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Biomedical subjects

Ed Palmer

Publications and source records attributed to Ed Palmer.

At least 19 recordsLinked to original sources
PubMed2006

Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling.

A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.

Animals↗
PubMed2006

Lck regulates the threshold of activation in primary T cells, while both Lck and Fyn contribute to the magnitude of the extracellular signal-related kinase response.

The src family kinases p56lck (Lck) and p59fyn (Fyn) are the most proximal signaling molecules to be activated downstream of the T-cell receptor. Using an inducible transgenic model, we can regulate the expression of Lck in primary T cells and ask how the signaling cascade and differentiation potential are affected by the absence or the presence of reduced levels of Lck. We show that in naïve T cells, Lck controls the threshold of activation by preferentially regulating multiple signaling pathways that result in the mobilization of Ca2+ through activation of phospholipase C-gamma and protein kinase C as well as activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Fyn is also able to stimulate the ERK/MAPK pathway in primary T cells but has little influence on the mobilization of Ca2+. Only Lck efficiently stimulates production of diacylglycerol and therefore RasGRP1 recruitment to the plasma membrane and phosphorylation of Shc, suggesting that Fyn activates ERK via a different upstream signaling route. Finally, we show that signals through Lck are essential for the development of T-cell-effector potential, particularly for effective cytokine transcription.

Adaptor Proteins, Signal Transducing↗
PubMed2006

The T-cell antigen receptor: a logical response to an unknown ligand.

The immune system can be roughly divided into innate and adaptive compartments. The adaptive compartment includes the B and T lymphocytes, whose antigen receptors are generated by recombination of gene segments. The consequence is that the creation of self-reactive lymphocytes is unavoidable. For the host to remain viable, the immune system has evolved a strategy for removing autoimmune lymphocytes during development. This review discusses how T lymphocytes are generated, how they recognize antigens, and how their antigen receptor directs the removal of self-reactive T cells.

Animals↗
PubMed2005

The CD3epsilon proline-rich sequence, and its interaction with Nck, is not required for T cell development and function.

The CD3epsilon proline-rich sequence (PRS) binds to the cytosolic adaptor molecule Nck after TCR ligation. It has been proposed that this interaction is essential for immunological synapse formation and T cell activation. To assess the physiological importance of the CD3epsilon PRS, we have generated mice that lack this motif (CD3epsilon.PRS(M)). Pull-down experiments demonstrated the inability of Nck to bind to the CD3epsilon PRS in thymocytes from mutant mice after TCR ligation. Surprisingly, no differences were observed in the number and percentage of T cell subsets in the thymus and spleen, and there was no apparent defect in positive or negative selection. Furthermore, the proliferative response of CD3epsilon.PRS(M) T cells to staphylococcal enterotoxin B and anti-CD3 Ab was normal. TCR surface expression, constitutive internalization, and Ag-induced down-modulation were also normal. These data suggest that the interaction between the CD3epsilon PRS and Nck, or any other Src homology 3 domain-containing molecule, is not essential for T cell development and function.

Adaptor Proteins, Signal Transducing↗
PubMed2005

T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes.

The T cell receptor (TCR) can recognize a variety of cognate peptide/major histocompatibility complex (pMHC) ligands and translate their affinity into distinct cellular responses. To achieve this, the nonsignaling alphabeta heterodimer communicates ligand recognition to the CD3 signaling subunits by an unknown mechanism. In thymocytes, we found that both positive- and negative-selecting pMHC ligands expose a cryptic epitope in the CD3 complex upon TCR engagement. This conformational change is induced in vivo and requires the expression of cognate MHC. We conclude that TCR engagement with a cognate pMHC ligand induces a conformational change in the CD3 complex of thymocytes and propose that this marks an initial event during thymic selection that signals the recognition of self-antigen.

Animals↗
PubMed2005

Distinct temporal programming of naive CD4+ T cells for cell division versus TCR-dependent death susceptibility by antigen-presenting macrophages.

Naive T cells become programmed for clonal expansion and contraction during the early hours of antigenic signaling. Recent studies support an 'autopilot' model, wherein the commitment to proliferate and the magnitude of the proliferative response are simultaneously determined during a single, brief period of antigen exposure. Here, we have examined whether the proliferation of naive CD4+ T cells must occur on 'autopilot', or whether extended periods of antigenic signaling can impact primary proliferative responses to antigen-presenting macrophages (macrophage APC). We found that a single exposure to antigen (18 h) simultaneously committed T cells to (1) up-regulate surface TCR above the level expressed on naive T cells, (2) undergo minimal cell division, and (3) acquire susceptibility to TCR-dependent activation-induced cell death. However, continued antigenic signaling between 18 and 72 h was required to amplify the number of daughter cells derived from the already committed T cells. Thus, a discrete commitment time was followed by a 'tuning' period, where extended antigenic signaling determined the volume of the proliferative response. We conclude that T cell commitment to full clonal expansion versus TCR-dependent death susceptibility represent two separate programming events whose timing can be segregated by macrophage APC.

Animals↗
PubMed2004

A chimeric T cell receptor with super-signaling properties.

A key question yet to be resolved concerns the structure and function relationship of the TCR complex. How does antigen recognition by the TCR-alphabeta chains result in the activation of distinct signal transduction pathways by the CD3-gammadeltaepsilon/zeta complex? To investigate which part of the TCR-beta chain is involved in TCR signaling, we exchanged different domains of the constant regions of the TCR-beta chain with the corresponding TCR-gamma chain domains. We show here that hybridoma cells expressing a chimeric TCR-beta chain (betaIII) containing intracellular and transmembrane TCR-gamma amino acids, together with a wild-type TCR-alpha (alphawt) chain, were 10 times more sensitive to antigenic stimulation compared to cells expressing TCR-alphawt/betawt chains. This super-signaling phenotype of the betaIII chain was observed in two different TCRs. One specific for an alloantigen (I-A(bm12)) and one for an autoantigen (I-A(b)/MOG(35-55)). We found that this chimeric alphawt/betaIII TCR had normal association with CD3-gammadeltaepsilon and zeta chains. To investigate the effect of the chimeric betaIII chain in transgenic T cells, we made MOG(35-55)-specific TCR transgenic mice expressing either the alphawt/betawt or chimeric alphawt/betaIII TCR. Similar to what was observed in hybridoma cells, transgenic alphawt/betaIII T cells showed a super-signaling phenotype upon antigenic stimulation. Further studies may help us understand the effect of increased TCR signaling on autoimmunity and may lead to the identification of signaling molecules that can be targeted to stop the progression of autoimmune disorders such as multiple sclerosis.

Animals↗
PubMed2004

T cell division and death are segregated by mutation of TCRbeta chain constant domains.

We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.

Amino Acid Sequence↗
PubMed2003

The beta1 and beta3 integrins promote T cell receptor-mediated cytotoxic T lymphocyte activation.

Recognition by CD8+ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that beta1 and beta3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC.peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling.

Animals↗
PubMed2003

Signaling life and death in the thymus: timing is everything.

T lymphocytes are generated in the thymus, where developing thymocytes must accept one of two fates: They either differentiate or they die. These fates are chiefly determined by signals that originate from the T cell receptor (TCR), a single receptor complex with a remarkable capacity to decide between distinct cell fates. This review explores TCR signaling in thymocytes and focuses on the kinetic aspects of ligand binding, coreceptor involvement, protein phosphorylation, and mitogen-activated protein kinase (MAPK) activation. Understanding the logic of TCR signaling may eventually explain how thymocytes and T cells distinguish self from nonself, a phenomenon that has fascinated immunologists for 50 years.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Mutational analysis of conserved amino acids in the T cell receptor alpha-chain transmembrane region: a critical role of leucine 112 and phenylalanine 127 for assembly and surface expression.

Correct assembly of all TCR complex polypeptides is essential for its cell surface expression and function. The transmembrane region of the TCRalpha chain is highly conserved and to gain insight into the structural and functional role of these residues, single amino acid substitutions were introduced and surface expression and signaling ability studied in T hybridoma cells. Introduction of acid residues within the TCRalpha chain transmembrane region were mostly tolerated, indicating that the net charge within this region of the TCR complex is not crucial to either assembly or signaling. However, mutations of leucine 112 or phenylalanine 127 to aspartic acids (L112D or F127D, respectively) resulted in dramatic loss of surface expression and, therefore, their signaling ability. Intracellular flow cytometry showed that the mutant TCRalpha polypeptides were present at levels comparable to wild-type, indicating that the reduced surface expression was not a consequence of impaired protein survival. The defect was characterized by immunoprecipitation and showed that residues L112 and F127 were involved in early interactions with the CD3 complex. A large proportion of the TCRalpha chain mutants L112D and F127D consisted of immature protein, indicative of a problem during early assembly of the TCR. Our findings provide evidence for the involvement of the conserved L112 and F127 residues of the TCRalpha chain transmembrane region in the assembly process of the TCR complex.

Amino Acid Sequence↗
PubMed2003

Allograft rejection in a new allospecific CD4+ TCR transgenic mouse.

The application of TCR transgenic mice to transplantation immunology is hampered by the limited lines available. Recently, we reported CD4+ T cell receptor (TCR) transgenic mice specific for I-Abm12 expressed on B6.C.H-2bm12 mice. Here, we characterized rejection of skin and vascularized cardiac allografts in these mice, which we term ABM (for anti-bm12). In vivo proliferative experiments reveal that all CD4 T cells in ABM mice react to bm12 antigens. Surprisingly, while ABM mice have accelerated (compared to B6 recipients) rejection of bm12 skin allografts, they, like B6 recipients, fail to acutely reject bm12 cardiac allografts. This is not due to lack of immunogenicity of bm12 hearts, as these grafts are acutely rejected by primed ABM recipients, although not by primed B6 recipients. Lastly, long-term surviving bm12 grafts in ABM recipients are relatively free from chronic rejection (compared with B6 recipients), which may be due to skewing of the CD4 repertoire towards direct alloreactivity, and consequent lack of CD4 mediated indirect allorecognition as evidenced by the lack of IgG deposition in those grafts. The results indicate that a complex interplay between responder frequency, priming and repertoire dictates the occurrence, or lack thereof, of acute and chronic rejection.

Animals↗
PubMed2003

Negative selection--clearing out the bad apples from the T-cell repertoire.

Dead cells are a prominent feature of the thymic landscape as only 5% of developing thymocytes are exported as mature T cells. The remaining thymocytes die by one of two mechanisms; most thymocytes die because they are not positively selected and do not receive a survival signal, whereas a minority of thymocytes undergo T-cell receptor (TCR)-mediated apoptosis, a process known as negative selection. Negative selection is extremely important for establishing a functional immune system, as it provides an efficient mechanism for ridding the T-cell repertoire of self-reactive and potentially autoimmune lymphocytes. This review discusses several cellular and molecular aspects of negative selection.

Apoptosis↗
PubMed2003

Surface T-cell antigen receptor expression and availability for long-term antigenic signaling.

It is important to understand how T-cell antigen receptor (TCR) engagement and signaling are regulated throughout an immune response. This review examines the dynamics of surface TCR expression and signaling capacity during thymic and effector T-cell development. Although the TCR can undergo vast changes in surface expression, T cells remain capable of sustaining TCR engagement for long periods of time. This may be achieved by a combination of mechanisms that involve (a) controlling the quantity of surface TCR available for ligand interaction and (b) controlling the quality of surface TCR expression during T-cell activation. TCR signaling itself appears to be one of the main quantitative modulators of surface TCR expression, and it can cause both downregulation and upregulation at different times of T-cell activation. Recent studies indicate that the degree of upregulation is tunable by the strength of antigenic stimulation. There is evidence that qualitatively distinct forms of the TCR exist, and their potential role in sustained antigenic signaling is also discussed. A goal of future studies will be to better characterize these modulations in surface TCR expression and to clarify their impact on the regulation of immune responses.

Animals↗
PubMed2003

New TCR transgenic model for tracking allospecific CD4 T-cell activation and tolerance in vivo.

We have developed an adoptive transfer model system to visualize the dynamics of alloantigen-specific CD4+ T-cell activation in vivo. Using TCR-transgenic (tg) mice reactive to I-Ab(m12), we studied the clonal expansion and differentiation of alloreactive T cells by tracking the fate of adoptively transferred TCR-tg CD4+ T cells in syngeneic mice transplanted with skin grafts expressing I-Ab(m12). Following transplantation, alloantigen-specific TCR-tg CD4+ T-cell expansion was observed initially in the draining lymph nodes followed by the spleen. TCR-tg CD4+ T cells up-regulated CD69 and CD25 expression, developed an effector/memory surface phenotype and produced IFN-gamma in response to alloantigen ex vivo. Furthermore, we validate the model system as a means for studying the effects of tolerogenic regimens on alloreactive CD4+ T cells, demonstrating that CTLA4Ig inhibits alloantigen-dependent clonal expansion and effector function of TCR-tg CD4+ T cells in vivo. We describe the first model for tracking alloreactive CD4+ T-cell activation in vivo. It provides a powerful tool for studying CD4+ T-cell mediated alloimmune responses and mechanisms of tolerance induction in vivo.

Adoptive Transfer↗
PubMed2002

A role for the alpha-chain connecting peptide motif in mediating TCR-CD8 cooperation.

To generate peripheral T cells that are both self-MHC restricted and self-MHC tolerant, thymocytes are subjected to positive and negative selection. How the TCR discriminates between positive and negative selection ligands is not well understood, although there is substantial evidence that the CD4 and CD8 coreceptors play an important role in this cell fate decision. We have previously identified an evolutionarily conserved motif in the TCR, the alpha-chain connecting peptide motif (alpha-CPM), which allows the TCR to deliver positive selection signals. Thymocytes expressing alpha-CPM-deficient receptors do not undergo positive selection, whereas their negative selection is not impaired. In this work we studied the ligand binding and receptor function of alpha-CPM-deficient TCRs by generating T cell hybridomas expressing wild-type or alpha-CPM-deficient forms of the T1 TCR. This K(d)-restricted TCR is specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide(252-260) IASA-YIPSAEK(ABA)I and is therefore amenable to TCR photoaffinity labeling. The experiments presented in this work show that alpha-CPM-deficient TCRs fail to cooperate with CD8 to enhance ligand binding and functional responses.

Amino Acid Motifs↗
PubMed2002

CD3 delta establishes a functional link between the T cell receptor and CD8.

T cells expressing T cell receptor (TCR) complexes that lack CD3 delta, either due to deletion of the CD3 delta gene, or by replacement of the connecting peptide of the TCR alpha chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8 beta or tailless CD8 beta, we examined whether CD3 delta serves to couple TCR.CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252-260 peptide in the context of H-2K(d). We report that, in thymocytes and hybridomas expressing the T1 TCR.CD3 complex, CD8 alpha beta associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8 alpha alpha or a CD3 delta(-) variant of the T1 TCR. CD3 delta was selectively co-immunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3 delta. Because CD8 alpha beta is a raft constituent, due to this association a fraction of TCR.CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3 delta couples TCR.CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8(+) T cells.

Affinity Labels↗