Benjamin E. Willcox

Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G

Ig-like transcript 4 (ILT4) (also known as leukocyte Ig-like receptor 2, CD85d, and LILRB2) is a cell surface receptor expressed mainly on myelomonocytic cells, whereas ILT2 (also known as leukocyte Ig-like receptor 1, CD85j, and LILRB1) is expressed on a wider range of immune cells including subsets of natural killer and T cells. Both ILTs contain immunoreceptor tyrosine-based inhibitory receptor motifs in their cytoplasmic tails that inhibit cellular responses by recruiting phosphatases such as SHP-1 (Src homology 2 domain containing tyrosine phosphatase 1). Although these ILTs have been shown to recognize a broad range of classical and nonclassical human MHC class I molecules (MHCIs), their precise binding properties remain controversial. We have used surface plasmon resonance to analyze the interaction of soluble forms of ILT4 and ILT2 with several MHCIs. Although the range of affinities measured was quite broad (Kd = 2–45 μM), some interesting differences were observed. ILT2 generally bound with a 2- to 3-fold higher affinity than ILT4 to the same MHCI. Furthermore, ILT2 and ILT4 bound to HLA-G with a 3- to 4-fold higher affinity than to classical MHCIs, suggesting that ILT/HLA-G recognition may play a dominant role in the regulation of natural killer, T, and myelomonocytic cell activation. Finally, we show that ILT2 and ILT4 effectively compete with CD8 for MHCI binding, raising the possibility that ILT2 modulates CD8+ T cell activation by blocking the CD8 binding as well as by recruiting inhibitory molecules through its immunoreceptor tyrosine-based inhibitory receptor motif.

Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA.

The major histocompatibility complex (MHC) class I homolog, MICA, is a stress-inducible ligand for NKG2D, a C-type lectin–like activating immunoreceptor. The crystal structure of this ligand-receptor complex that we report here reveals an NKG2D homodimer bound to a MICA monomer in an interaction that is analogous to that seen in T cell receptor–MHC class I protein complexes. Similar surfaces on each NKG2D monomer interact with different surfaces on either the α1 or α2 domains of MICA. The binding interactions are large in area and highly complementary. The central section of the α2-domain helix, disordered in the structure of MICA alone, is ordered in the complex and forms part of the NKG2D interface. The extensive flexibility of the interdomain linker of MICA is shown by its altered conformation when crystallized alone or in complex with NKG2D.

TCR Binding to Peptide-MHC Stabilizes a Flexible Recognition Interface

The binding of TCRs to their peptide-MHC ligands is characterized by a low affinity, slow kinetics, and a high degree of cross-reactivity. Here, we report the results of a kinetic and thermodynamic analysis of two TCRs binding to their peptide-MHC ligands, which reveal two striking features. First, significant activation energy barriers must be overcome during both association and dissociation, suggesting that conformational adjustments are required. Second, the low affinity of binding is a consequence of highly unfavorable entropic effects, indicative of a substantial reduction in disorder upon binding. This is evidence that the TCR and/or peptide-MHC have flexible binding surfaces that are stabilized upon binding. Such conformational flexibility, which may also be a feature of primary antibodies, is likely to contribute to cross-reactivity in antigen recognition.

Crystal structure of HLA-A2 bound to LIR-1, a host and viral major histocompatibility complex receptor.

Leukocyte immunoglobulin-like receptor 1 (LIR-1), an inhibitory receptor expressed on monocytes, dendritic cells and lymphocytes, regulates cellular function by binding a broad range of classical and nonclassical major histocompatibility complex (MHC) class I molecules, and the human cytomegalovirus MHC class I homolog UL18. Here we describe the 3.4-Å crystal structure of a complex between the LIR-1 D1D2 domains and the MHC class I molecule HLA-A2. LIR-1 contacts the mostly conserved β2-microglobulin and α3 domains of HLA-A2. The LIR-1 binding site comprises residues at the interdomain hinge, and a patch at the D1 tip. The structure shows how LIR-1 recognizes UL18 and diverse MHC class I molecules, and indicates that a similar mode of MHC class I recognition is used by other LIR family members.

T Cell Receptor and Coreceptor CD8αα Bind Peptide-MHC Independently and with Distinct Kinetics

The T cell surface glycoprotein CD8 enhances T cell antigen recognition by binding to MHC class I molecules. We show that human CD8 alphaalpha binds to the MHC class I molecule HLA-A2 with an extremely low affinity (Kd approximately 0.2 mM at 37 degrees C) and with kinetics that are between 2 and 3 orders of magnitude faster than reported for T cell receptor/peptide-MHC interactions. Furthermore, CD8 alphaalpha had no detectable effect on a T cell receptor (TCR) binding to the same peptide-MHC class I complex. These binding properties provide an explanation as to why the CD8/MHC class I interaction is unable to initiate cell-cell adhesion and how it can enhance TCR recognition without interfering with its specificity.

Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E.

The crystal structure of the nonclassical human class lb MHC molecule HLA-E has been determined in complex with a prototypic ligand, the nonamer peptide (VMAPRTVLL), derived from the highly conserved residues 3-11 of the human MHC class la leader sequence. The mode of peptide binding retains some of the standard features observed in MHC class la complexes, but novel features imply that HLA-E has evolved to mediate specific binding to a tightly defined set of almost identical hydrophobic peptides from the highly conserved class l leader sequences. These molecular adaptations make HLA-E a rigorous checkpoint at the cell surface reporting on the integrity of the antigen processing pathway to CD94/NKG2 receptor-bearing natural killer cells.

Molecular competition for NKG2D: H60 and RAE1 compete unequally for NKG2D with dominance of H60.

NKG2D is a potent activating receptor on natural killer cells, T cells, and macrophages. Mouse NKG2D interacts with two cell surface ligands related to class I MHC molecules: RAE1 and H60. We used soluble versions of NKG2D, RAE1, and H60 to characterize their interactions. RAE1 and H60 each bind NKG2D with nanomolar affinities, indicating tighter binding than most cell surface immune interactions, but NKG2D binds to H60 with approximately 25-fold higher affinity than to RAE1. RAE1 and H60 compete directly for occupancy of NKG2D, and, thus, NKG2D can be occupied by only one ligand at a time. The NKG2D-H60 interaction is more temperature dependent and makes greater use of electrostatic interactions than the NKG2D-RAE1 interaction. The distinct thermodynamic profiles provide insights into the different molecular mechanisms of the binding interactions.

Cytotoxic T lymphocytes recognize structurally diverse, clade‐specific and cross‐reactive peptides in human immunodeficiency virus type‐1 gag through HLA‐B53

Human immunodeficiency virus type‐1 (HIV‐1) cytotoxic T lymphocyte (CTL) epitopes have largely been defined in Caucasian populations infected with clade B virus. Identification of potentially protective CTL epitopes in non‐B clade‐infected African subjects is important for vaccine development. In a study of CTL responses in clade A‐infected Gambians, using cytotoxicity, interferon‐γ (IFN‐γ) enzyme‐linked immunospot (ELISpot) and HLA‐B53‐peptide tetramer assays, we identified three HLA‐B53‐restricted epitopes in HIV‐1 gag p24. CTL specific for an epitope in a highly immunogenic region of the p24 protein showed no cross‐reactivity to other HIV‐1 clades. Two of the epitopes would not have been predicted from the peptide‐binding motif due to the absence of a proline anchor at position 2. Structural analysis of HLA‐B53 and its relative, HLA B35, enabled us to re‐define the peptide‐binding motif to include other P2 anchors. These results demonstrate the value of combined immunological and structural analyses in defining novel CTL epitopes and have implications for HIV‐1 vaccine design.

Crystal structure of LIR-2 (ILT4) at 1.8 Å: differences from LIR-1 (ILT2) in regions implicated in the binding of the Human Cytomegalovirus class I MHC homolog UL18

BackgroundLeukocyte Immunoglobulin-like Receptor-1 (LIR-1) and LIR-2 (also known as ILT2 and ILT4 respectively) are highly related cell surface receptors that bind a broad range of class I MHC molecules with low (μM) affinities. Expressed on monocytic cells and macrophages, both molecules transmit inhibitory signals after binding ligands. In addition to binding host class I MHC, the LIR-1 molecule, which is also expressed on lymphoid tissues, binds with a high (nM) affinity to UL18, a class I MHC homolog encoded by Human Cytomegalovirus (HCMV). In comparison, LIR-2 binds UL18 only weakly (μM KD). To understand how HCMV preferentially targets the more broadly expressed LIR-1 molecule, we determined the crystal structure of a ligand-binding fragment of LIR-2, and compared this to the existing high-resolution crystal structure of LIR-1.ResultsRecombinant LIR-2 (domains 1 and 2) was produced in E. coli and crystallized using streak seeding to optimize the crystal morphology. A data set complete to 1.8 Å was collected at 100 K from a single crystal in the P41212 spacegroup. The structure was solved by molecular replacement, using a search model based on the LIR-1 structure.ConclusionsThe overall structure of LIR-2 D1D2 resembles both LIR-1, and Killer Inhibitory Receptors, in that the A strand in each domain forms hydrogen bonds to both β sheets, and there is a sharp angle between the two immunoglobulin-like domains. However, differences from LIR-1 are observed in each domain, with two key changes apparent in the ligand-binding domain, D1. The region corresponding to the residue 44–57 helix of LIR-1 adopts a topology distinct from that of both LIR-1 and the KIR structures, involving a shortened 310 helix. Secondly, the predicted UL18 binding region of LIR-1 is altered substantially in LIR-2: the 76–84 loop mainchain is displaced 11 Å with respect to LIR-1, and Tyrosine 38 adopts an alternative rotamer conformation. In summary, the structure of LIR-2 has revealed significant differences to LIR-1, including ones that may help to explain the >1000-fold lower affinity of LIR-2 for UL18.