Erin J. Adams

Analysis of a Successful Immune Response against Hepatitis C Virus

To investigate the type of immunity responsible for resolution of hepatitis C virus (HCV) infection, we monitored antibody and intrahepatic cytotoxic T lymphocyte (CTL) responses during acute (<20 weeks) infection in chimpanzees. Two animals who terminated infection made strong CTL but poor antibody responses. In both resolvers, CTL targeted at least six viral regions. In contrast, animals developing chronic hepatitis generated weaker acute CTL responses. Extensive analysis of the fine specificity of the CTL in one resolver revealed nine peptide epitopes and restriction by all six MHC class I allotypes. Every specificity shown during acute hepatitis persisted in normal liver tissue more than 1 yr after resolution. These results suggest that CD8+CTL are better correlated with protection against HCV infection than antibodies.

The Origins of HLA‐A,B,C Polymorphism

Polymorphism of HLA-A,B and C genes determines the repertoire of receptor specificities expressed by cytolytic CDS T lymphocytes (CTL) and the response of natural killer (NK) cells. In the performance of these functions HLA-A,B,C heavy chains interact with ^i-microglobulin (/A-m), a multitude of peptides of 810 amino acids, diverse T-cell receptors (TcR), the CD8 co-receptor, class I receptors of NK cells, the TAP peptide transporter, and the chaperonins calnexin and heavy chain binding protein (BIP). Polymorphism modulates all these interactions and in aggregate affects profoundly the cytolytic lymphocyte response in persons with different HLA-A,B,C types. Pairwise comparison of unrelated individuals in modern human populations shows they rarely have the same HLA-A,B,C type. As a consequence the human cytoiytic lymphocyte response to infectious agents, transformed cells, transplanted tissues and autoantigens varies from one person to another. There is no wild-type the normal individual on which research can be focused. So, to gain understanding of the human immune response and facilitate the prevention and cure of disease, it becomes necessary to study all HLA-A,B,C types. The further this endeavor has been pursued the greater the diversity of HLA-A,B and C that has been found. Although a monumental headache for the biological or clinical reductionist, the tremendous diversity of HLA-A,B,C make these genes wonderful tools for those interested in the evolution of human populations and their immune relationship to diverse pathogens.

Evidence that structural rearrangements and/or flexibility during tcr binding can contribute to t cell activation

While in many cases the half-life of T cell receptor (TCR) binding to a particular ligand is a good predictor of activation potential, numerous exceptions suggest that other physical parameter(s) must also play a role. Accordingly, we analyzed the thermodynamics of TCR binding to a series of peptide-MHC ligands, three of which are more stimulatory than their stability of binding would predict. Strikingly, we find that during TCR binding these outliers show anomalously large changes in heat capacity, an indicator of conformational change or flexibility in a binding interaction. By combining the values for heat capacity (DeltaCp) and the half-life of TCR binding (t(1/2)), we find that we can accurately predict the degree of T cell stimulation. Structural analysis shows significant changes in the central TCR contact residue of the peptide-MHC, indicating that structural rearrangements within the TCR-peptide-MHC interface can contribute to T cell activation.

Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human γδ T-cell subset.

Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.

Recognition of Lyso-Phospholipids by Human Natural Killer T Lymphocytes

By identifying the lipid LPC as an endogenous antigen, recognized by the invariant subset of human NKT cells, this study establishes a novel link between these immunoregulatory cells and an inflammatory lipid mediator.

The Intracellular B30.2 Domain of Butyrophilin 3A1 Binds Phosphoantigens to Mediate Activation of Human Vγ9Vδ2 T Cells

In humans, Vγ9Vδ2 T cells detect tumor cells and microbial infections, including Mycobacterium tuberculosis, through recognition of small pyrophosphate containing organic molecules known as phosphoantigens (pAgs). Key to pAg-mediated activation of Vγ9Vδ2 T cells is the butyrophilin 3A1 (BTN3A1) protein that contains an intracellular B30.2 domain critical to pAg reactivity. Here, we have demonstrated through structural, biophysical, and functional approaches that the intracellular B30.2 domain of BTN3A1 directly binds pAg through a positively charged surface pocket. Charge reversal of pocket residues abrogates binding and Vγ9Vδ2 T cell activation. We have also identified a gain-of-function mutation within this pocket that, when introduced into the B30.2 domain of the nonstimulatory BTN3A3 isoform, transfers pAg binding ability and Vγ9Vδ2 T cell activation. These studies demonstrate that internal sensing of changes in pAg metabolite concentrations by BTN3A1 molecules is a critical step in Vγ9Vδ2 T cell detection of infection and tumorigenesis.

Species‐specific evolution of MHC class I genes in the higher primates

Summary: Humans express three highly polymorphic ‘classical’ (HLA‐A,B and C) and three conserved ‘non‐classical’ (HLA‐E, F and G) MHC class I genes. Their comparison with the MHC class I genes of apes and monkeys reveals the differential extent to which MHC class I genes have been preserved during primate evolution. African apes have orthologues of all six human genes, and although allelic lineages of the A and C loci are shared, these species share none of the human alleles. In Asian apes, several MHC class I genes show significant differences from the human genes, a trend which continues with the Old World monkeys, and even more so in the New World monkeys, where E and F are the only human gene orthologues. The C locus is confined to humans and apes. Multiple A‐related and B‐related loci have been identified in apes and Old World monkeys showing that duplication of these loci has been a common event during primate evolution. Certain of the daughter loci exhibit low polymorphism, suggesting they have adopted a non‐classical function. The differing rates at which MHC class I genes have evolved during primate evolution likely reflects their differing functions in the immune response.

CK-1, a putative chemokine of rainbow trout (Oncorhynchus mykiss).

Summary: Chemokines arc small inducible proteins that direct the migration of leukocytes. While chemokines are well characterised in mammals, they have yet to be identified in fish. We have isolated a cDNA clone from rainbow trout (Oncorhynchus mykiss) which encodes a protein (CK‐1) having structural features typical of chemokines. Amino‐acid residues that define the β‐chemokines of mammals are conserved in CK‐1, including the paired cysteine motif, CC. Further similarities are shared with the C6 subfamily of β‐chemokines. In contrast, the organisation of the CK‐f gene is closer to that of mammalian α‐chemokine genes than β‐chemokine genes. The CK‐1 gene is present in all four salmonid species examined and the nucleotide sequences of the exons are highly conserved. CK‐1 has characteristics in common with mammalian α and β‐chemokine suggesting that this salmonid chemokine gene preserves traits once present in the ancestral chemokine gene from which modern mammalian chemokine genes evolved.

Crystal Structure of Vδ1 T Cell Receptor in Complex with CD1d-Sulfatide Shows MHC-like Recognition of a Self-Lipid by Human γδ T Cells

The nature of the antigens recognized by γδ T cells and their potential recognition of major histocompatibility complex (MHC)-like molecules has remained unclear. Members of the CD1 family of lipid-presenting molecules are suggested ligands for Vδ1 TCR-expressing γδ T cells, the major γδ lymphocyte population in epithelial tissues. We crystallized a Vδ1 TCR in complex with CD1d and the self-lipid sulfatide, revealing the unusual recognition of CD1d by germline Vδ1 residues spanning all complementarity-determining region (CDR) loops, as well as sulfatide recognition separately encoded by nongermline CDR3δ residues. Binding and functional analysis showed that CD1d presenting self-lipids, including sulfatide, was widely recognized by gut Vδ1+ γδ T cells. These findings provide structural demonstration of MHC-like recognition of a self-lipid by γδ T cells and reveal the prevalence of lipid recognition by innate-like T cell populations.

NK Cell Receptors of the Orangutan (Pongo pygmaeus): A Pivotal Species for Tracking the Coevolution of Killer Cell Ig-Like Receptors with MHC-C

CD94, NKG2, Ly49, and killer cell Ig-like receptor (KIR) expressed by orangutan peripheral blood cells were examined by cloning and sequencing cDNA from a panel of individuals. Orthologs of human CD94, NKG2A, D, and F were defined. NKG2C and E are represented by one gene, Popy-NKG2CE, that is equidistant from the two human genes. Several Popy-CD94, NKG2A, and NKG2CE alleles were defined. Popy-Ly49L is expressed in cultured NK cells and has a sequence consistent with it encoding a functional receptor. Orangutan KIR corresponding to the three KIR lineages expressed in humans and chimpanzees were defined. Popy-KIR2DL4 of lineage I is the only ortholog of a human or chimpanzee KIR, but in all individuals examined, the transcripts of this gene produced premature termination, either in the D2 domain or at the beginning of the cytoplasmic domain. Ten Popy-KIR3DL and one Popy-KIR3DS of lineage II are all closely related, but represent the products of at least two genes. The two Popy-KIR2DL and four Popy-KIR2DS of lineage III also represent two genes, both being more related to KIR2DS4 than to other human and chimpanzee KIR of lineage III. The Popy-KIR2D include ones predicted to be specific for the C1 epitope of MHC-C, but none specific for C2. This correlates with the observation that all orangutan MHC-C allotypes examined have the C1 motif.