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K Kishihara

Publications and source records attributed to K Kishihara.

At least 55 records · Page 3Linked to original sources
PubMed1996

T cell receptor V alpha and V beta gene usage by tumour-infiltrating lymphocytes in oral squamous cell carcinoma.

Oral squamous cell carcinomas (SCC) are often infiltrated by a large number of T lymphocytes. To clarify the nature of the tumour-infiltrating lymphocytes (TIL), we examined T cell receptor (TCR) V alpha and V beta gene usage by TIL and peripheral blood mononuclear cells (PBMC) obtained from 10 patients with oral SCC. We obtained RNA from TIL and PBMC, synthesized complementary DNA, and used the polymerase chain reaction (PCR) method with a panel of primers specific for the V gene segment subfamily (V alpha 1-18/V beta 1-20). We thus found that TIL showed more restricted usage of V beta gene families in contrast to PBMC of the same patients while two unique V beta gene (V beta 6 and V beta 5.2) segment transcripts were overexpressed in the TIL of more than half of the patients. On the other hand, no major difference was observed in the V alpha gene usage between the TIL and PBMC of most patients. To characterize these T cell subpopulations with unique V beta gene segment transcripts further, we sequenced the complementarity-determining region 3 in V beta 6-C beta and V beta 5.2-C beta PCR products derived from TIL and PBMC of two selected patients in each case. Although no usage of the conserved amino acid sequence by TIL was detected, the frequent use of V beta 6/J beta 1.1 in one patient and the V beta 6/J beta 2.7 gene segments in another patient was observed. Regarding the V beta 5.2 transcripts, obtained from the other two patients, no preferential usage of specific J beta gene segments by TIL was observed. These results suggest that the unique T cell populations are amplified in patients with oral SCC, possibly as a consequence of an in situ immune reaction.

Adult↗
PubMed1996

CD4 expression is important but not essential for infection with exogenous mouse mammary tumor virus.

We studied local events in the popliteal lymph nodes of CD4-deficient mice following foot pad injection with an MMTV strain which carries the gene for a V beta 14-specific superantigen. Injection of the V beta 14-specific MMTV induced vigorous expansion of V beta 14+ CD4+ T cells and B cells in their lymph nodes of CD4+/- heterozygous control mice. On the other hand, CD4-/- mice injected with the MMTV showed a proliferation of V beta 14+ T cells among the population of TCR alpha beta + CD4-CD8- T cells, although to a lesser extent. This phenomenon was not accompanied by vigorous B cell expansion. A PCR assay revelated that the MMTV definitely infected the lymph nodes cells of the CD4-/- mouse. However, the infectivity of the MMTV in CD4-/- mice was approximately 20 times lower than that in CD4+/- mice. These findings indicate that, in MMTV infection of CD4-deficient mice, the superantigen-reactive T cells among the population of TCR alpha beta +CD4-CD8- T cells substitute for the superantigen-reactive CD4- T cells of normal mice, and that the absence of CD4 molecules decreased the infectivity of MMTV because of insufficient expansion of the superantigen-reactive T cells.

Animals↗
PubMed1996

Evidence for down-regulation of highly expressed TCR by CD4 and CD45 on non-selected CD4+CD8+ thymocytes.

Immature CD4+CD8+ double-positive (DP) thymocytes are positively selected for further development if they express TCR reacting with thymic ligands of low affinity. However, the majority of DP thymocytes express low TCR levels. This low level of TCR may be insufficient to recognize thymic ligands. To understand the basis for the low expression of TCR on DP thymocytes, we determined the density of TCR expression at various stages of their development using TCR transgenic (TCR-Tg) mice. We found that TCR expression was high in the thymocytes that had recently transited into the DP stage but then gradually decreased on DP cells if they were not selected by TCR interaction with MHC molecules. However, such TCR suppression was not observed in positively selected DP cells and in the non-selected DP cells obtained from CD45 deficient mice or from mice receiving anti-CD4 mAb. These findings suggest that the once highly expressed TCR at the DP stage is suppressed by CD45 and/or CD4 on non-selected thymocytes. Furthermore, TCR suppression is prevented by TCR-mediated signals. The maintenance of high TCR levels on positively selected DP thymocytes may facilitate their selection.

Animals↗
PubMed1995

Generation of T cells with differential responses to alloantigens in CD45 exon 6-deficient mice.

We characterized the CD45+ population in the CD45 exon 6-deficient mice and addressed a question as to whether the T cells keep responsive properties to alloantigens. The peripheral CD45+ T cells from the untreated knockout mice showed a decreased expression level of CD45 but increased expression levels of LFA-1 alpha and CD44, indicating that the T cells are possibly in an activation state. The CD45 exon 6-deficient mice could readily reject fully allogeneic skin grafts but not non-MHC-disparate skin grafts. The skin allograft-rejected CD45 exon 6-deficient mice showed an increased number of peripheral CD8+ T cells expressing an isoform of CD45RB (CD45 zeta). In vitro assays including mixed lymphocyte reaction, CTL, and cytokine productions were consistent to the in vivo results. Furthermore, the effector cells shown in these assays were CD45+CD8+ T cells expressing a CD45RB isoform, CD45 zeta. Moreover, the CD45+ T cells had an abnormal regulation of CD45 expression level, as they could not increase their CD45 expression level after in vitro allogeneic stimulation. These results indicate that abnormal mature T cells in the periphery of CD45 exon 6-deficient mice cannot respond to nominal Ags presented by MHC but can react to allo-MHC. The reason that such functionally impaired T cells can be generated in the mice is still unknown but these results imply that there is differential requirement of CD45 function for alloreactive T cells in a thymic selection(s) and in effector functions.

Animals↗
PubMed1995

Impaired development of V gamma 3 dendritic epidermal T cells in p56lck protein tyrosine kinase-deficient and CD45 protein tyrosine phosphatase-deficient mice.

To determine whether p56lck protein tyrosine kinase and CD45 protein tyrosine phosphatase are involved in the signal transduction during intrathymic differentiation of gamma/delta T cells, we have examined the development of T cells expressing V gamma 3 T cell receptor (TCR) in mice deficient for either protein. The skin from both mice contained significantly reduced numbers of dendritic epidermal T cells expressing decreased levels of V gamma 3 TCR at the cell surface. Analysis of the fetal thymus from these mice suggested that maturation of V gamma 3 thymocytes was blocked at the immature stage that was characterized by the low level of V gamma 3 TCR and the high level of heat stable antigen. These results imply that both p56lck and CD45 are involved in the signal transduction during maturation of V gamma 3 T cells in the fetal thymus.

Animals↗
PubMed1995

T lymphocyte development in p56lck deficient mice: allelic exclusion of the TcR beta locus is incomplete but thymocyte development is not restored by TcR beta or TcR alpha beta transgenes.

The protein tyrosine kinase, p56lck, is involved in signal transduction in mature T cells and in the molecular events controlling early thymocyte differentiation. Thymuses of mice deficient for p56lck expression (p56lck-/-) consist of immature CD4-CD8- double-negative (DN) and CD4+CD8+ double-positive (DP) thymocytes and are severely reduced in total cell number. In this report we have studied DN thymocytes from p56lck-/- mice and found an increase in the proportion of the CD44-CD25+ subset, suggesting that transit through this stage, which is known to require T cell receptor (TcR) beta expression, may be delayed in the absence of p56lck expression. In addition, the expression of a transgenic TcR beta chain or TcR alpha beta pair did not restore thymic development in p56lck-/- mice. However, in contrast to mice expressing a dominant negative isoform of p56lck in which DP thymocytes do not develop, DP thymocytes still develop in nontransgenic and TcR transgenic p56lck-/- mice. These results demonstrate that expansion of the DP subset is impaired in p56lck-/- mice. In contrast, allelic exclusion is not severely compromised. Although there was an increase in the number of peripheral T cells expressing more than one V beta chain in TcR transgenic p56lck-/- mice, we found that inhibition of endogenous TcR beta gene rearrangement was almost complete in thymocytes of V beta transgenic p56lck-/- mice and we could not detect any peripheral T cells that expressed more than one V beta chain in non-transgenic p56lck-/- mice.

Alleles↗
PubMed1995

Differential requirements of CD45 for lymphocyte development and function.

CD45 exon 6-deficient mice show a profound block of thymocyte development at a transitional differentiation step from immature CD4+ CD8+ to mature single-positive cells. Only a few T lymphocytes are observed in the periphery of such animals, but B cell development is not affected. We investigated whether mature B lymphocytes in CD45 exon 6-deficient mice have normal functions in the absence of CD45 expression and show here that CD45-defective B lymphocytes from CD45 exon 6-deficient mice have intact B lymphocyte functions, including T-dependent and T-independent antigen-specific antibody production and class switching in vivo if normal CD4+ T lymphocytes were adoptively transferred into the mutant mice. From these results, we conclude that CD45 expression on B lymphocytes is not essential for B cell responses following antigen stimulation in vivo. However, CD4+ T helper function was severely diminished in CD45 exon 6-deficient mice, suggesting that the requirements of CD45 molecules in antigen-specific response and development are different between T and B lymphocytes.

Animals↗
PubMed1995

Intestinal T cells in CD8 alpha knockout mice and T cell receptor transgenic mice.

Intraepithelial lymphocytes (IEL) refer to the T cells located at the epithelium of the intestines. Unlike the T cells in other peripheral lymphoid organs, the majority of IEL express the CD8 cell surface protein. To study the role of CD8 in the ontogeny and the function of IEL, phenotypic analysis of IEL from CD8 alpha knockout mice and normal mice was performed. The CD8 alpha gene in CD8 alpha knockout mice was disrupted by homologous recombination. These mice are defective in thymic maturation of cytotoxic T cells. In normal mice, alpha beta T cells that were CD8 alpha alpha+ or CD4+ CD8 alpha alpha+, and gamma delta T cells that were CD8 alpha alpha+, were the distinct populations found only in IEL. In CD8 alpha knockout mice, the population size of IEL remained normal, but the majority of IEL were CD4- CD8- T cells expressing alpha beta or gamma delta T cell receptors. IEL from the H-Y transgenic mice2, which express the male H-Y antigen specific T cell receptor in a normal and in a CD8 alpha-null background, were also studied. In contrast to thymic derived T cells, CD8 alpha alpha+ IEL with the autoreactive transgenic T cell receptor were not deleted, but clonally expanded in the male transgenic mice. Interestingly, no pathological symptoms were observed in the intestines of these mice. In the absence of CD8 alpha expression, the H-Y specific autoreactive IEL did not accumulate in the intestines. The results suggest that CD8 alpha alpha+ IEL are derived extra-thymically and their responses towards antigens require the CD8 accessory molecule.

Animals↗
PubMed1994

Characteristics of fetal thymus-derived T cell receptor gamma delta intestinal intraepithelial lymphocytes.

We have previously demonstrated that grafting of CBF1 (H-2b/d) fetal thymus (FTG) under the kidney capsule of congenitally athymic nude mice of BALB/c background (H-2d) generates a substantial number of T cell receptor (TCR) gamma delta intestinal intraepithelial lymphocytes (IEL) that were of FTG origin (H-2b+) (see accompanying report). Here we investigated the characteristics of these FTG-derived TCR gamma delta IEL and compared them to the extrathymically derived TCR gamma delta IEL found in nude mice. Phenotypically, FTG-derived TCR gamma delta IEL were similar to their extrathymically derived counterparts in that most were Thy-1-, CD5- and CD8 alpha alpha (homodimer). V gamma and V delta gene usage in thymus-derived and extrathymically derived TCR gamma delta IEL were found to be virtually the same. Functionally, FTG-derived TCR gamma delta IEL were similar to the TCR gamma delta IEL found in euthymic mice as both were relatively anergic to TCR cross-linking in vitro. However, FTG-derived TCR gamma delta IEL differed slightly from extrathymically derived TCR gamma delta IEL, which were completely nonresponsive to the same in vitro stimulation. Overall, these findings support the view that FTG-derived and extrathymically derived TCR gamma delta IEL are almost indistinguishable. Lastly, we demonstrate that despite their thymic origin, development of FTG-derived TCR gamma delta IEL partially takes place extrathymically; that is positive selection of FTG-derived V delta 4 IEL occurs extrathymically. In addition, we demonstrate that the CD8 molecule is not necessary for development and homing of FTG-derived TCR gamma delta IEL. This later finding suggests that the CD8 alpha alpha molecule develops extrathymically for FTG-derived CD8 alpha alpha TCR gamma delta IEL.

Animals↗
PubMed1994

Streptomyces ATP nucleotide 3'-pyrophosphokinase-gene cloning and sequence analysis.

Streptomyces ATP nucleotide 3'-pyrophosphokinase is an extracellular enzyme that transfers 5'-beta, gamma-pyrophosphoryl groups of ATP to a variety of nucleotides at the 3'-OH site. The enzyme gene was cloned from partially Sau3AI-digested chromosomal DNA of S. morookaensis in S. lividans TK24/pIJ699 and then in E. coli JM83/pUC12. Some transformants produced the active enzyme. The gene was sequenced by the dideoxynucleotide termination procedure. Its GC content was 72%. Its putative promoter regions, showing little homology to that of the Streptomyces consensus type, were pointed out. No sequence homology was found between the pyrophosphokinase and any other known genes including those of the most mechanistically similar bacterial stringent factor and related proteins. Northern hybridization analysis showed that the gene is constitutionally polycistronic and expressed under transcriptional control. Nuclease S1 mapping indicated that the gene transcription starts from its translation initiation site.

Amino Acid Sequence↗
PubMed1993

The induction of experimental autoimmune myocarditis in mice lacking CD4 or CD8 molecules [corrected].

Experimental induction of most autoimmune diseases appears to depend on the activation of CD4+ T helper cells, while CD8+ lymphocytes may have a role in disease progression. To study the role of CD4+ and CD8+ T cell subsets in T cell-dependent autoimmunity, mice lacking CD4 or CD8 molecules after gene targeting were injected with cardiac myosin to induce organ specific autoimmune myocarditis. Mice homozygous for the CD8 mutation (CD8-/-) developed significantly more severe disease as compared to CD4+/-CD8+/- controls. Surprisingly, CD4-/- mice developed autoimmune myocarditis with infiltration of TCR alpha beta +CD4-CD8- T cells in the heart tissue and appearance of autoantibodies. These data demonstrate that the lack of CD4+ or CD8+ T cells has no significant influence on the initiation of autoimmune myocarditis. CD4+ and CD8+ cells regulate disease severity and these results may explain the occurrence of autoimmunity in CD4 immunodeficiencies.

Animals↗
PubMed1993

Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development.

Interferon regulatory factor 1 (IRF-1), a transcriptional activator, and its antagonistic repressor, IRF-2, were originally identified as regulators of the type I interferon (IFN) system. We have generated mice deficient in either IRF-1 or IRF-2 by gene targeting in embryonic stem cells. IRF-1-deficient fibroblasts lacked the normally observed type I IFN induction by poly(I):poly(C), while they induced type I IFN to similar levels as the wild type following Newcastle disease virus (NDV) infection. In contrast, IRF-2-deficient fibroblasts showed up-regulated type I IFN induction by NDV infection. A profound reduction of TCR alpha beta+CD4-CD8+ T cells in IRF-1-deficient mice, with a thymocyte developmental defect, reveals a critical role for IRF-1 in T cell development. IRF-2-deficient mice exhibited bone marrow suppression of hematopoiesis and B lymphopoiesis and mortality following lymphocytic choriomeningitis virus infection.

Animals↗
PubMed1993

Differential T cell costimulatory requirements in CD28-deficient mice.

T cell receptor stimulation without costimulation is insufficient for the induction of an optimal immune response. It is thought that engagement of the CD28 molecule with its ligand B7 provides an essential costimulatory signal without which full activation of T cells cannot occur. A mouse strain with a defective CD28 gene was established. Development of T and B cells in the CD28-deficient mice appeared normal. However, T lymphocytes derived from CD28-/- mutant mice had impaired responses to lectins. Lectin stimulation did not trigger interleukin-2 (IL-2) production, IL-2 receptor alpha expression was significantly decreased, and exogenous IL-2 only partially rescued the CD28 defect. Basal immunoglobulin (Ig) concentrations in CD28-deficient mice were about one-fifth of those found in wild-type controls, with low titers of IgG1 and IgG2b but an increase in IgG2a. In addition, activity of T helper cells in CD28-/- mice was reduced and immunoglobulin class switching was diminished after infection with vesicular stomatitis virus. However, cytotoxic T cells could still be induced and the mice showed delayed-type hypersensitivity after infection with lymphocytic choriomeningitis virus. Thus, CD28 is not required for all T cell responses in vivo, suggesting that alternative costimulatory pathways may exist.

Animals↗
PubMed1993

Normal B lymphocyte development but impaired T cell maturation in CD45-exon6 protein tyrosine phosphatase-deficient mice.

The transmembrane tyrosine phosphatase CD45 is expressed in multiple isoforms on all nucleated hematopoietic cells, resulting from alternative splicing of variable exons. We generated mice with a mutation in the variable CD45 exon 6, using homologous recombination. In mice homozygous for the CD45-exon6 mutation, B cells and most T cells did not express CD45. Development of B cells appeared normal, although Ig mu-induced proliferation was completely abrogated. Thymocyte maturation was blocked at the transitional stage from immature CD4+CD8+ to mature CD4+ or CD8+ cells, and only a few T cells could be detected in peripheral lymphoid organs. Clonal deletion of superantigen-reactive T cells still occurred. Cytotoxic T cell responses to lymphocytic choriomeningitis virus were absent in CD45-exon6-/- mice. These data imply that CD45 is differentially required for the development and function of B and T lymphocytes.

Animals↗
PubMed1993

Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection.

The multiple biological activities of tumor necrosis factor (TNF) are mediated by two distinct cell surface receptors of 55 kd (TNFRp55) and 75 kd (TNFRp75). Using gene targeting, we generated a TNFRp55-deficient mouse strain. Cells from TNFRp55-/-mutant mice lack expression of TNFRp55 but display normal numbers of high affinity TNFRp75 molecules. Thymocyte development and lymphocyte populations are unaltered, and clonal deletion of potentially self-reactive T cells is not impaired. However, TNF signaling is largely abolished, as judged by the failure of TNF to induce NF-kappa B in T lymphocytes from TNFRp55-deficient mice. The loss of TNFRp55 function renders mice resistant to lethal dosages of either lipopolysaccharides or S. aureus enterotoxin B. In contrast, TNFRp55-deficient mice are severely impaired to clear L. monocytogenes and readily succumb to infection. Thus, the 55 kd TNFR plays a decisive role in the host's defense against microorganisms and their pathogenic factors.

Animals↗
PubMed1993

Requirement for tyrosine kinase p56lck for thymic development of transgenic gamma delta T cells.

The Src-related protein tyrosine kinase p56lck is essential for antigen-specific signal transduction and thymic maturation of T cells that have an alpha beta T cell receptor (TCR), presumably by physical association with CD4 or CD8 molecules. To evaluate the requirement for p56lck in the development of T cells that have gamma delta TCRs, which generally do not express CD4 or CD8, p56lck mutant mice were bred with TCR gamma delta transgenic mice. Few peripheral cells that carried the transgenes could be detected in p56lck-/- mice, although 70 percent of thymocytes were transgenic. Development of transgenic gamma delta+ thymocytes was blocked at an early stage, defined by interleukin-2 receptor alpha expression. However, extrathymic development of CD8 alpha alpha+ TCR gamma delta+ intestinal intraepithelial lymphocytes appeared to be normal. Thus, p56lck is crucial for the thymic, but not intestinal, maturation of gamma delta T cells and may function in thymic development independently of CD4 or CD8.

Animals↗