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B Malissen

Publications and source records attributed to B Malissen.

At least 37 records · Page 2Linked to original sources
PubMed1999

Crippling of CD3-zeta ITAMs does not impair T cell receptor signaling.

We evaluated the importance of CD3-zeta ITAMs in T cell responses by breeding the P14 transgenic TCR into mice in which CD3-zeta chains lacking all or part of their ITAMs were genetically substituted for wild-type CD3-zeta chains. In contrast to the H-Y TCR, the P14 TCR permitted the development of peripheral CD8+ T cells harboring signaling-defective CD3-zeta subunits. The absence of functional CD3-zeta ITAMs did not reduce the spectrum of activation events and effector functions that constitute the normal attributes of mature CD8+ T cells. The only detectable differences were quantitative and noted only when T cells were challenged with suboptimal peptide concentrations. Therefore, the ITAMs present in the CD3-gammadeltaepsilon module are sufficient for qualitatively normal TCR signaling and those present in CD3-zeta have no exclusive role during T cell activation.

Animals↗
PubMed1998

Natural and engineered disorders of lymphocyte development.

Mammals have evolved complex developmental pathways to generate a large repertoire of B and T lymphocytes capable of mounting effective immune responses. Analysis of natural and engineered immunodeficiencies constitutes a powerful approach to delineating these pathways and identifying the molecular sensors that couple the survival of developing lymphocytes to the achievement of successful gene rearrangements at the loci coding for B and T cell antigen receptors. Besides identifying cytokines, growth factors, and transcription factors involved in lymphocyte development, genetic analysis also makes it possible to organize most of these protagonists into gene networks that control critical events in the life of developing lymphocytes.

Animals↗
PubMed1998

The CD3-gammadeltaepsilon and CD3-zeta/eta modules are each essential for allelic exclusion at the T cell receptor beta locus but are both dispensable for the initiation of V to (D)J recombination at the T cell receptor-beta, -gamma, and -delta loci.

The pre-T cell receptor (TCR) associates with CD3-transducing subunits and triggers the selective expansion and maturation of T cell precursors expressing a TCR-beta chain. Recent experiments in pre-Talpha chain-deficient mice have suggested that the pre-TCR may not be required for signaling allelic exclusion at the TCR-beta locus. Using CD3-epsilon- and CD3-zeta/eta-deficient mice harboring a productively rearranged TCR-beta transgene, we showed that the CD3-gammadeltaepsilon and CD3-zeta/eta modules, and by inference the pre-TCR/CD3 complex, are each essential for the establishment of allelic exclusion at the endogenous TCR-beta locus. Furthermore, using mutant mice lacking both the CD3-epsilon and CD3-zeta/eta genes, we established that the CD3 gene products are dispensable for the onset of V to (D)J recombination (V, variable; D, diversity; J, joining) at the TCR-beta, TCR-gamma, and TCR-delta loci. Thus, the CD3 components are differentially involved in the sequential events that make the TCR-beta locus first accessible to, and later insulated from, the action of the V(D)J recombinase.

Alleles↗
PubMed1998

Quantitative aspects of T-cell recognition: from within the antigen-presenting cell to within the T cell.

T lymphocytes circulate continually throughout the peripheral lymphoid organs, where they scrutinize the surface of cells to detect the presence of nonself protein fragments. During the last years, many facets of T-cell function have been unravelled. After being bound by major histocompatibility complex (MHC) molecules, peptides derived from nonself as well as from self proteins are delivered to the cell surface. A few copies of a nonself peptide "presented" at the cell surface in the context of an MHC molecule can be detected by specific T cells, and suffice to trigger T-cell activation. This paper reviews the requirements imposed on T cells to fulfill this exquisite sensitivity.

Antigen-Presenting Cells↗
PubMed1998

CD8 expression allows T cell signaling by monomeric peptide-MHC complexes.

Physiologically, TCR signaling is unlikely to result from the cross-linking of TCR-CD3 complexes, given the low density of specific peptide-MHC complexes on antigen-presenting cells. We therefore have tested directly an alternative model for antigen recognition. We show that monomers of soluble peptide-MHC trigger Ca2+ responses in CD8alphabeta+ T cells. This response is not observed in CD8- T cells and when either the CD8:MHC or CD8:Lck interactions are prevented. This demonstrates that an intact CD8 coreceptor is necessary for effective TCR signaling in response to monomeric peptide-MHC molecules. We propose that this heterodimerization of TCR and CD8 by peptide-MHC corresponds to the physiological event normally involved during antigen-specific signal transduction.

Amino Acid Sequence↗
PubMed1998

Glimpses at the recognition of peptide/MHC complexes by T-cell antigen receptors.

More than a decade after the first description of the primary structure of a T-cell antigen receptor (TCR), the recent determination of the crystal structure of several unliganded TCR ectodomains and of two TCRs complexed to peptide-MHC ligand provides a structural basis for understanding the initial event that triggers T-cell activation. This review focuses on the topology of the variable (V) domains found in TCRs and immunoglobulins and attempts to delineate the structural features that may render the TCR complementarity-determining regions particularly suited to dock on the peptide/MHC surface. Finally, the available TCR structures provide an opportunity to re-evaluate the molecular basis for intrathymic positive selection as well as the mechanisms that make a given TCR neither infinitely specific, nor so flexible that it engages productively any MHC-binding peptides.

Amino Acid Sequence↗
PubMed1997

CD45 and RPTPalpha display different protein tyrosine phosphatase activities in T lymphocytes.

To examine the substrate specificity and function of two receptor protein tyrosine phosphatases, CD45 and RPTPalpha, RPTPalpha was expressed in a CD45(-), T-cell receptor (TCR)+, BW5147 T-lymphoma cell. High levels of expression of RPTPalpha did not fully restore either proximal or distal TCR-mediated signalling events. RPTPalpha was unable to reconstitute the phosphorylation of CD3zeta and did not increase the expression of the activation marker, CD69, on stimulation with TCR/CD3. RPTPalpha did not significantly alter the phosphorylation state or kinase activity of two CD45 substrates, p56(lck) or p59(fyn), suggesting that RPTPalpha does not have the same specificity or function as CD45 in T-cells. Further comparison of the two phosphatases indicated that immunoprecipitated RPTPalpha was approx. one-seventh to one-tenth as active as CD45 when tested against artificial substrates. This difference in activity was also observed in vitro with purified recombinant enzymes at physiological pH. Additional analysis with Src family phosphopeptides and recombinant p56(lck) as substrates indicated that CD45 was consistently more active than RPTPalpha, having both higher Vmax and lower Km values. Thus CD45 is intrinsically a much more active phosphatase than RPTPalpha, which provides one reason why RPTPalpha cannot effectively dephosphorylate p56(lck) and substitute for CD45 in T-cells. This work establishes that these two related protein tyrosine phosphatases are not interchangeable in T-cells and that this is due, at least in part, to quantitative differences in phosphatase activity.

Animals↗
PubMed1997

The common cytokine receptor gamma chain controls survival of gamma/delta T cells.

We have investigated the role of common gamma chain (gamma c)-signaling pathways for the development of T cell receptor for antigen (TCR)-gamma/delta T cells. TCR-gamma/delta-bearing cells were absent from the adult thymus, spleen, and skin of gamma c-deficient (gamma c-) mice, whereas small numbers of thymocytes expressing low levels of TCR-gamma/delta were detected during fetal life. Recent reports have suggested that signaling via interleukin (IL)-7 plays a major role in facilitating TCR-gamma/delta development through induction of V-J (variable-joining) rearrangements at the TCR-gamma locus. In contrast, we detected clearly TCR-gamma rearrangements in fetal thymi from gamma c- mice (which fail to signal in response to IL-7) and reduced TCR-gamma rearrangements in adult gamma c thymi. No gross defects in TCR-delta or TCR-beta rearrangements were observed in gamma c- mice of any age. Introduction of productively rearranged TCR V gamma 1 or TCR V gamma 1/V delta 6 transgenes onto mice bearing the gamma c mutation did not restore TCR-gamma/delta development to normal levels suggesting that gamma c-dependent pathways provide additional signals to developing gamma/delta T cells other than for the recombination process. Bcl-2 levels in transgenic thymocytes from gamma c- mice were dramatically reduced compared to gamma c+ transgenic littermates. We favor the concept that gamma c-dependent receptors are required for the maintenance of TCR-gamma/delta cells and contribute to the completion of TCR-gamma rearrangements primarily by promoting survival of cells committed to the TCR-gamma/delta lineage.

Animals↗
PubMed1997

The three-dimensional structure of a T-cell antigen receptor V alpha V beta heterodimer reveals a novel arrangement of the V beta domain.

The crystal structure of a mouse T-cell antigen receptor (TCR) Fv fragment complexed to the Fab fragment of a specific anti-clonotypic antibody has been determined to 2.6 A resolution. The polypeptide backbone of the TCR V alpha domain is very similar to those of other crystallographically determined V alphas, whereas the V beta structure is so far unique among TCR V beta domains in that it displays a switch of the c" strand from the inner to the outer beta-sheet. The beta chain variable region of this TCR antigen-binding site is characterized by a rather elongated third complementarity-determining region (CDR3beta) that packs tightly against the CDR3 loop of the alpha chain, without leaving any intervening hydrophobic pocket. Thus, the conformation of the CDR loops with the highest potential diversity distinguishes the structure of this TCR antigen-binding site from those for which crystallographic data are available. On the basis of all these results, we infer that a significant conformational change of the CDR3beta loop found in our TCR is required for binding to its cognate peptide-MHC ligand.

Amino Acid Sequence↗
PubMed1997

A CD8 genomic fragment that directs subset-specific expression of CD8 in transgenic mice.

Helper and cytotoxic T cell subsets require the expression of different coreceptors (CD4 and CD8, respectively) for their development and function. We have cloned the CD8 gene locus from genomic cosmid and P1 libraries and analyzed the region around the CD8alpha and CD8beta genes for gene expression regulatory elements. DNase I (DNase I) hypersensitivity analysis of 80 kb in the CD8 locus identified four clusters of putative regulatory regions, three of which are thymocyte specific. Transgenic mice carrying the cloned CD8alphabeta genomic locus and containing the identified DNase I-hypersensitive site clusters express the transgenic CD8 in a developmentally regulated, tissue-specific, and CD8 T cell subset-specific manner.

Animals↗
PubMed1997

The single positive T cells found in CD3-zeta/eta-/- mice overtly react with self-major histocompatibility complex molecules upon restoration of normal surface density of T cell receptor-CD3 complex.

CD3-zeta/eta-deficient mice have small thymuses containing cells that show a profound reduction in the surface levels of T cell receptors and terminate their differentiation at the CD4+CD8+ stage. Rather unexpectedly, CD3- or very low single positive T cells accumulate over time in the spleen and lymph nodes of CD3-zeta/eta-deficient mice after a process dependent on MHC expression. Fusion of these peripheral T cells with a CD3-zeta-positive derivative of the BW5147 TCR-alpha-/beta- thymoma resulted in hybridomas that do express an heterogeneous set of T cell receptor alpha/beta dimers at their surface and at density comparable to those found in hybridomas derived from wild-type peripheral T cells. We have investigated the specificities of these T cell receptors using spleen cells from congenic and mutant mouse strains, and showed that the majority of them readily recognized self-MHC class I or class II molecules. These results demonstrate that by increasing the density and/or output of the T cell receptors expressed in peripheral T cells, one can confer them with the capacity to respond to normal density of self-MHC molecules.

Animals↗
PubMed1997

Qualitatively distinct signaling through T cell antigen receptor subunits.

T cell antigen receptors (TCR) contain several subunits including CD3gamma, delta, and epsilon, and TCRzeta and eta which are capable of mediating signal transduction. It is unclear whether the signaling function of these subunits is completely redundant. To assess the relative signaling capabilities of TCR subunits, we compared proximal events in signal transduction by wild-type TCR complexes and TCR devoid of functional zeta subunits, as well as chimeric receptors containing the cytoplasmic domains of TCRzeta or CD3epsilon. Results demonstrate that in BW5147 wild-type TCR, tail-less zeta TCR, CD3epsilon, and TCRzeta transduce signals leading to tyrosine phosphorylation of similar sets of cellular substrates, including the receptor subunits, Fyn, ZAP-70, and phospholipase Cgamma1 (PLCgamma1). Surprisingly, unlike wild-type TCR, tail-less zeta TCR, and CD3epsilon, TCRzeta was incapable of transducing signals resulting in inositol triphosphate (IP3) generation or intracellular free calcium ([Ca2+]i) mobilization. These data indicate that tyrosine phosphorylation of PLCgamma1 is not sufficient to drive IP3 production and [Ca2+]i mobilization. Most importantly, data presented indicate that TCRzeta and CD3epsilon engage partially distinct signaling pathways.

Amino Acid Sequence↗
PubMed1997

CD2-mediated signaling in T cells lacking the zeta-chain-specific immune receptor tyrosine-based activation (ITAM) motif.

T lymphocytes can be activated via the T cell receptor (TCR) or by triggering through a number of other surface structures, including the CD2 co-receptor molecule. Signaling through the CD2 molecule was shown previously to be dependent on the TCR-associated zeta-chain. Here, we show that CD2-induced activation also functions in T cells which express zeta-chains lacking a functional immune-receptor tyrosine-based activation motif (ITAM). TCR-positive T cells that express only the transmembrane part of the zeta-chain protein and thus lack a functional zeta-derived ITAM readily produce interleukin (IL)-2 when cross-linked with CD2-specific monoclonal antibodies (mAb). TCR-negative T cell hybridomas expressing minimal receptors consisting of an extracellular CD25 and an intracellular zeta-chain-derived segment were effectively stimulated via CD2-specific mAb. For CD2-mediated co-stimulation of TCR-negative cells, two zeta-chain-derived ITAM were sufficient to induce IL-2 when the CD2 molecules were co-cross-linked with the chimeric CD25-zeta molecules. Taken together, our results show that CD2-induced signaling does not necessarily employ the zeta-chain in TCR-positive cells and that CD2-dependent co-stimulation in TCR-negative cells can be mediated via two functional zeta-chain-derived ITAM.

Animals↗
PubMed1997

Analysis of immunoreceptor tyrosine-based activation motif (ITAM) binding to ZAP-70 by surface plasmon resonance.

The signaling function of the T cell antigen receptor (TCR) is mediated via CD3 polypeptides, the cytoplasmic sequences of which bear conserved immunoreceptor tyrosine-based activation motifs (ITAM). ITAM are defined by two YxxL/I sequences separated by a six-eight amino acid long spacer. Upon antigen recognition, ITAM become phosphorylated on both tyrosine residues, creating a high affinity binding site for the tandem SH2 domains found in the protein tyrosine kinase ZAP-70. Using surface plasmon resonance, we further dissected the sequences required for the binding of ZAP-70 to each TCR-associated ITAM. First, we generated protein tyrosine phosphatase-resistant ITAM peptide analogs, in which difluorophosphonomethyl phenylalanyl (F2p) replaced both phosphotyrosines, and showed that those protein tyrosine phosphatase-resistant analogs bind ZAP-70 with high affinity, establishing a rational strategy for the design of novel pharmacological tools capable of interfering with TCR signaling function. Second, we substituted the five amino acids separating the two YxxL/I sequences of the CD3 zeta 1 ITAM with a non-peptidic linker made up of gamma-amino butyric acid units and demonstrated that the length of this intervening sequence rather than its chemical composition is essential for high affinity binding of phosphorylated ITAM to the ZAP-70 SH2 domains.

Amino Acid Sequence↗
PubMed1996

CD8 beta increases CD8 coreceptor function and participation in TCR-ligand binding.

To study the role of CD8 beta in T cell function, we derived a CD8 alpha/beta-(CD8-/-) T cell hybridoma of the H-2Kd-restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma was transfected either with CD8 alpha alone or together with CD8 beta. All three hybridomas released interleukin 2 upon incubation with L cells expressing Kd-peptide derivative complexes, though CD8 alpha/beta cells did so more efficiently than CD8 alpha/alpha and especially CD8-/- cells. More strikingly, only CD8 alpha/beta cells were able to recognize a weak agonist peptide derivative variant. This recognition was abolished by Fab' fragments of the anti-Kd alpha 3 monoclonal antibody SF1-1.1.1 or substitution of Kd D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR-ligand binding on CD8 alpha/beta cells was approximately 5- and 20-fold more avid than on CD8 alpha/a and CD8-/- cells, respectively. SF1-1.1.1 Fab' or Kd mutation D227K reduced the TCR photoaffinity labeling on CD8 alpha/beta cells to approximately the same low levels observed on CD8-/- cells. These results indicate that CD8 alpha/beta is a more efficient coreceptor than CD8alpha/alpha, because it more avidly strengthens TCR-ligand binding.

Amino Acid Sequence↗