Radiation-induced-thymic lymphoma occurs in young,but not in old mice

Young mice exposed to fractionated whole-body irradiation develop thymic lymphoma. By using young and old mice, we examined the effect of age on the occurrence of radiation-induced thymic lymphoma in mice. In the first experiment, young and old mice were grafted with newborn thymus under kidney capsule and then treated with fractionated whole-body irradiation (FWI). In the second and third experiments, four combinations of bone marrow chimeras were constructed by transplanting bone marrow cells from young and old mice into young and old mice. Then these chimera mice were grafted with newborn thymus and treated with fractionated whole-body irradiation. The results in the present study indicate that the incidence of thymic lymphoma is influenced by age factors of thymic microenvironment, bone marrow, and host environment. If they are all young, the incidence of thymic lymphoma is high. If one of these is old, the incidence definitely decreases. Thymic lymphoma never occurred in old thymic environment even in the presence of young thymocytes. In conclusion, age advantage is present in the induction of thymic lymphoma after the treatment with FWI and the incidence definitely decreases in the presence of old factors.     

Immunohistology of T cell differentiation in the thymus of H-Y-specific T cell receptor alpha/beta transgenic mice

We examined the immunohistological aspects of the H-Y specific T cell receptor (TcR) alpha/beta transgene expression in the thymus of male and female transgenic (Tg) mice. Virtually all thymocytes expressed the beta transgene in both the male and female thymus. Expression of accessory molecules (co-receptors) in Tg mice deviated from control mice. In the male Tg thymus, CD8 expression was either low or absent on both cortical and medullary thymocytes. In contrast, in the thymus of female mice, CD8+ cells were found both in the cortex and in the medulla. The majority of medullary thymocytes was bright CD8+. This is in clear contrast to the CD8 distribution in control B6 mice, where only a few percent of medullary cells are CD8+. Similarly, the proportion of cells expressing CD4 antigens was reduced in the cortex and medulla of the thymus from male Tg mice, as compared to the thymus of female Tg mice and B6 control mice. Comparative analysis of the stromal cell types of the thymic microenvironments in the three groups of mice revealed that the cortical thymic microenvironment of male Tg mice differed, compared to that of female Tg mice. In particular, the deep cortex showed a closely packed meshwork of epithelial reticular cells. Moreover, H-2Db molecules (which are the restricting elements for the Tg TcR alpha/beta) were abnormally expressed in the thymic cortex of male mice. The cortical microenvironment in female mice, on the other hand, appeared normal. Together, the data indicate that TcR alpha/beta transgene expression in male mice leads to an aberrant co-receptor expression in both cortical and medullary lymphoid cells as well as an abnormal composition of the cortical microenvironment. Both phenomena may be the consequence of "negative selection" of developing H-Y-specific T cells, as it occurs only in the male Tg thymus. The absence of the H-Y antigen, but presence of the restricting element H-2Db in the thymic cortex of female mice, leads to accumulation of CD8+ in the medulla, a phenomenon interpreted as "positive selection".     

T cell subset-specific expression of antigen receptor beta chains in alpha chain-transgenic mice.

A large number of V beta 8 gene-encoded cDNA were analyzed from peripheral CD4+ and CD8+ T cell subsets of T cell receptor (TcR) alpha chain-transgenic mice. This analysis demonstrates that a limited repertoire of TcR beta chains are co-expressed with the transgenic alpha chain. Most importantly, certain V beta 8-J beta combinations were found exclusively in one of the subsets and, in some cases, subset-specific differences were localized to the VDJ junctional region of the beta chain genes. In contrast, CD4-CD8- transgenic T cells, as well as CD4+ and CD8+ T cells from normal littermate controls, were found to express diverse beta chain repertoires. The present study suggests that beta chains with distinct structural characteristics are expressed in the CD4+ and CD8+ subsets, respectively. Moreover, the data suggest that the same structural constraints do not apply to the population of CD4-CD8- transgenic T cells.     

Rearrangements of T-cell antigen receptor gamma and delta chain genes are detected in the long-term cultured bone marrow cells of athymic nude mice but not in those of euthymic mice.

We have previously shown that extrathymic rearrangements of T-cell receptor (TcR) gamma and delta chain genes occur in the peripheral lymphoid tissues of athymic nude mice. To further determine where the TcR gene rearrangements occur in nude mice, we investigated the rearrangement and expression of the TcR genes in the long-term cultured bone marrow (LTBM) cells which were homogenous in developments without mature T cells as assessed by FACS analysis. The LTBM derived from euthymic mice contained TcR gamma and delta chain genes in germline configuration, while gene rearrangements of both locus were detected in the LTBM cells from nude mice. These results suggested that gamma and delta gene rearrangements do occur in the bone marrow cells of nude mice and that the T-cell precursors in bone marrow may be increased in frequency in such animals.     

Inhibition of abnormal T cell development and autoimmunity in gld mice by transgenic T cell receptor beta chain.

Mice homozygous for the gld (generalized lymphoproliferative disease) mutation developed systemic autoimmune disease and severe lymphadenopathy due to an age-related accumulation in the peripheral lymphoid organs of polyclonal T cells bearing a unique phenotype (CD4-CD8-TCR alpha beta+B220+). These T cells overexpress T cell receptor (TcR) alpha beta chain RNA, proto-oncogenes c-myb and fyn, and proliferate poorly in response to TcR-mediated stimulation. The origin of these T cells is poorly understood. To study the influence of a functionally rearranged TcR beta chain on the T cell developmental abnormality of the gld mutation and autoimmunity, we have backcrossed TcR V beta 8.1-transgenic mice to C3H-gld/gld to homozygosity (transgenic gld mice). In transgenic gld mice, lymphadenopathy was markedly inhibited and the accumulation of CD4-CD8- T cells did not occur, although the remaining T cells overexpressed c-myb and proliferated poorly in response to TcR occupancy. These features indicate that the pattern of proto-oncogene expression and abnormal function persist in phenotypically normal T cells in transgenic gld mice, and that these characteristics can be dissociated from the accumulation of CD4-CD8- T cells. The hypergammaglobulinemia and anti-double-stranded DNA (anti-dsDNA) antibody production was partially improved in transgenic gld mice, supporting the critical role of T cells in abnormal B cell activation described in autoimmunity-prone mice. To investigate further the mechanisms underlying the inhibition of CD4-CD8- T cell accumulation in transgenic gld mice, the fetal ontogeny of T cells in transgenic mice was compared with that of non-transgenic mice. In transgenic thymus, development of TcR alpha beta+ cells was accelerated as detected by earlier expression of CD4, CD8 and TcR in fetal thymus. In contrast, the number of TcR gamma delta+ cells was reduced. We suggest that altered T cell development in transgenic mice directly or indirectly inhibits the accumulation of abnormal T cells in gld mice.     

Frequent deletion of the transgene in T cell receptor {beta} chain transgenic mice

TCR V_ß8.1 transgenic mice were generated using a genomicTCR V_ß gene construct under the control of its promoterand enhancer. Among three lines of transgenic mice, one lineexpressed the transgenic TCR on only 70% of peripheral T cells,while the other two lines expressed it on almost all matureT cells. T cells which lacked expression of the transgenic TCRß chain expressed endogenous TCR ß chains.The molecular basis underlying the lack of transgene expressionin T cells of this line of transgenic mice was investigated.The transgenic TCR^– cells were isolated by two methods.First, Thy-1⁺ V_ß8.1/8.2^– cells were purifiedfrom peripheral T cells using cell sorting. Second, transgenicTCR^– T cell clones were established. In both cases, Southernblotting indicated that V_ß8.1^– T cells had deletedthe transgenic TCR gene. Thus, deletion of the transgenic TCRcan occur in a high proportion of T cells, which allows rearrangementand expression of endogenous TCR ß chains.     

alpha beta T cell receptor ligand-specific oligomerization revisited.

The mechanism of T cell receptor signaling is unclear. Included among models for TCR signaling is ligand-induced oligomerization in a fashion analogous to other cell surface receptors. Published kinetic, saturation binding, and light scattering experiments have been interpreted to suggest a propensity for soluble alpha beta TCR/peptide/MHC ectodomain complexes to oligomerize. Upon performing these experiments with soluble ectodomains of human class I and class II restricted alpha beta TCRs, we find no evidence for dimerization or oligomerization of complexes. Apparently, oligomerization in solution to a detectable extent is not a general property of soluble alpha beta TCRs or their complexes with ligand. Our results suggest that membrane-anchored, fully assembled TCRs should be studied to determine the role oligomerization plays in T cell signaling.     

Sequences and repertoire of the human T cell receptor alpha and beta chain variable region genes in thymocytes

To compare and contrast the human T cell antigen receptor (TcR) alpha and beta chain messages found in human thymocytes to those previously isolated from human peripheral blood T lymphocytes and other nonthymic sources, 13 TcR alpha and 13 TcR beta cDNA were isolated from a human thymocyte library and the nucleotide sequences were determined. The data indicate that, as was found in the peripheral T lymphocytes, the majority of the TcR alpha and TcR beta chain thymocyte cDNA were derived from potentially functional messages. Although the thymocyte-derived TcR cDNA do not contain any unique structural features when compared to TcR cDNA from mature T lymphocytes, 4 new J alpha segments, 17 new V-gene segments (9 V alpha; 8 V beta) and 7 additional V-gene families (4 V alpha and 3 V beta) and sequences had been identified. The exon C beta O, found in many murine thymocyte TcR beta messages, was not found in over 75 human beta chain messages. Based on these new data, a revised estimate of human TcR V alpha, J alpha and V beta repertoires is calculated. The most significant change has been the increase in the estimated number of human TcR V beta-gene segments to a total of about 100 distributed among about 18 families. The V alpha families are now revised upward to 16, with a total number of V alpha segments of 50. The estimate of the J alpha segments in humans remains between 50-100.     

T cell receptor beta chain gene rearrangements and V beta gene usage in horse cytochrome c-specific T cell hybridomas

Comparison of T cell receptor alpha and beta-chain genes in murine major histocompatibility complex (MHC) class I and class II-restricted T cell clones and hybridomas recognizing different antigens indicates that no simple correlation exists between the observed antigen/MHC specificity and the expression of certain alpha and beta-chain heterodimers. We have attempted to establish a possible correlation by analyzing T cell receptor beta chain gene rearrangements and V beta gene usage in five T cell hybridomas with identical antigen/MHC specificity and another hybridoma recognizing a different antigenic determinant in association with the same restriction molecule. We report here that in each of the five clones a uniquely rearranged beta chain gene is expressed in combination with at least two different V beta gene segments. The presence of the differently rearranged T cell receptor beta chain genes correlated with the finding of distinct fine specificity pattern of antigen recognition in each of the hybridomas. Interestingly, two hybridomas specific for different epitopes showed identical beta chain D-J rearrangements indicating that the differences might be encoded by the alpha chain gene or/and the V beta gene element.     

T cell receptor beta chain usage in myelin basic protein-specific rat T lymphocytes

Myelin basic protein (MBP)-specific T cell lines and clones have been established from rats of the major histocompatibility complex (MHC)-compatible Lewis and BS strains. All lines and clones are MHC class II restricted and share the CD4+ phenotype. The cells proliferate specifically in response to either a peptide representing amino acids #68-88 of guinea pig MBP, to residues #47-67 or to an unidentified myelin antigen which is distinct from MBP. All lines and clones specific for MBP express the same T cell receptor (TcR) variable (V) beta chain element, which is homologous to the mouse V beta 8.2 gene segment. Three lines/clones with the same antigen fine specificity have identical V beta D beta J beta junctions on the protein level, a region which represents part of the potential antigen-binding portion of the TcR; two of the lines express members of the V alpha 2 family. These results suggest biased usage of TcR V beta elements in rat T cells specific for MBP. Our findings broaden the basis for a rational therapeutic strategy to specifically intervene in the rodent model system of experimental allergic encephalomyelitis.     

Polymorphism in human T cell receptor alpha chain variable region genes

Polymorphism of human T cell antigen receptor (TCR)alpha genes was detected by restriction fragment length polymorphism (RFLP). Individual TCR alpha gene segments showed limited polymorphism in that few restriction enzymes revealed polymorphism in genomic DNA samples and when RFLP were detected only two or three allelic forms were observed. A rabbit TCR alpha cDNA clone (VJR5) detected polymorphism in human DNA samples digested with PvuII or MspI. In order to characterize the human V alpha genes detected by the rabbit probe, genomic clones hybridizing with the VJR5 probe were isolated and characterized. A probe derived from a human genomic clone (HUTAVR5) hybridized with some but not all fragments detected by the rabbit VJR5 probe. The data suggest that the rabbit probe hybridized with two distinct human TCR alpha V genes and that polymorphism of each gene was detectable by only one restriction enzyme. In contrast to the limited polymorphism of TCR alpha genes detected by individual markers, extensive heterogeneity of TCR alpha genes was observed in the combination of markers present in haplotypes. Six RFLP were used as TCR alpha markers to define haplotypes in 20 parents and 53 offspring from 10 families and 17 different combinations of markers were observed. The observation that TCR alpha haplotypes include numerous combinations of markers that individually show limited polymorphism suggests that recombination may occur frequently within the TCR alpha gene complex.     

Generation of monoclonal antibodies against a human T cell receptor beta chain expressed in transgenic mice.

The generation of a panel of monoclonal antibodies specific for different variable (V) regions of human T cell receptors will be of great importance in the study of T cell-mediated diseases. However, relatively few such reagents exist, due in part to the poor immunogenicity of TcRs on the surface of human T cells. We have employed a strategy in which T cells from a transgenic mouse line expressing a human V beta 3 C beta 1 TcR were used to immunise syngeneic conventional mice to generate two monoclonal antibodies specific for human T cell receptors. Binding of antibody JOVI.3, which stained approximately 5% of human peripheral blood CD3 positive T cells, correlated with the expression of the human TcR V beta 3 gene segment. Antibody JOVI.1 recognised a determinant on the majority of TcRs, staining 50-75% of peripheral blood T cells and T cell lines expressing different V beta regions. Some TcRs, however, failed to react with this antibody. Both antibodies immunoprecipitated detergent-solubilised TcR molecules and were capable of inducing proliferation of peripheral blood T cells.     

Expression and role of interleukin-2 receptor beta chain on CD4-CD8- T cell receptor alpha beta+ cells [corrected]

Using anti-murine interleukin-2 receptor beta chain (IL-2R beta) monoclonal antibody (mAb), we have examined the expression of IL-2R beta on murine thymocyte subpopulations. We found that it was constitutively expressed on 1%-4% of thymocytes in an almost mutually exclusive fashion with IL-2R alpha. The expression of IL-2R beta is developmentally regulated. While it is expressed mainly on T cell receptor gamma delta+ (TcR gamma delta+) cells during fetal age, the major subpopulation expressing IL-2R beta in adult mouse shifts to CD4-CD8-TcR alpha beta+ thymocytes. A considerable portion of CD4-CD8- TcR alpha beta+ cells in other organs, including spleen, bone marrow and liver, was also found to express IL-2R beta. In fetal thymus organ culture, the above thymocyte subset was induced to expand in response to exogeneous IL-2, and the expansion was inhibited by addition of anti-IL-2R beta mAb, suggesting that IL-2R beta is functional in this subpopulation. However, in vivo blockade of the IL-2/IL-2R pathway with the mAb did not exert any effects on the appearance of CD4-CD8- TcR alpha beta+ cells both in the thymus and the periphery. This indicates that the development of CD4-CD8- TcR alpha beta+ cells is not solely controlled by IL-2 but also by other complex elements.     

Intra- and inter-allelic ordering of T cell receptor beta chain gene assembly

Allelic exclusion at the TCRbeta locus mandates that gene assembly be regulated in a manner that permits feedback inhibition of further complete TCRbeta rearrangements upon pre-TCR expression. Here we show that assembly of TCRbeta chain genes from Vbeta, Dbeta and Jbeta gene segments is intra-allelically ordered, proceeding primarily through DJbeta, and not VDbeta, intermediates. This ensures that Vbeta to DJbeta rearrangement, which can be feedback inhibited, is the final step in the assembly process. A newly assembled VDJbeta rearrangement must be tested to determine if it is in-frame before Vbeta to DJbeta rearrangement is permitted on the alternate allele. This inter-allelic ordering may occur through a general inefficiency of Vbeta to DJbeta rearrangement and/or through static differences in accessibility of the two TCRbeta alleles. However, we find that within the regulatory context of allelic exclusion, Vbeta to DJbeta rearrangement proceeds to completion on both alleles. Furthermore, all possible VDJbeta rearrangements are not completed on one allele before Vbeta to DJbeta rearrangement is initiated on the alternate allele. Together, these data support a dynamic model of inter-allelic accessibility that permits the ordered and efficient assembly of complete variable region genes on both TCRbeta alleles during T cell development.     

MHC-restricted T cell receptor signaling is required for alpha beta TCR replacement of the pre T cell receptor

A developmental block is imposed on CD25(+)CD44(-) thymocytes at the beta-selection checkpoint in the absence of the pre T cell receptor (preTCR) alpha-chain, pTalpha. Early surface expression of a transgenic alphabeta TCR has been shown to partially circumvent this block, such that thymocytes progress to the CD4(+)CD8(+) double-positive stage. We wanted to analyze whether a restricting MHC element is required for alphabeta TCR-expressing double-negative (DN) thymocytes to overcome the developmental block in pTalpha-deficient animals. We used the HY-I knock-in model that endows thymocytes with alphabeta TCR expression in the DN compartment but has the advantage of physiological expression levels, in contrast to conventional TCR transgenes. On a pTalpha-deficient background, this HY-I TCR transgene 'rescued' CD25(+)CD44(-) thymocytes from apoptosis and enabled progression to later differentiation stages. On a non-selecting MHC background, however, pTalpha-deficient HY-I mice presented a pronounced reduction in numbers of splenocytes and thymocytes when compared to animals of selecting MHC genotype, showing that MHC restriction is necessary to drive HY-TCR-mediated rescue of pTalpha-deficient thymocytes.     

Involvement of both T cell receptor V alpha and V beta variable region domains and alpha chain junctional region in viral antigen recognition.

We have studied the lymphocytic choriomeningitis virus (LCMV)-specific cytotoxic T cell response in transgenic mice expressing either the T cell receptor (TcR) alpha (V alpha 2/J alpha TA31) or the corresponding TcR beta (V beta 8.1/D beta/J beta 2.4) chain originally isolated from the LCMV glycoprotein specific (residues 32-42), H-2Db-restricted T cell clone P14. The expression of single transgenic TcR chains did not influence the corresponding endogenous TcR V gene usage in unstimulated T cells indicating that one particular TcR alpha or beta chain can randomly pair with different V beta or V alpha chains without any obvious bias. However, upon infection with LCMV, reactive cytotoxic T lymphocytes (CTL) from P14 beta-transgenic mice were predominantly V alpha 2+ whereas CTL from P14 alpha-transgenic mice preferentially expressed V beta 8.1 and unexpectedly also V beta 8.3 (but not V beta 8.2). Correspondingly, the LCMV-specific CTL response in both alpha and beta TcR-transgenic mice was strongly biased to the original P14 T cell epitope (LCMV glycoprotein residues 32-42). Sequence analysis of a large panel of LCMV-reactive "half-transgenic" TcR from P14 single receptor chain-transgenic mice revealed a highly conserved VJ alpha and a more diverse VDJ beta junctional region. This report demonstrates that the antigen specificity of the studied TcR depends on the specific combination of both TcR alpha and beta chains which implies that amino acids located in the TcR V alpha and V beta segments as well as in the junctional region are involved in binding of the viral antigenic fragment.