Stefan Meuer

An alternative pathway of T-cell activation: A functional role for the 50 kd T11 sheep erythrocyte receptor protein

A series of seven monoclonal antibodies was produced against the T-lineage-specific 50 kd T11 sheep erythrocyte rosette (SRBC) receptor protein in order to define the function of the molecule. Three distinct epitopes were detected: T11(1), the SRBC binding site expressed on all T lymphocytes and thymocytes; T11(2), an epitope unrelated to the SRBC binding site but with a similar distribution; and T11(3), a neo-epitope expressed only upon T-cell activation. Simultaneous triggering of T11(2) and T11(3) epitopes by monoclonal antibodies induces T lymphocytes to proliferate and mediate their functional programs in the absence of antigen and/or antigen-presenting cells. This antigen-independent mode of triggering is distinct from that involving the T3-Ti antigen receptor complex and represents an alternate pathway of T-cell activation. Given that T11 is the earliest T-lineage surface glycoprotein to appear in thymic ontogeny and is thus expressed before T3-Ti, the former may be involved in clonal expansion and/or differentiation during early development.

Antigen recognition by human T lymphocytes is linked to surface expression of the T3 molecular complex

Four distinct surface molecules on human T cells are defined by the monoclonal antibodies anti-T1, anti-T3 (anti-T3A), anti-T11 and anti-T12. Following cell binding, anti-T3 (anti-T3A) and anti-T1 induce independent modulation of their respective ligands, whereas anti-T11 and anti-T12 do not. To explore the biological consequences of this modulation, we used cloned populations of T4 and T8 cytotoxic T lymphocytes. Anti-T3 (anti-T3A), but not anti-T1, inhibits cytotoxic T lymphocyte effector function by T4 and T8 clones as well as antigen-specific T cell recognition. The latter is not secondary to a generalized inhibitory effect since responsiveness to interleukin 2 is maintained. Moreover, after modulation, cytotoxic T lymphocytes recover cytolytic function in parallel with reexpression of surface T3 molecules. We provide evidence for a direct linkage between antigen recognition by T lymphocytes and surface expression of the T3 molecular complex.

Expression of interleukin‐12‐related cytokine transcripts in inflammatory bowel disease: Elevated interleukin‐23p19 and interleukin‐27p28 in Crohn's disease but not in ulcerative colitis

Background: It has been suggested that Crohns disease (CD) is associated with an exaggerated T‐helper 1 cytokine response manifested by increased production of interleukin (IL)‐12. IL‐12 is a heterodimeric protein comprising 2 disulfide‐linked subunits designated p35 and p40. Recently, IL‐12‐related cytokines, IL‐23 and IL‐27, were described. Biologically active IL‐23 is a heterodimer whose p40 subunit is identical to IL‐12p40 whereas its p19 subunit is distantly related to IL‐12p35. IL‐27 consists of EBI3, an IL‐12p40‐related protein, and p28, a newly discovered IL‐12p35‐related polypeptide. Aim: We sought to determine whether mucosal expression of IL‐23p19 and IL‐27p28 transcripts correlate with the inflammatory activity in inflammatory bowel disease (IBD). Patients/Methods: Messenger RNA expression in colonic mucosa from patients with Crohns disease (CD; n = 37) and ulcerative colitis (UC; n = 19), and in non‐IBD control subjects (specific colitis [SC]; n = 16) and normal, nondiseased control patients (n = 12) was measured by reverse‐transcribed real‐time polymerase chain reaction. Results: IL‐23p19 was significantly increased in inflamed mucosa in CD (P = 0.0377) and to a lesser extent also in UC patients but not in SC patients. Elevation of IL‐23p19 transcript levels in CD correlated with the severity of endoscopic lesions. IL‐27p28 transcripts and EBI3 transcripts were significantly elevated only in active CD. Discussion: IL‐23p19, IL‐27p28, and EBI3 transcripts are strongly up‐regulated in CD. The stimulatory effects of these cytokines on naive T cells in addition to a strongly synergistic action with IL‐12 to trigger interferon‐&ggr; production may contribute to the perpetuation of the inflammatory process in patients with CD. Notably, increased expression of IL‐23 and IL‐27 transcripts in CD suggests a T helper 1‐dominated immunologic function in this disease.

The human T cell receptor: appearance in ontogeny and biochemical relationship of α and β subunits on IL-2 dependent clones and T cell tumors

Abstract The human T cell receptor for antigen (Ti) has recently been identified on IL-2 dependent T cell clones as a 90 kd disulfide-linked heterodimer comprised of one 49–51 kd alpha (α) and one 43 kd beta (β) chain. These subunits are noncovalently associated with a monomorphic 20–25 kd T3 molecule. Here, we produce monoclonal antibodies to a human tumor (REX) derived from an earlier stage of thymic differentiation in order to determine whether clonotypic structures are expressed and to define the ontogeny of Ti. The results of SDS-PAGE and peptide map analyses indicate that an homologous T3-associated heterodimer is synthesized and expressed by REX. This glycoprotein shares several peptides in common with clonotypic structures on an IL-2 dependent T cell clone. In addition, similar Ti related molecules appear during intrathymic ontogeny in parallel with surface T3 expression. The latter findings provide the structural basis for the immunological competence observed exclusively within the T3+ thymocyte compartment.

The human T-cell receptor.

Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to the identification of the human T-cell antigen receptor as a surface complex comprised of a clonotypic 90-kD Ti heterodimer and the invariant 20- and 25-kD T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunologic competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate genes. Moreover, the presence of unique peptides following proteolysis of different Ti molecules isolated by non-cross-reactive anticlonotypic monoclonal antibodies supports the notion that variable regions exist within both the alpha and the beta subunits. N-Terminal amino acid sequencing and molecular cloning of the Ti beta subunit further show that it bears an homology to the first V-region framework of immunoglobulin light chains and represents the product of a gene that rearranges specifically in T lymphocytes. Triggering of the T3-Ti receptor complex gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors. The implications of these findings for understanding human T-cell growth and its regulation in disease states are discussed.

The serine phosphatases PP1 and PP2A associate with and activate the actin‐binding protein cofilin in human T lymphocytes

Cofilin, an actin‐depolymerizing protein, is essential for the functional dynamics of the actin cytoskeleton and for cell viability. In unstimulated human peripheral blood T lymphocytes cofilin is phosphorylated and localized in the cytoplasm. Following co‐stimulation through accessory receptors (e.g. CD2 or CD28) – however, not following TCR/CD3 stimulation alone – cofilin undergoes dephosphorylation. The subcellular localization as well as the actin‐binding activity of cofilin are regulated by the phosphorylation state of serine‐3. Thus, only the dephosphorylated form of cofilin associates with the actin cytoskeleton and possesses the capability to translocate into the nucleus. Recently, LIM‐kinase 1 was shown to inactivate cofilin through phosphorylation. Here, we have identified the functional counterparts of LIM‐kinase 1: the serine/threonine phosphatases of type 1 and type 2A not only associate with cofilin but also dephosphorylate this 19‐kDa protein and thereby mediate cofilin activation. In malignant T lymphoma cells, activation of these phosphatases occurs spontaneously, independent of external stimuli. In untransformed human peripheral blood T lymphocytes, these phosphatases function through a cyclosporin A/FK506‐resistant co‐stimulatory signaling pathway which is common for the accessory receptors CD2 and CD28. This co‐stimulatory signaling pathway is also not affected by a series of other clinically established immunosuppressive drugs (i.e. rapamycin, dexamethasone, leflunomide or mycophenolic acid).

In Situ Expression of Interleukin-10 in Noninflamed Human Gut and in Inflammatory Bowel Disease

A dysregulated secretion of contra-inflammatory cytokines such as interleukin-10 (IL-10) could play a role in the pathogenesis of inflammatory bowel disease (IBD). We have investigated the expression of IL-10 in gut tissues from patients with Crohns disease (CD), ulcerative colitis (UC) and controls by mRNA in situ hybridization and immunohistochemistry. Intestinal epithelial cells were found to express IL-10 mRNA and IL-10 protein in all of the tissues investigated without any major differences in the expression patterns. However, compared with noninflamed gut, significantly increased numbers of mononuclear cells (MNCs) producing IL-10 were present in inflamed gut, both in CD and UC. This cytokine was expressed most prominently by inflammatory infiltrates enriched in macrophages, although T cells seem to contribute to its production as well. Elevated IL-10 expression in IBD was mainly detected in the submucosa, whereas IL-10 production by lamina propria cells remained comparably low. In contrast, the expression of IL-1beta mRNA was preferentially increased in the lamina propria. Our data argue against a general deficiency in IL-10 production in IBD. The results suggest rather that the local production of IL-10 by mucosal MNCs in IBD is insufficient to down-regulate pro-inflammatory cytokines such as IL-1beta in the lamina propria compartment.

The delineation of antigen receptors on human T lymphocytes

Monoclonal antibodies have identified several surface molecules involved in target cell recognition by cytotoxic human T cells. In this article it is proposed that T cells have two recognition units: a complex composed of the T3 molecule and a clonally unique glycoprotein which binds antigen associated with polymorphic MHC gene product; and the T4 or T8 molecule which binds to a constant region of an MHC gene product.

The human T cell receptor: analysis with cytotoxic T cell clones

Employing human T lymphocyte clones and monoclonal antibodies to their surface glycoproteins, the antigen receptors on these cells have been defined. Based on functional and biochemical data, it is shown that each T cell displays two major recognition units on its surface. One structure consists of the antigen-binding region (Tin-T3) which views antigen X in the context of a polymorphic region of an MHC molecule. A second (T8 or T4) serves as an associative recognition structure for a constant region of the class I or class II molecule. The antigen-binding structure is a heterodimer of disulfide linked 49KD and 43KD subunits, which contains clonally unique variable regions. These are non-covalently associated with the 20/25KD monomorphic T3 molecule expressed on all mature human T lymphocytes. The associative-recognition element on an individual clone is either T8 or T4, depending on its subset derivation. It is likely that these glycoproteins bind to constant regions of class I or class II molecules, respectively, and are independent of the Tin-T3 complex complex.

Activation and Signaling Status of Human Lamina Propria T Lymphocytes

In this study, proliferative responses of human lamina propria T lymphocytes were examined in vitro. The response of lamina propria T lymphocytes to Sepharose-bound anti-CD3 antibody plus interleukin 2 was significantly lower than the response of autologous peripheral blood T lymphocytes, whereas the responses of lamina propria T lymphocytes to anti-T11(2/3) antibodies plus sheep erythrocytes or anti-CD28 antibody plus interleukin 2 were largely preserved. After coculture with mucosa supernatant, peripheral blood T lymphocytes showed a similar pattern of reactivity as lamina propria T lymphocytes. This reduced reactivity to T-cell antigen receptor stimulation appears to exist at the level of signal transduction, because triggering of CD3 induces low amounts of intracellular inositol 1,4,5-triphosphate and no free calcium increase in lamina propria T lymphocytes when compared with peripheral blood T lymphocytes. This study indicates that the antigen receptor-dependent activation pathway of lamina propria T lymphocytes for proliferation is down-regulated by intestinal mucosa derived factor(s) and that the alternative pathways mediated by CD2 or CD28 are largely preserved. Based on previous data that lamina propria T lymphocytes can provide help to B cells, it is possible that these alternative activation pathways play an important role in T-B cell interaction in the gut.