Pamela J. Bjorkman

T-cell antigen receptor genes and T-cell recognition

The four distinct T-cell antigen receptor polypeptides (α, β, γ, δ) form two different heterodimers (α:β and γ:δ) that are very similar to immunoglobulins in primary sequence, gene organization and modes of rearrangement. Whereas antibodies have both soluble and membrane forms that can bind to antigens alone, T-cell receptors exist only on cell surfaces and recognize antigen fragments only when they are embedded in major histocompatibility complex (MHC) molecules. Patterns of diversity in T-cell receptor genes together with structural features of immunoglobulin and MHC molecules suggest a model for how this recognition might occur. This view of T-cell recognition has implications for how the receptors might be selected in the thymus and how they (and immunoglobulins) may have arisen during evolution.

Sequence and Structural Convergence of Broad and Potent HIV Antibodies That Mimic CD4 Binding

Anti-HIV broadly neutralizing antibodies with similar specificities and modes of binding were found in multiple HIV-infected individuals. Passive transfer of broadly neutralizing HIV antibodies can prevent infection, which suggests that vaccines that elicit such antibodies would be protective. Thus far, however, few broadly neutralizing HIV antibodies that occur naturally have been characterized. To determine whether these antibodies are part of a larger group of related molecules, we cloned 576 new HIV antibodies from four unrelated individuals. All four individuals produced expanded clones of potent broadly neutralizing CD4-binding-site antibodies that mimic binding to CD4. Despite extensive hypermutation, the new antibodies shared a consensus sequence of 68 immunoglobulin H (IgH) chain amino acids and arise independently from two related IgH genes. Comparison of the crystal structure of one of the antibodies to the broadly neutralizing antibody VRC01 revealed conservation of the contacts to the HIV spike.

Crystal Structure of the Hemochromatosis Protein HFE and Characterization of Its Interaction with Transferrin Receptor

HFE is an MHC-related protein that is mutated in the iron-overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR) and reduces its affinity for iron-loaded transferrin, implicating HFE in iron metabolism. The 2.6 A crystal structure of HFE reveals the locations of hemochromatosis mutations and a patch of histidines that could be involved in pH-dependent interactions. We also demonstrate that soluble TfR and HFE bind tightly at the basic pH of the cell surface, but not at the acidic pH of intracellular vesicles. TfR:HFE stoichiometry (2:1) differs from TfR:transferrin stoichiometry (2:2), implying a different mode of binding for HFE and transferrin to TfR, consistent with our demonstration that HFE, transferrin, and TfR form a ternary complex.

HIV therapy by a combination of broadly neutralizing antibodies in humanized mice

Human antibodies to human immunodeficiency virus-1 (HIV-1) can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection. Previous work showed that antibodies exert selective pressure on the virus but escape variants emerge within a short period of time. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice. Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy, the longer half-life of antibodies led to control of viraemia for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in humanized mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals.

The Inhibitory Receptor LIR-1 Uses a Common Binding Interaction to Recognize Class I MHC Molecules and the Viral Homolog UL18

LIR-1 is a class I MHC receptor related to natural killer inhibitory receptors (KIRs). Binding of LIR-1 or KIRs to class I molecules results in inhibitory signals. Unlike individual KIRs, LIR-1 recognizes many class I alleles and also binds UL18, a human cytomegalovirus class I MHC homolog. Here, we show that LIR-1 interacts with the relatively nonpolymorphic alpha3 domain of class I proteins and the analogous region of UL18 using its N-terminal immunoglobulin-like domain. The >1000-fold higher affinity of LIR-1 for UL18 than for class I illustrates how a viral protein competes with host proteins to subvert the host immune response. LIR-1 recognition of class I molecules resembles the CD4-class II MHC interaction more than the KIR-class I interaction, implying a functional distinction between LIR-1 and KIRs.

Direct Assessment of MHC Class I Binding by Seven Ly49 Inhibitory NK Cell Receptors

Mouse NK cells express at least seven inhibitory Ly49 receptors. Here we employ a semiquantitative cell-cell adhesion assay as well as class I/peptide tetramers to provide a comprehensive analysis of specificities of Ly49 receptors for class I MHC molecules in eight MHC haplotypes. Different Ly49 receptors exhibited diverse binding properties. The degree of class I binding was related to the extent of functional inhibition. The tetramer studies demonstrated that neither glycosylation nor coreceptors were necessary for class I binding to Ly49 receptors and uncovered peptide-specific recognition by a Ly49 receptor. The results provide a foundation for interpreting and integrating many existing functional studies as well as for designing tests of NK cell development and self-tolerance.

Crystal structure of the hereditary haemochromatosis protein HFE complexed with transferrin receptor.

HFE is related to major histocompatibility complex (MHC) class I proteins and is mutated in the iron-overload disease hereditary haemochromatosis. HFE binds to the transferrin receptor (TfR), a receptor by which cells acquire iron-loaded transferrin. The 2.8 Å crystal structure of a complex between the extracellular portions of HFE and TfR shows two HFE molecules which grasp each side of a twofold symmetric TfR dimer. On a cell membrane containing both proteins, HFE would ‘lie down’ parallel to the membrane, such that the HFE helices that delineate the counterpart of the MHC peptide-binding groove make extensive contacts with helices in the TfR dimerization domain. The structures of TfR alone and complexed with HFE differ in their domain arrangement and dimer interfaces, providing a mechanism for communicating binding events between TfR chains. The HFE–TfR complex suggests a binding site for transferrin on TfR and sheds light upon the function of HFE in regulating iron homeostasis.

Increasing the potency and breadth of an HIV antibody by using structure-based rational design.

Structural analysis of an HIV antibody reveals residues important for neutralization breadth and potency. Antibodies against the CD4 binding site (CD4bs) on the HIV-1 spike protein gp120 can show exceptional potency and breadth. We determined structures of NIH45-46, a more potent clonal variant of VRC01, alone and bound to gp120. Comparisons with VRC01-gp120 revealed that a four-residue insertion in heavy chain complementarity–determining region 3 (CDRH3) contributed to increased interaction between NIH45-46 and the gp120 inner domain, which correlated with enhanced neutralization. We used structure-based design to create NIH45-46G54W, a single substitution in CDRH2 that increases contact with the gp120 bridging sheet and improves breadth and potency, critical properties for potential clinical use, by an order of magnitude. Together with the NIH45-46–gp120 structure, these results indicate that gp120 inner domain and bridging sheet residues should be included in immunogens to elicit CD4bs antibodies.

Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies

Broadly neutralizing HIV antibodies (bNAbs) can recognize carbohydrate-dependent epitopes on gp120. In contrast to previously characterized glycan-dependent bNAbs that recognize high-mannose N-glycans, PGT121 binds complex-type N-glycans in glycan microarrays. We isolated the B-cell clone encoding PGT121, which segregates into PGT121-like and 10-1074–like groups distinguished by sequence, binding affinity, carbohydrate recognition, and neutralizing activity. Group 10-1074 exhibits remarkable potency and breadth but no detectable binding to protein-free glycans. Crystal structures of unliganded PGT121, 10-1074, and their likely germ-line precursor reveal that differential carbohydrate recognition maps to a cleft between complementarity determining region (CDR)H2 and CDRH3. This cleft was occupied by a complex-type N-glycan in a “liganded” PGT121 structure. Swapping glycan contact residues between PGT121 and 10-1074 confirmed their importance for neutralization. Although PGT121 binds complex-type N-glycans, PGT121 recognized high-mannose-only HIV envelopes in isolation and on virions. As HIV envelopes exhibit varying proportions of high-mannose- and complex-type N-glycans, these results suggest promiscuous carbohydrate interactions, an advantageous adaptation ensuring neutralization of all viruses within a given strain.

The transferrin receptor modulates Hfe-dependent regulation of hepcidin expression.

Hemochromatosis is caused by mutations in HFE, a protein that competes with transferrin (TF) for binding to transferrin receptor 1 (TFR1). We developed mutant mouse strains to gain insight into the role of the Hfe/Tfr1 complex in regulating iron homeostasis. We introduced mutations into a ubiquitously expressed Tfr1 transgene or the endogenous Tfr1 locus to promote or prevent the Hfe/Tfr1 interaction. Under conditions favoring a constitutive Hfe/Tfr1 interaction, mice developed iron overload attributable to inappropriately low expression of the hormone hepcidin. In contrast, mice carrying a mutation that interferes with the Hfe/Tfr1 interaction developed iron deficiency associated with inappropriately high hepcidin expression. High-level expression of a liver-specific Hfe transgene in Hfe-/- mice was also associated with increased hepcidin production and iron deficiency. Together, these models suggest that Hfe induces hepcidin expression when it is not in complex with Tfr1.