Canonical T cell receptor docking on peptide-MHC is essential for T cell signaling.

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRß-variable (TRBV) 17+ TCRs from the naïve mouse CD8+ T cell repertoire that recognizes the H-2Db-NP366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints.

Combining Three-Dimensional Modeling with Artificial Intelligence to Increase Specificity and Precision in Peptide-MHC Binding Predictions.

The reliable prediction of the affinity of candidate peptides for the MHC is important for predicting their potential antigenicity and thus influences medical applications, such as decisions on their inclusion in T cell-based vaccines. In this study, we present a rapid, predictive computational approach that combines a popular, sequence-based artificial neural network method, NetMHCpan 4.0, with three-dimensional structural modeling. We find that the ensembles of bound peptide conformations generated by the programs MODELLER and Rosetta FlexPepDock are less variable in geometry for strong binders than for low-affinity peptides. In tests on 1271 peptide sequences for which the experimental dissociation constants of binding to the well-characterized murine MHC allele H-2Db are known, by applying thresholds for geometric fluctuations the structure-based approach in a standalone manner drastically improves the statistical specificity, reducing the number of false positives. Furthermore, filtering candidates generated with NetMHCpan 4.0 with the structure-based predictor led to an increase in the positive predictive value (PPV) of the peptides correctly predicted to bind very strongly (i.e., K d < 100 nM) from 40 to 52% (p = 0.027). The combined method also significantly improved the PPV when tested on five human alleles, including some with limited data for training. Overall, an average increase of 10% in the PPV was found over the standalone sequence-based method. The combined method should be useful in the rapid design of effective T cell-based vaccines.

A Tolerogenic Artificial APC Durably Ameliorates Experimental Autoimmune Encephalomyelitis by Directly and Selectively Modulating Myelin Peptide-Autoreactive CD4+ and CD8+ T Cells.

In this study, a tolerogenic artificial APC (TaAPC) was developed to directly and selectively modulate myelin-autoreactive CD4+ and CD8+ T cells in the myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced experimental autoimmune encephalomyelitis in C57BL/6J mice. Cell-sized polylactic-coglycolic acid microparticles were generated to cocouple target Ags (MOG40-54/H-2Db-Ig dimer, MOG35-55/I-Ab multimer), regulatory molecules (anti-Fas and PD-L1-Fc), and "self-marker" CD47-Fc and encapsulate inhibitory cytokine (TGF-ß1). Four infusions of the TaAPCs markedly and durably inhibited the experimental autoimmune encephalomyelitis progression and reduced the local inflammation in CNS tissue. They circulated throughout vasculature into peripheral lymphoid tissues and various organs, but not into brain, with retention of 36 h and exerted direct effects on T cells in vivo and in vitro. Two infusions of the TaAPCs depleted 65-79% of MOG35-55-specific CD4+ and 46-62% of MOG40-54-specific CD8+ T cells in peripheral blood, spleen, and CNS tissues in an Ag-specific manner and regulatory molecule-dependent fashion; induced robust T cell apoptosis; inhibited the activation and proliferation of MOG peptide-reactive T cells; reduced MOG peptide-reactive Th1, Th17, and Tc17 cells; and expanded regulatory T cells. They also inhibited IFN-γ/IL-17A secretion and elevated IL-10/TGF-ß1 production in splenocytes but not in CNS tissue. More importantly, the TaAPCs treatment did not obviously suppress the overall immune function of host. To our knowledge, this study provides the first experimental evidence for the capability of TaAPCs to directly modulate autoreactive T cells by surface presentation of multiple ligands and paracrine release of cytokine, thus suggesting a novel Ag-specific immunotherapy for the T cell-mediated autoimmune diseases.

The Immunogenicity of a Proline-Substituted Altered Peptide Ligand toward the Cancer-Associated TEIPP Neoepitope Trh4 Is Unrelated to Complex Stability.

Human cancers frequently display defects in Ag processing and presentation allowing for immune evasion, and they therefore constitute a significant challenge for T cell-based immunotherapy. We have previously demonstrated that the antigenicity of tumor-associated Ags can be significantly enhanced through unconventional residue modifications as a novel tool for MHC class I (MHC-I)-based immunotherapy approaches. We have also previously identified a novel category of cancer neo-epitopes, that is, T cell epitopes associated with impaired peptide processing (TEIPP), that are selectively presented by MHC-I on cells lacking the peptide transporter TAP. In this study, we demonstrate that substitution of the nonanchoring position 3 into a proline residue of the first identified TEIPP peptide, the murine Trh4, results in significantly enhanced recognition by antitumor CTLs toward the wild-type epitope. Although higher immunogenicity has in most cases been associated with increased MHC/peptide complex stability, our results demonstrate that the overall stability of H-2Db in complex with the highly immunogenic altered peptide ligand Trh4-p3P is significantly reduced compared with wild-type H-2Db/Trh4. Comparison of the crystal structures of the H-2Db/Trh4-p3P and H-2Db/Trh4 complexes revealed that the conformation of the nonconventional methionine anchor residue p5M is altered, deleting its capacity to form adequate sulfur-π interactions with H-2Db residues, thus reducing the overall longevity of the complex. Collectively, our results indicate that vaccination with Thr4-p3P significantly enhances T cell recognition of targets presenting the wild-type TEIPP epitope and that higher immunogenicity is not necessarily directly related to MHC/peptide complex stability, opening for the possibility to design novel peptide vaccines with reduced MHC/peptide complex stability.

Kinetic and thermodynamic studies of the interaction between activating and inhibitory Ly49 natural killer receptors and MHC class I molecules.

Natural killer (NK) cells are lymphocytes of the innate immune system that eliminate virally infected or malignantly transformed cells. NK cell function is regulated by diverse surface receptors that are both activating and inhibitory. Among them, the homodimeric Ly49 receptors control NK cell cytotoxicity by sensing major histocompatibility complex class I molecules (MHC-I) on target cells. Although crystal structures have been reported for Ly49/MHC-I complexes, the underlying binding mechanism has not been elucidated. Accordingly, we carried out thermodynamic and kinetic experiments on the interaction of four NK Ly49 receptors (Ly49G, Ly49H, Ly49I and Ly49P) with two MHC-I ligands (H-2Dd and H-2Dk). These Ly49s embrace the structural and functional diversity of the highly polymorphic Ly49 family. Combining surface plasmon resonance, fluorescence anisotropy and far-UV circular dichroism (CD), we determined that the best model to describe both inhibitory and activating Ly49/MHC-I interactions is one in which the two MHC-I binding sites of the Ly49 homodimer present similar binding constants for the two sites (∼106 M-1) with a slightly positive co-operativity in some cases, and without far-UV CD observable conformational changes. Furthermore, Ly49/MHC-I interactions are diffusion-controlled and enthalpy-driven. These features stand in marked contrast with the activation-controlled and entropy-driven interaction of Ly49s with the viral immunoevasin m157, which is characterized by strong positive co-operativity and conformational selection. These differences are explained by the distinct structures of Ly49/MHC-I and Ly49/m157 complexes. Moreover, they reflect the opposing roles of NK cells to rapidly scan for virally infected cells and of viruses to escape detection using immunoevasins such as m157.

Protective T Cell Responses Featured by Concordant Recognition of Middle East Respiratory Syndrome Coronavirus-Derived CD8+ T Cell Epitopes and Host MHC.

The coordinated recognition of virus-derived T cell epitopes and MHC molecules by T cells plays a pivotal role in cellular immunity-mediated virus clearance. It has been demonstrated that the conformation of MHC class I (MHC I) molecules can be adjusted by the presented peptide, which impacts T cell activation. However, it is still largely unknown whether the conformational shift of MHC I influences the protective effect of virus-specific T cells. In this study, utilizing the Middle East respiratory syndrome coronavirus-infected mouse model, we observed that through the unusual secondary anchor Ile5, a CD8+ T cell epitope drove the conformational fit of Trp73 on the α1 helix of murine MHC I H-2Kd In vitro renaturation and circular dichroism assays indicated that this shift of the structure did not influence the peptide/MHC I binding affinity. Nevertheless, the T cell recognition and the protective effect of the peptide diminished when we made an Ile to Ala mutation at position 5 of the original peptide. The molecular bases of the concordant recognition of T cell epitopes and host MHC-dependent protection were demonstrated through both crystal structure determination and tetramer staining using the peptide-MHC complex. Our results indicate a coordinated MHC I/peptide interaction mechanism and provide a beneficial reference for T cell-oriented vaccine development against emerging viruses such as Middle East respiratory syndrome coronavirus.

Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C.

Murine natural killer (NK) cells are regulated by the interaction of Ly49 receptors with major histocompatibility complex class I molecules (MHC-I). Although the ligands for inhibitory Ly49 were considered to be restricted to classical MHC (MHC-Ia), we have shown that the non-classical MHC molecule (MHC-Ib) H2-M3 was a ligand for the inhibitory Ly49A. Here we establish that another MHC-Ib, H2-Q10, is a bona fide ligand for the inhibitory Ly49C receptor. H2-Q10 bound to Ly49C with a marginally lower affinity (∼5 µm) than that observed between Ly49C and MHC-Ia (H-2K(b)/H-2D(d), both ∼1 µm), and this recognition could be prevented by cis interactions with H-2K in situ To understand the molecular details underpinning Ly49·MHC-Ib recognition, we determined the crystal structures of H2-Q10 and Ly49C bound H2-Q10. Unliganded H2-Q10 adopted a classical MHC-I fold and possessed a peptide-binding groove that exhibited features similar to those found in MHC-Ia, explaining the diverse peptide binding repertoire of H2-Q10. Ly49C bound to H2-Q10 underneath the peptide binding platform to a region that encompassed residues from the α1, α2, and α3 domains, as well as the associated ß2-microglobulin subunit. This docking mode was conserved with that previously observed for Ly49C·H-2K(b) Indeed, structure-guided mutation of Ly49C indicated that Ly49C·H2-Q10 and Ly49C·H-2K(b) possess similar energetic footprints focused around residues located within the Ly49C ß4-stand and L5 loop, which contact the underside of the peptide-binding platform floor. Our data provide a structural basis for Ly49·MHC-Ib recognition and demonstrate that MHC-Ib represent an extended family of ligands for Ly49 molecules.

MHC-NP: predicting peptides naturally processed by the MHC.

We present MHC-NP, a tool for predicting peptides naturally processed by the MHC pathway. The method was part of the 2nd Machine Learning Competition in Immunology and yielded state-of-the-art accuracy for the prediction of peptides eluted from human HLA-A*02:01, HLA-B*07:02, HLA-B*35:01, HLA-B*44:03, HLA-B*53:01, HLA-B*57:01 and mouse H2-D(b) and H2-K(b) MHC molecules. We briefly explain the theory and motivations that have led to developing this tool. General applicability in the field of immunology and specifically epitope-based vaccine are expected. Our tool is freely available online and hosted by the Immune Epitope Database at http://tools.immuneepitope.org/mhcnp/.

Proline substitution independently enhances H-2D(b) complex stabilization and TCR recognition of melanoma-associated peptides.

The immunogenicity of H-2D(b) (D(b)) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T-cell cross-reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D(b) in complex with an optimized version of the melanoma-associated epitope gp10025-33 . Furthermore, the p3P modification directly increased the affinity of the D(b)/gp10025-33 -specific T-cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D(b)/gp10025-33 complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition.