Re-Directing CD4(+) T Cell Responses with the Flanking Residues of MHC Class II-Bound Peptides: The Core is Not Enough.
ABSTRACT: Recombinant ?? T cell receptors, expressed on T cell membranes, recognize short peptides presented at the cell surface in complex with MHC molecules. There are two main subsets of ?? T cells: CD8(+) T cells that recognize mainly cytosol-derived peptides in the context of MHC class I (pMHC-I), and CD4(+) T cells that recognize peptides usually derived from exogenous proteins presented by MHC class II (pMHC-II). Unlike the more uniform peptide lengths (usually 8-13mers) bound in the MHC-I closed groove, MHC-II presented peptides are of a highly variable length. The bound peptides consist of a core bound 9mer (reflecting the binding motif for the particular MHC-II type) but with variable peptide flanking residues (PFRs) that can extend from both the N- and C-terminus of the MHC-II binding groove. Although pMHC-I and pMHC-II play a virtually identical role during T cell responses (T cell antigen presentation) and are very similar in overall conformation, there exist a number of subtle but important differences that may govern the functional dichotomy observed between CD8(+) and CD4(+) T cells. Here, we provide an overview of the impact of structural differences between pMHC-I and pMHC-II and the molecular interactions with the T cell receptor including the functional importance of MHC-II PFRs. We consider how factors such as anatomical location, inflammatory milieu, and particular types of antigen presenting cell might, in theory, contribute to the quantitative (i.e., pMHC ligand frequency) as well as qualitative (i.e., variable PFR) nature of peptide epitopes, and hence offer a means of control and influence of a CD4(+) T cell response. Lastly, we review our recent findings showing how modifications to MHC-II PFRs can modify CD4(+) T cell antigen recognition. These findings may have novel applications for the development of CD4(+) T cell peptide vaccines and diagnostics.
Project description:Major histocompatibility complex (MHC) class II molecules (MHC-II) function by binding antigenic peptides and displaying these peptides on the surface of antigen presenting cells (APCs) for recognition by peptide-MHC-II (pMHC-II)-specific CD4 T cells. It is known that cell surface MHC-II can internalize, exchange antigenic peptides in endosomes, and rapidly recycle back to the plasma membrane; however, the molecular machinery and trafficking pathways utilized by internalizing/recycling MHC-II have not been identified. We now demonstrate that unlike newly synthesized invariant chain-associated MHC-II, mature cell surface pMHC-II complexes internalize following clathrin-, AP-2-, and dynamin-independent endocytosis pathways. Immunofluorescence microscopy of MHC-II expressing HeLa-CIITA cells, human B cells, and human DCs revealed that pMHC enters Arf6(+)Rab35(+)EHD1(+) tubular endosomes following endocytosis. These data contrast the internalization pathways followed by newly synthesized and peptide-loaded MHC-II molecules and demonstrates that cell surface pMHC-II internalize and rapidly recycle from early endocytic compartments in tubular endosomes.
Project description:Fluorochrome-conjugated peptide-MHC (pMHC) class I multimers are staple components of the immunologist's toolbox, enabling reliable quantification and analysis of Ag-specific CD8(+) T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4(+) T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II-bound peptides, can enhance TCR-pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4(+) T cells, highlighting an unappreciated feature of TCR-pMHC-II interactions.
Project description:T cell receptors (TCRs) recognize peptides presented by MHC molecules (pMHC) on an antigen-presenting cell (APC) to discriminate foreign from self-antigens and initiate adaptive immune responses. In addition, T cell activation generally requires binding of this same pMHC to a CD4 or CD8 co-receptor, resulting in assembly of a TCR-pMHC-CD4 or TCR-pMHC-CD8 complex and recruitment of Lck via its association with the co-receptor. Here we review structural and biophysical studies of CD4 and CD8 interactions with MHC molecules and TCR-pMHC complexes. Crystal structures have been determined of CD8?? and CD8?? in complex with MHC class I, of CD4 bound to MHC class II, and of a complete TCR-pMHC-CD4 ternary complex. Additionally, the binding of these co-receptors to pMHC and TCR-pMHC ligands has been investigated both in solution and in situ at the T cell-APC interface. Together, these studies have provided key insights into the role of CD4 and CD8 in T cell activation, and into how these co-receptors focus TCR on MHC to guide TCR docking on pMHC during thymic T cell selection.
Project description:CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide-flanking residues (PFRs) can extend beyond the limits of the HLA binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence T-cell receptor (TCR) affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide-binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak TCR-binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111-130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak affinity TCR-pHLA-II interactions.
Project description:Epitope recognition by major histocompatibility complex II (MHC-II) is essential for the activation of immunological responses to infectious diseases. Several studies have demonstrated that this molecular event takes place in the MHC-II peptide-binding groove constituted by the ? and ? light chains of the heterodimer. This MHC-II peptide-binding groove has several pockets (P1-P11) involved in peptide recognition and complex stabilization that have been probed through crystallographic experiments and in silico calculations. However, most of these theoretical calculations have been performed without taking into consideration the heavy chains, which could generate misleading information about conformational mobility both in water and in the membrane environment. Therefore, in absence of structural information about the difference in the conformational changes between the peptide-free and peptide-bound states (pMHC-II) when the system is soluble in an aqueous environment or non-covalently bound to a cell membrane, as the physiological environment for MHC-II is. In this study, we explored the mechanistic basis of these MHC-II components using molecular dynamics (MD) simulations in which MHC-II was previously co-crystallized with a small epitope (P7) or coupled by docking procedures to a large (P22) epitope. These MD simulations were performed at 310 K over 100 ns for the water-soluble (MHC-IIw, MHC-II-P(7w), and MHC-II-P(22w)) and 150 ns for the membrane-bound species (MHC-IIm, MHC-II-P(7m), and MHC-II-P(22m)). Our results reveal that despite the different epitope sizes and MD simulation environments, both peptides are stabilized primarily by residues lining P1, P4, and P6-7, and similar noncovalent intermolecular energies were observed for the soluble and membrane-bound complexes. However, there were remarkably differences in the conformational mobility and intramolecular energies upon complex formation, causing some differences with respect to how the two peptides are stabilized in the peptide-binding groove.
Project description:Antigen presentation is a cellular process that involves a number of steps, beginning with the production of peptides by proteolysis or aberrant synthesis and the delivery of peptides to cellular compartments where they are loaded on MHC class I (MHC-I) or MHC class II (MHC-II) molecules. The selective loading and editing of high-affinity immunodominant antigens is orchestrated by molecular chaperones: tapasin/TAP-binding protein, related for MHC-I and HLA-DM for MHC-II. Once peptide/MHC (pMHC) complexes are assembled, following various steps of quality control, they are delivered to the cell surface, where they are available for identification by ?? receptors on CD8+ or CD4+ T lymphocytes. In addition, recognition of cell surface peptide/MHC-I complexes by natural killer cell receptors plays a regulatory role in some aspects of the innate immune response. Many of the components of the pathways of antigen processing and presentation and of T cell receptor (TCR)-mediated signaling have been studied extensively by biochemical, genetic, immunological, and structural approaches over the past several decades. Until recently, however, dynamic aspects of the interactions of peptide with MHC, MHC with molecular chaperones, or of pMHC with TCR have been difficult to address experimentally, although computational approaches such as molecular dynamics (MD) simulations have been illuminating. Studies exploiting X-ray crystallography, cryo-electron microscopy, and multidimensional nuclear magnetic resonance (NMR) spectroscopy are beginning to reveal the importance of molecular flexibility as it pertains to peptide loading onto MHC molecules, the interactions between pMHC and TCR, and subsequent TCR-mediated signals. In addition, recent structural and dynamic insights into how molecular chaperones define peptide selection and fine-tune the MHC displayed antigen repertoire are discussed. Here, we offer a review of current knowledge that highlights experimental data obtained by X-ray crystallography and multidimensional NMR methodologies. Collectively, these findings strongly support a multifaceted role for protein plasticity and conformational dynamics throughout the antigen processing and presentation pathway in dictating antigen selection and recognition.
Project description:CD8(+) CTL responses directed toward the HLA-B*51:01-restricted HIV-RT128-135 epitope TAFTIPSI (TI8) are associated with long-term nonprogression to AIDS. Clonotypic analysis of responses to B51-TI8 revealed a public clonotype using TRAV17/TRBV7-3 TCR genes in six out of seven HLA-B*51:01(+) patients. Structural analysis of a TRAV17/TRBV7-3 TCR in complex with HLA-B51-TI8, to our knowledge the first human TCR complexed with an 8-mer peptide, explained this bias, as the unique combination of residues encoded by these genes was central to the interaction. The relatively featureless peptide-MHC (pMHC) was mainly recognized by the TCR CDR1 and CDR2 loops in an MHC-centric manner. A highly conserved residue Arg(97) in the CDR3? loop played a major role in recognition of peptide and MHC to form a stabilizing ball-and-socket interaction with the MHC and peptide, contributing to the selection of the public TCR clonotype. Surface plasmon resonance equilibrium binding analysis showed the low affinity of this public TCR is in accordance with the only other 8-mer interaction studied to date (murine 2C TCR-H-2K(b)-dEV8). Like pMHC class II complexes, 8-mer peptides do not protrude out the MHC class I binding groove like those of longer peptides. The accumulated evidence suggests that weak affinity might be a common characteristic of TCR binding to featureless pMHC landscapes.
Project description:Major histocompatibility complex class II (MHC II) expressed on the surface of antigen-presenting cells (APCs) displays peptides to CD4? T cells. Depletion of membrane cholesterol from APCs by methyl ?-cyclodextrin treatment compromises peptide-MHC II complex formation coupled with impaired binding of conformational antibody, which binds close to the peptide binding groove of MHC II. Interestingly, the total cell surface of MHC II remains unaltered. These defects can be corrected by restoring membrane cholesterol. In silico docking studies with a three-dimensional model showed the presence of a cholesterol binding site in the transmembrane domain of MHC II (TM-MHC-II). From the binding studies it was clear that cholesterol, indeed, interacts with the TM-MHC-II and alters its conformation. Mutation of cholesterol binding residues (F240, L243, and F246) in the TM-MHC-II decreased the affinity for cholesterol. Furthermore, transfection of CHO cells with full-length mutant MHC II, but not wild-type MHC II, failed to activate antigen-specific T cells coupled with decreased binding of conformation-specific antibodies. Thus, cholesterol-induced conformational change of TM-MHC-II may allosterically modulate the peptide binding groove of MHC II leading to T cell activation.
Project description:BACKGROUND: Identification of antigenic peptide epitopes is an essential prerequisite in T cell-based molecular vaccine design. Computational (sequence-based and structure-based) methods are inexpensive and efficient compared to experimental approaches in screening numerous peptides against their cognate MHC alleles. In structure-based protocols, suited to alleles with limited epitope data, the first step is to identify high-binding peptides using docking techniques, which need improvement in speed and efficiency to be useful in large-scale screening studies. We present pDOCK: a new computational technique for rapid and accurate docking of flexible peptides to MHC receptors and primarily apply it on a non-redundant dataset of 186 pMHC (MHC-I and MHC-II) complexes with X-ray crystal structures. RESULTS: We have compared our docked structures with experimental crystallographic structures for the immunologically relevant nonameric core of the bound peptide for MHC-I and MHC-II complexes. Primary testing for re-docking of peptides into their respective MHC grooves generated 159 out of 186 peptides with Cα RMSD of less than 1.00 Å, with a mean of 0.56 Å. Amongst the 25 peptides used for single and variant template docking, the Cα RMSD values were below 1.00 A for 23 peptides. Compared to our earlier docking methodology, pDOCK shows upto 2.5 fold improvement in the accuracy and is ~60% faster. Results of validation against previously published studies represent a seven-fold increase in pDOCK accuracy. CONCLUSIONS: The limitations of our previous methodology have been addressed in the new docking protocol making it a rapid and accurate method to evaluate pMHC binding. pDOCK is a generic method and although benchmarks against experimental structures, it can be applied to alleles with no structural data using sequence information. Our outcomes establish the efficacy of our procedure to predict highly accurate peptide structures permitting conformational sampling of the peptide in MHC binding groove. Our results also support the applicability of pDOCK for in silico identification of promiscuous peptide epitopes that are relevant to higher proportions of human population with greater propensity to activate T cells making them key targets for the design of vaccines and immunotherapies.
Project description:Peptide exchange technologies are essential for the generation of pMHC-multimer libraries for probing diverse, polyclonal TCR repertoires in various settings. Here we report, using the molecular chaperone TAPBPR, a robust method for the capture of stable, empty MHC-I molecules, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange ‘goldilocks’ peptides with high-affinity peptides of interest in an overnight reaction. Using the same system, we describe high-throughput assays to validate binding of multiple candidate peptides on the empty MHCI groove. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T-cell transcription profiles together with their cognate pMHC-I specificities in a single experiment. Overall design: spleen, jurkat, and neuroblastoma samples