Regulation of Thy-1 gene expression in transgenic mice

Genomic DNA fragments encompassing the human Thy-1 or mouse Thy-1.1 gene have been microinjected into pronuclei of mouse embryos homozygous for the Thy-1.2 allele. In the resulting transgenic mice, the human gene is expressed in a pattern characteristic of normal human tissues, and is not influenced by the pattern of endogenous mouse Thy-1 expression. The mouse Thy-1.1 gene fragment is expressed in a pattern typical of mouse Thy-1, although it is more limited in its distribution. The results indicate the presence of multiple cis-acting regulators of Thy-1 gene expression that have changed in both their character and arrangement over the course of Thy-1 gene evolution.     

T cell autoimmunity in Ig transgenic mice.

Autoantibodies directed at a diverse group of proteins of the U1/Sm ribonucleoprotein (snRNP) are characteristic of systemic lupus erythematosus and are found in the MRL murine model of this disease. This study examines the role of transgenic B lymphocytes in the regulation of autoreactive T cells to the snRNP autoantigen. Transgenic mice were developed bearing an Ig heavy chain gene specific for the D protein component of murine snRNP. B lymphocytes in these mice are neither deleted nor anergic and are of an immature (heat-stable Aghigh) phenotype. T lymphocytes from anti-snRNP transgenic mice were examined using a recombinant form of the D protein of the murine snRNP complex. Our results revealed that transgenic anti-snRNP B cell APCs stimulated CD4 T cells from wild-type C57BL/6 and MRL lpr/lpr mice, while nonspecific APCs failed to stimulate CD4 T cells. This study demonstrates that autoreactive T cells are not deleted from wild-type mice, although their activation is facilitated by autoantigen-specific APCs. The snRNP-reactive T cells in C57BL/6 transgenic mice are tolerized, in contrast to those T cells from MRL lpr/lpr transgenic mice. These studies implicate a role for autoreactive B lymphocytes in the in vivo activation and/or diversification of autoreactive T cells.     

Ectopic expression of Thy-1 in the kidneys of transgenic mice induces functional and proliferative abnormalities

Hybrid human--mouse Thy-1.1 genes were injected into pronuclei of Thy-1.2 mice to produce transgenic animals. A hybrid gene composed of the 5' part of the mouse Thy-1.1 gene combined with the 3' human untranslated regions was expressed abnormally in the kidney podocytes, which resulted in severe protein-uria and subsequent death in several founder mice. A hybrid Thy-1 gene composed of the human coding region with the 5' and 3' flanking regions of the mouse gene was expressed abnormally in a different part of the kidney (the tubular epithelia), which resulted in a proliferative kidney disorder. In addition, a neoplasm was found in the brain of one of these mice. These results show that the Thy-1 protein can play an important role in the activation, proliferation, and differentiation of many different cell types.     

Identification of the transgenic integration site in 2C T cell receptor transgenic mice

2C T cell receptor (TCR) transgenic mice have been long used to study the molecular basis of TCR binding to peptide/major compatibility complexes and the cytotoxicity mechanism of cytotoxic T lymphocytes (CTLs). To study the role of variable gene promoters in allelic exclusion, we previously constructed mutant mice in which the Vβ13 promoter was deleted (P13 mice). Introduction of 2C transgene into P13 mice accelerated the onset of systemic CD8 T cell lymphoma between 14 and 27 weeks of age, although parental P13 mice appeared to be normal. This observation suggests that the lymphoma development may be linked to features of 2C transgene. To identify the integration site of 2C transgene, Southern blotting identified a 2C-specific DNA fragment by 3′ region probe of 2C TCR α transgene, and digestion-circularization-polymerase chain reaction (DC-PCR) amplified the 2C-specific DNA fragment with inverse primers specific to the southern probe. Sequence analysis revealed that DC-PCR product contained the probe sequences and the junction sequences of integration site, indicating that 2C TCR α transgene is integrated into chromosome 1. Further genomic analysis revealed cytosolic phospholipase A2 group IVA (cPLA2) as the nearest gene to the integration site. cPLA2 expression was upregulated in the normal thymi and T cell lymphomas from 2C transgenic mice, although it was not altered in the lymph nodes of 2C transgenic mice. The result is the first report demonstrating the integration site of 2C TCR transgene, and will facilitate the proper use of 2C transgenic mice in studies of CTLs.     

Antibodies to nonpolymorphic determinants of the Thy-1 molecule inhibit T cell proliferation

The effect of anti-Thy-1 monoclonal antibodies on murine mixed lymphocyte reactions and concanavalin A-induced mitogenesis were investigated. It is demonstrated that rat antibodies against nonpolymorphic determinants of the murine Thy-1 antigen inhibited cell proliferation in the absence of complement. In contrast, antibodies against polymorphic determinants of Thy-1 had no effect on T cell activation. Inhibition of T cell proliferation did not depend on the isotype of the blocking antibody, because both IgM and IgG antibodies against monomorphic determinants were inhibitory, whereas IgM or IgG antibodies against allotypic determinants were inactive. In addition, the blocking activity could not be attributed to the xenogeneic (rat) origin of the antibodies to nonpolymorphic Thy-1 determinants, because rat anti-Thy-1.2 antibodies had no effect on cell activation. Thus, the efficacy of anti-Thy-1 antibodies as T cell inhibitors was determined by the antibody specificity. The suppressive mechanism of anti-Thy-1 antibodies was effective throughout the entire course of mixed lymphocyte reactions. Addition of antibodies at any time point during the first 90 hr of a 120-hr mixed lymphocyte culture resulted in significant suppression of the proliferative response. However, in some cases an early enhancement preceded suppression of the response. The modulation of proliferative responses by anti-Thy-1 did not result from a nonspecific mitogenic effect of the antibodies on T lymphocytes, because no effects were observed when antibodies were added to responder cells alone. These results suggest that the Thy-1 molecule, or a molecule that is located on the cell membrane in close proximity to the Thy-1 antigen, is involved in the activation of T lymphocytes.     

Detection of a Thy-1 precursor in human T lymphoma cell lines

In an attempt to gain insight into the biosynthesis and processing of human Thy-1, rabbit antisera (R alpha TP) were raised against a synthetic peptide (TP) of 13 amino acid residues of identical amino acid sequence to that of residues 110-122 of the putative Thy-1 precursor deduced from the cDNA sequencing. Immunoblotting assay showed that the IgG fraction of R alpha TP (R alpha TP-IgG) recognized the synthetic peptide without demonstrable cross-reactivity with isolated human brain Thy-1 and detergent-solubilized membrane proteins of human brain cells. Immunohistochemical studies showed that both R alpha TP-IgG and HB-2S-1 (a monoclonal antibody which reacts with both membrane-bound Thy-1 on viable cells and detergent-solubilized Thy-1) stained cells of two human T lymphoma cell lines (HUT-78 and HUT-102) but they did not stain cells of a human B lymphoma cell line (Raji). In contrast, in cell surface indirect immunofluorescence assay, HB-2S-1 reacted with HUT-78 and HUT-102 cells while R alpha TP-IgG did not. Taken together, these data indicate the existence of a precursor form of human Thy-1 which is processed prior to anchoring to the cell surface.     

Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene.

The nef gene, which encodes related cytoplasmic proteins in both human (HIV) and simian (SIV) immunodeficiency viruses is dispensable for viral replication in vitro. In contrast, in vivo experiments have revealed that SIV nef is required for efficient viral replication and development of AIDS in SIV infected rhesus monkeys, thus indicating that nef plays an essential role in the natural infection. We show that expression of the Nef protein from the HIV-1 NL43 isolate in transgenic mice perturbs development of CD4+ T cells in the thymus and elicits depletion of peripheral CD4+ T cells. Thymic T cells expressing NL43 Nef show altered activation responses. In contrast, Nef protein of the HIV-1 HxB3 isolate does not have an overt effect on T cells when expressed in transgenic animals. The differential effects of the two HIV-1 nef alleles in transgenic mice correlate with down-regulation of CD4 antigen expression on thymic T cells. The differential interactions of the NL43 and HxB3 nef alleles with CD4 were reproduced in a transient assay in human CD4+ CEM T cells. Down-regulation of CD4 by nef in both human and transgenic murine T cells indicates that the relevant interactions are conserved in these two systems and suggests that the consequences of Nef expression on the host cell function can be analyzed in vivo in the murine system. Our observations from transgenic mice suggest that nef-elicited perturbations in T cell signalling play an important role in the viral life cycle in vivo, perhaps resulting in elimination of infected CD4+ T cells.     

Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter

Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis might be reducing liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4⁺ T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.     

Intrabodies against the EVH1 domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells

Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single-chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (V(H)) leader signal sequence at the N-terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott-Aldrich syndrome protein (WASP). WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin (IL)-2 synthesis in the T-cell receptor (TCR) signaling pathway. It has been suggested that an EVH1 domain in the N-terminal region of WASP may participate in IL-2 synthesis. In transgenic mice expressing anti-EVH1 scFvs derived from hybridoma cells producing WASP-EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti-EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL-2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T-cell defects in WASP transgenic mice overexpressing the EVH1 domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 domain function that regulates IL-2 synthesis signaling without affecting the overall domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.     

Evidence for CD8-independent T cell maturation in transgenic mice

Double-negative (CD4-/CD8-) T cells expressing the alpha/beta transgenic TCR from the 2C cell line (anti-H-2Ld) were examined in the periphery of animals whose MHC type produces positive, negative, or no selection for differentiation of the TCR on single positive (CD8+) cells. Regardless of the selection haplotype the CD4-/CD8- cells are capable of activation by anticlonotypic mAb indicating that negative selection does not inactivate the "forbidden" TCR. Rather, the lack of response to H-2Ld in the negative haplotype is likely to absence of CD8 required to produce a functional response to H-2Ld. The similarity of surface phenotype and functional activity of these CD4-/CD8- cells maturing in different haplotypes suggests they may arise by an alternative lineage which, unlike the dominant TCR alpha/beta pathway, does not require coexpression of CD4/CD8.     

T cell receptor alpha-chain influences reactivity to Mls-1 in V beta 8.1 transgenic mice.

Most, but not all, V beta 8.1+ T cells respond to M1s-1 and are clonally deleted in the thymus of M1s-1-expressing animals. To formally examine the role of the TCR alpha-chain in reactivity and tolerance to M1s-1, we have analyzed M1s-1 reactivity in a large panel of CD4+ hybridomas generated from TCR V beta 8.1 transgenic mice, that express an identical, potentially M1s-1-reactive beta-chain. The data show that the alpha-chain strongly influences the M1s-1 reactivity of the hybridomas and that the differences in reactivity had relevance for tolerance. Thus, V alpha 11+ hybridiomas were biased toward M1s-1 reactivity and V alpha 11+ T cells were correspondingly absent from the peripheral repertoire of M1s-1-expressing transgenic mice. V alpha 2+ hybridomas, on the other hand, were biased against M1s-1 reactivity, and V alpha 2+ T cells were correspondingly amplified in the M1s-1-expressing transgenic mice. Structural analysis of the alpha-chains revealed that the M1s-1 reactivity of the V alpha 11+ hybridomas segregated precisely with family member, such that V alpha 11.1+ hybridomas were M1s-1-reactive and V alpha 11.3+ hybridomas were not M1s-1-reactive. On the other hand, there was not a clear correlation between family member and M1s-1 reactivity in the V alpha 2+ hybridomas. The hybridomas also showed striking variation in their reactivity to staphylococcal enterotoxin B (SEB), and the SEB reactivity of the V alpha 11+ hybridomas correlated precisely with family member and with M1s-1 reactivity. In contrast, there was not a clear correlation with V alpha 2+ alpha-chain structure and SEB reactivity. Also, there was no correlation between M1s-1 reactivity and SEB reactivity in individual V alpha 2+ hybridomas, suggesting that the recognition of the two superantigens by the same TCR is not equivalent. Taken together, these data define a role for the TCR alpha-chain in superantigen reactivity and T cell tolerance, and provide a structural explanation for the different fates of M1s-1-reactive T cells in normal and transgenic mice.     

T cell receptor gamma delta expression of Thy-1+ dendritic epidermal cells--an update

Thy-1+ dendritic epidermal cells (Thy-1+DEC) are present in the murine epidermis. They are morphologically dendritic and express Thy-1, CD3 and asialoGM1, but not CD4 or CD8. T cell receptor (TCR) of Thy-1+DEC is TCR gamma delta. Allison et al and Tonegawa et al recently found that TCR of Thy-1+DEC is V gamma 5 J gamma C gamma -V delta 1D2J2C delta and has no junctional diversity. This TCR gamma delta of Thy-1+DEC is identical to TCR expressed on the earliest fetal thymocytes. It is distinct from that of other epithelial associated lymphocytes or other thymocytes. The ligand of Thy-1+DEC is not known, although TCR gamma delta of adult type could recognize allogenic major histocompatibility complex(MHC) class I or class II and mycobacterium antigen, especially heat shock protein. The TCR of Thy-1+DEC may not be the homing receptor to epidermis. The further studies are needed to elucidate the ligands or functions of Thy-1+DEC.     

Attenuation of Th1 response in decoy receptor 3 transgenic mice

The soluble decoy receptor 3 (DcR3) is a member of the TNFR superfamily. Because DcR3 is up-regulated in tumor tissues and is detectable in the sera of cancer patients, it is regarded as an immunosuppressor to down-regulate immune responses. To understand the function of DcR3 in vivo, we generated transgenic mice overexpressing DcR3 systemically. In comparison with HNT-TCR (HNT) transgenic mice, up-regulation of IL-4 and IL-10 and down-regulation of IFN-gamma, IL-12, and TNF-alpha were observed in the influenza hemagglutinin(126-138) peptide-stimulated splenocytes of HNT-DcR3 double-transgenic mice. When infected with Listeria monocytogenes, DcR3 transgenic mice show attenuated expression of IFN-gamma as well as increased susceptibility to infection. The Th2 cell-biased phenotype in DcR3 transgenic mice is attributed to decreased IL-2 secretion by T cells, resulting in the suppression of IL-2 dependent CD4(+) T cell proliferation. This suggests that DcR3 might help tumor growth by attenuating the Th1 response and suppressing cell-mediated immunity.     

Thy-1-negative and Ly-1-negative variants of T cells produce interleukin 2 in response to mitogens

IL 2 production by T cell variants, which lack the Thy-1 or Ly-1 surface glycoproteins, was studied. Cross-linking of the Thy-1 molecule resulted in IL 2 production by the EL4 thymoma and by a T cell hybridoma, suggesting that Thy-1 may play a role in T lymphocyte triggering. To further study the functional role of this molecule, Thy-1-negative variants were selected and analyzed for IL 2 production in response to phorbol-12-myristate-13-acetate (PMA) or to Con A. It was demonstrated that in spite of their failure to express Thy-1, the Thy-1-negative clones were capable of IL 2 production. These results indicated that although Thy-1 cross-linking triggers cell activation, a signal provided by Thy-1 is not indispensable for cell activation by mitogens. The T cell tumor line LBRM331A5 responds synergistically to IL 1 and PHA by releasing IL 2. It was demonstrated that anti Ly-1 monoclonal antibodies and PHA co-stimulated LBRM331A5 cells, as did IL 1 plus PHA. Thus, anti Ly-1 antibodies mimic the effect of IL 1, suggesting a role for Ly-1 antigen in T cell activation, perhaps by serving as an IL 1 receptor or as an associated molecule. To further study the functional role of Ly-1 and its relation to IL 1 receptor, Ly-1-negative variants of the LBRM331A5 cell line were selected and analyzed for IL 2 production in response to PHA plus IL 1. It was demonstrated that the Ly-1-negative clones were capable of IL 2 production as efficiently as Ly-1-positive clones. These results indicate that the Ly-1 and IL 1 receptor are distinct molecules, which are involved in different activation pathways.     

Genetic controls of T cell-independent Thy-1 alloantibody responses

Early and late primary IgM antibody responses of mice to Thy-1.1 antigens showed different antigenic and cellular requirements. We studied genetic controls of the early primary responses, which could be induced by subcellular thymocyte antigens independently of host T-cell activity. All Thy-1.2 mouse strains of Igh ^a(BALB/c and BC8), Igh-V ^aC^b(BAB14), Igh ^d(AKR/Cum), Igh ^j(CBA/J, C3H/HeN, C3H.SW, and C3H.JK), and Igh ⁿ(NZB) definitely responded early to Thy-1.1 antigens from AKR/J (Igh ^d), A.Thy-1.1 (Igh ^e), or B10.Thy-1.1 (Igh ^b) mice or SD rats, whereas all strains of Igh ^b(C57BL/6, C57BL/10, B10.D2, B10.BR, B10.A, CB20 and CWB), Igh ^c(DBA/2), Igh ^e(A/J), and Igh ^o(C.AL20) responded poorly to the same antigens. This contrasts with the observation that both strains of Igh ^j(C3H/HeN) and Igh ^b(B10.BR) responded well at later times. As was the case for late responses, the matching of H-2 between donor and recipient resulted in early responses of exceptional quality in high-responder strains. It was concluded that under the influence of H-2, whose incompatibility between donor and recipient partially interferes with responses, early but not late primary Thy-1.1-specific antibody responses are selectively controlled by Igh-V or closely linked Ir gene(s) as a new V _Hmarker.Abbreviations used in this paper Tl T cell-independent - TD T cell-dependent - PFC plaque-forming cell(s) - Igh immunoglobulin heavy chain - V _H variable region of heavy chain - C _H constant region of heavy chain     

Effects of overexpression of growth hormone on T cell activity in transgenic mice

Growth hormone plays a key role in the maturation and maintenance of the immune response, however, the effects of chronic high circulating concentrations of the hormone on the immune system is poorly understood. Transgenic mice overexpressing bovine growth hormone (b-GH) gene, fused to the rat phosphoenolpyruvate carboxykinase promoter (PEPCK), with very high plasma concentration of heterologous b-GH and their littermate normal siblings were used. Spleen cellularity, percentages of total T lymphocytes, CD4+ and CD8+ cells, ratio of T cell subpopulations, mitogen-induced lymphocyte proliferation and natural killer (NK) cell activity were examined in male transgenic mice and normal littermate mice at 2 and 6 months of age. The number of splenic lymphocytes was greater in transgenic mice than in matched normal littermates at both ages. The NK cell activity was lower in transgenic mice than in the matched normal littermates at both ages, with the lowest values found in older mice. The b-GH transgenic mice had lower percentages of T cells at both ages, however, in young transgenic mice, the percentage of CD4+ cells was reduced while percentage of CD8+ cells was increased in comparison to normal controls. Both basal and mitogen-induced proliferation capacity of splenocytes were reduced in PEPCK-b-GH-25 mice as compared to normal littermates of both ages. Proliferative indexes in response to concanavalin A and phytohemagglutinin were markedly decreased in 6 month old PEPCK-b-GH-25 mice as compared to littermate controls or younger mice. These results indicate that overexpression of b-GH in mice is associated with decreased T cell function and that these abnormalities are age-dependent.