Differential expression of two distinct T-cell receptors during thymocyte development

The product of the T-cell receptor (TCR) gamma-gene has recently been found to be expressed on a subset of both peripheral cells and thymocytes. As an initial approach to understanding the role of this gamma-chain of TCR (TCR gamma) in T-cell development, we have studied the ontogeny of TCR expression at the protein level in the developing murine thymus. We show here that the first T3-associated TCR to be expressed in the developing thymus is a disulphide-linked heterodimer composed of a gamma-chain of relative molecular mass 35,000 (Mr 35K) and a 45K partner (termed TCR delta). This TCR gamma delta is first detected approximately two days before the appearance of cell-surface TCR alpha beta heterodimers. We report that N-glycosidase digestions reveal that all of the gamma-protein expressed on fetal thymocytes, as in adult CD4-8-(L3T4-, Lyt2-) thymocytes, bear N-linked carbohydrate side chains. The major gamma-gene transcribed in mature, alpha beta-bearing T cells (V gamma 1.2C gamma 2)encodes no N-linked glycosylation site so these results suggest that the fetal gamma delta receptor defines a distinct T-cell lineage whose development in the thymus precedes classical alpha beta-bearing cells.     

CD3delta couples T-cell receptor signalling to ERK activation and thymocyte positive selection

Thymocytes from mice lacking the CD3delta chain of the T-cell receptor (TCR), unlike those of other CD3-deficient mice, progress from a CD4- CD8- double-negative to a CD4+ CD8+ double-positive stage. However, CD3delta-/- double-positive cells fail to undergo positive selection, by which double-positive cells differentiate into more mature thymocytes. Positive selection is also impaired in mice expressing inactive components of the Ras/mitogen activated protein (MAP) kinase signalling pathway. Here we show that CD3delta-/- thymocytes are defective in the induction of extracellular signal-regulated protein kinase (ERK) MAP kinases upon TCR engagement, whereas activation of other MAP kinases is unaffected. The requirement for CD3delta maps to its extracellular or transmembrane domains, or both, as expression of a tail-less CD3delta rescues both ERK activation and positive selection in CD3delta-/- mice. Furthermore, the defect correlates with severely impaired tyrosine phosphorylation of the linker protein LAT, and of the CD3zeta chain that is localized to membrane lipid rafts upon TCR engagement. Our data indicate that the blockade of positive selection of CD3delta-/- thymocytes may derive from defective tyrosine phosphorylation of CD3zeta in lipid rafts, resulting in impaired activation of the LAT/Ras/ERK pathway.     

Distinct sequence of negative or positive selection implied by thymocyte T-cell receptor densities

Recent evidence suggests that positive and negative selection of thymocytes bearing alpha beta T-cell receptors occurs during the predominant double-positive (CD4+CD8+) stage. But the sequence or stage at which positive or negative selection occurs during thymocyte maturation has not been well defined. Here we use transgenic mice to show that the CD4+CD8+ stage might be further subdivided into CD3lo (low) and CD3in (intermediate) stages. The CD3in stage could represent T cells that have been positively selected, as this stage is dependent on the presence of the appropriate major histocompatibility complex restriction element. In addition, we use two different tolerizing antigens to show that negative selection may occur either before or after this CD3in stage.     

Histamine regulates T-cell and antibody responses by differential expression of H1 and H2 receptors

Many pathological processes, including those causing allergies and autoimmune diseases, are associated with the presence of specialized subsets of T helper cells (TH1 and TH2) at the site of inflammation. The diversity of TH1 and TH2 function is not predetermined but depends on signals that drive the cells towards either subset. Histamine, released from effector cells (mast cells and basophils) during inflammatory reactions can influence immune response. Here we report that histamine enhances TH1-type responses by triggering the histamine receptor type 1 (H1R), whereas both TH1- and TH2-type responses are negatively regulated by H2R through the activation of different biochemical intracellular signals. In mice, deletion of H1R results in suppression of interferon (IFN)-gamma and dominant secretion of TH2 cytokines (interleukin (IL)-4 and IL-13). Mutant mice lacking H2R showed upregulation of both TH1 and TH2 cytokines. Relevant to T-cell cytokine profiles, mice lacking H1R displayed increased specific antibody response with increased immunoglobulin-epsilon (IgE) and IgG1, IgG2b and IgG3 compared with mice lacking H2R. These findings account for an important regulatory mechanism in the control of inflammatory functions through effector-cell-derived histamine.     

Homing of a gamma delta thymocyte subset with homogeneous T-cell receptors to mucosal epithelia

In mice gamma delta T-cell populations with distinct T-cell receptor (TCR) repertoires and homing properties have been identified. Diversified populations are found in lymphoid organs and intestinal epithelia. By contrast, the gamma delta T-cells that have been found in the murine skin are homogeneous. They express a TCR consisting of one particular V gamma 5 and one particular V delta 1 chain and seem to originate from early fetal thymocytes. We have now systematically analysed many tissues by immunohistochemistry and TCR gene sequencing aided by the polymerase chain reaction. These studies revealed a second homogeneous gamma delta T-cell subset in epithelia not of the intestine and skin, but of the vagina, uterus and tongue. The TCR expressed by this gamma delta T-cell subset consists of the same V delta 1 chain. Cells that express this particular TCR have previously been shown to be positively selected in the late fetal thymus.     

Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages.

Analysis of mice carrying mutant T-cell antigen receptor (TCR) genes indicates that TCR-beta gene rearrangement or expression is critical for the differentiation of CD4-CD8- thymocytes to CD4+CD8+ thymocytes, as well as for the expansion of the pool of CD4+CD8+ cells. TCR-alpha is irrelevant in these developmental processes. The development of gamma delta T cells does not depend on either TCR-alpha or TCR-beta.     

Cbl-b regulates the CD28 dependence of T-cell activation

Whereas co-stimulation of the T-cell antigen receptor (TCR) and CD28 triggers T-cell activation, stimulation of the TCR alone may result in an anergic state or T-cell deletion, both possible mechanisms of tolerance induction. Here we show that T cells that are deficient in the adaptor molecule Cbl-b (ref. 3) do not require CD28 engagement for interleukin-2 production, and that the Cbl-b-null mutation (Cbl-b(-/-)) fully restores T-cell-dependent antibody responses in CD28-/- mice. The main TCR signalling pathways, such as tyrosine kinases Zap-70 and Lck, Ras/mitogen-activated kinases, phospholipase Cgamma-1 and Ca2+ mobilization, were not affected in Cbl-b(-/-) T cells. In contrast, the activation of Vav, a guanine nucleotide exchange factor for Rac1/Rho/CDC42, was significantly enhanced. Our findings indicate that Cbl-b may influence the CD28 dependence of T-cell activation by selectively suppressing TCR-mediated Vav activation. Mice deficient in Cbl-b are highly susceptible to experimental autoimmune encephalomyelitis, suggesting that the dysregulation of signalling pathways modulated by Cbl-b may also contribute to human autoimmune diseases such as multiple sclerosis.     

Cloning of complementary DNA encoding T-cell replacing factor and identity with B-cell growth factor II

Proliferation and maturation of antigen-stimulated B cells are regulated by several soluble factors derived from macrophages and T cells. These soluble factors are functionally divided into two groups: B-cell growth factor (BCGF), thought to be involved in B-cell proliferation; and B-cell differentiation factor (BCDF), responsible for maturation of activated B cells into immunoglobulin-secreting cells. This classification needs to be re-examined in the light of the recent cloning of complementary DNA encoding IgG1 induction factor (interleukin-4, IL-4) from the 2.19 mouse T-cell line. Recombinant IL-4 has BCGF and BCDF activities and affects B cells, T cells and mast cells (refs 7, 8; our unpublished data). Another well-characterized B-cell factor is T-cell replacing factor (TRF), which, when secreted by the murine T-cell hybridoma B151K12, is defined by two activities: induction of IgM secretion by BCL1 leukaemic B-cell line; and induction of secondary anti-dinitrophenol (DNP) immunoglobulin G (IgG) synthesis in vitro by DNP-prime B cells. Although TRF from B151K12 was classified as BCDF, purified TRF has BCGF-II activity. To elucidate the molecular properties of TRF we isolated cDNA encoding TRF from the 2.19 T-cell line and report here the structure and multiple activities of this lymphokine.     

A sphingosine-1-phosphate receptor regulates cell migration during vertebrate heart development

Coordinated cell migration is essential in many fundamental biological processes including embryonic development, organogenesis, wound healing and the immune response. During organogenesis, groups of cells are directed to specific locations within the embryo. Here we show that the zebrafish miles apart (mil) mutation specifically affects the migration of the heart precursors to the midline. We found that mutant cells transplanted into a wild-type embryo migrate normally and that wild-type cells in a mutant embryo fail to migrate, suggesting that mil may be involved in generating an environment permissive for migration. We isolated mil by positional cloning and show that it encodes a member of the lysosphingolipid G-protein-coupled receptor family. We also show that sphingosine-1-phosphate is a ligand for Mil, and that it activates several downstream signalling events that are not activated by the mutant alleles. These data reveal a new role for lysosphingolipids in regulating cell migration during vertebrate development and provide the first molecular clues into the fusion of the bilateral heart primordia during organogenesis of the heart.     

Monoclonal suppressor T-cell factor displaying V H restriction and fine antigenic specificity

The production of stable T-cell clones is essential for the study of T-cell-derived, specific immunoregulatory products and of specific T-cell receptors. T-cell clones have been established by radiation leukaemia virus (RadLV)-induced transformation of suppressor T lymphocytes specific for hen egg white lysozyme (HEL). We report here that culture supernatant obtained from these T-cell clones can, when injected into mice, specifically suppress the anti-HEL antibody response. This monoclonal T-cell product suppresses the antibody response induced by HEL and human lysozyme, but not that induced by ring-necked pheasant egg white lysozyme (REL), thus displaying fine antigenic specificity probably restricted to an epitope involving phenylalanine at amino acid residue 3, present in the N-terminal region of HEL and shared by human lysozyme but absent in REL. The suppression induced by this monoclonal T-cell product is restricted by both H-2 and Igh-1 genes whereas anti-HEL antibodies bearing a predominant idiotype are induced in all mice strains tested, irrespective of their H-2 haplotype or Igh-1 allotype.     

A monoclonal antibody against antigen-specific helper factor augments T-cell help

Antigen-specific molecules, commonly termed 'factors', have been shown to be released from helper and suppressor T cells. These factors mimic the activity of the cells that secrete them and there is much speculation about the relationship of antigen-specific factors to T-cell receptors for antigen. We have raised a variety of antisera in rabbits which were shown to react against conserved 'constant' determinants on either helper or suppressor factors independently of antigenic specificity or mouse strain of origin of the factor. In contrast, syngeneic mouse antisera were found to react with 'variable' factor determinants in an antigen-specific and mouse strain-dependent manner. These antisera thus define two regions on factor molecules, one 'variable' (related to antigen specificity) and the other 'constant' (related to function). However, potential contaminants in these antisera have limited their usefulness. Thus, we are now generating monoclonal antibodies against T-cell factors and report here the properties of a monoclonal antibody (AF3.44.4) which reacts with antigen-specific helper factors. This antibody also binds to helper T cells and, in the presence of antigen, augments helper cell induction in vitro, which, in turn, leads to enhanced antibody production in vitro. These characteristics suggest that AF3.44.4 recognizes a determinant shared by helper factor and the antigen receptor on helper T cells.