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PubMed1997

Expression and signal transduction of T-cell antigen receptor (TCR)/CD3 complexes on fresh or in vitro expanded T lymphocytes from patients with Hodgkin's and non-Hodgkin's lymphomas.

T-cell responses against soluble antigens, alloantigens and mitogens are frequently diminished in patients with certain types of cancer. In the present study, the authors investigated possible mechanisms for the partial T-cell immunodeficiency in patients with Hodgkin's or non-Hodgkin's lymphomas. It was found that T-cells from lymphoma patients had significantly reduced proliferative responses to EBV-transformed B-cell lines and to anti-TCR/CD3 MoAb; a 30-50% reduction of cells expressing membrane T-cell receptor (TCR) complexes; and a significantly reduced signal transduction function. Long-term in vitro culture conditions were developed to expand T cells in TCR/CD3-dependent or TCR/CD3-independent manners. With such methods, it was found that the decreased T-cell responses in patients with Hodgkin's and non-Hodgkin's lymphomas appeared to be an intrinsic T-cell defect (not at the antigen presenting cell level), and the T-cell responses could be recovered after only a few days in culture. Thus, it is suggested that the T-cell response-defect in Hodgkin or non-Hodgkin lymphoma patients is a reversible phenomenon, dependent on the patient's tumour-bearing environment.

B-Lymphocytes↗

PubMed1998

Transcription of thyroid autoantigens in non-expressing tissues.

Reverse transcriptase- polymerase chain reaction (RT-PCR) enhances the probability of detecting rare transcripts in complex mixtures of mRNA. Using thyroid autoantigens and the controversy about the role of the TSH-receptor (TSH-R) in thyroid-associated ophthalmopathy as an example, this study demonstrates the problems of interpreting RT-PCR results in typically non-expressing tissues resulting from the extremely high sensitivity of the method. Unexpected transcripts for thyroperoxidase, thyroglobulin, TSH-R (exon 1-4, 354 bp), FSH-receptor, or insulin fragments were demonstrated in a number of thyroid or orbit-derived as well as unrelated tissues or cell types. Unexpected transcripts were most prevalent in fibroblasts, irrespective of the tissue of origin and most likely caused by ectopic transcription. To establish a physiological significance of rare transcripts such as the TSH-R in orbital tissues, demonstration of the protein in addition to the positive RT-PCR results is needed.

Autoantigens↗

PubMed1990

Heterogeneity of the regulation of phospholipase C by phorbol esters in T lymphocytes.

In many cells, protein kinase C (PKC) activation inhibits cellular phospholipase C thereby preventing receptor-mediated phosphatidylinositol (PI) metabolism. In T lymphocytes, the T cell antigen receptor (Ti)/CD3 complex regulates PI hydrolysis and we have examined the consequences of PKC activation on Ti/CD3-mediated PI metabolism in human peripheral blood-derived T lymphocytes (T lymphoblasts) and the leukemic T cell line Jurkat. In Jurkat cells, PI metabolism after Ti/CD3 stimulation, is inhibited by PKC activation. PKC activation also inhibits calcium-induced PI metabolism in permeabilized Jurkat cells. In marked contrast, PI metabolism after Ti/CD3 stimulation in T lymphoblasts, is not inhibited by PKC activation. Moreover, in permeabilized T lymphoblasts PI metabolism can be induced by calcium in synergy with guanine 5'-O-(3-thiotrisphosphate) via a PKC-insensitive mechanism. The different effect of PKC stimulation on PI metabolism in Jurkat cells and T lymphoblasts reveals heterogeneity of PLC regulation in T lymphocytes. The data also indicate that the role of PKC as a regulator of Ti/CD3 signal transduction can differ depending on cell type.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1994

[Study of the membrane expression of TcR/CD3 complex using somatic cell mutants].

T lymphocytes express membrane antigen receptor TcR/CD3 complexes only when all subunits are correctly assembled. Studies on TcR/CD3 membrane negative T cell variants containing all necessary subunits intracellularly, may allow to identify amino acids important for different subunit interactions. In this review, we summarize our recent work on TcR/CD3 negative variants of the human T cell line Jurkat. We found two critical amino acids in the TcR-alpha and TcR-beta extracellular constant regions (phenylalanine n. 216 and intrachain disulfide cysteine n. 212) involved in TcR-alpha beta/CD3- gamma epsilon, delta epsilon intermediary complex/zeta 2 homodimer interactions: (1) amino acid exchanges of phenylalanine demonstrated the importance of an aromatic amino acid residue at this position; (2) the intrachain disulfide bond assures a tertiary structure of the constant domain that is necessary for association with zeta 2 homodimers.

Amino Acid Sequence↗

PubMed1992

Heteroconjugated antibodies enhance lymphocyte-mediated tumour cell lysis in vitro and in vivo.

Covalent linkage of an antitumour antibody specific for a tumour cell surface antigen to an antilymphocyte antibody specific for the T lymphocyte receptor complex produces a heteroconjugated antibody that can activate and redirect cytotoxic T lymphocytes to lyse tumour cells. The ability of an antilymphocyte-antitumour heteroconjugate (500A2 x 96.5) to direct the lysis of murine melanoma cells by cultured murine lymphocytes was tested in vitro using a 4-h chromium release assay and in vivo with a tumour neutralization assay. In vitro, the addition of heteroconjugated antibody significantly increased tumour lysis by murine C3H/HeN lymphocytes (median specific lysis 82.7 per cent with lymphocytes plus heteroconjugate versus 9.5 per cent for lymphocytes alone, P less than 0.001). In vivo, treatment with heteroconjugated antibody plus lymphocytes significantly reduced the development of pulmonary metastases after intravenous tumour administration (median number of pulmonary metastases 28.5 for combined treatment versus 250 for heteroconjugate or lymphocytes alone, P less than 0.001).

Animals↗

PubMed2001

T Lymphocytes infiltrating various tumour types express the MHC class II ligand lymphocyte activation gene-3 (LAG-3): role of LAG-3/MHC class II interactions in cell-cell contacts.

The product of the Lymphocyte Activation Gene-3 (LAG-3, CD223) is a high affinity MHC class II ligand expressed by activated CD4(+) and CD8(+) T cells, which can associate with the T cell receptor (TCR) and downregulate TCR signalling in vitro. We have also reported that a soluble mLAG-3Ig fusion protein works as a vaccine adjuvant in vivo in mice, enhancing Th1 and CD8 T cell responses. Here, we report that LAG-3 expression was found, using fluorescent activated cell sorting (FACS) analysis, on 11-48% of human tumour-infiltrating lymphocytes (TILs) isolated from eight freshly dissociated renal cell carcinomas (RCCs), and was restricted mostly to CD8(+) cells. Immunohistochemical analysis confirmed LAG-3 expression by TILs in 9/11 RCCs, as well as in tumours of different origins, such as melanomas (3/5) and lymphomas (7/7). Since not only antigen presenting cells (APCs), but also TILs themselves strongly express major histocompatibility complex (MHC) class II, we firstly investigated whether LAG-3/MHC class II T-T cell contacts might influence tumour cell recognition. However, cytotoxicity inhibition was not observed in two RCC-specific CD8(+) T cell clones in the presence of the LAG-3-specific MAb, and there was also no observed difference in the recognition of LAG-3-transfected or wild-type RCC by these cytotoxic T lymphocytes (CTLs). In contrast, MHC class II engagement by LAG-3Ig was found to enhance the capacity of immature dendritic cells to stimulate naive T cell proliferation and IL-12-dependent IFN-gamma production by T cells in vitro. These results therefore provide support for a role for TIL-expressed LAG-3 in the engagement of class II molecules on APCs, thereby contributing to APC activation and Th1/Tc1 commitment, without downregulating cytotoxicity.

Antigens, CD↗

PubMed1998

Three-dimensional segregation of supramolecular activation clusters in T cells.

Activation of T cells by antigen-presenting cells (APCs) depends on the complex integration of signals that are delivered by multiple antigen receptors. Most receptor-proximal activation events in T cells were identified using multivalent anti-receptor antibodies, eliminating the need to use the more complex APCs. As the physiological membrane-associated ligands on the APC and the activating antibodies probably trigger the same biochemical pathways, it is unknown why the antibodies, even at saturating concentrations, fail to trigger some of the physiological T-cell responses. Here we study, at the level of the single cell, the responses of T cells to native ligands. We used a digital imaging system and analysed the three-dimensional distribution of receptors and intracellular proteins that cluster at the contacts between T cells and APCs during antigen-specific interactions. Surprisingly, instead of showing uniform oligomerization, these proteins clustered into segregated three-dimensional domains within the cell contacts. The antigen-specific formation of these new, spatially segregated supramolecular activation clusters may generate appropriate physiological responses and may explain the high sensitivity of the T cells to antigen.

Animals↗

PubMedDate not supplied

The delta- and epsilon-chains of the human T3/T-cell receptor complex are distinct polypeptides.

The T3/T-cell receptor complex on the surface of human thymus-derived lymphocytes consists of four glycoproteins: the alpha-chain of relative molecular mass (Mr) 40,000-50,000 (40-50K), the beta-chain (37-45K); the gamma-chain (25K) and the delta-chain (20K). The T3 alpha- and beta-chains have been identified as clonotypic T-cell receptors, but functionally the T3/T-cell receptor chains seem to form a single complex: monoclonal antibodies directed at the 20K T3 components are mitogenic for normal human T lymphocytes and, at higher concentrations, anti-clonotypic and anti-20K reagents block T-cell function. Recently, Zanders et al. showed that incubation of human T-helper clones with high concentrations of antigen abolishes antigen-specific proliferation and induces disappearance of T3 from the cell surface. Thus, the T3/T-cell receptor complex consists of two variable subunits, the T3 alpha- and beta-chains, which interact with antigen and the monomorphic 20K/25K T3 chains. Recently, the existence of a fifth polypeptide chain, the unglycosylated T3 epsilon-chain, has been postulated. Here we confirm that a 20K epsilon-chain does exist. The T3 epsilon-chain differs from the T3 delta-chain in primary structure as judged by N-terminal amino acid sequencing, peptide mapping and immunoblotting with anti-T3-delta and anti-T3-epsilon antibodies. Treatment with endoglycosidase F revealed two nonglycosylated T3 delta polypeptide backbone chains (16K and 14K) with identical amino termini. Together with previous pulse-chase experiments this observation suggests that the 14K T3 polypeptide is derived from the 16K T3 precursor by proteolytic processing near the C-terminus of the molecule.

Amino Acid Sequence↗

PubMed2004

Mechanisms of bacillus Calmette-Guerin mediated natural killer cell activation.

PURPOSE: Natural killer (NK) cells are of crucial importance for bacillus Calmette-Guerin (BCG) mediated antitumor effects. We defined the mechanisms of BCG mediated NK cell activation in vitro. MATERIALS AND METHODS: A standard Cr release assay was used to measure the cytotoxicity of BCG activated NK cells. Using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany) we depleted various immune cell subpopulations from BCG stimulated peripheral blood mononuclear cells to phenotype activated NK cells. During the stimulation process anticytokine antibodies and recombinant cytokines were added to define their role in NK cell activation. For costimulation studies peripheral blood mononuclear cells were separated into lymphocytes and monocytes by counterflow-centrifugation (elutriation). Inhibitory NK cell receptor expression on activated NK cells was measured by flow cytometry by antiCD3, antiCD56 and anti-inhibitory NK cell receptor triple staining. RESULTS: The accessory function of monocytes was indispensable for BCG mediated NK cell activation. However, the stimulatory potential of monocytes did not require direct cell-cell contact to NK cells or major histocompatibility complex dependent antigen presentation to T cells. Monocyte derived interleukin (IL)-12 and to a lesser extent interferon (IFN)-alpha were key mediators for stimulating BCG induced NK cell cytotoxicity and IFN-gamma production. In contrast, IL-10 inhibited NK cell cytotoxicity and IL-18 did not show any effect. Exogenous recombinant IFN-alpha and IL-12 enhanced BCG mediated secretion of IFN-gamma and yet BCG induced NK cell cytotoxicity remained unchanged. While the CD158a and CD158b subsets did not have a significant role, NKG2A cells represented the predominant cytolytic subset in BCG activated NK cells. CONCLUSIONS: Following BCG stimulation the monocyte derived TH1 cytokines IL-12 and IFN-alpha activate tumor cytotoxic CD3/CD56/NKG2A NK cells. Our results elucidate NK activating mechanisms that are operative during BCG immunotherapy for bladder cancer and are relevant for an early, innate antimycobacterial immune response.

BCG Vaccine↗

PubMed1994

Abnormal signal transduction by T cells of mice with parental tumors is not seen in mice bearing IL-2-secreting tumors.

There is considerable evidence to demonstrate that immune function is abnormal in tumor-bearing mice, perhaps accounting, at least in part, for progressive tumor growth. In an attempt to generate an antitumor response, we used retroviral vectors to express IL-2 cDNA in CMS5, a murine fibrosarcoma. Mice inoculated with unmodified tumor cells suffered progressive tumor growth, whereas tumors secreting IL-2 were rejected or grew slowly. Animals bearing unmodified but not IL-2-secreting tumors also were immunosuppressed. On the basis of these observations, we were interested in how IL-2 secretion by the tumor cells prevented the onset of hyporesponsiveness. To identify biochemical differences between T cells of mice with parental vs slowly growing IL-2-secreting tumors, we examined signal transduction after activation through the CD3/TCR complex. Protein tyrosine phosphorylation was altered and calcium flux was reduced in cells of mice with parental tumors compared with animals with slowly growing IL-2-secreting tumors. In addition, levels of protein for the tyrosine kinases p56lck and p59fyn, as well as the TCR-zeta-chain, were reduced. These differences in signal transduction were observed for T cells of mice with parental and IL-2-secreting tumors of the same size, demonstrating that differences in tumor size alone could not explain our findings. Thus, IL-2 secretion by tumors seems to be able to prevent immunosuppression by maintaining normal signal transduction in T cells, facilitating the generation of antitumor responses.

Animals↗

PubMed1997

Ligation of the T cell antigen receptor induces tyrosine phosphorylation of p105CasL, a member of the p130Cas-related docking protein family, and its subsequent binding to the Src homology 2 domain of c-Crk.

p105CasL (CasL) is a recently identified signaling molecule closely related to the p130Cas (Crk-associated substrate) docking protein. CasL has a single Src homology (SH) 3 domain in its N-terminal portion followed by multiple consensus motifs for binding to SH2 domains. Like original p130Cas, CasL undergoes tyrosine phosphorylation upon integrin-mediated cell adhesion. In the present report, we provide direct evidence that CasL is also involved in T cell antigen receptor (TcR)-mediated signal transduction. In binding studies in vitro using glutathione-S-transferase fusion proteins, we have identified 105- and 120-kDa phosphotyrosyl proteins (pp105 and pp120, respectively) tightly bound to the SH2 domain of the Crk adapter protein in the H9 human T cell line after stimulation through the CD3/TcR complex. pp120, but not pp105, also bound to the SH3 of another adapter protein, Ash/Grb2. Immunoblotting with specific antibodies revealed that pp120 and pp105 were identical to the c-cbl proto-oncogene product (p120cbl) and CasL, respectively. Association between Crk and tyrosine-phosphorylated CasL after TcR stimulation was also confirmed in vivo. CasL phosphorylation induced by TcR ligation reached maximal levels within 2 min and rapidly declined thereafter, whereas the integrin-dependent response occurred slowly and was more prolonged. Finally, we demonstrated that Crk/CasL association occurred in peripheral blood T lymphocytes in response to TcR engagement. Our findings suggest that CasL is involved in T cell activation signals and resides at a point where two distinct receptor-mediated signaling pathways converge. This provides one mechanism by which integrins may mediate T cell co-stimulation.

Adaptor Proteins, Signal Transducing↗

PubMed1991

Down-regulation of T cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction.

By generating two types of transgenic mice we have investigated how extrathymic events can contribute to self tolerance. The major histocompatibility complex class I gene Kb was expressed under the control of the glial fibrillary acidic protein promoter in cells of neuroectodermal origin outside the thymus. These mice were tolerant to Kb. When crossed to transgenic mice expressing a Kb-specific T cell receptor (TCR), clonotype+, CD8+CD4- mature T cells could be detected in normal numbers in the thymus of the double-transgenic mice but were strongly reduced in spleen and lymph nodes in comparison with TCR single-transgenic mice. After isolation of clonotype negative splenic T cells and activation in vitro, reappearance of the clonotype+, CD8+CD4- cells was observed. These results indicate that down-regulation of TCR and CD8 molecules on the antigen-specific T cells is a novel mechanism, by which peripheral tolerance to this antigen can occur.

Animals↗

PubMed1994

Modulation of T cell proliferative response by accessory cell interactions.

Antigen-specific activation of the T cell is accomplished by engagement of the T cell receptor (TCR) by an antigen (Ag)/MHC complex presented on the surface of an antigen- presenting cell (APC). However, it has been demonstrated that engagement of the TCR by Ag/HC complexes alone is normally insufficient to lead to a proliferative response and the development of effector function. Thus it has been proposed that the APC also provides additional signals which serve to modulate the T cell's response. These second or costimulatory signals are thought to be critical in the generation of a T cell-driven immune response. Several receptors have been proposed to be capable of serving as costimulatory receptors. Candidate molecules include CD28 and LFA-1 as well as other receptors. In this review the studies that we have performed to clarify the role of both LFA-1 and CD28 in providing costimulatory activity for T cell activation are discussed. In addition, we present evidence that under certain conditions, TCR signalling alone can be sufficient to lead to T cell proliferation.

Animals↗

PubMed1990

Cutaneous response to recombinant interleukin 2 in human immunodeficiency virus 1-seropositive individuals.

We report that 11 human immunodeficiency virus 1 (HIV-1)-seropositive patients, including three AIDS patients, were able to generate a cellular immune response to the intradermal injection of low doses (2-10 micrograms) of recombinant interleukin 2 (rIL-2). A dose-dependent zone of induration appeared at the site of injection, peaked at 24 hr, and was accompanied by the local accumulation of T cells, monocytes, and Langerhans cells. Despite the reductions in the CD4+ T-cell counts in the peripheral blood of most patients, CD4+ T-cells could still be mobilized with rIL-2 injections into the skin. The total number of immigrant cells was equivalent to those in HIV-1-seronegative patients, although the CD4+/CD8+ ratio of the dermal population was reduced. In response to rIL-2, major histocompatibility complex (MHC) class II antigen was expressed on the surface of keratinocytes, Langerhans cells, lymphocytes, and macrophages. In addition, the gamma interferon (IFN-gamma)-induced protein IP-10 rapidly appeared in dermal inflammatory cells and keratinocytes. A majority of HIV-1-seropositive patients demonstrated low or absent responses to common skin-test antigens. Those with positive zones of induration were often defective in the cellular expression of the IFN-gamma-induced MHC class II antigen. The simultaneous administration of rIL-2 and soluble antigen at widely separated cutaneous sites led to an enhancement of skin-test antigen reactivity in seropositive patients. The results suggest that local administration of rIL-2 to seropositive patients may act systemically, stimulating cellular immunity to recall antigens, and thus may be of potential benefit in the defense against opportunistic pathogens encountered in HIV-1 infection.

Antigens, CD↗

PubMed2004

Recruitment of the actin-binding protein HIP-55 to the immunological synapse regulates T cell receptor signaling and endocytosis.

Actin cytoskeleton dynamics critically regulate T cell activation. We found that the cytoplasmic adaptor HIP-55, a Src/Syk-kinases substrate and member of the drebrin/Abp1 family of actin-binding proteins, localized to the T cell-antigen-presenting cell (APC) contact site in an antigen-dependent manner. Using green fluorescent protein fusion proteins, both Src homology 3 (SH3) and actin binding domains were found necessary for recruitment at the T cell-APC interface. HIP-55 was not implicated in conjugate formation and actin polymerization but regulated distal signaling events through binding and activation of hematopoietic progenitor kinase 1 (HPK1), a germinal center kinase (GCK) family kinase involved in negative signaling in T cells. Using RNA interference and overexpression experiments, the HIP-55-HPK1 complex was found to negatively regulate nuclear factor of activated T cell (NFAT) activation by the T cell antigen receptor. Moreover, we show that HIP-55, which partly co-localized with early endocytic compartments, promoted both basal and ligand-dependent T cell receptor (TCR) down-modulation, resulting in a decreased TCR expression. SH3 and actin-depolymerizing factor homology domains were required for this function. As controls, the expression of CD28 and the glycosylphosphatidylinositol-linked protein CD59 was not affected by HIP-55 overexpression. These results suggest that, in addition to binding to HPK1, HIP-55 might negatively regulate TCR signaling through down-regulation of TCR expression. Our findings show that HIP-55 is a key novel component of the immunological synapse that modulates T cell activation by connecting actin cytoskeleton and TCRs to gene activation and endocytic processes.

Actins↗

PubMed2000

Functional requirement for class I MHC in CNS development and plasticity.

Class I major histocompatibility complex (class I MHC) molecules, known to be important for immune responses to antigen, are expressed also by neurons that undergo activity-dependent, long-term structural and synaptic modifications. Here, we show that in mice genetically deficient for cell surface class I MHC or for a class I MHC receptor component, CD3zeta, refinement of connections between retina and central targets during development is incomplete. In the hippocampus of adult mutants, N-methyl-D-aspartate receptor-dependent long-term potentiation (LTP) is enhanced, and long-term depression (LTD) is absent. Specific class I MHC messenger RNAs are expressed by distinct mosaics of neurons, reflecting a potential for diverse neuronal functions. These results demonstrate an important role for these molecules in the activity-dependent remodeling and plasticity of connections in the developing and mature mammalian central nervous system (CNS).

Animals↗

PubMed1987

Human thymocyte subpopulations: maturational stages defined by the expression of the T3-T cell receptor complex.

Using sensitive fluorescence flow-cytometric techniques, human thymocyte subpopulations were fractionated according to their surface expression of the T3-T cell receptor (T3-Ti) complex. Two major subpopulations, one expressing low (immature subpopulation) and one high T3 antigen surface density (mature fraction) were characterized in detail with respect to surface antigen expression, right-angle scatter and proliferative capacity. Thymocyte subpopulations were activated through the T11 molecule (alternate pathway) and compared with regard to interleukin-2 (IL-2) receptor expression, changes in right-angle scatter and 3H-thymidine incorporation. We report that both populations could be activated through the T11 pathway to undergo nuclear activation and express IL-2 receptors. Moreover, in the absence of accessory cells, only the most mature population, expressing high T3 density, could be induced to proliferate, whereas immature cortical thymocytes required accessory cells for proliferation. These findings suggest that the cellular microenvironment may have a critical role in regulating the activation of immature cortical thymocytes.

Antigen-Presenting Cells↗

PubMed1984

Triggering of the T3-Ti antigen-receptor complex results in clonal T-cell proliferation through an interleukin 2-dependent autocrine pathway.

Human T-cell clones and anti-T-cell-receptor antibodies (clonotypic) directed at surface receptors for antigen (T3-Ti molecular complex) as well as anti-interleukin 2 (IL-2) and anti-IL-2-receptor antibodies were utilized to investigate the mechanism by which alloantigens or antigen plus self-major histocompatibility complex (MHC) (i.e., physiologic ligand) trigger specific clonal proliferation. Soluble or Sepharose-bound anti-Ti monoclonal antibodies, like physiologic ligand, enhanced proliferative responses to purified IL-2 by inducing a 6-fold increase in surface IL-2 receptor expression. In contrast, only Sepharose-bound anti-Ti or physiologic ligand triggered endogenous clonal IL-2 production and resulted in subsequent proliferation. The latter was blocked by antibodies directed at either the IL-2 receptor or IL-2 itself. These results suggest that induction of IL-2 receptor expression but not IL-2 release occurs in the absence of T3-Ti receptor cross-linking. Perhaps more importantly, the findings demonstrate that antigen-induced proliferation is mediated through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors.

Antibodies, Monoclonal↗