Shinji Ikemizu

A functional and structural basis for TCR cross-reactivity in multiple sclerosis

The multiple sclerosis (MS)-associated HLA major histocompatibility complex (MHC) class II alleles DRB1*1501, DRB5*0101 and DQB1*0602 are in strong linkage disequilibrium, making it difficult to determine which is the principal MS risk gene. Here we show that together the DRB1 and DRB5 loci may influence susceptibility to MS. We demonstrate that a T cell receptor (TCR) from an MS patient recognized both a DRB1*1501-restricted myelin basic protein (MBP) and DRB5*0101-restricted Epstein-Barr virus (EBV) peptide. Crystal structure determination of the DRB5*0101-EBV peptide complex revealed a marked degree of structural equivalence to the DRB1*1501–MBP peptide complex at the surface presented for TCR recognition. This provides structural evidence for molecular mimicry involving HLA molecules. The structural details suggest an explanation for the preponderance of MHC class II associations in HLA-associated diseases.

Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses.

Optimal immune responses require both an antigen-specific and a co-stimulatory signal. The shared ligands B7-1 and B7-2 on antigen-presenting cells deliver the co-stimulatory signal through CD28 and CTLA-4 on T cells. Signalling through CD28 augments the T-cell response, whereas CTLA-4 signalling attenuates it. Numerous animal studies and recent clinical trials indicate that manipulating these interactions holds considerable promise for immunotherapy. With the consequences of these signals well established, and details of the downstream signalling events emerging, understanding the molecular nature of these extracellular interactions becomes crucial. Here we report the crystal structure of the human CTLA-4/B7-1 co-stimulatory complex at 3.0 Å resolution. In contrast to other interacting cell-surface molecules, the relatively small CTLA-4/B7-1 binding interface exhibits an unusually high degree of shape complementarity. CTLA-4 forms homodimers through a newly defined interface of highly conserved residues. In the crystal lattice, CTLA-4 and B7-1 pack in a strikingly periodic arrangement in which bivalent CTLA-4 homodimers bridge bivalent B7-1 homodimers. This zipper-like oligomerization provides the structural basis for forming unusually stable signalling complexes at the T-cell surface, underscoring the importance of potent inhibitory signalling in human immune responses.

Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

Structure and dimerization of a soluble form of B7-1.

B7-1 (CD80) and B7-2 (CD86) are glycoproteins expressed on antigen-presenting cells. The binding of these molecules to the T cell homodimers CD28 and CTLA-4 (CD152) generates costimulatory and inhibitory signals in T cells, respectively. The crystal structure of the extracellular region of B7-1 (sB7-1), solved to 3 A resolution, consists of a novel combination of two Ig-like domains, one characteristic of adhesion molecules and the other previously seen only in antigen receptors. In the crystal lattice, sB7-1 unexpectedly forms parallel, 2-fold rotationally symmetric homodimers. Analytical ultracentrifugation reveals that sB7-1 also dimerizes in solution. The structural data suggest a mechanism whereby the avidity-enhanced binding of B7-1 and CTLA-4 homodimers, along with the relatively high affinity of these interactions, favors the formation of very stable inhibitory signaling complexes.

Topological Requirements and Signaling Properties of T Cell–activating, Anti-CD28 Antibody Superagonists

Full activation of naive T cells requires both engagement of the T cell antigen receptor (TCR; signal 1) and costimulatory signaling by CD28 (signal 2). We previously identified two types of rat CD28-specific monoclonal antibodies (mAbs): “conventional,” TCR signaling–dependent costimulatory mAbs and “superagonistic” mAbs capable of inducing the full activation of primary resting T cells in the absence of TCR ligation both in vitro and in vivo. Using chimeric rat/mouse CD28 molecules, we show that the superagonists bind exclusively to the laterally exposed C′′D loop of the immunoglobulin-like domain of CD28 whereas conventional, costimulatory mAbs recognize an epitope close to the binding site for the natural CD80/CD86 ligands. Unexpectedly, the C′′D loop reactivity of a panel of new antibodies raised against human CD28 could be predicted solely on the basis of their superagonistic properties. Moreover, mouse CD28 molecules engineered to express the rat or human C′′D loop sequences activated T cell hybridomas without TCR ligation when cross-linked by superagonistic mAbs. Finally, biochemical analysis revealed that superagonistic CD28 signaling activates the nuclear factor κB pathway without inducing phosphorylation of either TCRζ or ZAP70. Our findings indicate that the topologically constrained interactions of anti-CD28 superagonists bypass the requirement for signal 1 in T cell activation. Antibodies with this property may prove useful for the development of T cell stimulatory drugs.

The nature of molecular recognition by T cells.

Considerable progress has been made in characterizing four key sets of interactions controlling antigen responsiveness in T cells, involving the following: the T cell antigen receptor, its coreceptors CD4 and CD8, the costimulatory receptors CD28 and CTLA-4, and the accessory molecule CD2. Complementary work has defined the general biophysical properties of interactions between cell surface molecules. Among the major conclusions are that these interactions are structurally heterogeneous, often reflecting clear-cut functional constraints, and that, although they all interact relatively weakly, hierarchical differences in the stabilities of the signaling complexes formed by these molecules may influence the sequence of steps leading to T cell activation. Here we review these developments and highlight the major challenges remaining as the field moves toward formulating quantitative models of T cell recognition.

CD2 and the nature of protein interactions mediating cell-cell recognition

Summary: Rapid progress has recently been made in characterising the structures of leukocyte cell‐surface molecules. Detailed analyses of the structure and interactions of CD2 were the first involving a molecule that has not been directly linked to antigen recognition in the manner of antigen receptors or co‐receptors. It seems highly likely that the properties of ligand binding by CD2 are relevant to the general mechanisms of cell‐cell recognition. As an example of biological recognition, the defining characteristic of cell‐cell contact is that it involves che simultaneous interaction of hundreds, if not thousands, of molecules. Affinity and kinetic analyses of ligand binding by CD2 indicated that the protein interactions mediating cell‐cell contact, whilst highly specific, are much weaker than initially anticipated, probably due to the requirement that such contacts be easily reversible. Simultaneously, in addressing the mechanism of this mode of recognition, structural and mutational studies focussed on the role of charged residues clustered in the ligand‐binding face of CD2, yielding the concept that electrostatic complementarity, rather than surface‐shape complementarity, is the dominant feature of specific, low‐affinity protein recognition at die cell surface by CD2. The crystallographic analysis of the CD2‐binding domain of CD58 strongly supports this concept.

Structural basis of cell-cell adhesion by NCAM.

The neural cell adhesion molecule NCAM, a member of the immunoglobulin superfamily, mediates cell–cell recognition and adhesion via a homophilic interaction. NCAM plays a key role during development and regeneration of the nervous system and is involved in synaptic plasticity associated with memory and learning. The 1.85 Å crystal structure of the two N-terminal extracellular domains of NCAM reported here provides a structural basis for the homophilic interaction. The molecular packing of the two-domain structure reveals a cross shaped antiparallel dimer, and provides fundamental insight into trans-cellular recognition mediated by NCAM.

Structure and Interactions of the Human Programmed Cell Death 1 Receptor

Background: The inhibitory leukocyte receptor PD-1 binds two ligands, PD-L1 and PD-L2. Results: Nuclear magnetic resonance analysis and rigorous binding and thermodynamic measurements reveal the structure of, and the mode of ligand recognition by, PD-1. Conclusion: PD-L1 and PD-L2 bind differently to PD-1 and much more weakly than expected. Significance: Potent inhibitory signaling can be initiated by weakly interacting receptors. PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC′ sheet, which is flexible and completely lacks a C″ strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC′ sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1·ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3–4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.

Functionalization of Tumor Necrosis Factor-α Using Phage Display Technique and PEGylation Improves Its Antitumor Therapeutic Window

Purpose: In this study, the optimization of antitumor therapy with tumor necrosis factor-α (TNF-α) was attempted. Experimental Design: Using the phage display technique, we created a lysine-deficient mutant TNF-α (mTNF-K90R). This mutant had higher affinities to both TNF receptors, despite reports that certain lysine residues play important roles in trimer formation and receptor binding. Results: The mTNF-K90R showed an in vivo therapeutic window that was 13-fold higher than that of the wild-type TNF-α (wTNF-α). This was due to the synergistic effect of its 6-fold stronger in vitro bioactivity and its 2-fold longer plasma half-life derived from its surface negative potential. The reason why the mTNF-K90R showed a higher bioactivity was understood by a molecular modeling analysis of the complex between the wTNF-α and TNF receptor-I. The mTNF-K90R, which was site-specifically mono-PEGylated at the NH2 terminus (sp-PEG-mTNF-K90R), had a higher in vitro bioactivity and considerably longer plasma half-life than the wTNF-α, whereas the randomly mono-PEGylated wTNF-α had 6% of the bioactivity of the wTNF-α. With regard to effectiveness and safety, the in vivo antitumor therapeutic window of the sp-PEG-mTNF-K90R was 60-fold wider than that of the wTNF-α. Conclusions: These results indicated that this functionalized TNF-α may be useful not only as an antitumor agent but also as a selective enhancer of vascular permeability in tumors for improving antitumor chemotherapy.