The protein interactions of the immunoglobulin receptor family tyrosine-based activation motifs present in the T cell receptor ζ subunits and the CD3 γ,δ and ϵ chains

Immunoglobulin family tyrosine-based activation motifs (ITAM), which define the conserved signaling sequence EX₂YX₂L/IX₇YX₂L/I, couple the T cell antigen receptor (TCR) to cellular proteins including protein tyrosine kinases (PTK) and adapter molecules. The TCR is a multichain complex with four invariant chains CD3γ, δ and ? that each contain a single ITAM and the TCR ζ chain that contains three ITAM. The present study explores the protein interactions of the doubly phosphorylated CD3 γ, δ, ? ITAM to determine whether they have common or unique biochemical properties. The data show that the doubly phosphorylated ITAM all bind the PTK ZAP-70, but the ITAM also variably bind the PTK p59fyn and the adapters Shc, Grb-2 and the p85 regulatory subunit of phosphoinositol 3′ kinase. The CD3 and ζ ITAM display a hierarchy of ZAP-70 binding: ζ1 = γ = δ > ζ3 > ζ2 = ?. Shc, Grb-2 and p85 could bind the ζ ITAM and the CD3 γ and δ ITAM, but not the CD3 ? ITAM. There were also subtle differences in the hierarchy of reactivity of these adapters for the CD3 γ,δ and ζ ITAM that show that the ζ, CD3 γ, δ and ? ITAM have different binding properties. The present study thus shows that the different ITAM of the TCR/CD3 complex can interact with different cytosolic effectors, indicating that differential ITAM phosphorylation during T cell activation could be a mechanism to generate signaling diversity by the TCR complex.     

The dynamic TCRδ: TCRδ chains in the amphibian Xenopus tropicalis utilize antibody‐like V genes

The content and organization of the Xenopus tropicalis TCRα/δ locus was determined. This locus is highly conserved among tetrapods, with the genes encoding the TCRδ chains embedded with those encoding TCRα. However, the frog TCRα/δ is unusual in that it contains V genes that appear indistinguishable from those in the IgH locus (VH). These V genes, termed VHδ, make up 70% of the V genes at the TCRδ locus and are expressed exclusively in TCRδ chains. Finding TCRδ chains that use antibody‐like V domains in frogs is similar to the situation in shark TCRδ variants and TCRμ in marsupials. These results suggest that such unconventional TCR may be more widespread across vertebrate lineages than originally thought and raise the possibility of previously unrealized subsets of T cells. We also revealed close linkage of TCRα/δ, IgH, and Igλ in Xenopus which, in combination with linkage analyses in other species, is consistent with the previous models for the emergence of these antigen receptor loci.     

T cell receptor γδ repertoire is skewed in cerebrospinal fluid of multiple sclerosis patients: molecular and functional analyses of antigen-reactive γδ clones

To study the relevance of γδ T cells in multiple sclerosis (MS) we analyzed the T cell receptor (TCR) γδ repertoire and the antigen reactivity of γδ clones isolated from cerebrospinal fluid (CSF). In T cell cultures derived from CSF we found an increased percentage of Vδ1⁺ cells as compared to peripheral blood of the same donors. Phenotypic analysis of cells from MS CSF with Vγ- and Vγ-specific monoclonal antibodies (mAb) showed that the Vγ1 chain is most frequently associated with γ chains belonging to the VγI family. Sequence analysis of TCR genes revealed heterogeneity of junctional regions in both δ and γ genes indicating polyclonal expansion. γδ clones were established and some recognized glioblastoma, astrocytoma or monocytic cell lines. Stimulation with these targets induced serine esterase release and lymphokine expression characteristic of the T_H0-like phenotype. Remarkably, these tumor-reactive γδ cells were not detected in the peripheral blood using PCR oligotyping, but were found in other CSF lines independently established from the same MS patient. Altogether, these results demonstrate that in the CSF there is a skewed TCR γδ repertoire and suggest that γδ cells reacting against brain-derived antigens might have been locally expanded.     

The T cell antigen receptor α and β chains interact via distinct regions with CD3 chains

Selective pairwise interactions between CD3 chains and the clonotypic T cell antigen receptor (TCR)-α, -β chains has recently been established. In this study, the region of interaction between clonotypic and CD3 chains involved with assembly was examined. To determine the site of protein interaction a variety of genetically altered TCR chains were constructed. These included: truncated proteins, lacking transmembrane and or cytosolic domains; chimeric proteins, in which extracellular, transmembrane or cytosolic domains were replaced with similar domains derived from either the Tac antigen or CD4; and point mutagenized TCR chains. COS-1 cells were transfected with cDNA, metabolically labeled, and immunoprecipitates analyzed using non-equilibrium pH gel electrophoresis (NEPHGE)-SDS/PAGE. The results demonstrated that assembly between TCR-α and TCR-β chains occurred at the extracellular level. Assembly of the TCR-α chain with CD3-δ, and CD3-ε was localized to an eight-amino acid motif within the transmembrane domain of TCR-α. Site-specific mutations of the TCR-α charged residues within this motif ( arginine, lysine) to leucine and similar point mutations of the transmembrane CD3-ε and CD3-δ charge groups resulted in the abrogation of assembly. In contast, TCR-β and CD3-ε binary complexes interacted via their extracellular domain. Analogous to TCR-α, the site of TCR-β and CD3-δ assembly was at the transmembrane region. Despite multiple genetic manipulations on CD3-γ and ζ; these proteins failed to assemble with TCR-α. Similarly, there was no interaction between TCR-β and ζ. These findings when coupled with the information on pairwise interactions and formation of higher order subcomplexes extend our model for the structure of the TCR complex.     

Mechanisms determining cell membrane expression of different γδ TCR chain pairings

We investigated the ability of the most common TCR‐γ and δ chains to express on the cell surface. Vγ1Cγ4 and Vγ7Cγ1 chains paired with all TCR‐δ chains tested, whereas Vγ4Cγ1 chains were found with Vδ4 and Vδ5, but not with Vδ2 or Vδ6 chains, and Vγ2Cγ2 chains were expressed only with Vδ5. Mapping studies showed that up to four polymorphic residues influence the different co‐expressions of Vγ1 and Vγ2 chains with Vδ chains. Unexpectedly, these residues are not located in the canonical γ/δ interface, but in the outer part of the γδ TCR complex exposed to the solvent. Expression of functional Vδ4 or Vδ6 chains in Vγ2/Vδ5⁺ cells or of functional Vγ2Cγ2 in Vγ1⁺ cells reduced cell‐surface expression of the γδ TCR. Taken together, these data show that (i) the Vγ/Vδ repertoire of mouse γδ T cells is reduced by physical constraints in their associations. (ii) Lack of Vγ2/Vδ expression is due to the formation of aberrant TCR complexes, rather than to an intrinsic inability of the chains to pair and (iii) despite not being expressed at the cell surface, the presence of a functionally rearranged Vγ2 chain in γδ T cells results in reduced TCR levels.     

Assembly of the TCR/CD3 complex: CD3ϵ/δ and CD3ϵ/γ dimers associate indistinctly with both TCRα and TCRβ chains. Evidence for a double TCR heterodimer model

The TCR/CD3 complex is composed of six subunits which are expressed on the cell surface in a coordinate fashion after assembly in the endoplasmic reticulum (ER). The TCR/CD3 complex is assembled after a series of pairwise interactions involving the formation of dimers of CD3ϵ with either CD3γ or CD3δ. These dimers assemble with TCRα and TCRβ chains, and finally, the CD3ζ homodimer is added to allow export of the full complex from the ER. A model has been proposed suggesting that during assembly the CD3ϵ/CD3γ dimer interacts exclusively with TCRβ and the CD3ϵ/CD3δ dimer with TCRα to form a complex with a single TCRα/β heterodimer. We show in this study, by immunoprecipitation and two-dimensional gel electrophoresis, that in the human T cell line Jurkat as well as in total human thymocytes, this preferential interaction does not occur and instead, the CD3ϵ/CD3γ and CD3ϵ/CD3δ dimers associate with both TCR chains simultaneously and indistinctly. These data are confirmed by the analysis of the TCRα-negative T cell line MOLT-4 in which TCRβ is found separately associated with CD3ϵ/CD3γ and with CD3ϵ/CD3δ dimers. Indirectly, our results support a model of stoichiometry in which two TCRα/β heterodimers are present in a TCR/CD3 complex. Furthermore, immunoprecipitation with anti-CD3γ and anti-CD3δ antibodies from 1 % NP40 and 1 % Brij96 cell lysates showed that these subunits form independent partial complexes which are cross-linked through the CD3ζ homodimer. This suggests that CD3ζ mediates the interaction between both TCRα/β heterodimers contained in the double TCR complex. Further proof for this hypothesis is obtained after analysis of a Jurkat cell transfectant containing a point mutation in the transmembrane domain of TCRβ that impairs the association of CD3ζ. In this mutant cell line, unlike a control line with wild-type TCRβ, the CD3γ- and CD3δ-containing complexes were found completely independent. Altogether, these results support a model of TCR/CD3 assembly and stoichiometry in which two TCR-α/β heterodimers form two hemicomplexes containing either CD3ϵ/γ or CD3ϵ/δ dimers which become associated via the CD3ζ homodimer.     

A TCR α chain transgene induces maturation of CD4− CD8− α β+ T cells from γ δ T cell precursors

The proportion of CD4⁻ CD8⁻ double-negative (DN) α β T cells is increased both in the thymus and in peripheral lymphoid organs of TCR α chain-transgenic mice. In this report we have characterized this T cell population to elucidate its relationship to α β and γ δ T cells. We show that the transgenic DN cells are phenotypically similar to γ δ T cells but distinct from DN NK T cells. The precursors of DN cells have neither rearranged endogenous TCRα genes nor been negatively selected by the Mls^a antigen, suggesting that they originate from a differentiation stage before the onset of TCR α chain rearrangements and CD4/CD8 gene expression. Neither in-frame VδDδJδ nor VγJγ rearrangements are over-represented in this population. However, since peripheral γ δ T cells with functional TCRβ gene rearrangements have been depleted in the transgenics, we propose that the transgenic DN population, at least partially, originates from the precursors of those cells. The present data lend support to the view that maturation signals to γ δ lineage-committed precursors can be delivered via TCR α β heterodimers.     

Characterization of γ/δ T cell clones isolated from human fetal liverand thymus

The origin and development of T cells bearing γ/δ T cell receptors (TcR) has been extensively studied in the mouse. By contrast, little is known about development patterns and diversity of the human γ/δ T cell lineage. To study therepertoire of human γ/δ⁺ T cells during T cell ontogeny, wehave isolated clonal populations of γ/δ⁺ T cells from 14-week fetal thymus and liver and characterized the molecular compositionof their TcR. The technique of in situ hybridization was used to identify cells expressing TcR genes in fetal liver and thymus. A panel of clones representative of developing T cell populations found in vivo was subsequently isolated from both tissues and clones expressing cell surface γ/δ receptors were identified. Although both the liver-derived γ/δ⁺ T cell clone, L2, and the thymus-derived γ/δ⁺ T cell clone, T6, had similar cell surface phenotypes, namely CD3⁺, CD7⁺, CD45⁺ and CD8^?, their reactivity with anti-CD2 and -CD4 antibodies was different. L2 was CD2^high, CD4^? whereas T6 was CD2^low, CD4^low. Both clones possessed effector functions similar to those of adult T cells as demonstrated by the synthesis and secretion of cytokines in response to stimulation through the CD3/TcR complex. Analysis of the TcR composition of the fetal clones showed both clones to possess similar or identical γ chain components, C_γ1, J_γ1/2, V_γ8, and both utilize V_δ gene segments other than V_δ1. This TcR genotype has not been previously reported in the analysis of adult γ/δ⁺ T cells. Our studies have identified a unique population of human γ/δ⁺ T cells that may be derived extrathymically and appear to be preferentially and perhaps transiently expressed during fetal life.     

T cell receptor heterogeneity in γδ T cell clones from intestinal biopsies of patients with celiac disease

In celiac disease large numbers of γδ T lymphocytes infiltrate the intestinal epithelia. We have isolated intestinal γδ T cell clones from patients with celiac disease and have analyzed their T cell receptor repertoire. T cell lines and clones were obtained from jejunal biopsies of 14 celiac patients and 12 individuals without celiac disease. These were analyzed by staining with monoclonal antibodies against CD3, αβ and γδ T cell receptor, by Southern blot with γ and δ specific probes and by polymerase chain reaction using Vδ-specific oligonucleotides. Intestinal γδ cells from patients with celiac disease differed from those of controls with normal jejunal histology in that Vδ1⁺ cells and Vδ Vδ2 cells were significantly increased. There was no evidence of the expansion of one or more clones expressing particular types of γδ T cell receptor.     

A novel subset of adult γδ thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire

We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of γδ thymocytes, initially defined by expressing low levels of Thy-1, that represents around 5 % and 30 % of total γδ thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS-sorted Thy-1^dull γδ thymocytes from DBA/2 mice with anti-γδ monoclonal antibodies in the presence of interleukin-2 (IL-2) results in the secretion of high levels of several cytokines, including interferon-γ (IFN-γ), IL-4, IL-10, and IL-3. In contrast, only IFN-γ was detected in parallel cultures of Thy-1^bright γδ thymocytes. Virtually all Thy-1^dull γδ thymocytes express high levels of CD44 and low levels of the heat-stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy-1^dull γδ thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy-1^dull γδ thymocytes from DBA/2 mice express TCR encoded by the Vγ1 gene and a novel Vδ6 gene named Vδ6.4. Sequence analysis of these functionally rearranged γ and δ genes revealed highly restricted Vδ-Dδ-Jδ junctions, and somewhat more diverse Vγ-Jγ junctions. We conclude that Thy-1^dull γδ thymocytes exhibit properties that are equivalent to those of natural killer TCRαβ T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.     

Transendothelial chemotaxis of human αβ and γδ T lymphocytes to chemokines

Two subpopulations of human T lymphocytes expressing different antigen receptors, α / β and γ / δ, emigrate into inflamed tissues in distinctive patterns. We compared the transmigration of α / β and γ / δ T cells to C-C and C-X-C chemokines using an in vitro transendothelial chemotaxis assay. The C-C chemokines monocyte chemoattractant protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1α and MIP-1β stimulated similar, dose-dependent chemotaxis of purified γ / δ T cells, whereas MCP-1, RANTES, and MIP-1α pro duced greater chemotaxis of purified α / β T cells than MIP-1β. In contrast, the C-X-C chemokines interleukin (IL)-8 and interferon-γ inducible protein-10 (IP-10) did not promote chemotaxis of either α / β or γ / δ T cells. Three γ / δ T cell clones with differing CD4 and CD8 phenotypes also migrated exclusively to C-C chemokines. Phenotypic analysis of mononuclear cells that transmigrated from an input population of unfractionated peripheral blood mono nuclear cells confirmed the results with purified γ / δ T cells. Our data demonstrate that human peripheral blood α / β and γ / δ T cells can transmigrate to MCP-1, RANTES, MIP-1α, and MIP-1β, and suggest that both T lymphocyte subpopulations share the capacity to emigrate in response to C-C chemokines during inflammation.     

CD48 May serve as an accessory molecule for the activation of a subset of human γ/δ T cells

To further assess the role of CD48 in the interaction of human γ/δ T cells with their specific target, we generated two series of alloreactive clones, L and K. These clones express a V1-D-J1-C δ chain associated to V3-J2-C2 (L) or V2-J2-C2 (K) γ chain. Functionally they were CTLs able to lyse the sensitizing B-cell line E418. The cytotoxicity of the L and K clones toward E418 was inhibited by anti-CD48 mAb. That of the L clones was also inhibited by anti-HLA class I mAbs. Variation in L and K lysis profile was observed against a panel of CD48 targets, further strengthening the argument that they display distinct specificities and suggesting that they do not recognize CD48. Heterogeneity in TCR gene segment usage, MHC-dependent recognition of E418 by the L clones, and resistance of some CD48⁴ targets strongly suggest that CD48 itself does not interact with L and K TCR. Transfection of CHO cells with CD48 induced killing by the K clones. This killing was inhibited by anti-CD48 mAbs. Taking into account the recent reports on CD48 as an accessory molecule, our results suggest that by binding to CD2 (and/or an unknown ligand), CD48 may serve to strengthen E/T interaction and may contribute to the activation of a minor subset of γ/δ T cells.     

The human natural killer cell receptor for major histocompatibility complex class I molecules. Surface modulation of p58 molecules and their linkage to CD3 ζ chain,FcϵRI γ chain and the p56lck kinase

The natural killer cell (NK)-specific p58 surface molecules, recognized by the GL183 and EB6 monoclonal antibodies (mAb), have been shown to represent the putative NK receptor for HLA-C molecules. The interaction between p58 receptors and HLA-C results in inhibition of the NK-mediated target cell lysis. In this study, GL183^?EB6⁺ clones (Cw4-specific), after mAb-induced surface modulation of EB6 molecules, acquired the ability to lyse the Cw4^? C1R cells. In NK clones co-expressing both GL183 and EB6 molecules and unable to kill Cw3-protected target cells, the mAb-induced modulation of EB6 molecules resulted both in selective co-modulation of GL183 molecules and in the lysis of Cw3-transfected P815 murine cells. In line with the co-modulation experiments we also show that the GL183 and EB6 molecules can be co-immunoprecipitated from GL183⁺/EB6⁺ clones after cell lysis in the presence of digitonin. The p58 receptor also revealed an association with molecules belonging to the ζ family (i.e. CD3 ζ and Fc?RI γ chains). Two-dimensional diagonal gel analysis of the p58 complex immunoprecipitated from polyclonally activated p58⁺ NK cells indicated a preferential association with CD3 ζ chains either in the form of covalently linked ζ-γ homodimers or in the form of ζ-γ heterodimers, while γ-γ homodimers were detectable in low amounts. However, p58⁺ clones displaying a unique association with γ-γ homodimers could also be isolated. Probing the immunoprecipitated p58 complex with anti-p56^lck antibody also revealed an association with this member of the src family. In addition, mAb-mediated signaling of NK clones via p58 molecules induced increments of p58/p56^lck association. However, under the same experimental conditions that induced optimal in vivo tyrosine phosphorylation of the CD16-associated CD3 ζ chains, no tyrosine phosphorylation was detected in the p58-associated CD3 ζ, chains. In these in vivo experiments neither anti-CD16 nor anti-p58 mAb could induce tyrosine phosphorylation of the γ chains. Finally, the anti-p58-mediated inhibition of the NK cell triggering via CD16 molecules was not accompained by a down-regulation of the tyrosine phosphorylation of the CD16-associated CD3 ζ chains.