Yili Li

Structure of the human activating natural cytotoxicity receptor NKp30 bound to its tumor cell ligand B7-H6

As revealed by the first crystal structure of a natural cytotoxicity receptor bound to its ligand, NKp30 engages B7-H6 in a manner structurally distinct from that of other CD28 family members.

Structure of a TCR with high affinity for self-antigen reveals basis for escape from negative selection

The failure to eliminate self‐reactive T cells during negative selection is a prerequisite for autoimmunity. To escape deletion, autoreactive T‐cell receptors (TCRs) may form unstable complexes with self‐peptide–MHC by adopting suboptimal binding topologies compared with anti‐microbial TCRs. Alternatively, escape can occur by weak binding between self‐peptides and MHC. We determined the structure of a human autoimmune TCR (MS2‐3C8) bound to a self‐peptide from myelin basic protein (MBP) and the multiple sclerosis‐associated MHC molecule HLA‐DR4. MBP is loosely accommodated in the HLA‐DR4‐binding groove, accounting for its low affinity. Conversely, MS2‐3C8 binds MBP–DR4 as tightly as the most avid anti‐microbial TCRs. MS2‐3C8 engages self‐antigen via a docking mode that resembles the optimal topology of anti‐foreign TCRs, but is distinct from that of other autoreactive TCRs. Combined with a unique CDR3β conformation, this docking mode compensates for the weak binding of MBP to HLA‐DR4 by maximizing interactions between MS2‐3C8 and MBP. Thus, the MS2‐3C8–MBP–DR4 complex reveals the basis for an alternative strategy whereby autoreactive T cells escape negative selection, yet retain the ability to initiate autoimmunity.

Structural basis for self-recognition by autoimmune T-cell receptors

T‐cell receptors (TCRs) recognize peptides presented by major histocompatibility complex molecules (pMHC) to discriminate between foreign and self‐antigens. Whereas T‐cell recognition of foreign peptides is essential for protection against microbial pathogens, recognition of self‐peptides by T cells that have escaped negative selection in the thymus can lead to autoimmune disease. Structural studies of autoimmune TCR–pMHC complexes have provided insights into the mechanisms underlying self‐recognition and escape from thymic deletion. Two broad categories of self‐reactive TCRs can be clearly distinguished: (i) TCRs with altered binding topologies to self‐pMHC and (ii) TCRs that bind self‐pMHC in the canonical diagonal orientation, but where there are structural defects or suboptimal anchors in the self‐ligand. For both categories, however, the overall stability of the autoimmune TCR–pMHC complex is markedly reduced compared to anti‐microbial complexes, allowing the autoreactive T cells to evade negative selection, yet retain the ability to be activated by self‐antigens in target organs. Additionally, the structures provide insights into TCR cross‐reactivity, which can contribute to autoimmunity by increasing the likelihood of self‐pMHC recognition. Efforts are now underway to understand the impact of structural alterations in autoimmune TCR–pMHC complexes on higher order assemblies involved in TCR signaling, as well as on immunological synapse formation.

Redundancy in Antigen-Presenting Function of the HLA-DR and -DQ Molecules in the Multiple Sclerosis-Associated HLA-DR2 Haplotype

The three HLA class II alleles of the DR2 haplotype, DRB1*1501, DRB5*0101, and DQB1*0602, are in strong linkage disequilibrium and confer most of the genetic risk to multiple sclerosis. Functional redundancy in Ag presentation by these class II molecules would allow recognition by a single TCR of identical peptides with the different restriction elements, facilitating T cell activation and providing one explanation how a disease-associated HLA haplotype could be linked to a CD4+ T cell-mediated autoimmune disease. Using combinatorial peptide libraries and B cell lines expressing single HLA-DR/DQ molecules, we show that two of five in vivo-expanded and likely disease-relevant, cross-reactive cerebrospinal fluid-infiltrating T cell clones use multiple disease-associated HLA class II molecules as restriction elements. One of these T cell clones recognizes >30 identical foreign and human peptides using all DR and DQ molecules of the multiple sclerosis-associated DR2 haplotype. A T cell signaling machinery tuned for efficient responses to weak ligands together with structural features of the TCR-HLA/peptide complex result in this promiscuous HLA class II restriction.

Structural Basis for Recognition of Cellular and Viral Ligands by NK Cell Receptors

Natural killer (NK) cells are key components of innate immune responses to tumors and viral infections. NK cell function is regulated by NK cell receptors that recognize both cellular and viral ligands, including major histocompatibility complex (MHC), MHC-like, and non-MHC molecules. These receptors include Ly49s, killer immunoglobulin-like receptors, leukocyte immunoglobulin-like receptors, and NKG2A/CD94, which bind MHC class I (MHC-I) molecules, and NKG2D, which binds MHC-I paralogs such as the stress-induced proteins MICA and ULBP. In addition, certain viruses have evolved MHC-like immunoevasins, such as UL18 and m157 from cytomegalovirus, that act as decoy ligands for NK receptors. A growing number of NK receptor–ligand interaction pairs involving non-MHC molecules have also been identified, including NKp30–B7-H6, killer cell lectin-like receptor G1–cadherin, and NKp80–AICL. Here, we describe crystal structures determined to date of NK cell receptors bound to MHC, MHC-related, and non-MHC ligands. Collectively, these structures reveal the diverse solutions that NK receptors have developed to recognize these molecules, thereby enabling the regulation of NK cytolytic activity by both host and viral ligands.

Structural Basis for Penetration of the Glycan Shield of Hepatitis C Virus E2 Glycoprotein by a Broadly Neutralizing Human Antibody

Background: HCV uses glycan shielding to mask epitopes recognized by neutralizing antibodies. Results: The structure of a human antibody bound to an HCV E2 epitope revealed how it penetrates a shield created by glycosylation shifting. Conclusion: Antibody binding induces an epitope conformation that accommodates multiple glycans. Significance: The structure provides a template for engineering E2 to elicit protective antibodies able to overcome glycosylation shifting. Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. A challenge for HCV vaccine development is to identify conserved epitopes able to elicit protective antibodies against this highly diverse virus. Glycan shielding is a mechanism by which HCV masks such epitopes on its E2 envelope glycoprotein. Antibodies to the E2 region comprising residues 412–423 (E2412–423) have broadly neutralizing activities. However, an adaptive mutation in this linear epitope, N417S, is associated with a glycosylation shift from Asn-417 to Asn-415 that enables HCV to escape neutralization by mAbs such as HCV1 and AP33. By contrast, the human mAb HC33.1 can neutralize virus bearing the N417S mutation. To understand how HC33.1 penetrates the glycan shield created by the glycosylation shift to Asn-415, we determined the structure of this broadly neutralizing mAb in complex with its E2412–423 epitope to 2.0 Å resolution. The conformation of E2412–423 bound to HC33.1 is distinct from the β-hairpin conformation of this peptide bound to HCV1 or AP33, because of disruption of the β-hairpin through interactions with the unusually long complementarity-determining region 3 of the HC33.1 heavy chain. Whereas Asn-415 is buried by HCV1 and AP33, it is solvent-exposed in the HC33.1-E2412–423 complex, such that glycosylation of Asn-415 would not prevent antibody binding. Furthermore, our results highlight the structural flexibility of the E2412–423 epitope, which may serve as an immune evasion strategy to impede induction of antibodies targeting this site by reducing its antigenicity.

Structural Basis for the Presentation of Tumor-Associated MHC Class II-Restricted Phosphopeptides to CD4+ T Cells

Dysregulated protein phosphorylation is a hallmark of malignant transformation. Transformation can generate major histocompatibility complex (MHC)-bound phosphopeptides that are differentially displayed on tumor cells for specific recognition by T cells. To understand how phosphorylation alters the antigenic identity of self-peptides and how MHC class II molecules present phosphopeptides for CD4(+) T-cell recognition, we determined the crystal structure of a phosphopeptide derived from melanoma antigen recognized by T cells-1 (pMART-1), selectively expressed by human melanomas, in complex with HLA-DR1. The structure revealed that the phosphate moiety attached to the serine residue at position P5 of pMART-1 is available for direct interactions with T-cell receptor (TCR) and that the peptide N-terminus adopts an unusual conformation orienting it toward TCR. This structure, combined with measurements of peptide affinity for HLA-DR1 and of peptide-MHC recognition by pMART-1-specific T cells, suggests that TCR recognition is focused on the N-terminal portion of pMART-1. This recognition mode appears to be distinct from that of foreign antigen complexes but is remarkably reminiscent of the way autoreactive TCRs engage self- or altered self-peptides, consistent with the tolerogenic nature of tumor-host immune interactions.

Structural and Biophysical Insights into the Role of CD4 and CD8 in T Cell Activation

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 CD8αα and CD8αβ 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.

Structure of NKp65 bound to its keratinocyte ligand reveals basis for genetically linked recognition in natural killer gene complex.

The natural killer (NK) gene complex (NKC) encodes numerous C-type lectin-like receptors that govern the activity of NK cells. Although some of these receptors (Ly49s, NKG2D, CD94/NKG2A) recognize MHC or MHC-like molecules, others (Nkrp1, NKRP1A, NKp80, NKp65) instead bind C-type lectin-like ligands to which they are genetically linked in the NKC. To understand the basis for this recognition, we determined the structure of human NKp65, an activating receptor implicated in the immunosurveillance of skin, bound to its NKC-encoded ligand keratinocyte-associated C-type lectin (KACL). Whereas KACL forms a homodimer resembling other C-type lectin-like dimers, NKp65 is monomeric. The binding mode in the NKp65–KACL complex, in which a monomeric receptor engages a dimeric ligand, is completely distinct from those used by Ly49s, NKG2D, or CD94/NKG2A. The structure explains the exceptionally high affinity of the NKp65–KACL interaction compared with other cell–cell interaction pairs (KD = 6.7 × 10−10 M), which may compensate for the monomeric nature of NKp65 to achieve cell activation. This previously unreported structure of an NKC-encoded receptor–ligand complex, coupled with mutational analysis of the interface, establishes a docking template that is directly applicable to other genetically linked pairs in the NKC, including Nkrp1–Clr, NKRP1A–LLT1, and NKp80–AICL.

Structure-Based Design of Altered MHC Class II-Restricted Peptide Ligands with Heterogeneous Immunogenicity.

Insights gained from characterizing MHC–peptide–TCR interactions have held the promise that directed structural modifications can have predictable functional consequences. The ability to manipulate T cell reactivity synthetically or through genetic engineering might thus be translated into new therapies for common diseases such as cancer and autoimmune disorders. In the current study, we determined the crystal structure of HLA-DR4 in complex with the nonmutated dominant gp100 epitope gp10044–59, associated with many melanomas. Altered peptide ligands (APLs) were designed to enhance MHC binding and hence T cell recognition of gp100 in HLA-DR4+ melanoma patients. Increased MHC binding of several APLs was observed, validating this approach biochemically. Nevertheless, heterogeneous preferences of CD4+ T cells from several HLA-DR4+ melanoma patients for different gp100 APLs suggested highly variable TCR usage, even among six patients who had been vaccinated against the wild-type gp100 peptide. This heterogeneity prevented the selection of an APL candidate for developing an improved generic gp100 vaccine in melanoma. Our results are consistent with the idea that even conservative changes in MHC anchor residues may result in subtle, yet crucial, effects on peptide contacts with the TCR or on peptide dynamics, such that alterations intended to enhance immunogenicity may be unpredictable or counterproductive. They also underscore a critical knowledge gap that needs to be filled before structural and in vitro observations can be used reliably to devise new immunotherapies for cancer and other disorders.