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K Kishihara

Publications and source records attributed to K Kishihara.

At least 73 records · Page 4Linked to original sources
PubMed1992

CD45RA and CD45RBhigh expression induced by thymic selection events.

CD45 is a protein tyrosine phosphatase involved in T and B cell signaling. While peripheral T cells switch CD45 isoforms upon activation, events leading to exon switching during T cell development in the thymus have not been determined. The expression of high molecular weight isoforms of CD45 was examined on thymocytes from nontransgenic and T cell receptor (TCR) transgenic mice. All thymocytes from nontransgenic mice were CD45RB+ as assessed by staining with MB23G2, an anti-CD45RB-specific monoclonal antibody. Interestingly, there was a small population (1-3%) of thymocytes that displayed a higher intensity of staining with MB23G2, CD45RBhigh. CD45RBhigh thymocytes were found in all subsets defined by CD4 and CD8 expression and were also present within the TCR-alpha/beta high population. To analyze whether or not CD45 expression correlated with thymic selection events, expression of CD45RBhigh and a second isoform, CD45RA, was examined on thymocytes from H-Y and 2C TCR transgenic mice and found to correlate with positive and negative selection events but did not occur in nonselecting backgrounds. CD45RA and CD45RBhigh upregulation was also not observed in transgenic mice backcrossed into CD8-deficient mice, a scenario in which there is no positive selection of transgene-expressing thymocytes. These data suggest that modulation of CD45 isoform expression may be involved in thymic selection events.

Animals↗
PubMed1992

Profound block in thymocyte development in mice lacking p56lck.

The protein Lck (p56lck) has a relative molecular mass of 56,000 and belongs to the Src family of tyrosine kinases. It is expressed exclusively in lymphoid cells, predominantly in thymocytes and peripheral T cells. Lck associates specifically with the cytoplasmic domains of both CD4 and CD8 T-cell surface glycoproteins and interacts with the beta-chain of the interleukin-2 receptor, which implicates Lck activity in signal transduction during thymocyte ontogeny and activation of mature T cells. Here we generate an lck null mutation by homologous recombination in embryonic stem cells to evaluate the role of p56lck in T-cell development and activation. Lck-deficient mice show a pronounced thymic atrophy, with a dramatic reduction in the double-positive (CD4+CD8+) thymocyte population. Mature, single-positive thymocytes are not detectable in these mice and there are only very few peripheral T cells. These results illustrate the crucial role of this T-cell-specific tyrosine kinase in the thymocyte development.

Animals↗
PubMed1992

Cloning and expression of an inducible lymphoid-specific, protein tyrosine phosphatase (HePTPase).

Tyrosine phosphorylation and dephosphorylating events have been shown to be central to the process of growth regulation and signal transduction. We report here, the identification of a new gene with a tyrosine phosphatase domain (EC 3.1.3.48) which is expressed exclusively in thymus and spleen. A cDNA of 2760 bp encodes a 339-amino acid, intracellular, single-domain tyrosine phosphatase. When expressed as a glutathionine-S-transferase fusion protein, efficient lysis of p-nitrophenyl phosphate is noted, indicating in vitro enzymatic activity of the cloned gene product. Normal mouse lymphocytes increase mRNA expression 10-15-fold upon stimulation with phytohemagglutinin, concanavalin A, lipopolysaccharide or anti-CD3 monoclonal antibody. This new hematopoietic tyrosine phosphatase, (HePTP), may play a role in the regulation of T and B lymphocyte development and signal transduction.

Amino Acid Sequence↗
PubMed1990

Clonal deletion of self-reactive T cells at the early stage of T cell development in thymus of radiation bone marrow chimeras.

Sequential appearance of T cell subpopulations occurs in the thymocytes of irradiated C3H/He mice (H-2k, Mls-1b2a, Thy-1.2) after transplantation with bone marrow cells of AKR/J mice (H-2k, Mls-1a2b, Thy-1.1) (AKR----C3H chimeras). The donor-derived thymocytes of AKR----C3H chimeras on day 14 after bone marrow transplantation (BMT) contained a large number of blastlike CD4+CD8+ cells which represent relatively immature thymocytes, whereas those on day 21 after BMT consisted of small sized CD4+,CD8+ cells which represent a great part in normal thymocytes. To define the developmental stage at which clonal deletion of self-reactive T cells occurs in adult thymus, we followed the fate of V beta 6- or V beta 11-bearing T cells in the donor-derived thymocytes at the early stage of AKR----C3H chimeras. Mature thymocytes expressing high intensity of V beta 6 or V beta 11, which are involved in recognition of Mls-1a or MHC I-E gene products, respectively, were deleted from the donor-derived thymocytes on day 21. Immature thymocytes expressing low intensity of V beta 6 in CD3low thymocyte fraction decreased in proportion, whereas those expressing low intensity of V beta 11 rather increased in proportion in the donor-derived thymocytes of AKR----C3H chimeras from day 14 to day 21 after BMT. These results suggest that the clonal deletion of V beta 6-positive cells occurs just at the stage of immature CD3lowCD4+CD8+ cells, whereas the clonal deletion of V beta 11-positive cells may begin at the transitional stage from CD3lowCD4+CD8+ cells to CD3high single positive cells. Timing of negative selection of thymocytes may vary in distinct T cells capable of recognizing different self-Ag.

Animals↗
PubMed1990

Deletion of self-reactive T cells in nude mice grafted with neonatal allogeneic thymus.

The cellular basis of tolerance induction has been investigated in BALB/c(H-2d, thy 1.2, M1s1b2a) nude mice grafted with thymus of neonatal AKR/J mice(H-2k,Thy1.1,M1s1a2b). The spleen cells from nude mice grafted with AKR/J thymus showed a significantly decreased level of primary cytotoxic T cell response when stimulated with AKR/J cells, although these cells lysed well target cells of a third party C57BL/6 when stimulated with C57BL/6 cells. Consistent with CTL responses, T cells bearing V beta 6, that is important for recognizing M1s1a-encoded products of the thymic phenotype, were virtually abolished in the spleen and lymph node cells of nude mice 8 wk after grafting with AKR/J thymus. However, a substantial number of V beta 6-bearing T cells were detected in the peripheral organs of nude mice 23 wk after grafting with AKR/J thymus and in those of nude mice grafted with AKR/J fetal thymus depleted of macrophages/dendritic cells by incubating with 2'-deoxyguanosine in vitro before grafting. On the other hand, T cells bearing V beta 3, which are selectively related to M1s2a-encoded products of the host phenotype, were expressed neither on the peripheral T cells of nude mice grafted with AKR/J thymus at any stage after grafting nor on those of nude mice grafted with 2'-deoxyguanosine-treated AKR/J thymus. These data suggested that both V beta 6 and V beta 3 T cells were eliminated in the thymus of nude mice grafted with AKR/J thymus, presumably on the basis of interaction with both of graft-derived persisting and host-derived hemopoietic cells in the thymus and that thymic epithelium appears to have little capacity to eliminate T cells reactive to minor lymphocyte stimulating-encoded products.

Animals↗
PubMed1990

Stimulation of all T cells bearing V beta 1, V beta 3, V beta 11 and V beta 12 by staphylococcal enterotoxin A.

To determine the molecular mechanisms of T cell stimulation by staphylococcal enterotoxin A (SEA), we examined the expression of T cell receptor (TcR) V beta on the T cells from four strains of mice stimulated in vitro with SEA, using flow cytometric analysis for the number of T cells bearing V beta 3, V beta 6, V beta 8, V beta 11 and RNA blotting analysis for the amount of transcripts of V beta 1, V beta 5 and V beta 12. The number of T cell blasts bearing V beta 1, V beta 3, V beta 1 or V beta 12 were increased in the T cell blasts proliferating in vitro in response to SEA in C57BL/6 mice. In AKR/J mice, which contain few V beta 11- or V beta 12-bearing T cells due to a tolerance to the self-MHC class II IE-antigens, T cells bearing V beta 1 or V beta 3 responded to SEA. SEA enriched only V beta 1-bearing T cells in BALB/c mice carrying Mls-2a which lack Mls-1a-reactive V beta 3-bearing T cells as well as V beta 11- and V beta 12-bearing T cells. In spite of the presence of V beta 1-bearing T cells, C3H/He T cells exhibited a very low responsiveness to SEA. T cell repertoires skewed by clonal deletion of self-reactive T cells may in part account for the different sensitivity to SEA among the different strains. A tolerance to SEA can be established in C57BL/6 mice which have been primed i.v. with SEA and treated i.p. with 200 mg/kg of cyclophosphamide 2 days later. All mature T cells bearing V beta 3 or V beta 11 were virtually abolished in the periphery of tolerant mice. These results suggest that most T cells reactive to SEA bear V beta 1, V beta 3, V beta 11 or V beta 12 and that clonal deletion of mature T cells reactive to SEA may account for the cellular mechanisms for cyclophosphamide-induced tolerance to SEA.

Animals↗
PubMed1990

Clonal anergy in self-reactive alpha/beta T cells is abrogated by heat-shock protein-reactive gamma/delta T cells in aged athymic nude mice.

Although T cells proliferate and differentiate primarily in the thymus, athymic nude mice contain an appreciable level of T cell receptor alpha/beta and gamma/delta T cells, suggesting the existence of the extrathymic pathway in the development of both T cells. Recent studies with nude mice indicate that clonal deletion of self-reactive T cells does not occur extrathymically. In the present study, we have investigated the responsiveness of self-reactive T cells differentiating along an extrathymic pathway in aged BALB/c (H-2d, Mls-1b2a, I-E+, 7-8 month old) nude mice. Consistent with recent reports, T cells bearing V beta 3 or V beta 11, which are important for recognizing proteins encoded by the Mls-2a or the I-E allele, respectively, are readily detected in age nude mice. The V beta 3- or V beta 11-bearing T cells, however, do not proliferate in response to staphylococcal enterotoxin A which specifically stimulates V beta 3- or V beta 11-bearing T cells. When exogenous recombinant interleukin 2 was added to the culture, the V beta 3-bearing T cells in aged nude mice significantly proliferated in response to staphylococcal enterotoxin A. Aged nude mice also contained a substantial level of gamma/delta T cells which account for 15.6% of all Thy-1.2+ cells. The gamma/delta T cells proliferated and produced a significant level of interleukin 2 in response to the 65-kDa mycobacterial heat-shock protein, which is highly homologous to its eukaryotic counterpart. These results suggest that unresponsiveness of self-reactive T cells may be reversed by T cells responding to stress proteins expressed by the invading microbes and/or the stressed autologous cells.

Aging↗
PubMed1990

Expression of T cell receptor V gamma 5 in the adult thymus of irradiated mice after transplantation with fetal liver cells.

T cell receptor (TcR) gamma/delta displays limited diversity and its diversity is distinct in different stages of ontogeny and in different anatomical sites. The V gamma 5 and V delta 1 gene products are preferentially expressed on the early fetal thymocytes and on Thy-1+ dendritic epidermal cells, whereas the V gamma 4 and V delta 5 gene products are abundantly expressed on the adult thymocytes. To elucidate whether the developmentally ordered appearance of thymocytes expressing TcR gamma/delta is dependent on the source of T cell precursors or is controlled by the thymic environment where T cells develop, we compared the expression of V gamma 5 on the early-appearing thymocytes between irradiated mice after transplantation with fetal liver (FL) cells and those after transplantation with bone marrow (BM) cells. Sequential appearance of thymocyte subpopulations was observed in the thymus of radiation FL chimeras similar to that seen in radiation BM chimeras. A substantial number of thymocytes bearing V gamma 5 appeared in the thymus at the early stage of radiation FL chimeras, whereas few, if any, of such V gamma 5-bearing thymocytes were detected in the thymus at any stage of radiation BM chimeras. These results suggested that the ordered expression of V gamma repertoire may depend on the origin of the T cell precursors but not on the thymic environment.

Animals↗
PubMed1990

Different expression of T-cell receptor beta-chain variable region genes in lymph nodes of lpr mice with different alleles of the major histocompatibility complex.

In order to search for a possible role of abnormally proliferating T cells in developing autoimmune disease in lpr mice, and to define the difference of the T cells among various lpr-congeneic mice with different clinicopathological findings, the T-cell receptor (TcR) V beta gene expression in the enlarged lymph nodes (LN) of C3H/HeJ-lpr/lpr (C3H-lpr), C57BL/6-lpr/lpr (B6-lpr) and MRL/Mp-lpr/lpr (MRL-lpr) mice was analysed. A RNA blot analysis using several V beta-specific probes showed that the V beta 3 gene, whose products are important for recognizing Mlsb/a, was used in B6-lpr and MRL-lpr with the Mlsb/b but not in C3H-lpr with the Mlsb/a. The V beta 5 gene, which is selectively related to I-E molecules, was predominantly used in B6-lpr(I-E-) but not in C3H-lpr(I-E+) nor MRL-lpr(I-E+). Similarly, the V12 gene was also expressed in B6-lpr but not in C3H-lpr. To compare in detail in V beta repertoire among lpr mice with different major histocompatibility complex (MHC) backgrounds, the V beta gene sequences in the cDNA libraries from LN cells of C3H-lpr were analysed, following the recent investigation of B6-lpr mice (Ohga et al., 1989). Eleven beta-chain cDNA out of 32 beta cDNA in B6-lpr and 24 beta-chain cDNA out of 55 beta cDNA in C3H-lpr were found to contain sequences with open reading frames that potentially encode functional TcR beta-chain. The frequencies of the messages in the cDNA libraries from these mice were consistent with the RNA blot analysis using V beta 3- and V beta 5-specific probes. It was notable that 36% of the functional beta-chain mRNA in B6-lpr and 50% of the beta mRNA in C3H-lpr expressed the V beta 8 gene family. When the TcR V beta gene expression was compared between the LN cells in C3H-lpr, B6-lpr and MRL-lpr, as reported by Singer et al. (1986), the usage of V beta genes other than the V beta 8 gene family in B6-lpr (H-2b) LN cells differed significantly from those in C3H-lpr (H-2k) and MRL-lpr (H-2k). The results presented here indicate that the usage of V beta genes is heavily influenced by the genetic background of lpr mice, similar to normal mice, but with preferential usage of the V beta 8 gene family as a common structural feature in lpr gene-induced cell populations.

Alleles↗
PubMed1989

The expression and sequences of T cell antigen receptor beta-chain genes in the thymus at an early stage after sublethal irradiation.

The sequential appearance of the thymocyte subpopulations and TCR gene messages occurred in the thymus of AKR mice (H-2k, Mlsa) from 7 to 14 days after sublethal irradiation. The thymocytes on day 7 after irradiation were composed of a large number of CD4+CD8+ blast-like cells and a relatively high proportion of CD4-CD8- cells (15 to 25%) but few CD3highCD4+CD8-/CD4-CD8+ cells. Approximately 22% of the CD4-CD8- cells were CD3high and -27% of the CD3highCD4-CD8- cells (-6% of whole CD4-CD8- cells) were F23.1+. The thymocytes on day 7 expressed a large amount of gamma- and delta-chain gene transcripts but reduced levels of alpha- and beta-chain gene transcripts. The V gene repertoire of 18 functional beta-chain cDNA derived from the thymocytes on day 7 was compared with those of 20 functional beta-chain cDNA derived from the thymocytes on day 14 which were composed of a large number of CD3lowCD4+CD8+ small-sized cells and a small number of CD3highCD4+CD8- cells. It is noteworthy that the distribution of V beta genes expressed in the thymocytes on day 7 was much the same as that in the thymocytes on day 14 but significantly different from that in normal BALB/c thymocytes as previously described. Interestingly, neither V beta 8.1 nor V beta 6 genes, which are important for recognition of the product of the Mlsa locus, was detected in these two cDNA libraries. These results suggest that clonal selection of TCR V beta repertoire, irrespective of positive or negative selection, appears to occur at the early stage of T cell differentiation, i.e., on the blast-like CD4+CD8+ thymocytes.

Amino Acid Sequence↗
PubMed1989

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.

Animals↗
PubMed1989

Expression and sequences of T cell receptor beta-chain variable genes in the enlarged lymph nodes of C57BL/6-lpr/lpr mice.

An autosomal recessive gene, lpr, is responsible for lymphoproliferation and autoimmunity of lpr-mice, in which background genes are also known to influence the development of autoimmune disease. To define the differences in abnormally proliferating T cells between C57BL/6-lpr/lpr and MRL/Mp-lpr/lpr mice, and to try and understand the influence of background in the differing expression of autoimmune disease in both strains, we analysed the sequences of T cell antigen receptor V beta genes expressed in the cells from the enlarged lymph nodes of C57BL/6-lpr/lpr mice. Eleven beta cDNAs out of the 38 C beta-specific cDNAs contained sequences with open reading frames from the beginning of the variable region to the expected termination codons at the end of the constant regions. Notably, 36% of the functional beta-chain mRNAs expressed V beta 8.3 genes, whereas V beta 8.1 and V beta 8.2 genes were not found. These results are consistent with a relatively lower frequency of the V beta 8.1 or V beta 8.2 expressing cells in the hypertrophic lymph nodes of C57BL/6-lpr/lpr mice, detected by KJ16-133 monoclonal antibody. Interestingly, other V beta genes expressed in these mice were completely distinct from those in MRL/Mp-lpr/lpr mice as described by Singer et al. (1986). The different distribution of V beta genes expressed in C57BL/6-lpr/lpr from that in MRL/Mp-lpr/lpr mice might be related to the differences in the genetic background and the expression of lpr gene-associated autoimmunity.

Amino Acid Sequence↗
PubMed1989

Abnormal rearrangements of T-cell receptor genes occur in long-term cultured bone marrow cells of lpr/lpr mice.

To search the abnormality in prethymic T-cell precursors in lpr/lpr(lpr) mice, rearrangement and expression of T-cell receptor (TcR) genes were investigated in long-term cultured bone marrow (LTBM) cells of lpr mice, in which the developmental steps of T-cell precursors may be better synchronized than those in bone marrow (BM) cells. Neither rearrangment nor expression of TCR gamma and delta genes were detected in the LTBM cells from +/+ control mice, whereas some gamma gene rearrangements were detected in those derived from lpr mice, irrespective of the genetic background. When BM cells or LTBM cells from lpr mice were transplanted into supralethally irradiated +/+ mice the lpr-derived BM cells appeared earlier in the thymus of the recipient mice than +/+-derived BM cells and the recipients suffered from lethal wasting syndrome. In addition, the lpr-derived BM cells showed higher activity in colony-forming unit spleen (CFUs) than the +/+-derived BM cells. These results suggest that the T-cell progenitors in the BM of lpr mice may be different not only in quantity but also in quality from those of +/+ mice.

Animals↗
PubMed1988

Expression of T cell antigen receptor genes in the thymus of irradiated mice after bone marrow transplantation.

Sequential appearance of the expression of T cell antigen receptor genes was investigated in the thymus of irradiated mice at the early stage after transplantation of Thy-1 congeneic H-2 compatible allogeneic bone marrow cells. The first cells to repopulate the thymus on day 7 after bone marrow transplantation were intrathymic radioresistant T cell precursors, which expanded mainly to CD4+CD8+ host-type thymocytes by day 14. A high level of gamma gene expression but a much reduced level of alpha and beta gene expression were detected in the host-type thymocytes on day 7. During regeneration of these cells, gamma-chain messages fell to low level and alpha and beta mRNA levels increased. The thymus of the recipients began to be repopulated by donor-derived T cells about 2 wk after bone marrow transplantation and was almost completely replaced by the third week. An ordered expression of gamma then beta and alpha-chain gene transcript was also observed in the donor-type thymocytes at the early stage after bone marrow transplantation. The use of thymocytes at early stage in whole-body irradiated bone marrow chimera provides a pertinent source for investigating the molecular mechanism of T cell differentiation in adult thymus.

Animals↗
PubMed1988

"Radioresistant" intrathymic T cell precursors express T cell receptor C gamma 4- and C delta-specific gene messages.

We have studied the expression and sequences of T cell receptor gamma and delta chain gene messages in intrathymic T cell precursors of mice irradiated with 600 rads. On day 7 after irradiation a high level of expression of gamma and delta chain messages was detected in thymocytes which were composed of a relatively high proportion of CD3+CD4-CD8- thymocytes. During further development of the precursors from day 7 to day 14 after irradiation, gamma and delta chain messages fell to low levels and alpha and beta mRNA levels increased. Nucleotide sequence analysis of 14 gamma and 10 delta chain complementary DNA (cDNA) in the thymocytes on day 7 revealed that there were 7 functional gamma chain transcripts composed of V gamma 2-J gamma 2-C gamma 2 or V gamma 1-J gamma 4-C gamma 4 gene segments, and only 1 functional delta chain transcript composed of the V delta M23-D delta 1-D delta 2-J delta 1-C delta gene segments. The repertoire of gamma chain and delta chain genes used in "radioresistant" intrathymic T cell precursors of adult mice appears to be limited.

Amino Acid Sequence↗
PubMed1988

Functional T cell receptor delta chain gene messages in athymic nude mice.

The rearrangement and expression of T cell antigen receptor (TcR) delta chain genes were investigated in congenitally athymic nude mice. The lymphoid cells derived from nude mice showed evidence of rearranged delta chain genes and a relatively high level of delta chain gene messages. Nucleotide sequence analysis revealed that nude mice contained an in-frame delta chain transcript, composed of V delta 5-D delta 1-D delta 2-J delta 1-C delta genes in the spleen. We have previously described functionally rearranged TcR gamma chain genes in the spleen of nude mice. Taken together, T cell precursors appear to proliferate and differentiate along the extrathymic pathway into TcR gamma/delta-bearing T cells in nude mice.

Amino Acid Sequence↗
PubMed1988

Age-associated increase in the expression of T cell antigen receptor gamma chain genes in mice.

T cell function generally declines with age. To determine the underlying cause of the age-related decline, we compared the expression levels of T cell antigen receptor genes encoding gamma, beta and alpha chains in the lymphoid tissues of young (8 wk old) vs. aged (40 wk old) mice. An age-associated increase in gamma chain gene transcripts was evident in the thymocytes, spleen cells and mesenteric lymph node cells. Aged mice had a relatively high proportion of CD3+CD4-CD8- cells but a reduced level of CD3+CD4-CD8+ cells in the lymphoid tissues, as compared with young counterparts. The allo-reactivity in the lymphoid cells, as assessed by mixed lymphocyte reaction (MLR) responses, decreased with advancing age. On the contrary, increased levels of syngeneic MLR and spontaneous cytolytic activity were noted in the lymphoid cells of aged mice, as compared with findings in their young counterparts. A remarkable increase in the number of CD3+CD4-CD8- cells and level of the gamma chain gene messages was also detected in the responder cells of day 4 syngeneic MLR culture from aged mice. An increase in the number of T cells bearing gamma chain may be related to the alterations in immunological functions in aged mice.

Aging↗