Immunodominance is altered in T cell receptor (beta-chain) transgenic mice without the generation of a hole in the repertoire.

Despite the tremendous plasticity of the TCR repertoire, T cells recognize a limited number of antigenic sites (frequently a single site, or immunodominant epitope) on a complex protein Ag. Current models suggest that the immunodominant epitope of a complex protein is the processed peptide that binds to the MHC molecule with the highest affinity. Conversely, the inability of the T cell population to recognize a specific epitope, termed a "hole" in the repertoire, can prevent the immunodominance of a peptide despite efficient processing and MHC binding of the peptide. The role of specific TCR alpha- or beta-chains in determining MHC restriction and recognizing specific epitopes is complex and incompletely understood. To evaluate the contribution of each TCR chain to the functional diversity of the T cell repertoire, we investigated in vivo the T cell response to phage lambda-repressor protein in transgenic mice expressing a single rearranged beta-chain gene (C57L beta mice) in association with the complete germline alpha-chain repertoire. Our results demonstrate that expression of the TCR beta-chain transgene alters the immunodominant epitope recognized by T cells. However, after immunization with the appropriate peptide the transgenic mice can also respond to the nonimmunodominant epitope; thus, the expression of the TCR beta-chain transgene does not create a hole in the repertoire. These data indicate that the primary site, or immunodominant epitope, of an Ag recognized by T cells can be altered by the preimmune TCR repertoire independent of antigen processing and MHC affinity.     

Thymic and postthymic regulation of diabetogenic CD8 T cell development in TCR transgenic nonobese diabetic (NOD) mice

Natural development of diabetes in nonobese diabetic (NOD) mice requires both CD4 and CD8 T cells. Transgenic NOD mice carrying alphabeta TCR genes from a class I MHC (Kd)-restricted, pancreatic beta cell Ag-specific T cell clone develop diabetes significantly faster than nontransgenic NOD mice. In these TCR transgenic mice, a large fraction of T cells express both transgene derived and endogenous TCR beta chains. Only T cells expressing two TCR showed reactivity to the islet Ag. Development of diabetogenic T cells is inhibited in mice with no endogenous TCR expression due to the SCID mutation. These results demonstrate that the expression of two TCRs is necessary for the autoreactive diabetogenic T cells to escape thymic negative selection in the NOD mouse. Further analysis with MHC congenic NOD mice revealed that diabetes development in the class I MHC-restricted islet Ag-specific TCR transgenic mice is still dependent on the presence of the homozygosity of the NOD MHC class II I-Ag7.     

Tolerance induction by veto CTLs in the TCR transgenic 2C mouse model. II. Deletion of effector cells by Fas-Fas ligand apoptosis

The direct assay of veto CTLs in the 2C mouse model enables monitoring, by FACS, the fate of the TCR transgenic effector CD8(+) T cells, the transgene of which can be stained with clonotypic Ab 1B2. After the addition of veto cells, CD8(+)1B2(+) effector cells increasingly express annexin V, and maximal apoptosis is attained 72 h after initiation of MLR. This veto activity can be partially blocked by anti-CD8 Abs directed against the allele expressed by the veto CTLs, but not by the effector cells. When effector CD8(+) T cells were from 2C mice, which lack Fas expression ((2CX lpr)F(2)), deletion of effector cells was not exhibited by veto cells. The protein levels of the apoptosis inhibitors FLIP and Bcl2 in purified CD8(+)1B2(+) effector cells at different time points after MLR showed an initial up-regulation of these inhibitors, with marked reduction of FLIP, but not of Bcl2, by 48 h after initiation of culture. Taken together, these results are in accordance with a Fas-FasL-based mechanism in which prolonged binding between the effector cell and the veto cell might be required to allow FLIP to be down-regulated. Such prolonged interaction might be afforded through the interaction of CD8 molecules on the veto cell with the alpha3 domain of H2 class 1 on the effector cell.     

Abnormal thymocyte development and production of autoreactive T cells in T cell receptor transgenic autoimmune mice.

Development of a C57BL/6-+/+ TCR transgenic mouse containing the rearranged TCR alpha- and beta-chain specific for the Db + HY male Ag results in production of a nearly monoclonal population of early thymocytes expressing the Db + HY reactive TCR. These thymocytes are autoreactive in H-2Db male mice and undergo clonal deletion and down-regulation of CD8. To study the effect of the lpr gene on development of autoreactive T cells, these transgenic mice were backcrossed with C57BL/6-lpr/lpr mice. T cell populations in the thymus and spleen were analyzed by three-color flow cytometry for expression of CD4, CD8, and TCR. The thymus of TCR transgenic H-2b/b lpr/lpr male mice had an increase in percent and absolute number of CD8dull thymocytes compared to TCR transgenic H-2b/b +/+ male mice. However, there was not a complete defect in clonal deletion, because clonal deletion and down-regulation of CD8 was apparent in both +/+ and lpr/lpr H-2Db HY+ male mice compared to H-2Db HY- female mice. The phenotype of splenic T cells was almost identical in TCR transgenic +/+ and lpr/lpr males with about 50% CD4-CD8- T cells and 50% CD8+ T cells. However, there was a dramatic increase in the SMLR proliferative response of splenic T cells from TCR transgenic lpr/lpr males compared to TCR transgenic +/+ males. To determine the specificity of this response, spleen cells from TCR transgenic lpr/lpr and +/+ mice were cultured with irradiated H-2b/b and H-2k/k male and female spleen cells. T cells from TCR transgenic C57BL/6-lpr/lpr male mice had an increased proliferative response to H-2b/b male spleen cells compared to T cells from TCR transgenic C57BL/6(-)+/+ male mice, but both lpr/lpr and +/+ mice had a minimal response to irradiated H-2b/b female or H-2k/k male or female stimulator cells. The splenic T cells from TCR transgenic lpr/lpr mice also had an increased specific cytotoxic activity against H-2b/b male target cells compared to TCR transgenic +/+ mice. These results demonstrate that there is a defect in negative selection of self-reactive T cells in the thymus of lpr/lpr mice and a defect in induction or maintenance of clonal anergy of self-reactive T cells in the periphery of lpr/lpr mice.     

Preferential activation of an IL-2 regulatory sequence transgene in TCR gamma delta and NKT cells: subset-specific differences in IL-2 regulation

A transgene with 8.4-kb of regulatory sequence from the murine IL-2 gene drives consistent expression of a green fluorescent protein (GFP) reporter gene in all cell types that normally express IL-2. However, quantitative analysis of this expression shows that different T cell subsets within the same mouse show divergent abilities to express the transgene as compared with endogenous IL-2 genes. TCR gamma delta cells, as well as alpha beta TCR-NKT cells, exhibit higher in vivo transgene expression levels than TCR alpha beta cells. This deviates from patterns of normal IL-2 expression and from expression of an IL-2-GFP knock-in. Peripheral TCR gamma delta cells accumulate GFP RNA faster than endogenous IL-2 RNA upon stimulation, whereas TCR alpha beta cells express more IL-2 than GFP RNA. In TCR gamma delta cells, IL-2-producing cells are a subset of the GFP-expressing cells, whereas in TCR alpha beta cells, endogenous IL-2 is more likely to be expressed without GFP. These results are seen in multiple independent transgenic lines and thus reflect functional properties of the transgene sequences, rather than copy number or integration site effects. The high ratio of GFP: endogenous IL-2 gene expression in transgenic TCR gamma delta cells may be explained by subset-specific IL-2 gene regulatory elements mapping outside of the 8.4-kb transgene regulatory sequence, as well as accelerated kinetics of endogenous IL-2 RNA degradation in TCR gamma delta cells. The high levels and percentages of transgene expression in thymic and splenic TCR gamma delta and NKT cells, as well as skin TCR gamma delta-dendritic epidermal T cells, indicate that the IL-2-GFP-transgenic mice may provide valuable tracers for detecting developmental and activation events in these lineages.     

Cytotoxic T lymphocytes generated by short-term in vitro TCR stimulation in the presence of IL-4 are therapeutically effective against B16 melanoma

P14 TCR transgenic CD8+ T cells (LCMV gp33-specific) were activated by antigen in the presence of either IL-2 or IL-2+IL-4 to generate effector cytotoxic T lymphocytes (CTLs). The therapeutic effectiveness of such IL-2- or IL-2+IL-4-grown CTLs was tested in mice that had received intravenous inoculations of B16.gp33 melanoma cells 7 days previously. Administration of P14 CTLs activated by antigen +IL-2+IL-4 was significantly more effective at reducing melanoma colony formation in the lung than those grown in the presence of antigen +IL-2. Highly significant improvement in survival was observed with 80% of B16.gp33-inoculated mice showing long-term survival after therapy with 10×10⁶ antigen +IL-2+IL-4-activated P14 CTLs. Similar therapeutic effectiveness of antigen +IL-2+IL-4-activated P14 CTLs against subcutaneously inoculated B16.gp33 melanoma cells was also found. There was significant reduction in P14 CD8+ T cells in the peripheral blood of B16.gp33-inoculated mice than in mice that did not receive B16.gp33 melanoma cells, indicating possible homing of P14 CD8+ T cells to the site of tumor growth or antigen-induced apoptotic cell death. These results may have implications in tumor therapy using CTLs grown ex vivo, especially during early stages of tumor formation. They also support the concept that the therapeutic effectiveness of CTLs can be governed by the cytokine context in which they are activated.     

Evidence for a role for T cell receptors (TCR) in the effector phase of acute bone marrow graft rejection. TCR V beta 5 transgenic mice lack effector cells able to cause graft rejection.

Lethally irradiated mice reject within 24 h certain marrow grafts, a phenomenon called either allogeneic or hybrid resistance. The cells responsible for this rejection (NK1+ CD3+ cells (TNK) express Ag of NK cells as well as the TCR-associated CD3 complex. This raises the question whether TCR participate in the function of these cells during graft rejection. By using flow cytometry it is shown that the majority of TNK cells expresses the TCR-alpha/beta chains and by using adoptive cell transfer assays evidence is presented that it is the TCR-alpha/beta expressing cells that cause rejection. To explore whether any particular TCR chains have to be expressed on these cells, C57L mice were assayed and found to be responders suggesting that the V beta chains deleted in these mice are not obligatory. However, introduction of a specific TCR V beta 5 chain into C57BL/6 mice as a transgene leads to inability to transfer resistance. TNK cells of V beta 5 transgenic mice express the introduced gene suggesting that it is the transgenic TCR that is responsible for the lack of function. In assessing T cell functions in V beta 5 transgenic mice it is shown that although these mice generate CTL specific for H-2d targets there is a deficiency to recognize H-2Dd, i.e., of determinants presumed to be recognized in the acute rejection mechanism. Thus TNK cells and CTL share the inability to recognize H-2Dd epitopes due to expression of the V beta 5 transgene. The notion that TCR on TNK cells play a role in the acute rejection process makes it necessary to postulate a receptor selection mechanism for these cells.     

Tolerance induction by veto CTLs in the TCR transgenic 2C mouse model. I. Relative reactivity of different veto cells

Several bone marrow cells and lymphocyte subpopulations, known as veto cells, were shown to induce transplantation tolerance across major histocompatibility Ags. Due to the low frequency of the effector T cells against which the veto cells inhibitory activity is aimed, the fate of the effector cells was traditionally followed indirectly by functional limiting dilution assays, which are cumbersome and depend on numerous parameters. In the present study the fate of the effector T cells was monitored directly by FACS, using TCR transgenic mouse CD8(+) T cells in which the transgene is directed against H-2(d) (the 2C model). This assay is validated by demonstrating the potency, selectivity, radiation sensitivity, and contact dependency of anti-third-party CTLs previously demonstrated by the limiting dilution assay. In contrast to veto CTLs, nonactivated CD8(+) T cells lack veto activity. Comparison by FACS in the 2C model revealed a hierarchy of veto cells, in the order of veto CTLs activated NK cells, activated CD4(+) T cells, and activated B cells. The latter cells as well as nonactivated CD4(+) or NK cells were shown to be completely devoid of veto activity.     

T cell tolerance to Mlsa encoded antigens in T cell receptor V beta 8.1 chain transgenic mice.

To study T cell tolerance, transgenic mice were generated that expressed the Mlsa-reactive T cell receptor (TCR) beta chain V beta 8.1 (cDNA) under the control of the H-2Kb promoter/immunoglobulin heavy chain enhancer on approximately 90% of peripheral T cells. In transgenic mice bearing Mlsa, thymocytes expressing the TCR at a high density were deleted and the percentage of Thy 1.2+ lymph node cells was reduced. The CD4/CD8 ratio of mature T cells was reversed in Mlsa and Mlsb transgenic mice independent of the H-2. RNA analysis and immunofluorescence with TCR V beta-specific antibodies revealed that expression of endogenous TCR beta genes was suppressed. Both Mlsa and Mlsb TCR beta chain transgenic mice mounted a T-cell-dependent IgG response against viral antigens, whereas the capacity to generate alloreactive and virus-specific cytotoxic T cells was impaired in TCR beta chain transgenic Mlsa, but not in transgenic Mlsb mice.     

The structure of the human CD2 gene and its expression in transgenic mice.

We report the genomic organization of the human CD2 gene and its expression in transgenic mice. A 28.5 kb segment of DNA consisting of 4.5 kb 5' flanking sequences, 15 kb containing the gene's five exons and 9 kb of 3' flanking sequences can direct the expression of the CD2 gene only on thymocytes, circulating T cells and megakaryocytes of the transgenic mice. The expression of each copy of the human CD2 transgene appears to be as high as the endogenous mouse CD2 gene and as high as the expression on the surface of human T lymphocytes, independent of the site of integration and dependent on the copy number of genes that have integrated.     

Expression of a hybrid immunoglobulin-T cell receptor protein in transgenic mice

We have constructed a hybrid immunoglobulin (VDJH)-T cell receptor (C alpha) gene using the VDJH exon from a digoxin-specific antibody. This gene was used to make a line of transgenic mice. The hybrid VDJH-C alpha protein is expressed on a subset of T cells in these mice, and we have shown that it forms part of a functional TCR complex by the criteria of coprecipitation and comodulation of CD3 and TCR beta chain components and T cell activation with anti-idiotypic antibodies or digoxin. Furthermore, in cells expressing the hybrid protein, there is allelic exclusion of endogenous TCR alpha genes. We discuss the implications for the comparative structure of T cell receptors and immunoglobulins.     

Spontaneous organ-specific Th2-mediated autoimmunity in TCR transgenic mice

CD4(+) T cells that lead to autoimmune gastritis (AIG) in BALB/c mice are either Th1 or Th2 cells. To test whether the phenotype of disease is related to the particular TCR expressed by the pathogenic cell, we have generated several lines of TCR transgenic mice using receptors cloned from pathogenic Th1 or Th2 cells. We previously described spontaneous inflammatory AIG in A23 mice, caused by the transgenic expression of the TCR from a Th1 clone, TXA23. In this study we describe the generation of A51 mouse lines, transgenic for the TCR of a CD4(+) self-reactive Th2 clone, TXA51. A proportion of A51 mice spontaneously develop AIG by 10 wk of age, with a disease characterized by eosinophilic infiltration of the gastric mucosa and Th2 differentiation of transgenic T cells in the gastric lymph node. The Th2 phenotype of this autoimmune response seems to be related to a low availability of MHC class II-self peptide complexes. This in vivo model of spontaneous Th2-mediated, organ-specific autoimmunity provides a unique example in which the clonotypic TCR conveys the Th2 disease phenotype.     

Structural features of the integration site of foreign DNA in the transgenic mouse genome

The structure of the transgenic mouse DNA region containing an integrated transgene (fragment of pBR322 sequence) was analysed. In one of the sequences flanking the transgene, short direct and inverted overlapping repeats were revealed at a distance of 60 bp from the integration site. In the same flanking sequence, there is an extended sequence (3.5 kbp) 0.3-1 kbp away from the transgene. It repeats 100-300 times in the mouse genome and is highly conservative (the homologs of the repeat have been revealed in other mammalian, bird, fish and insect genomes). This up-to-date unknown family of highly-conserved dispersed repeats has been denoted by T1. We believe that both the revealed short inverted repeats capable of forming hairpins with loops and the T1 repeat are structures involved in the process of non-homologous insertion of foreign DNA into the region of the transgenic mouse genome.     

Analysis of TCRalphabeta combinations used by simian immunodeficiency virus-specific CD8+ T cells in rhesus monkeys: implications for CTL immunodominance

Immunodominance is a common feature of Ag-specific CTL responses to infection or vaccines. Understanding the basis of immunodominance is crucial to understanding cellular immunity and viral evasion mechanisms and will provide a rational approach for improving HIV vaccine design. This study was performed comparing CTLs specific for the SIV Gag p11C (dominant) and SIV Pol p68A (subdominant) epitopes that are consistently generated in Mamu-A*01(+) rhesus monkeys exposed to SIV proteins. Additionally, vaccinated monkeys were used to prevent any issues of antigenic variation or dynamic changes in CTL responses by continuous Ag exposure. Analysis of the TCR repertoire revealed the usage of higher numbers of TCR clones by the dominant p11C-specific CTL population. Preferential usage of specific TCRs and the in vitro functional TCR-alpha- and -beta-chain-pairing assay suggests that every peptide/MHC complex may only be recognized by a limited number of unique combinations of alpha- and beta-chain pairs. The wider array of TCR clones used by the dominant p11C-specific CTL population might be explained by the higher probability of generating those specific TCR chain pairs. Our data suggest that Ag-specific naive T cell precursor frequency may be predetermined and that this process dictates immunodominance of SIV-specific CD8(+) T cell responses. These findings will aid in understanding immunodominance and designing new approaches to modulate CTL responses.     

Mea2/Golga3 gene is disrupted in a line of transgenic mice with a reciprocal translocation between Chromosomes 5 and 19 and is responsible for a defective spermatogenesis in homozygotes

A line of transgenic mouse T604 transmitted a transgene to progeny together with a set of chromosomes with a reciprocal translocation. The transgene was integrated at a single site in the translocated chromosomes, as revealed by fluorescence in situ hybridization. The transgenic hemizygous males, also heterozygous for the translocation of chromosomes, showed apparently normal spermatogenesis, while the males homozygous for the transgene as well as for the translocated chromosomes showed a defect in spermatogenesis. Considering that the genetic rearrangement by either insertion of the transgene or the chromosome translocation in the T604 mouse line might have caused a recessive mutation in a gene indispensable for spermatogenesis, we have mapped the transgene integration site and the translocation breakpoints in mouse chromosomes. Linkage analysis with SSLP markers showed that the loci for the transgene and the translocation breakpoints were closely located to D5Mit24 on Chromosome (Chr) 5, and to a region between D19Mit19 and D19Jpk2 on Chr 19. Mea2 gene, mapped only 2 cM from D5Mit24 and known to show male-specific enhanced expression in the testis, was analyzed as a candidate for the gene disrupted in T604 transgenic mice. Southern blot analysis revealed that Mea2 gene was indeed disrupted in T604 mice, and Northern blot analysis of the testis RNA showed that the expression of Mea2 was annihilated in the testis of T604 transgenic homozygotes. Received: 8 July 1998 / Accepted: 23 September 1998     

TCR-independent killing of B cell malignancies by anti-third-party CTLs: the critical role of MHC-CD8 engagement

We previously demonstrated that anti-third-party CTLs (stimulated under IL-2 deprivation against cells with an MHC class I [MHC-I] background different from that of the host and the donor) are depleted of graft-versus-host reactivity and can eradicate B cell chronic lymphocytic leukemia cells in vitro or in an HU/SCID mouse model. We demonstrated in the current study that human allogeneic or autologous anti-third-party CTLs can also efficiently eradicate primary non-Hodgkin B cell lymphoma by inducing slow apoptosis of the pathological cells. Using MHC-I mutant cell line as target cells, which are unrecognizable by the CTL TCR, we demonstrated directly that this killing is TCR independent. Strikingly, this unique TCR-independent killing is induced through lymphoma MHC-I engagement. We further showed that this killing mechanism begins with durable conjugate formation between the CTLs and the tumor cells, through rapid binding of tumor ICAM-1 to the CTL LFA-1 molecule. This conjugation is followed by a slower second step of MHC-I-dependent apoptosis, requiring the binding of the MHC-I α2/3 C region on tumor cells to the CTL CD8 molecule for killing to ensue. By comparing CTL-mediated killing of Daudi lymphoma cells (lacking surface MHC-I expression) to Daudi cells with reconstituted surface MHC-I, we demonstrated directly for the first time to our knowledge, in vitro and in vivo, a novel role for MHC-I in the induction of lymphoma cell apoptosis by CTLs. Additionally, by using different knockout and transgenic strains, we further showed that mouse anti-third-party CTLs also kill lymphoma cells using similar unique TCR-independence mechanism as human CTLs, while sparing normal naive B cells.     

成骨细胞特异性表达Cre重组酶转基因小鼠的建立

构建了含有骨钙素基因启动子、Cre重组酶基因和人生长激素基因polyA的转基因载体pOC-Cre, 以显微注射的方法将4.6 kb的转基因片段OC-Cre导入小鼠受精卵。16只子代小鼠中经PCR和Southern杂交鉴定, 有2只小鼠携带外源基因, 整合率为12.5%。为了检测OC-Cre在转基因小鼠中表达的组织特异性, 将转基因首建者小鼠与基因组上携带有LoxP位点的条件性Smad4基因敲除小鼠交配, PCR结果显示, 仅在子代纯合型小鼠骨组织基因组中扩增出了Cre介导重组后的片段。将OC-Cre转基因小鼠与ROSA26报告小鼠交配, 利用LacZ染色对双转基因阳性子代小鼠进行检测, 结果显示Cre重组酶在成骨细胞中特异性表达并介导ROSA基因座LoxP位点间的重组。所有这些结果说明：所建立的OC-Cre转基因小鼠在成骨细胞中特异性表达Cre重组酶, 并能在体内介导成骨细胞基因组上LoxP位点间的重组, 是一种理想的研制成骨细胞特异性基因敲除小鼠的工具小鼠。     

Identification of the transgenic integration site in immunodeficient tgε26 human CD3ε transgenic mice

A strain of human CD3ε transgenic mice, tgε26, exhibits severe immunodeficiency associated with early arrest of T cell development. Complete loss of T cells is observed in homozygous tgε26 mice, but not in heterozygotes, suggesting that genomic disruption due to transgenic integration may contribute to the arrest of T cell development. Here we report the identification of the transgenic integration site in tgε26 mice. We found that multiple copies of the human CD3ε transgene are inserted between the Sstr5 and Metrn loci on chromosome 17, and that this is accompanied by duplication of the neighboring genomic region spanning 323 kb. However, none of the genes in this region were abrogated. These results suggest that the severe immunodeficiency seen in tgε26 mice is not due to gene disruption resulting from transgenic integration.