Preferential recognition of TCR hypervariable regions by human anti-idiotypic T cells induced by T cell vaccination

T cell responses to myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). Immunization with irradiated MBP-reactive T cells (T cell vaccination) induces anti-idiotypic T cell responses that suppress circulating MBP-reactive T cells. This T cell-T cell interaction is thought to involve the recognition of TCR expressed on target T cells. The study was undertaken to define the idiotypic determinants responsible for triggering CD8+ cytotoxic anti-idiotypic T cell responses by T cell vaccination in patients with MS. A panel of 9-mer synthetic TCR peptides corresponding to complementarity-determining region 2 (CDR2) and CDR3 of the immunizing MBP-reactive T cell clones were used to isolate anti-idiotypic T cell lines from immunized MS patients. The resulting TCR-specific T cell lines expressed exclusively the CD8 phenotype and recognized preferentially the CDR3 peptides. CDR3-specific T cell lines were found to lyze specifically autologous immunizing MBP-reactive T cell clones. The findings suggest that CDR3-specific T cells represented anti-idiotypic T cell population induced by T cell vaccination. In contrast, the CDR2 peptides were less immunogenic and contained cryptic determinants as the CDR2-specific T cell lines did not recognize autologous immunizing T cell clones from which the peptide sequence was derived. The study has important implications in our understanding of in vivo idiotypic regulation of autoimmune T cells and the regulatory mechanism underlying T cell vaccination.     

Decreased binding of peptides-MHC class I (pMHC) multimeric complexes to CD8 affects their binding avidity for the TCR but does not significantly impact on pMHC/TCR dissociation rate

The CD8 coreceptor plays a crucial role in both T cell development in the thymus and in the activation of mature T cells in response to Ag-specific stimulation. In this study we used soluble peptides-MHC class I (pMHC) multimeric complexes bearing mutations in the CD8 binding site that impair their binding to the MHC, together with altered peptide ligands, to assess the impact of CD8 on pMHC binding to the TCR. Our data support a model in which CD8 promotes the binding of TCR to pMHC. However, once the pMHC/TCR complex is formed, the TCR dominates the pMHC/TCR dissociation rates. As a consequence of these molecular interactions, under physiologic conditions CD8 plays a key role in complex formation, resulting in the enhancement of CD8 T cell functions whose specificity, however, is determined by the TCR.     

Structure of a fully assembled tumor-specific T cell receptor ligated by pMHC

1. Download : Download high-res image (173KB)
2. Download : Download full-size image

     

Flexibility in MHC and TCR recognition: degenerate specificity at the T cell level in the recognition of promiscuous Th epitopes exhibiting no primary sequence homology

Recognition of peptide Ags by T cells through the TCR can be highly specific. In this report we show the degeneracy of Ag recognition at both MHC and TCR levels. We present evidence that unrelated promiscuous Th cell epitopes from various protein sources exhibit sufficient structural homology, despite minimal structural identity, to elicit cross-reactive proliferative responses at the bulk T cell level. This epitopic mimicry was also observed when peptide (CS.T3(378-395) and TT(830-844))-specific CD4+ T cell lines and T cell hybridoma clones were used in proliferation and Ag presentation assays. A scrambled CS.T3(378-395) peptide did not show any proliferation, indicating that the specificity of the cross-reactive responses may be linked with the primary structure of the peptides. Blocking of CS.T3(378-395)-specific CD4+ T cell proliferation by anti-MHC class II mAb showed that recognition of promiscuous T cell epitopes is largely in association with MHC class II molecules. These findings suggest that promiscuous Th epitopes may be useful in designing peptide-based vaccine constructs. At the same time these results show that at the T cell level there may be a great deal of immunological cross-reactivity between heterologous pathogens, and because of this the host's response to a pathogen may be modified by its previous experience with other unrelated pathogens.     

T cell antigen receptor (TCR) transmembrane peptides colocalize with TCR,not lipid rafts,in surface membranes

We have previously shown that a synthetic peptide termed core peptide (CP), which corresponds to a sequence within the transmembrane domain of the alpha chain of the T cell antigen receptor (TCR), can inhibit IL-2 production in antigen-stimulated T cells and can suppress inflammation in several T cell-mediated animal models of disease. As the first step in determining the mechanism of CP action, we examined the association of CP with the plasma membrane of human T cells using confocal microscopy. A homogeneous distribution of CP was observed in the plasma membrane of human T cells. This membrane localization was dependent on the presence of positive charges in the CP sequence. CP analogs, containing either neutral or negatively charged amino acids in place of the positive amino acid charges, did not localize within TCR membranes. Following antibody-induced TCR clustering, there was specific colocalization of CP with surface TCR. No association was observed with other cell surface receptors when similarly clustered. Since TCR activation leads to an increased movement of the receptor complex to cholesterol/glycosphingolipid (GSL) plasma membrane microdomains (rafts) we examined whether the association of CP with TCR was raft-driven. TCR clustering led only to a partial colocalization of TCRs with raft GSL, ganglioside GM1, and a complete colocalization of CP with TCRs. We conclude that CP associates specifically with plasma membrane TCRs and not raft lipids.     

Force measurements of Myosin II waves at the yolk surface during Drosophila dorsal closure

The mechanical properties and the forces involved during tissue morphogenesis have been the focus of much research in the last years. Absolute values of forces during tissue closure events have not yet been measured. This is also true for a common force-producing mechanism involving Myosin II waves that results in pulsed cell surface contractions. Our patented magnetic tweezer, CAARMA, integrated into a spinning disk confocal microscope, provides a powerful explorative tool for quantitatively measuring forces during tissue morphogenesis. Here, we used this tool to quantify the in vivo force production of Myosin II waves that we observed at the dorsal surface of the yolk cell in stage 13 Drosophila melanogaster embryos. In addition to providing for the first time to our knowledge quantitative values on an active Myosin-driven force, we elucidated the dynamics of the Myosin II waves by measuring their periodicity in both absence and presence of external perturbations, and we characterized the mechanical properties of the dorsal yolk cell surface.     

CD2 can mediate TCR/CD3-independent T cell activation.

T lymphocytes can be activated clonotypically through TCR/CD3 complex or polyclonally via the CD2 molecule. Whether CD2-mediated activation is dependent on TCR/CD3 expression or signaling is controversial. We have re-explored this issue by using a series of CD2-transfected, TCR/CD3 surface membrane-negative human and mouse T cells. Our results clearly show that such T cells can be triggered for IL-2 secretion and increases in intracellular Ca2+ through the CD2 molecule in the absence of surface expression of TCR/CD3 complexes. These responses are only observed when cells express high levels of CD2 and there is a critical threshold of CD2 expression necessary for such activation in the absence of CD3. Concomitant expression of TCR/CD3 complex markedly lowers the level of CD2 required for activation via the latter pathway. These results provide a clear resolution of the controversy concerning the requirement for surface CD3 expression in T cell activation through CD2 and further suggest a possible role for CD2 in activation of TCR/CD3-negative cells.     

Peptide recognition by two HLA-A2/Tax11-19-specific T cell clones in relationship to their MHC/peptide/TCR crystal structures

The crystal structures of two human TCRs specific for a HTLV-I Tax peptide bound to HLA-A2 were recently determined, for the first time allowing a functional comparison of TCRs for which the MHC/peptide/TCR structures are known. Extensive amino acid substitutions show that the native Tax residues are optimal at each peptide position. A prominent feature of the TCR contact surface is a deep pocket that accommodates a tyrosine at position 5 of the peptide. For one of these TCRs, this pocket is highly specific for aromatic residues. In the other TCR structure, this pocket is larger, allowing many different residues to be accommodated. The CTL clones also show major differences in the specificity for several other peptide residues, including side chains that are not directly contacted by the TCR. Despite the specificity of these clones, peptides that are distinct at five or six positions from Tax11-19 induce CTL activity, indicating that substantial changes of the peptide surface are tolerated. Human peptides with limited sequence homology to Tax11-19 represent partial TCR agonists for these CTL clones. The distinct functional properties of these CTL clones highlight structural features that determine TCR specificity and cross-reactivity for MHC-bound peptides.     

Incorporation of transmembrane hydrophobic mutations in the TCR enhance its surface expression and T cell functional avidity

TCR-gene transfer represents an effective way to redirect the specificity of T lymphocytes for therapeutic purposes. Recent successful clinical trials have underscored the potential of this approach in which efficient expression of the exogenous TCR has been directly linked to the efficacy of T cell activity. It has been also demonstrated that the TCR exhibits a lack of stability associated with the presence of positively charged residues in its transmembrane (TM) region. In this study, we designed an original approach selectively to improve exogenous TCR stability by increasing the hydrophobic nature of the TCRα TM region. Incorporation of hydrophobic residues at evolutionarily permissive positions resulted in an enhanced surface expression of the TCR chains, leading to an improved cellular avidity and anti-tumor TCR activity. Furthermore, this strategy was successfully applied to different TCRs, enabling the targeting of human tumors from different histologies. We also show that the combination of these hydrophobic mutations with another TCR-enhancing approach further improved TCR expression and function. Overall, these findings provide information regarding TCR TM composition that can be applied for the improvement of TCR-gene transfer-based treatments.     

The INs and OUTs of pattern recognition receptors at the cell surface

Pattern recognition receptors (PRRs) enable plants to sense non-self molecules displayed by microbes to mount proper defense responses or establish symbiosis. In recent years the importance of PRR subcellular trafficking to plant immunity has become apparent. PRRs traffic through the endoplasmatic reticulum (ER) and the Golgi apparatus to the plasma membrane, where they recognize their cognate ligands. At the plasma membrane, PRRs can be recycled or internalized via endocytic pathways. By using genetic and biochemical tools in combination with bioimaging, the trafficking pathways and their role in PRR perception of microbial molecules are now being revealed.     

MPID-T2: a database for sequence-structure-function analyses of pMHC and TR/pMHC structures

Sequence-structure-function information is critical in understanding the mechanism of pMHC and TR/pMHC binding and recognition. A database for sequence-structure-function information on pMHC and TR/pMHC interactions, MHC-Peptide Interaction Database-TR version 2 (MPID-T2), is now available augmented with the latest PDB and IMGT/3Dstructure-DB data, advanced features and new parameters for the analysis of pMHC and TR/pMHC structures. AVAILABILITY: http://biolinfo.org/mpid-t2. CONTACT: shoba.ranganathan@mq.edu.au SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Comparison of a T cell clone and of T cells from a TCR transgenic mouse: TCR transgenic T cells specific for self-antigen are atypical

It has been widely assumed that T cells from TCR-transgenic (Tg) mice better represent the behavior of T cells from normal mice than do in vitro cultures of T cell clones. We have found that autoreactive T cells arising in the presumably more physiological environment of the BDC-2.5 TCR-Tg mouse, despite being apparently "naive" in surface phenotype, are highly activated functionally and do not resemble CD4(+) T cells from a spontaneously diabetic nonobese diabetic (NOD) mouse or the NOD-derived, diabetogenic CD4(+) T cell clone of origin, BDC-2.5. Our results suggest that autoreactive T cells cloned from the spontaneously diabetic NOD mouse more closely resemble effector T cells arising during the natural disease process.     

Generation of T cell help through a MHC class I-restricted TCR

CD4+ T cells that are activated by a MHC class II/peptide encounter can induce maturation of APCs and promote cytotoxic CD8+ T cell responses. Unfortunately, the number of well-defined tumor-specific CD4+ T cell epitopes that can be exploited for adoptive immunotherapy is limited. To determine whether Th cell responses can be generated by redirecting CD4+ T cells to MHC class I ligands, we have introduced MHC class I-restricted TCRs into postthymic murine CD4+ T cells and examined CD4+ T cell activation and helper function in vitro and in vivo. These experiments indicate that Ag-specific CD4+ T cell help can be induced by the engagement of MHC class I-restricted TCRs in peripheral CD4+ T cells but that it is highly dependent on the coreceptor function of the CD8beta-chain. The ability to generate Th cell immunity by infusion of MHC class I-restricted Th cells may prove useful for the induction of tumor-specific T cell immunity in cases where MHC class II-associated epitopes are lacking.     

Characterization of TCR gene rearrangements during adult murine T cell development

Development of the alphabeta and gammadelta T cell lineages is dependent upon the rearrangement and expression of the TCRalpha and beta or gamma and delta genes, respectively. Although the timing and sequence of rearrangements of the TCRalpha and TCRbeta loci in adult murine thymic precursors has been characterized, no similar information is available for the TCRgamma and TCRdelta loci. In this report, we show that approximately half of the total TCRdelta alleles initiate rearrangements at the CD44highCD25+ stage, whereas the TCRbeta locus is mainly in germline configuration. In the subsequent CD44lowCD25+ stage, most TCRdelta alleles are fully recombined, whereas TCRbeta rearrangements are only complete on 10-30% of alleles. These results indicate that rearrangement at the TCRdelta locus can precede that of TCRbeta locus recombination by one developmental stage. In addition, we find a bias toward productive rearrangements of both TCRdelta and TCRgamma genes among CD44highCD25+ thymocytes, suggesting that functional gammadelta TCR complexes can be formed before the rearrangement of TCRbeta. These data support a model of lineage commitment in which sequential TCR gene rearrangements may influence alphabeta/gammadelta lineage decisions. Further, because TCR gene rearrangements are generally limited to T lineage cells, these analyses provide molecular evidence that irreversible commitment to the T lineage can occur as early as the CD44highCD25+ stage of development.     

The influence of age on T cell generation and TCR diversity

The ability to mount protective immune responses depends on the diversity of T cells. T cell diversity may be compromised by the declining thymic output of new T cells. The aging process imposes a threat to diversity, because thymic function deteriorates. In this study we have examined the relationship between thymic production, homeostatic T cell proliferation and TCR beta-chain diversity in young (approximately 25 years), middle-aged ( approximately 60 years), and elderly adults (approximately 75 years). TCR excision circles (TREC) as a marker of thymic output exponentially decreased by >95% between 25 and 60 years of age. The frequency of Ki67(+) cycling CD4 T cells remained steady, and surprisingly, the diversity of the naive CD4 T cell repertoire was maintained at approximately 2 x 10(7) different TCR beta-chains. After the age of 70 years, TRECs only slightly declined, but homeostatic proliferation doubled. The diversity of the T cell pool drastically contracted to 200,000 TCR beta-chains. Also, the phenotypic distinction between naive and memory CD4 T cells became fuzzy. The collapse in CD4 T cell diversity during the seventh and eighth decades indicates substantial T cell loss and implies that therapeutic measures to improve vaccine responses will have to include strategies for T cell replenishment.