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PubMed1998

Cytoskeletal polarization of T cells is regulated by an immunoreceptor tyrosine-based activation motif-dependent mechanism.

Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell-cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex-dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-zeta and CD3epsilon, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-zeta and once in CD3epsilon, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements.

Actins↗

PubMed1997

Retinol (vitamin A) is a cofactor in CD3-induced human T-lymphocyte activation.

Immunomodulatory effects of different retinoids have been demonstrated, both in vivo and in vitro, in different cellular lineages including human and murine thymocytes, human lung fibroblasts, Langerhans' cells, tumoral cells and natural killer (NK) cells; however, any attempt to demonstrate the effect of retinoids on human peripheral blood mononuclear cells (PBMC) resulted in negative results. In the present work, it is shown that retinol and retinoic acid induce a marked increase of proliferation on human PBMC from 32 unrelated healthy individuals, which had previously been stimulated with anti-CD3 antibodies 48 hr before. Serum-free medium, specific retinoid concentration (10(-7) M) and a particular timing of retinol addition to the cultures (48 hr after CD3 stimulation) was necessary clearly to detect this retinol-enhancing effect. The increased proliferative response is specifically mediated via the clonotipic T-cell receptor-CD3 complex and correlates with the up-regulation of certain adhesion/activation markers on the T-lymphocyte surface: CD18, CD45RO and CD25; also Th1-type of cytokines (interleukin-2 and interferon-gamma) are found concordantly increased after retinoid costimulation, both measured by a direct protein measurement and by a specific mRNA increase. In addition, it is shown that the in vitro retinol costimulation is only present in immunodeficient patients who have no defect on CD3 molecules and activation pathway. The fact that retinol costimulate lymphocytes only via CD3 (and not via CD2 or CD28) and the lack of response enhancement in immunodeficients with impaired CD3 activation pathway indicates that retinoids may be used as therapeutic agents in immune system deficiencies that do not affect the clonotypic T-cell receptor.

CD3 Complex↗

PubMed1992

A VLA-4 alpha-chain specific monoclonal antibody enhances CD3-induced IL-2/IL-2 receptor-dependent T-cell proliferation.

Cross-linking the T-cell receptor-associated CD3 complex using the immobilized monoclonal antibody OKT3 can induce low levels of proliferation of purified resting T cells. The effect of coimmobilizing a monoclonal antibody 19H8 specific for the alpha-chain of the integrin VLA-4 on T-cell activation was evaluated. The level of proliferation induced by coimmobilization of the anti-VLA-4 with OKT3 was about 2- to 3-fold over proliferation induced by maximal OKT3 stimulation. The costimulatory activity of 19H8 was dependent on CD3 stimulation since immobilized 19H8 by itself did not induce proliferation. IL-2 secretion was found to be increased over 2-fold with 19H8 costimulation. Addition of exogenous IL-2 resulted in enhanced proliferation of both OKT3 and OKT3 plus 19H8-stimulated cells, but T cells coactivated with 19H8 exhibited a greater capacity to proliferate in response to exogenously supplied IL-2. Analysis of IL-2 receptor expression by flow cytometry revealed that the percentage of CD25-positive cells activated with either OKT3 or OKT3 plus 19H8 is comparable, but the mean fluorescence of cells coactivated with 19H8 is about 3-fold over cells stimulated with OKT3 alone. Dependency of the 19H8 enhanced proliferation on the IL-2/IL-2 receptor system was established by using IL-2-specific neutralizing antisera that reduced the proliferation of T cells activated with OKT3 alone or OKT3 plus 19H8 to comparable levels.2+hese results demonstrate that adhesion molecules may operate at the level of cytokine production and expression of its receptors to modulate the activation state of a cell.

Antibodies, Monoclonal↗

PubMed1990

Transport and secretion of truncated T cell receptor beta-chain occurs in the absence of association with CD3.

The T cell receptor (TCR) beta-chain is produced in the endoplasmic reticulum where it associates with the TCR alpha-chain and the members of the CD3 complex to form the complete receptor. When the other chains of the complex are not available, the beta-chain is rapidly degraded within the endoplasmic reticulum. When incomplete TCR.CD3 complexes are formed, they are transported through the Golgi apparatus and degraded in lysosomes. In this study, a truncated form of the TCR beta-chain has been made by removal of the transmembrane and cytoplasmic segments. Unlike the normal beta-chain, the truncated molecule is stable and is transported through the Golgi apparatus and secreted. This process occurs at a similar rate in both T and B cells, indicating that it is not affected by the presence or absence of CD3 components. These data suggest that an element in the transmembrane or cytoplasmic region of the beta-chain confers sensitivity to the degradative control mechanisms that regulate TCR expression.

Acetylglucosaminidase↗

PubMed1989

High toxic efficiency of ricin immunotoxins specific for the T-cell antigen receptor of a human leukemia T-cell line.

Immunotoxins (ITs) were prepared by covalently coupling ricin to monoclonal antibodies (MAbs) directed against: (a) 2 different epitopes of the T-cell receptor (TcR) expressed by the Jurkat leukemia T-cell line (JTi2 and JTi4 MAb), (b) 2 epitopes of the CD3 complex (SpV-T3b and 11D8 MAb), (c) the CD2 and the CD8 cell-surface molecules. Conjugates were assayed for their cytotoxic activity by pre-incubating the Jurkat cell line with different concentrations (10-250 ng/ml) of each IT for 2 hr at 37 degrees C in the presence of 0.1 M lactose. After washing, cells were cultured for 24 hr and their protein synthesis and proliferative capacities were assessed. Dose-response experiments indicated that JTi2, JTi4 and anti-CD3 (11D8) ITs inhibited by greater than 90% the cell line proliferation at 50 ng/ml, a 5-fold lower concentration than that required to achieve a similar effect when anti-CD2 and anti-CD3 (SpVT3b) were used. After 4 hr of culture subsequent to treatment with JTi2 or JTi4 ITs (250 ng/ml), protein synthesis was inhibited (greater than 80%). By limiting dilution analysis (LDA) we estimated that the frequency of proliferating Jurkat cells (1/1.5) was reduced to 1/20, 1/460 and 1/300 after treatment with anti-CD3 (SpVT3b), JTi4 and JTi2 ITs, respectively. Phenotypic analysis of 13 clones derived from JTi2 IT-treated Jurkat cells showed that 50% were CD7+ CD3- JTi- variants. When bone-marrow mononuclear cells, previously mixed with low concentrations of Jurkat cells, were treated with anti-JTi ITs, the toxic efficiency estimated by LDA was maintained whereas the growth of CFU-GM remained unaltered.

Antibodies, Monoclonal↗

PubMed1990

T cell activation by a bispecific anti-CD3/anti-major histocompatibility complex class I antibody.

A bispecific anti-CD3/anti-major histocompatibility complex (MHC) class I antibody (Ab) that is able to activate human T cells in the absence of a second signal has been used to compare activation by this Ab to signaling by anti-CD3 Ab or by antigen (Ag). We have studied early, intermediate and late events that occur after triggering of a tetanus toxoid-specific T cell clone. While bivalent anti-CD3 Ab induce transient (less than 20 min) intracellular Ca2+ concentration increases, the bispecific Ab, like antigen-presenting cells plus Ag, produces sustained (greater than 2 h) elevated intracellular Ca2+ concentration. In addition, while the anti-CD3 Ab only induces low levels of c-myc mRNA lasting less than 3 h, the bispecific Ab induces high levels which are maintained for at least 8 h after triggering. Late events, such as interleukin 2 receptor expression and proliferation, occurring more than 20 h after activation, were seen only when T cells were stimulated by bispecific Ab or by antigen-presenting cell and Ag. Cells activated by linking T cell receptor (TcR)/CD3 to MHC class I antigens could not be activated by cross-linking the MHC and TcR/CD3 structures separately. Therefore, we propose that the bispecific Ab functions by modifying the membrane dynamics of the TcR/CD3 complex, rather than by the generation of a second signal through class I antigens.

Antigen-Presenting Cells↗

PubMed2004

Graft facilitating cells are derived from hematopoietic stem cells and functionally require CD3, but are distinct from T lymphocytes.

OBJECTIVE: We previously demonstrated that CD8(+)/TCR(-) bone marrow cells facilitate engraftment of HSC in allogeneic recipients without causing graft-vs-host disease. Whether facilitating cells (FC) develop from T cells or represent a distinct lineage has not been determined. METHODS: In the present studies, we characterized the lineage derivation of FC, defined the role for the CD3 complex in allogeneic facilitation, and demonstrated syngeneic facilitation by FC but not T cells. RESULTS: We demonstrate for the first time that FC development and function is independent of T cells and cannot be replaced by them. Purified GFP(+) HSC transplanted in syngeneic recipients produce GFP(+) FC, which facilitate in secondary transplants, confirming that FC are derived from HSC. In addition, FC, but not T cells, potently facilitate the engraftment of suboptimal numbers of HSC in syngeneic recipients. Notably, FC contain the transcripts for CD3 epsilon and CD3 delta, but not TCR alpha or TCR beta, excluding the possibility of T-cell contamination. Genetic mutations that generate a functional deficiency in CD3 signaling significantly impair FC function in allogeneic facilitation (p=0.006). CONCLUSION: Taken together, these data clearly distinguish FC from T cells. Moreover, they indicate that FC require the CD3 epsilon gene to facilitate allogeneic HSC engraftment. The unique function(s) of FC make them an attractive focus for new cell-based therapeutic approaches to enhance HSC engraftment while reducing toxicity, especially when limiting numbers of HSC are available.

Animals↗

PubMed1998

Differential effect on TCR:CD3 stimulation of a 90-kD glycoprotein (gp90/Mac-2BP), a member of the scavenger receptor cysteine-rich domain protein family.

We studied the effects of a 90-kD glycoprotein (gp90/Mac-2BP) belonging to the scavenger receptor family, present in normal serum and at increased levels in inflammatory disease and cancer patients, on some T cell function parameters. Whereas the lymphocyte proliferative response to non-specific mitogens such as phytohaemagglutinin (PHA) and concanavalin A (Con A), but not pokeweed mitogen (PWM), was strongly reduced, probably due to the lectin-binding properties of gp90/Mac-2BP, the response to T cell receptor (TCR) agonists such as superantigens and allogeneic cells was potentiated. When lymphocytes were stimulated with different anti-TCR:CD3 MoAbs, both in soluble and solid-phase form, gp90/Mac-2BP was able to down-regulate the proliferative response to anti-CD3 MoAb, whereas the response to anti-TCR alphabeta MoAb was enhanced. A similar differential effect was observed when a MoAb against CD5 (another member of the scavenger receptor superfamily) was added to anti-CD3 or anti-TCR-stimulated cells; anti-CD5 MoAb strongly down-modulated the CD3-mediated response, whereas its presence in culture was associated with potentiation of the response to TCR alphabeta agonists. gp90/Mac-2BP was able per se to up-regulate Ca2+ levels in freshly isolated lymphocytes; moreover, its presence in culture was associated with increased Ca2+ mobilization following stimulation with anti-TCR alphabeta, but not anti-CD3 MoAb. These data indicate that gp90/Mac-2BP could be able to influence some immune responses, possibly through multiple homologous interactions with other members of the scavenger receptor family; moreover, our findings suggest that signalling through the different components of the TCR:CD3 complex may follow distinct activation pathways into the cells.

3T3 Cells↗

PubMed1992

IP3 receptor: localization to plasma membrane of T cells and cocapping with the T cell receptor.

Immune responses in lymphocytes require cellular accumulation of large amounts of calcium (Ca2+) from extracellular sources. In the T cell tumor line Jurkat, receptors for the Ca(2+)-releasing messenger inositol 1,4,5-trisphosphate (IP3) were localized to the plasma membrane (PM). Capping of the T cell receptor-CD3 complex, which is associated with signal transduction, was accompanied by capping of IP3 receptors. The IP3 receptor on T cells appears to be responsible for the entry of Ca2+ that initiates proliferative responses.

Antigens, CD↗

PubMed1992

Intracellular events involved in CD5-induced human T cell activation and proliferation.

In this report we describe a novel pathway of human T cell activation and proliferation involving the CD5 surface Ag. The CD5-specific Cris1 mAb induces by itself monocyte-dependent proliferation of PBMC. Among a panel of CD5-specific mAb (Leu1, OKT1, LO-CD5a, F101-1C5, and F145GF3), only the F145GF3 mAb shared this property with Cris1. The analysis of the biochemical pathway involved in this activation showed the lack of detectable hydrolysis of inositol phosphates or early increments in the intracellular cytosolic calcium concentration, after triggering cells with the mitogenic CD5 mAb. However, stimulation with CD5 induces activation of protein kinase C, as measured by phosphorylation of a specific peptide substrate (peptide GS), which can be inhibited by a pseudosubstrate peptide inhibitor. Stimulation with CD5 mAb induces also tyrosine kinase activity, with a substrate pattern that differs from that induced after triggering lymphocytes through the TCR-CD3 complex. On the other hand the IL-2/IL-2R pathway seems involved in the CD5-mediated proliferation of PBMC because anti-IL-2R-specific mAb inhibits CD5-induced proliferation, and stimulation with mitogenic CD5 mAb induces production of IL-2 and expression of IL-2R alpha and beta chains. Therefore, the triggering of the CD5 Ag can induce IL-2- and monocyte-dependent human T cell proliferation by a biochemical pathway that differs, at least in the first stages, from the one that mediates TCR-CD3 complex-induced T cell activation.

Antibodies, Monoclonal↗

PubMed2001

A screen of mammalian antibodies on snapper (Pagrus auratus, Sparidae) peripheral blood leukocytes reveals cross reactivity of an anti-human CD3 antibody with a population of mIg(-) cells.

Detailed immunological studies of the teleosts have been hampered by a lack of antibodies against cell-specific markers. Furthermore, where antibodies have been raised, in many instances they have been found to be species-specific. In comparison, many monoclonal and polyclonal antibodies exist with specificities for mammalian proteins and glycoproteins that effectively differentiate leukocyte sub-populations. In this study, we have tested a panel of 54 commercial antibodies against human and murine cell surface receptors for their ability to bind leukocytes isolated from the peripheral blood of snapper (Pagrus auratus). From this panel, one antibody, A452, which is specific for the intracytoplasmic tail of the epsilon (epsilon) chain of the T cell receptor-associated CD3 complex (CD3epsilon) bound to a subpopulation of peripheral blood leukocytes. Mutually exclusive counterstaining was observed when this antibody was used in conjunction with a monoclonal anti-snapper immunoglobulin antibody. This suggests that A452 may be binding to putative snapper T cells.

Animals↗

PubMed1987

T cell activation by cross-linking anti-CD3 antibodies with second anti-T cell antibodies: dual antibody cross-linking mimics physical monocyte interaction.

The anti-CD3 antibody BMA030 (IgG2a isotype) induces T cell activation and proliferation if an interaction with monocytes is provided. In contrast to other anti-CD3 antibodies, it is unable to induce interleukin (IL)2 responsiveness through cross-linking by plastic-bound goat anti mouse Ig antibodies (panning). Cross-linking BMA030 with a second anti-T cell antibody is, however, able to induce IL 2 responsiveness in monocyte-depleted T cell cultures. In this report we show that a large number of different antibodies are suitable for this dual antibody stimulation, and that the extent of proliferation corresponds to the percentage of T cells expressing the respective T cell antigen. Proliferation induced by low concentrations (0.1-1 ng/ml) of other anti-CD3 antibodies requires also cross-linking with second anti-T cell antibodies. The proliferative response of monocyte-depleted T cells to two cross-linked anti-T cell antibodies plus added IL 2 is of the same magnitude as the one induced by anti-CD3 antibodies plus monocytes. On the other hand, if monocytes are present, soluble anti-CD2, -CD4, -CD8, -LFA-1 antibodies (IgG1 or F(ab')2 fragments) can inhibit OKT3 or BMA030-induced T cell activation. Anti-CD6 antibodies do not interfere with this monocyte-dependent T cell stimulation. We conclude that dual antibody stimulation mimics the physical contact of T cells with monocyte membranes, where the T cell receptor CD3 complex is cross-linked with neighboring structures (mainly so-called adhesion molecules) through the interaction with respective counter-structures on monocyte membranes. Dual antibody cross-linking bypasses this interaction and can be used to stimulate IL 2 responsiveness of antibody-defined T cells.

Adult↗

PubMed1993

Distinct roles for CD4 and CD8 as co-receptors in antigen receptor signalling.

The co-ordinated interactions of multiple membrane molecules with the T-cell receptor for antigen (the TCR-CD3) are prerequisite for T-cell activation. In this review we consider the involvement of CD4, CD8, and CD45 on the two lymphocyte lineages. Experiments from many laboratories have provided concordant results leading to the consensus that CD4 and CD8 are functional analogues, providing similar supplementary signals to those generated through the TCR-CD3 complex on MHC class-II- and MHC class-I-restricted T cells, respectively. However, recent results demonstrate striking differences in the coreceptor functions of CD4 and CD8. These differences reflect the distinct properties of the molecules themselves, which in turn are associated with CD45 involvement in the activation of CD4+ and CD8+ T cells.

Animals↗

PubMed2000

Chronic PMA treatment of Jurkat T lymphocytes results in decreased protein tyrosine phosphorylation and inhibition of CD3- but not Ti-dependent antibody-triggered Ca2+ signaling.

We have treated Jurkat T lymphocytes with a concentration (160 nM) of phorbol myristyl acetate (PMA) that down-regulates conventional and novel protein kinase C (PKC) isozymes and we have investigated the effects on Ca2+ signaling and protein tyrosine phosphorylation using mAb (C305) directed against the beta-subunit of the Ti heterodimer or the epsilon/delta-component of the CD3 complex (mAb Leu 4 or OKT 3). The levels of expression of PKC alpha, betaI, betaII, and delta were reduced by 90% or more in PMA-treated cells, whereas the expression of PKCtheta decreased by approximately 30%. In contrast, the chronic treatment with PMA increased the expression of PKCepsilon and PKCzeta. There was a lack of Ca2+ response and myo-inositol trisphosphate (IP3) production in PMA-treated cells when they were exposed to mAb Leu 4 but the cells responded to mAb C305. The treatment with PMA did not affect the surface expression of Ti or CD3. The overall levels of tyrosine-phosphorylated proteins were markedly reduced in PMA-treated cells. We investigated whether these observations were related to defects in signal transduction related to protein tyrosine kinase (PTK) of the src and syk families. The electrophoretic mobilities of p59(fyn) or ZAP-70 were not changed in PMA-treated cells but p56(Ick) migrated as a large band of M(r) 60-62 kDa. The decreased mobility of p56(Ick) was related to a state of hyperphosphorylation. The activity of modified p56(Ick) was not up-regulated in activated Jurkat cells. Our data suggest that clonotypic Ti can trigger Ca2+ mobilization independently of conventional PKC isoforms. Our observations further suggest that conventional PKC isoforms are involved early in the cascade of events associated with Jurkat T lymphocyte activation.

CD3 Complex↗

PubMed1990

Intact antigen receptor-mediated generation of inositol phosphates and increased intracellular calcium in CD4 CD8 T lymphocytes from MRL lpr mice.

The predominant T lymphocytes that accumulate in the peripheral lymphoid tissues of mice homozygous for the lpr gene bear the phenotype CD3+CD4-CD8-. By certain functional criteria these cells would appear to have impaired CD3-mediated signal transduction, in that they do not respond to alloantigen and produce little if any detectable IL-2 or other lymphokines. However, the signal pathway appears adequate for achieving other T cell functions, including induction of high affinity IL-2R, and thymic deletion. To clarify the basis of this seeming discrepancy, we examined transmembrane signal transduction in T cell subsets of lpr/lpr (lpr) and +/+ mice, as defined by increased [Ca2+]i and the generation of inositol phosphates (InsPs). Stimulation of lpr CD4-CD8- cells with anti-CD3 antibody produced prompt and sustained increases in the concentration of [C2+]i and in InsPs. Similar responses occurred in mature T cells from lpr and +/+ mice, except for the somewhat slower kinetics of their increased [Ca2+]i. In marked distinction to the anti-CD2-mediated response, Con A, even in high doses, could not stimulate any increase of [Ca2+]i in lpr CD4-CD8- cells, and only modest increases in InsPs. Mature T cells, whether of lpr or +/+ origin, yielded normal increased [Ca2+]i with Con A. The reason for the differences in signal transduction between anti-CD3 and Con A stimulation of lpr CD4-CD8- cells may relate to the absence of surface structures on these immature T cells that are required for activation by Con A but not by anti-CD3. The data demonstrate that the CD3 complex in lpr CD4-CD8- T cells can couple to phospholipase C to hydrolyze phosphoinositides. These activation properties of lpr CD4-CD8- T cells have interesting functional parallels to thymocytes at the time of thymic selection, as well as tolerance induction of mature T lymphocytes.

Animals↗

PubMed1997

T cell receptor-zeta and granzyme B expression in mononuclear cell infiltrates in normal colon mucosa and colon carcinoma.

BACKGROUND: Whereas the presence of a lymphoid infiltrate has been associated with a favourable prognosis in colorectal carcinoma, the proliferative and cytotoxic responses of freshly isolated tumour infiltrating lymphocytes are frequently impaired. In mice, tumour induced immune suppression has been associated with a decreased expression of the zeta-chain of the T cell receptor (TCR)-CD3 complex, and loss of mRNA for granzyme B. AIM: To compare the expression of TCR-zeta and granzyme B in lymphocytes infiltrating normal colonic mucosa and Duke's A and D colorectal carcinomas. SPECIMENS: Paraffin wax embedded normal (n = 10) and malignant colonic mucosa (seven Dukes's A, nine Dukes's D). METHOD: Immunohistochemistry. RESULTS: The numbers of TCR-zeta + lymphocytes decreased from normal mucosa to Dukes's D carcinomas. In contrast, granzyme B+ lymphocytes were more frequent in Dukes's A carcinomas than in normal mucosa, but disappeared from advanced stage tumours. Granzyme B expressing cells were mainly CD3- (natural killer/lymphokine activated killer cells) in normal mucosa, but CD3+ in tumours, indicating the presence of activated cytotoxic T lymphocytes. In vitro culture of tumour infiltrating lymphocytes rapidly restored the expression of both molecules. CONCLUSION: The frequency of TCR-zeta + and granzyme B+ lymphocytes is decreased in advanced stage colorectal carcinomas. The restoration of expression during in vitro stimulation suggests the presence of tumour derived suppressive factors in situ.

CD3 Complex↗

PubMed1994

Entry of CD4-CD8- immature thymocytes into the CD4/CD8 developmental pathway is controlled by tyrosine kinase signals that can be provided through TCR components.

Entry of thymus-migrated precursor cells into the CD4/CD8 developmental pathway was analyzed by using the short-term organ cultures of day 14 fetal mouse thymus lobes. Organ cultures of CD4-CD8- day 14 fetal thymocytes for 1-2 days resulted in the generation of CD4-CD8+ cells, which were mostly immediate precursor cells for CD4+CD8+ thymocytes. This differentiation of CD4-CD8- thymocytes into CD4-CD8+ cells was strongly enhanced by anti-CD3 antibodies. The anti-CD3- induced generation of CD4-CD8+ cells was even found in the immunodeficient scid fetal thymus cultures, and the cell surface CD3 expression on the scid fetal thymocytes could be directly visualized, indicating that functional CD3 could be expressed on CD4-CD8- immature thymocytes without being associated with rearranged TCR components. The anti-CD3-induced generation of CD4-CD8+ cells from scid and normal fetal thymus cultures was inhibited by tyrosine kinase inhibitors Herbimycin A and Tyrphostin. The generation of CD4-CD8+ cells in unstimulated normal fetal thymus cultures was also markedly inhibited by the tyrosine kinase inhibitors but not by Cyclosporin A, suggesting that tyrosine kinase-dependent but calcineurin-independent signals were essential for the differentiation of CD4-CD8- thymocytes. Interestingly, the generation of CD4-CD8+ cells from the normal fetal thymus cultures was modestly but consistently enhanced by anti-TCR beta antibody, suggesting that functional TCR beta in addition to CD3 was expressed on normal CD4-CD8- immature thymocytes. On the other hand, anti-TCR delta antibody did not affect this differentiation in the normal fetal thymus cultures and the generation of CD4-CD8+ cells from the fetal thymus cultures of TCR delta-deficient mice was still enhanced by anti-TCR beta or anti-CD3 antibodies, indicating that either TCR delta chains or TCR delta+ cells were not involved in the control of the differentiation into CD4-CD8+ cells. These results indicate that the entry of CD4-CD8- immature thymocytes into the CD4/CD8 developmental pathway is controlled by tyrosine kinase signals and that these signals can be provided through the engagement of TCR-CD3 complexes with or without TCR beta chains expressed on the CD4-CD8- immature thymocytes.

Animals↗

PubMed1998

Susceptibility and resistance to antigen-induced apoptosis in the thymus of transgenic mice.

Injection of TCR transgenic mice with antigenic peptide results in the deletion of immature thymocytes expressing the transgenic TCR. We have analyzed this process in mice transgenic for a TCR (F5) that recognizes a peptide from the influenza nucleoprotein (NP68). To determine whether deletion of immature thymocytes is the result of specific recognition of the antigenic peptide by the thymocytes or mature T cell activation, bone marrow chimeric mice were generated using a mixture of cells from F5 transgenic and nontransgenic mice. Injection of these mice with antigenic peptide leads to the preferential depletion of F5 transgenic thymocytes, whereas nontransgenic thymocytes remain largely unaffected. Furthermore, exposure of F5 fetal thymic lobes to peptide leads to thymocyte deletion even though no mature single positive T cells are present at this stage. These data suggest that Ag-induced death of immature thymocytes is due to peptide-specific recognition, although activated mature T cells appear to potentiate such deletion. Further administration of antigenic peptide to F5 mice results in the appearance of double-positive thymocytes that are resistant to Ag or anti-CD3-induced apoptosis. These data suggest a change in the ability of the cells to signal through the TCR-CD3 complex, resembling the state of anergy induced in peripheral T cells following chronic exposure to Ag.

Animals↗