Biomedical subjects

H S Teh

Publications and source records attributed to H S Teh.

At least 55 records · Page 3Linked to original sources

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↗

PubMed1995

The protein tyrosine kinase p56lck regulates TCR expression and T cell selection.

The role of the protein tyrosine kinase (PTK), p56lck, in T cell development was evaluated by mating TCR transgenic mice with transgenic mice that expressed lckF505, a constitutively activated form of p56lck which is under the control of the lck proximal promoter element. The TCR transgenic mice expressed either a receptor specific for the male antigen presented by Db (H-Y TCR) or a receptor specific for pigeon cytochrome c peptide presented by I-Ek class II MHC molecules (AND TCR). The lckF505 transgene caused lower TCR expression in immature CD4+CD8+ thymocytes from normal and TCR transgenic mice. Consistent with the conclusion that activated p56lck causes lower TCR expression, the PTK inhibitor, herbimycin A, was able to restore TCR expression to normal levels in CD4+CD8+ thymocytes from TCR/lckF505 doubly transgenic mice. However, despite lower TCR expression, calcium mobilization was only moderately reduced in CD4+CD8+ thymocytes from H-Y TCR/lckF505 doubly transgenic mice. Furthermore, negative selection of CD4+CD8+ thymocytes expressing the H-Y TCR occurred efficiently in H-Y TCR/lckF505 doubly transgenic male mice despite lower TCR levels. By contrast, analysis of H-Y TCR/lckF505 and AND TCR/lckF505 doubly transgenic mice showed that positive selection in these mice was reduced by 4- to 5-fold by the lckF505 transgene. The smaller proportion of cells that were positively selected in doubly transgenic lckF505 mice expressed normal levels of TCR but higher levels of the appropriate CD4 or CD8 co-receptor molecule. These results indicate that the positive selection of thymocytes is regulated by the enzymatic activity of p56lck.

Animals↗

PubMed1995

A mutation in the alpha 3 domain of Db that abrogates CD8 binding does not affect presentation of an immunodominant H-Y peptide.

The peptidic nature of the male (H-Y) antigen, a model minor histocompatibility antigen in H-2b mice, has recently been demonstrated. In this study we show that the H-Y peptide, which is recognized by PM-1, a Db-restricted cytotoxic T-lymphocyte (CTL) clone, is absent in male H-2d spleen cells but present in male H-2d spleen cells that also express a transgenic Db molecule under its endogenous promoter. This result indicates that both the H-Y and the Db gene products are essential and sufficient for production of the Db-restricted H-Y peptide. By comparing the ability of the PM-1 clone and bulk CTL generated in a secondary mixed lymphocyte culture to recognize H-Y peptidic material eluted from affinity-purified Db molecules and separated by reversed-phase high-performance liquid chromatography (HPLC), we provide evidence that there is an immunodominant H-Y epitope that is presented by the Db molecule. Furthermore, the presentation of this epitope is not affected by a mutation in the alpha 3 domain of Db (asp227 to lys227), which abrogates CD8 binding, since similar amounts of H-Y peptide were eluted from affinity-purified wild-type or mutant Db molecules. However, the generation of the H-Y epitope is dependent on the presence of beta 2-microglobulin, since it is absent in male H-2b mice that lack a functional beta 2-microglobulin gene. The implications of these findings on T-cell development are discussed.

Animals↗

PubMed1995

Effect of lithium and oral thyrotrophin-releasing hormone (TRH) on serum thyrotrophin (TSH) and radioiodine uptake in patients with well differentiated thyroid carcinoma.

Oral thyrotrophin-releasing hormone (TRH) and lithium were given to patients on follow-up for well-differentiated thyroid carcinoma to see their effect on serum thyrotrophin level (TSH) and radioiodine (I-131) uptake (RAIU). The study was randomised and doubled-blinded and consisted of a total of 19 patients in 3 groups. Group 1 received placebo and TRH, group 2 received lithium and placebo, and group 3 received lithium and TRH. Serum TSH and RAIU at 24 hours were measured before, and after treatment, with TRH, lithium, and/or placebo. In group 1, mean (+/-SEM) TSH increased from 48.9 (+/-15.2) mU/l to 148.2 (+/-48.0) mU/l (p < 0.05); in group 2, the change of 24.9 (+/- 15.9) mU/l to 31.7 (+/-14.1) mU/l in TSH was not statistically significant; and in group 3, TSH increased from 108.1 (+/-13.8) mU/l to 187.0 (+/-39.1) mU/l (p < 0.05). However, despite the significant change in TSH, there was no significant increase in I-131 uptake in any group: 7.70% to 10.43%, 7.15% to 7.43% and 2.49% to 2.61%, in groups 1, 2 and 3 respectively (p > 0.05). We conclude that while oral TRH will increase endogenous serum TSH significantly, there is no significant increase in I-131 uptake. Lithium was not an useful adjunct in increasing serum TSH or I-131 uptake in these patients.

Adenocarcinoma, Follicular↗

PubMed1994

Activation-induced cell death in proliferating T cells is associated with altered tyrosine phosphorylation of TCR/CD3 subunits.

The first signaling event that occurs after stimulation of the TCR is an increase in the tyrosine phosphorylation of a number of proteins including the TCR-zeta chain and CD3 subunits. It is known that proliferating T cells respond differently to TCR stimulation when compared with naive T cells. Stimulation of the TCR on proliferating T cells has been shown to result in cell death by apoptosis, a process referred to as activation-induced cell death (AICD). In this study we have determined the pattern of tyrosine phosphorylation of TCR-zeta and the CD3 subunits of naive and proliferating CD4- CD8+ T cells that express a transgenic TCR that is specific for the male Ag presented by Db class I molecules after TCR stimulation. AICD in proliferating T cells was mediated by stimulation with anti-TCR/CD3, but not anti-Thy-1, anti-CD8, or anti-Db mAbs. When compared with naive T cells, tyrosine phosphorylation of TCR-zeta was dramatically decreased in proliferating cells. Furthermore, whereas CD3 delta, CD3 epsilon, and CD3 gamma of naive T cells were phosphorylated in a 1:2:1 ratio after TCR stimulation, these subunits in proliferating T cells were tyrosine phosphorylated in a 1:1:0 ratio after TCR stimulation. Altered phosphorylation of these subunits was not caused by the lack of synthesis or amount of these proteins in proliferating cells. This is the first study implicating altered tyrosine phosphorylation of TCR/CD3 subunits in AICD.

Animals↗

PubMed1994

Production and characterization of monoclonal antibodies specific for the murine T cell receptor zeta chain.

The T cell receptor (TCR) comprises an antigen-specific alpha beta heterodimer non-covalently associated with the CD3 gamma delta epsilon and TCR zeta subunits. Both the CD3 and TCR zeta subunits are proposed to be responsible for the intracellular signal-transduction events. We report here the production of eight monoclonal antibodies (mAbs) that bind in an ELISA assay to a 113 amino acid synthetic peptide corresponding to the cytoplasmic domain of TCR zeta. Western blot analysis of anti-CD8 precipitates of lysates of transfectants expressing chimeric CD8/zeta constructs encoding increasing COOH-terminal truncations of TCR zeta indicates that four of these mAbs recognized the region of TCR zeta chain comprising the last 29 COOH-terminal residues. Thus, this region of TCR theta may encode an immunodominant epitope. Furthermore, one of these mAbs, G3, is capable of precipitating both non-phosphorylated and tyrosine phosphorylated TCR zeta. The G3 mAb should be useful for elucidating the structural and signalling characteristics of the TCR zeta chain.

Amino Acid Sequence↗

PubMed1994

Thymic CD45 tyrosine phosphatase regulates apoptosis and MHC-restricted negative selection.

The acquisition of immunologic self-tolerance is governed, in part, by selection mechanisms that occur during intrathymic T cell ontogeny. Although considerable data exist for the molecular basis of mature T cell signal transduction, the enzymes that participate in thymic TCR selection processes have remained unidentified. We report that augmented thymic expression of the CD45R0 protein tyrosine phosphatase increased the efficacy of TCR-mediated apoptosis and MHC-restricted negative selection of HY TCRs in vivo. Additionally, augmented CD45R0 expression resulted in the activation of endogenous p56lck tyrosine kinase in CD4+CD8+ thymocytes. These results identify a cellular enzyme, the CD45R0 protein tyrosine phosphatase, involved in the regulation of apoptosis and TCR selection mechanisms during CD4+CD8+ thymocyte differentiation.

Animals↗

PubMed1994

Role of infused CD8+ cells in the induction of peripheral tolerance.

The CD8 molecule when present on a cell recognized by a mature T cell has been shown to have an inhibitory effect on the development of an in vitro immune response by that T cell. To test whether CD8 has a role in the induction of peripheral tolerance in vivo, we have studied tolerance induction in B6 female mice carrying a transgenic TCR specific to male H-Y Ag. Previous studies have shown that male Ag reactive cells are inactivated in these mice after the i.v. injection of viable male lymphoid cells. Here, B6 female transgenic mice were injected with B6 male lymph node cells from CD4 or CD8 gene knockout mice, and the fate of male Ag-specific T cells was followed in vivo. Our results indicate that injection of male CD4 knockout (express CD8) lymphoid cells but not CD8 knockout (do not express CD8) lymphoid cells induced a significant reduction of male H-Y Ag reactive cells in the periphery, suggesting that CD8 plays an important role in the induction of peripheral tolerance of class I-restricted T cells. These results imply that peripheral tolerance can be induced not just by absence of signals required for activation but by the presence of additional signals.

Animals↗

PubMed1994

Specific CD45 isoforms differentially regulate T cell receptor signaling.

Multiple isoforms of T cell CD45 tyrosine phosphatase are expressed as a result of alternative RNA splicing among extracellular exons. To discern the presence and identity of distinct functions among CD45 isoforms, we compared thymic T cell activation responses by elevating expression of two CD45 isoforms normally found on quiescent T cells. We report that CD45RABC significantly increased CD4+ thymic T cell proliferation in both a mixed lymphocyte reaction and following anti-T cell receptor (TCR) antibody stimulation. Additionally, CD45RABC enhanced Ca2+ mobilization and phosphotyrosine accumulation, and suppressed the inhibitory effect of anti-CD4 antibodies. By contrast, CD45R0 did not enhance TCR signaling or phosphotyrosine levels in CD4+ thymic T cells and required a TCR co-stimulus to augment cellular proliferation. These studies provide genetic evidence that alternative CD45 isoforms are functionally distinct and disclose a unique mechanism by which T cell immunologic responsiveness can be modified.

Animals↗

PubMed1994

The T cell receptor repertoire of CD4-8+ thymocytes is altered by overexpression of the BCL-2 protooncogene in the thymus.

The bcl-2 gene encodes an intracellular, membrane-associated protein that protects immature cortical thymocytes from a wide variety of apoptotic stimuli, including glucocorticoids, radiation, and anti-CD3 treatment. Since cortical thymocytes are the primary target cells for thymic positive and negative selection processes, and since these processes are associated with cell death, we evaluated the role of bcl-2 in T cell development in two ways. In the first approach, transgenic mice expressing high levels of Bcl-2 in cortical thymocytes were mated with H-Y T cell receptor (TCR) transgenic mice, the latter being a well-defined system for the study of positive and negative selection of T cells. We found that the bcl-2 transgene had a dramatic effect on positive selection. This was manifested by a greatly increased production of mature thymocytes that were highly skewed towards the CD4-8+ lineage. The change involving CD4-8+ thymocytes occurred not only in bcl-2 transgenic mice, but was also observed in H-Y TCR/bcl-2 doubly transgenic mice, regardless of whether the H-Y TCR was expressed in the selecting (H-2b) or nonselecting (H-2d) environments. Furthermore, a large proportion of CD4-8+ thymocytes produced in H-2b H-Y TCR/bcl-2 doubly transgenic female mice expressed endogenous TCR alpha chains rather than the transgenic TCR alpha chain. These observations are consistent with the model that high expression of Bcl-2 in cortical thymocytes overrides the normal apoptotic pathway. This then allows the selection of CD4-8+ thymocytes expressing TCRs that are otherwise nonselectable. However, the bcl-2 transgene did not protect CD4+8+ thymocytes expressing the male-specific TCR from deletion in male doubly transgenic mice. In the second approach, we determined the level of bcl-2 mRNA expression in populations of thymocytes defined by their CD4/CD8 phenotypes using quantitative reversed transcriptase PCR techniques. Our results indicate that bcl-2 mRNA was expressed at a high level in immature CD4-8- thymocytes and in mature CD4+8- thymocytes. There is a dramatic downregulation of bcl-2 mRNA in CD4+8+ thymocytes, particularly those expressing a low level of TCR. CD4+8+ thymocytes that upregulated their TCR, likely as a result of receiving positive selection signals, also upregulated bcl-2 mRNA. This observation suggests that rescue of immature thymocytes from the programmed cell death pathway by positive selection signals is accompanied by the upregulation of bcl-2 mRNA.

Animals↗

PubMed1994

The kinetoplastid membrane protein 11 of Leishmania donovani and African trypanosomes is a potent stimulator of T-lymphocyte proliferation.

Kinetoplastid membrane protein 11 (KMP-11) from Leishmania donovani is an abundant 11-kDa surface membrane glycoprotein. Lymph node cells from mice of six different H-2 haplotypes immunized with KMP-11 or with L. donovani promastigotes were stimulated to proliferate in vitro KMP-11. Primed purified T cells required antigen presentation since they were not stimulated unless KMP-11-pulsed or L. donovani-infected macrophages were added. Promastigotes of a wide variety of Leishmania species and procyclic forms of African trypanosomes stimulated proliferation of KMP-11-primed or L. donovani promastigote-primed lymph node cells. All of the Leishmania promastigotes and African trypanosomes tested contained an 11-kDa protein, as detected by immunoblotting with KMP-11-specific monoclonal antibodies. The widespread distribution of the 11-kDa (KMP-11) molecules and their ability to stimulate strong T-lymphocyte proliferation in a non-H-restricted fashion suggest that they may be important molecules for induction of cell-mediated immune responses.

Animals↗

PubMed1993

Thymus-independent positive and negative selection of T cells expressing a major histocompatibility complex class I restricted transgenic T cell receptor alpha/beta in the intestinal epithelium.

We demonstrate that in the mouse intestinal epithelium the selection of T lymphocytes expressing a transgenic T cell receptor alpha/beta (TCR-alpha/beta) specific for male antigen (H-Y) in the context of H-2Db depends on the differential expression of H-Y and H-2Db in situ. In H-2Db transgenic males, there is no reduction in the number of intestinal intraepithelial lymphocytes (IEL), and the four main subsets of IEL expressing TCR-alpha/beta, defined by the differential expression of CD4, CD8 alpha, and CD8 beta, are present. Moreover, the level of expression of CD8 alpha and CD8 beta on CD8+ IEL subsets is unaltered. The frequency of CD8 alpha+ IEL expressing CD8 beta, in H-2Db male mice, however, is significantly decreased and these cells do not express the transgenic TCR. In contrast, virtually all CD8 alpha+beta- IEL in the same animals express the transgenic TCR. Still, these potentially autoreactive cells are refractile to H-Y/H-2Db stimulation in vitro. Both H-2Db and H-2Dd transgenic females contain high frequencies of cells expressing the transgenic TCR among CD8 alpha+beta- and CD8 alpha+beta+ IEL. However, two possibly related phenotypic features are peculiar to H-2Db female mice. The frequency of CD8 alpha+ IEL expressing CD8 beta is increased in these mice and, while in H-2Dd females the level of the transgenic TCR alpha chain expressed on CD8 alpha+beta+ IEL is uniformly low, some of the CD8 alpha+beta+ IEL in H-2Db females express a high level of both transgenic TCR chains. It is important to note, the ability of CD8 alpha+beta+ IEL to respond to H-Y/H-2Db stimulation in vitro is restricted to those coexpressing a high level of both transgenic TCR chains. The analysis of athymic radiation chimeras using adult thymectomized recipients of distinct H-Y/H-2 haplotypes, reconstituted with bone marrow from H-2Db transgenic females, demonstrates that all IEL subsets present in unmanipulated transgenic animals develop in the absence of a thymus. These IEL are phenotypically identical to those found in unmanipulated transgenic animals sharing the H-Y/H-2 haplotype of athymic recipients. Taken together, these results demonstrate that in the absence of male antigen, expression of H-2Db in the intestinal epithelium results in the positive selection of functional IEL specific for male antigen, in situ. When both H-Y and H-2Db are expressed in the intestinal epithelium, CD8 alpha+beta+ IEL expressing the transgenic TCR are negatively selected, while the frequency of nonfunctional CD8 alpha+beta- IEL expressing the transgenic CR is increased.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

CD8 inhibits signal transduction through the T cell receptor in CD4-CD8- thymocytes from T cell receptor transgenic mice reconstituted with a transgenic CD8 alpha molecule.

T cell repertoire selection processes involve intracellular signaling events generated through the TCR. The CD4 and CD8 coreceptor molecules can act as positive regulators of TCR signal transduction during these developmental processes. In this report, we have used TCR transgenic mice to determine whether TCR signaling can be modulated by the CD8 coreceptor molecule. These mice express on the majority of their T cells a TCR specific for the male (H-Y) Ag presented by the H-2Db MHC class I molecule. We show that CD4-CD8-, but not CD4-CD8+, thymocytes expressing the H-Y TCR responded with high intracellular calcium fluxes to TCR/CD3 stimulation without extensive receptor cross-linking. To examine the effects of CD8 expression on intracellular signaling responses in the CD4-CD8- cells, the H-Y TCR transgenic mice were mated with transgenic mice that constitutively expressed the CD8 alpha molecule on all T cells. The expression of the CD8 alpha alpha homodimer in the CD4-CD8-thymocytes led to impaired intracellular calcium responses and less efficient protein tyrosine phosphorylation of substrates after TCR engagement. In male H-2b H-Y transgenic mice, the majority of thymocytes have been deleted with the surviving cells expressing a high density of the transgenic TCR and exhibiting either a CD4-CD8- or CD4-CD8lo phenotype. It has been postulated that these cells escaped deletion by down-regulating the CD8 molecule. In the H-Y TCR/CD8 alpha double transgenic male mice, the CD4-CD8lo cells were completely eliminated as a result of CD8 alpha expression. However, the CD4-CD8- T cells were not deleted despite normal levels of the CD8 alpha transgene expression. These results suggest that the CD4-CD8- thymocytes may not be susceptible to the same deletional mechanisms as other thymocytes expressing TCR-alpha beta.

Animals↗

PubMed1993

CD8 is needed for positive selection but differentially required for negative selection of T cells during thymic ontogeny.

During thymic development, immature thymocytes expressing major histocompatibility complex (MHC) class I-restricted T cell receptors (TcR) differentiate into CD8+ T cells with cytolytic functions. To evaluate the role of CD8 in positive and negative selection during thymic ontogeny, mice rendered CD8-null by gene targeting were bred with three lines of transgenic mice expressing unique MHC class I-restricted TcR. In all three instances CD8 was required for positive selection of MHC class I-restricted transgenic T cells. The efficiency of positive selection decreased in accordance with a reduced level of CD8 expression on thymocytes. Surprisingly, there was a differential requirement for CD8 expression in negative selection of MHC class I-restricted thymocytes, depending on the antigen specificity of TcR. These observations show that CD8 is essential for positive selection but is differentially required for negative selection of MHC class I-restricted T cells. Thus thymic selection, at least for negative selection, can occur in the absence of the CD8 accessory molecule.

Animals↗

PubMed1993

The influence of rearranged T cell receptor alpha beta transgenes on early thymocyte development.

Most murine thymocytes and mature T cells originate from a numerically minor population of CD8-4- (double-negative, DN) thymocytes. In this study, we investigated the effects of rearranged T cell receptor (TcR) alpha and beta transgenes on early T cell development. We analyzed the precursor potential, the expression of CD25 and TcR at mRNA and/or protein level in DN thymocyte subsets in TcR transgenic (Tg) mice. We report the following observations: (i) despite a large overrepresentation of total DN cells in TcR Tg mice, precursor-containing CD25+ DN and CD8lo4lo thymocytes are reduced to a third of the nontransgenic control numbers; (ii) like in the normal mice, CD25+ DN and CD8lo4lo can, and TcR+ DN cells cannot generate other thymic subsets; (iii) TcR alpha mRNA and TcR alpha/beta protein levels are quantitatively increased, but their developmental expression is similar to that in normal mice; and (iv) surface TcR alpha beta expression becomes detectable as the thymocytes down-regulate CD25, paralleling the situation in normal mice. Our findings implicate stringent transcriptional control, rather than TcR gene rearrangement, as a decisive regulator of TcR alpha beta expression in early ontogeny.

Animals↗

PubMed1993

Allograft rejection by T cell receptor transgenic mice.

We have used a line of T cell receptor (TcR) transgenic mice, in which a significant portion of CD8+ T cells expresses a TcR that is specific for the minor histocompatibility antigen H-Y presented by the H-2Db Class I molecule, to examine the immune response to H-Y-incompatible skin or pancreatic islet allografts. Our results indicate that, in contrast to the conclusions of previous reports, pancreatic islet endocrine cells are invulnerable or only weakly vulnerable to an H-Y-directed immune response. An even more unexpected finding was that unlike normal female mice of the C57BL/6 background which consistently reject male skin within a few weeks, TcR transgene+ littermates reject male skin only infrequently. Our results are consistent with the conclusion that the inability of H-Y TcR females to reject male skin is due to a deficiency of cells with male-specific helper activity. Long-term acceptance of male grafts by H-Y TcR females leads to a state of T cell hyporesponsiveness to male skin grafts. In addition, T cells harvested from long-term skin acceptors were hyporesponsive to in vitro stimulation by a clonotype-specific monoclonal antibody. Transgenic mice with TcRs having antigenic specificity for defined transplantation antigens provide a unique model for study of the allograft response.

Animals↗

PubMed1993

Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta.

The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.

Animals↗

PubMed1992

B7 costimulates proliferation of CD4-8+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes.

In addition to TCR-derived signals, costimulatory signals derived from stimulation of the CD28 molecule by its natural ligand, B7, have been shown to be required for CD4+8- T cell activation. We investigate the ability of B7 to provide costimulatory signals necessary to drive proliferation and differentiation of virgin CD4-8+ T-cells that express a transgenic TCR specific for the male (H-Y) Ag presented by H-2Db class I MHC molecules. Virgin male-specific CD4-8+ T cells can be activated either with B7 transfected chinese hamster ovary (CHO) cells and T3.70, a mAb specific for the transgenic TCR-alpha chain that is associated with male-reactivity, or by male dendritic cells (DC). Activated CD4-8+ T cells proliferated in the absence of exogenously added IL-2. IL-2 activity was detected in supernatants of CD4-8+T3.70+ cells that were stimulated with T3.70 and B7+CHO cells. The response of CD4-8+T3.70+ cells to T3.70/B7+CHO or to male DC stimulation were inhibited by CTLA4Ig, a fusion protein comprising the extracellular portion of CTLA4 and human IgG C gamma 1. It has been previously shown that CTLA4Ig binds B7 with high affinity. Staining with CTLA4Ig revealed that DC express about 50 times more B7 than CD4-8+ T cells. CTLA4Ig also specifically blocked the proliferation of male-reactive cells in vivo. We have also used an in vitro deletion assay whereby immature CD4+8+ thymocytes expressing the transgenic male-specific TCR are deleted by overnight incubation with either immobilized T3.70 or male DC to investigate the participation of the CD28/B7 pathway in the negative selection of immature thymocytes. Staining with B7Ig established that both immature murine CD4+8+ and mature CD4-8+ thymocytes express a high level of CD28. However, despite the high expression of CD28 on CD4+8+ thymocytes, it was found that deletion of CD4+8+ thymocytes expressing the male-specific TCR by the T3.70 mAb was not inhibited by B7+CHO cells. Furthermore, the deletion of these thymocytes by DC also was not inhibited by CTLA4Ig. These findings provide evidence that although signaling through CD28 can costimulate a primary anti-male response in mature CD4-8+ T cells, the CD28/B7 pathway does not appear to participate in the negative selection of immature CD4+8+ thymocytes.

Animals↗