, the H-2Ld/P91A complex is expressed in isolation from other tumor-associated antigens, allowing definition of the immune response to a single defined tumor antigen, a situation closely analogous to events during tumorigenesis. We show that CD8+ T cell immune surveillance of P91A is ineffective without the introduction of a helper determinant operating through stimulation of CD4+ T cells. Recognition of the isolated P91A tumor antigen on normal tissue by CD8+ T cells is a tolerogenic process. Induction of T cell tolerance suggests tumor antigen-T cell interactions occurring during tumorigenesis may elicit T cell tolerance and hence confound some immunotherapeutic approaches.

The influence of positive selection on RAG expression in thymocytes.

The expression of recombination activating gene (RAG) products, responsible for T cell receptor (TcR) gene rearrangement, is shut off during positive selection of thymocytes. The precise stage at which this down-regulation occurs remains somewhat controversial. We have analyzed RAG-1 expression in thymocytes of TcR transgenic mice carried on selecting versus non-selecting genetic backgrounds, both by in situ hybridization on thymus sections and by polymerase chain reaction amplification of RNA from sorted cells. The data from several transgenic lines indicate that RAG expression is already reduced in immature, cortical, CD4+CD8+ cells in the presence of positively selecting major histocompatibility complex molecules, although complete shut-off is not achieved until the mature, medullary, single-positive stage. This finding has practical and theoretical significance for studies on the mechanism of positive selection.

A mouse Y chromosome gene encoded by a region essential for spermatogenesis and expression of male-specific minor histocompatibility antigens.

A new mouse Y chromosome gene, Smcy, has been isolated from the region encoding Spy, a spermatogenesis gene and Hya and Sdma, the genes that, respectively, control the expression of the male specific minor histocompatibility antigen H-Y, as measured by specific T-cell assays and the serologically detected male antigen SDMA. Smcy is well conserved on the Y in mouse, man and even marsupials. It is expressed in all adult male tissues tested and can also be detected during mouse development from as early as two cells. In addition, its human Y homologue, SMCY, is expressed in multiple tissues and maps to the same Yq deletion interval as the human H-Y antigen controlling locus, HY.

Clonal deletion of immature CD4+8+ thymocytes in suspension culture by extrathymic antigen-presenting cells.

One mechanism ensuring self tolerance of T cells is the clonal deletion of thymocytes bearing alpha beta T-cell receptors. The stage of thymocyte development at which the interaction with antigen-presenting cells (APCs) leads to deletion, however, has not been determined directly. Indirect evidence suggests that intrathymic APCs induce deletion of CD4+8+ thymocytes (which die by apoptosis) but deletion at less and more mature developmental stages has also been implied. It is also not clear if clonal elimination of thymocytes can be triggered by peripheral antigens carried on extrathymic APCs migrating through the thymus. Here we show antigen-specific induction of apoptosis in CD4+8+ thymocytes cultured in suspension, by thymic as well as splenic APCs. Thus the recognition of antigen by CD4+8+ thymocytes may lead to deletion, suggesting that this is the central mechanism of tolerance induction, which is not limited by the antigen-presenting ability of the thymic stroma.

Variable spread of X inactivation affecting the expression of different epitopes of the Hya gene product in mouse B-cell clones.

Cloned B-cell lines from a female T16H/XSxr mouse in which Tdy expression was suppressed due to X inactivation and from a male X/XSxr mouse, both of the (kxb)F1 haplotype, were examined for H-Y expression. This was determined both by their ability to act as targets for H-2k and H-2b-restricted H-Y-specific cytotoxic T cells and by their ability to stimulate the proliferation of H-2Kk, H-2Db (class I) and Ab (class II)-restricted T-cell clones. In B-cell clones from the T16H/XSxr mouse, expression of H-Y/Db exhibited partial X inactivation and only a proportion (congruent to 30%) of the cells were targets for or stimulated H-2Db-restricted H-Y-specific T cells. In contrast, H-Y epitopes restricted by H-2k (H-Y/Kk, H-Y/Dk) and Ab (H-Y/Ab) exhibited no X inactivation. Furthermore, no inactivation of H-Y/Db, H-Y/Ab, or H-Yk was observed in the male X/XSxr mouse. These results indicate that the T16H/XSxr female is a mosaic, as a result of the variable spread of X inactivation into the Sxr region. They further suggest that the H-Y antigen recognized in association with H-2k and H-2Db class I molecules and Ab class II molecules may be the product of more than one gene.