H-2 complementation in anti-H-Y cytotoxic T-cell responses can occur in chimeric mice.

Cytotoxic T-cell responses against the H-Y antigen in mice are under the control of major histocompatibility complex genes. Not only must cytotoxic T cells recognize both H-Y antigens and "self" H-2K/D molecules to lyse male target cells, but also "appropriate association" between H-Y antigens and H-2K/D antigens is required to induce such cytotoxic responses. Furthermore, it is suggested that appropriate association with H-2-I antigens may also be required to generate H-Y specific helper cells for the cytotoxic response. BALB/c(KdIdDd) mice are nonresponders against syngeneic H-Y antigens, because they lack appropriate associative H-2K/D antigens. This results in the failure of generation of anti H-Y cytotoxic cells, although helper cells may be induced. F1 hybrid mice (BALB/c X C3H/He)F1 or H-2 recombinant mice C3H-OH(KdIdDk) are responders, because H-2Dk (and H-2Kk in the F1) molecules offer appropriate association to H-Y antigens. We here report that allophenic chimeras (H-2d reversible H-2k) and irradiation bone marrow chimeras [H-2d + H-2k leads to F1(H-2d X H-2k)] generate anti-H-Y cytotoxic responses but that cells of the BALB/c(H-2d) genotype comprise most if not all of the cytotoxic cells. A working model is proposed to account for major histocompatibility complex control over anti-H-Y cytotoxic T-cell responses.     

Implications of TL Phenotype Changes in an H-2-Loss Variant of a Transplanted H-2b/H-2a Leukemia

An H-2-loss variant line, lacking H-2^a antigens, was obtained from an H-2^b/H-2^a (C57BL × A)F₁ transplanted leukemia by immunoselection. The TL phenotype of the unselected line was TL.1,2,3,4 and that of the variant TL.1,2,4, which is the phenotype of TL⁺ leukemias of C57BL. This finding, and the observed quantitative alterations of antigens TL.1,2 and 4, indicate that the Tla (Thymus leukemia antigen) locus was functionally deleted together with the H-2 locus in the process of variant formation, despite absence of selection against cells carrying TL antigens. Representation of H-2 antigens of the remaining haplotype, H-2^b, was quantitatively unchanged.     

Life-span, T-cell responses, and incidence of lymphomas in congenic mice.

Survival, T-cell functions, and postmortem histopathology were studied in H-2 congenic strains of mice bearing H-2b, H-2k, and H-2d haplotypes. Males lived longer than females in all homozygous and heterozygous combinations except for H-2d homozygotes, which showed no differences between males and females. Association of heterozygosity with longer survival was observed only with H-2b/H-2b and H-2b/H-2d mice. Analysis using classification and regression trees (CART) showed that both males and females of H-2b homozygous and H-2k/H-2b mice had the shortest life-span of the strains studied. In histopathological analyses, lymphomas were noted to be more frequent in females, while hemangiosarcomas and hepatomas were more frequent in males. Lymphomas appeared earlier than hepatomas or hemangiosarcomas. The incidence of lymphomas was associated with the H-2 haplotype--e.g., H-2b homozygous mice had more lymphomas than did mice of the H-2d haplotype. More vigorous T-cell function was maintained with age (27 months) in H-2d, H-2b/H-2d, and H-2d/H-2k mice as compared with H-2b, H-2k, and H-2b/H-2k mice, which showed a decline of T-cell responses with age.     

H-2 restriction as a consequence of intentional priming: T cells of fully allogeneic chimeric mice as well as of normal mice respond to foreign antigens in the context of H-2 determinants not encountered on thymic epithelial cells.

Fully allogeneic chimeras were able to develop in vitro alloantigen-specific, as well as H-2-restricted, Sendai virus-specific cytotoxic T-lymphocyte (CTL) response. Depending on the immunization regimen used, Sendai virus-specific CTL responses were restricted to the H-2 antigens of either the stem cell donor or the thymus. Similarly, unprimed splenic T cells of normal mice were found to contain CTL-precursor cells that specifically reacted against Sendai virus or trinitrophenyl derivatives in the context of allogeneic major histocompatibility complex determinants that had not been encountered during their thymic differentiation. A frequency analysis of allogeneically versus syngeneically restricted virus-specific CTL precursors present in splenic T cells showed a ratio of about 1 to 6. These results provide evidence that H-2 restriction of trinitrophenyl- or Sendai virus-specific T cells is dictated by the complex type of the antigen-presenting cell and thus appears to be independent of the type of thymus in which the T cells have undergone maturation.     

Expression of chicken liver cell adhesion molecule fusion genes in transgenic mice.

The tissue-specific expression of the chicken liver cell adhesion molecule (L-CAM) was studied by generating transgenic mice. The rat insulin II promoter was fused to a chicken L-CAM cDNA or to chicken genomic L-CAM sequences. Mice carrying the cDNA showed no expression of L-CAM. Mice carrying L-CAM genomic sequences showed expression in the beta cells of the pancreas, suggesting that sequences in introns or in flanking regions are required for expression. Murine L-CAM was undetectable in the beta cells of the pancreas of those transgenic mice expressing chicken L-CAM and thus appeared to be down-regulated, but expression of the mouse protein was not altered at other sites. Chicken L-CAM was also found in extrapancreatic tissues such as skin, kidney, liver, lung, intestine, blood vessels, and the choroid plexus and leptomeninges of the central nervous system. These findings raised the possibility that the chicken L-CAM gene contains cis regulatory elements that interfere with the specificity of a tissue-specific promoter such as the rat insulin promoter. To test this hypothesis, transgenic mice were produced with a construct containing the murine neurofilament promoter fused to genomic chicken L-CAM sequences. Chicken L-CAM was expressed in the brain and spinal cord, where L-CAM is not normally found, but it was also found in some nonneural tissues (kidney, liver, intestine, lung) in which L-CAM is normally expressed. The combined results suggest that tissue-specific cis-acting elements in the chicken L-CAM gene, when combined with heterologous promoters/enhancers, can generate novel patterns of gene expression.     

Expression of urinary H-2 odortypes by infant mice.

The extended H-2 complex of genes in the mouse includes at least three loci that independently specify distinctive body odors, "odortypes," whose differential recognition influences mating choice and affects the maintenance of early pregnancy. A prime experimental method of identifying H-2 odortypes is the specially designed Y-maze in which mice are trained, by water deprivation and reward, to distinguish odors conducted to the arms of the maze from H-2-dissimilar mice or their urines. It is confirmed that H-2-dissimilar infant mice, unlike adult mice, are not distinguished by trained mice in the Y-maze. However, a previous conclusion that infant mice do not express H-2 odortypes is shown to be incorrect, because the urines of H-2-dissimilar infant mice, even at 1 day of age, were distinguished in the Y-maze. Thus urine, ingested by the mother, clearly could suffice for her to distinguish her own from other H-2-dissimilar pups. Further, urine would seem to be a unique source of H-2 odortypes. If, as we believe, H-2 odortypes represent mostly compound odors composed by H-2 genetic variation in the urinary output of odorous metabolites, as distinct from simple odors that depend on chemical differences of single odorants, then the kidney, which is not responsible for H-2 odortype specificity, may nevertheless impart a unique character to urinary odortypes by virtue of differential excretion/resorption processing of various constituent odorous metabolites. In that case, various organs and tissues, among which the hematopoietic/lymphoid system is known to contribute to H-2 odortype specificity, may exhibit tissue-specific varieties of H-2 odortypes, their products having not yet been subjected to renal processing.     

Expression of p210bcr/abl by metallothionein promoter induced T-cell leukemia in transgenic mice

The p210bcr/abl chimeric protein is considered to be implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. To investigate its biologic function in vivo, we generated transgenic mice expressing p210bcr/abl driven by the metallothionein enhancer/promoter. Two of six founder mice and the transgenic progeny developed leukemias several months after birth. In the leukemic tissues, the expression of the p210bcr/abl transgene product was detected and the increased tyrosine-phosphorylation of cellular proteins was observed. The expressed p210bcr/abl transgene product was shown to possess an enhanced kinase activity. The leukemic cells showed rearrangements in the T-cell receptor loci, indicating that the leukemic cells were monoclonal and committed to the T-cell lineage. Polymerase chain reaction analysis for tissue distribution of p210bcr/abl expression showed that, in the transgenic line that reproducibly developed leukemias, p210bcr/abl was expressed in the hematopoietic tissues such as thymus and spleen; on the other hand, in the transgenic lines that have not developed leukemias, p210bcr/abl expression was detected only in the nonhematopoietic tissues such as the brain and kidney. These results suggest that the tumorigenicity of the p210bcr/abl chimeric protein is restricted to the hematopoietic tissues in vivo and that an event enhancing p210bcr/abl expression contributed a proliferative advantage to hematopoietic precursor cells and eventually developed T- cell leukemia in transgenic mice.     

Expression of cyclin D1 in epithelial tissues of transgenic mice results in epidermal hyperproliferation and severe thymic hyperplasia.

To study the involvement of cyclin D1 in epithelial growth and differentiation and its putative role as an oncogene in skin, transgenic mice were developed carrying the human cyclin D1 gene driven by a bovine keratin 5 promoter. As expected, all squamous epithelia including skin, oral mucosa, trachea, vaginal epithelium, and the epithelial compartment of the thymus expressed aberrant levels of cyclin D1. The rate of epidermal proliferation increased dramatically in transgenic mice, which also showed basal cell hyperplasia. However, epidermal differentiation was unaffected, as shown by normal growth arrest of newborn primary keratinocytes in response to high extracellular calcium. Moreover, an unexpected phenotype was observed in the thymus. Transgenic mice developed a severe thymic hyperplasia that caused premature death due to cardio-respiratory failure within 4 months of age. By 14 weeks, the thymi of transgenic mice increased in weight up to 40-fold, representing 10% of total body weight. The hyperplastic thymi had normal histology revealing a well-differentiated cortex and medulla, which supported an apparently normal T-cell developmental program based on the distribution of thymocyte subsets. These results suggest that proliferation and differentiation of epithelial cells are under independent genetic controls in these organs and that cyclin D1 can modulate epithelial proliferation without altering the initiation of differentiation programs. No spontaneous development of epithelial tumors or thymic lymphomas was perceived in transgenic mice during their first 8 months of life, although they continue under observation. This model provides in vivo evidence of the action of cyclin D1 as a pure mediator of proliferation in epithelial cells.     

T-cell receptor genes in autoimmune mice: T-cell subsets have unexpected T-cell receptor gene programs.

Two unique cell subsets have been identified in the autoimmune-prone MRL/MP lpr/lpr and C3H/HeJ gld/gld murine strains that have the Lyt-2-,L3T4-,Thy-1+, and Lyt-2-,L3T4-,Ia-,Thy-1- phenotypes, respectively. We have now found that these cells express T-cell receptor proteins on their surface. Our observations further indicate that the expression of the Thy-1 antigen does not correlate with the expression of alpha-chain and beta-chain T-cell receptor polypeptides. Interestingly, T-cell receptor gamma-chain RNA expression may be influenced or correlate with Thy-1 molecular expression. These studies indicate unusual relationships of different cell-surface structures that may reflect unexpected developmental programs.     

Immunopotentiation in mice bearing a spontaneous transplantable T-cell lymphoma: role of thymic extract

Progressive ascitic growth of a spontaneous transplantable T-cell lymphoma, designated as Dalton's lymphoma (DL), in a murine host has been shown to be associated with an involution of thymus accompanied by a massive depletion of the cortical region and an alteration in the distribution of thymocytes by a decrease of CD4+CD8+, CD4+CD8- and CD4-CD8+ phenotypes caused by an enhanced induction of apoptosis in thymocytes. Moreover, an inhibition of humoral and cell mediated immune responses involving non-specific as well as antigen-specific T cell proliferative and cytolytic abilities with a decrease in the production of interferon gamma (IFNg) by the T cells of DL bearing mice has been observed. Results of the present study show that the administration of total thymic extract (TE) in DL bearing mice results in an increased survival of the DL bearing mice alongwith a significant increase in the weight of thymus and the total number of thymocytes with a lesser number of percent apoptotic thymocytes as compared to that in untreated DL-bearing mice. It is also shown that TE administration has a positive immunomodulatory effect on T cell functions as T cells obtained from TE administered DL bearing mice show an increased IFNg, production and an improved antigen specific proliferative ability. Moreover, the study indicates that TE acts directly on T cells as in an in vitro assay TE antagonised DL growth-associated induction of thymocyte apoptosis. Taken together, the results support the immunomodulatory function of the adult thymus and utilization of thymus derived factors as a potential immunotherapeutic agent for reversing tumor growth-associated immunosuppression.     

Establishment and characterization of a mouse strain (TLL) that spontaneously develops T-cell lymphomas/leukemia

In this study, a mouse strain (TLL) that spontaneously develops T-cell lymphomas/leukemia with an early onset and high incidence was established and characterized. All tumors analyzed were found to express the alpha,beta T-cell receptor, and the majority of them had a mature, CD3+CD4+CD8- immunophenotype. In a few cases, tumors with a more immature CD3+CD4+CD8+ phenotype were isolated. Expanded phenotyping using a broad panel of lymphocyte differentiation markers confirmed the mature T-cell phenotype of the tumors. Histologic and cell cycle analysis of the tumors revealed an aggressive lymphoblastic malignancy with a very high proliferation rate and widespread engagement of bone marrow and lymphoid as well as nonlymphoid organs. Thus, the TLL mouse strain represents a unique model for the analysis of the oncogenesis and progression of mature T-cell tumors and for the development of therapeutic measures to combat such tumors.     

T-cell tolerance toward a transgenic beta-cell antigen and transcription of endogenous pancreatic genes in thymus.

Transgenic mice expressing T antigen (Tag) in pancreatic beta cells establish systemic tolerance toward this self-protein. The self-tolerance in two families of rat insulin promoter (RIP)-Tag mice, expressing different levels of Tag protein, has been characterized. These mice have impaired antibody responses to Tag, show diminished Tag-specific T-cell proliferation, and evidence an inability to generate Tag-specific cytotoxic T cells. The existence of systemic tolerance toward a beta-cell-specific protein motivated examination of transgene expression in the thymus. Indeed, low levels of Tag mRNA were detected intrathymically. Remarkably, this expression is a valid property of the insulin gene regulatory region, since insulin RNA was also expressed in the thymus of nontransgenic mice. RNA for other pancreatic genes was also detected in the thymus, thus raising the possibility that many tissue-specific genes could be expressed intrathymically during immunological development and induction of self-tolerance. These results raise important questions for future research into the role of the thymus in tolerance induction toward so-called tissue-specific antigens.     

Expression of CD94/NKG2A and killer immunoglobulin-like receptors in NK cells and a subset of extranodal cytotoxic T-cell lymphomas

Thirty-two natural killer (NK) and cytotoxic T-cell lymphomas and 14 noncytotoxic nodal T-cell lymphoma controls were immunostained with the use of monoclonal antibodies reactive against NK-cell receptor (NKR) molecules (CD94, NKG2A, p58.2, p58.1, p140, p70, p50.3). All NK-cell lymphomas (4 nasal/oral and 1 intestinal) expressed at least 1 NKR, the CD94/NKG2A complex. Two were positive for 1 or more killer immunoglobulin-like receptors. Of 15 extranodal cytotoxic T-cell lymphomas, 3 expressed CD94, including 2 intestinal and 1 hepatosplenic gammadelta T-cell lymphomas. In contrast, none of the nodal lymphomas were positive. Detection of NKRs may provide a useful tool to confirm the diagnosis of NK-cell lymphomas and to delineate a subgroup of cytotoxic T-cell lymphomas. Expression of NKRs only in extranodal cytotoxic T-cell lymphomas might reflect differences in the homing capabilities of cytotoxic T cells expressing NKRs in normal individuals and might be influenced in part by localized chronic immune reactions.     

Secretion of a soluble, chimeric gamma delta T-cell receptor-immunoglobulin heterodimer.

Soluble derivatives of T-cell antigen receptors (TCRs) should prove invaluable for studying the interaction of these receptors with antigens and major histocompatibility complex molecules, for structural studies, and for the identification of unknown ligands. We have engineered chimeric proteins, containing the extracellular domains of the mouse V gamma 1.1-C gamma 4 and V delta 6.2-C delta (V, variable; C, constant) TCR chains fused to the hinge region, CH2 (H, heavy), and CH3 domains of human IgG1 heavy chain, and expressed them by transient transfection in COS cells. We show here that TCR gamma-IgH and TCR delta-IgH chimeric chains are produced intracellularly in significant amounts, that the two chains can assemble correctly to form disulfide-linked, glycosylated heterodimers, and that a selective mechanism allows secretion of correctly paired receptor chains into the medium. Identity of the chimeric secreted TCR gamma delta-IgH heterodimer was confirmed by immunoblot analysis using V gamma 1-specific anti-peptide antiserum and immunoprecipitation analysis using the monoclonal antibody UC7, which is shown to be specific for the TCR delta chain. In addition, the soluble TCR gamma delta-IgH heterodimer can be immunoprecipitated with the anti-clonotypic monoclonal antibody F10/56, which suggests that the fusion protein likely has a structural conformation similar to that of the native TCR. The COS cell expression system may prove useful for the production of additional TCR-IgH fusion proteins.     

A nondeletional mechanism of peripheral tolerance in T-cell receptor transgenic mice.

To investigate tolerance to extrathymic self molecules, we produced two groups of transgenic mice: one expressed the major histocompatibility complex molecule H-2Kb in pancreatic beta cells, and the other expressed rearranged T-cell receptor genes encoding an anti-H-2Kb receptor. The transgenic T-cell receptor genes were shown to confer the correct specificity and to be expressed appropriately. T cells bearing this receptor were activated by H-2Kb in vitro and in vivo, and they underwent negative selection in mice expressing H-2Kb in the thymus. To determine the fate and function of these anti-H-2Kb T cells in mice expressing H-2Kb exclusively in the periphery, the two groups of transgenic mice were mated to produce double transgenic offspring. In these, transgene-expressing T cells were present in both thymus and periphery. Persisting T cells had not down-regulated either their antigen-specific receptors or their CD8 molecules. Despite the persistence of large numbers of potentially reactive T cells, the mice were tolerant of H-2Kb in that they could not reject H-2Kb-bearing skin grafts, although they did reject third-party grafts. The results show that peripheral T-cell tolerance, unlike that imposed in the thymus, does not involve deletion of T cells. The existence of T cells bearing receptors specific for self components raises the possibility that aberrant activation of such cells may lead to the development of autoimmune disease.     

Modulation of T-cell functions in KIR2DL3 (CD158b) transgenic mice.

In humans, a minor subset of T cells express killer cell Ig-like receptors (KIRs) at their surface. In vitro data obtained with KIR(+) alphabeta and gammadelta T-cell clones showed that engagement of KIR molecules can extinguish T-cell activation signals induced via the CD3/T-cell receptor (TCR) complex. We analyzed the T-cell compartment in mice transgenic for KIR2DL3 (Tg-KIR2DL3), an inhibitory receptor for HLA-Cw3. As expected, mixed lymphocyte reaction and anti-CD3 monoclonal antibody (MoAb)-redirected cytotoxicity exerted by freshly isolated splenocytes can be inhibited by engagement of transgenic KIR2DL3 molecules. In contrast, antigen and anti-CD3 MoAb-induced cytotoxicity exerted by alloreactive cytotoxic T lymphocytes cannot be inhibited by KIR2DL3 engagement. In double transgenic mice, Tg-KIR2DL3 x Tg-HLA-Cw3, no alteration of thymic differentiation could be documented. Immunization of double transgenic mice with Hen egg white lysozime (HEL) or Pigeon Cytochrome-C (PCC) was indistinguishable from immunization of control mice, as judged by recall antigen-induced in vitro proliferation and TCR repertoire analysis. These results indicate that KIR effect on T cells varies upon cell activation stage and show unexpected complexity in the biological function of KIRs in vivo.     

High-level erythroid expression of human alpha-globin genes in transgenic mice.

The human alpha 1-globin gene was fused downstream of two erythroid-specific DNase I super-hypersensitive sites that are normally located upstream of the human beta-globin locus. This construct was injected into fertilized mouse eggs, and expression was analyzed in 16-day fetal livers and brains. All 11 fetuses that contained intact copies of the transgene expressed correctly initiated human alpha-globin mRNA in the erythroid fetal liver but not in brain. Levels of expression ranged from 4% to 337% of endogenous mouse beta-globin mRNA. A human alpha-globin construct that did not contain super-hypersensitive sites was not expressed. These results demonstrate that human beta-globin locus activation sequences can stimulate high levels of human alpha-globin gene expression in erythroid tissue of transgenic mice. The results also provide a foundation for experiments designed to coexpress human alpha- and beta-globin genes in transgenic mice and suggest a feasible approach for production of a mouse model for human sickle cell disease.     

Isolation and characterization of a stage-specific transforming gene, Tlym-I, from T-cell lymphomas.

A cellular transforming gene detected by transfection of mouse T-cell lymphoma DNA has been isolated by molecular cloning. This gene (designated Tlym-I) is homologous to a small conserved family of sequences present in normal mouse and human DNAs but is not related to any of the previously described viral or cellular transforming genes.     

Transporters from H-2b, H-2d, H-2s, H-2k, and H-2g7 (NOD/Lt) haplotype translocate similar sets of peptides.

The TAP complex transports peptides from the cytosol into the lumen of the endoplasmic reticulum for presentation by major histocompatibility complex class I molecules. A limited degree of sequence polymorphism has been observed for the mouse TAP1 and TAP2 genes by restriction fragment length polymorphism and sequence analysis. However, functional polymorphism of the TAP transporter has thus far been observed for the rat only. Here we examine the effect of TAP polymorphism on ATP dependency and peptide specificity of TAP-mediated peptide transport and show that, in the mouse, polymorphism in TAP genes does not measurably alter the function of their gene products. We conclude that TAP polymorphism is unlikely to contribute to the development of autoimmune diseases and that, in the mouse, the specificity of the TAP transporter is matched to that of the F pocket of the class I molecules for which it provides the peptide substrates.