James Scott-Browne

Expansion and function of Foxp3-expressing T regulatory cells during tuberculosis

Mycobacterium tuberculosis (Mtb) frequently establishes persistent infections that may be facilitated by mechanisms that dampen immunity. T regulatory (T reg) cells, a subset of CD4+ T cells that are essential for preventing autoimmunity, can also suppress antimicrobial immune responses. We use Foxp3-GFP mice to track the activity of T reg cells after aerosol infection with Mtb. We report that during tuberculosis, T reg cells proliferate in the pulmonary lymph nodes (pLNs), change their cell surface phenotype, and accumulate in the pLNs and lung at a rate parallel to the accumulation of effector T cells. In the Mtb-infected lung, T reg cells accumulate in high numbers in all sites where CD4+ T cells are found, including perivascular/peribronchiolar regions and within lymphoid aggregates of granulomas. To determine the role of T reg cells in the immune response to tuberculosis, we generated mixed bone marrow chimeric mice in which all cells capable of expressing Foxp3 expressed Thy1.1. When T reg cells were depleted by administration of anti-Thy1.1 before aerosol infection with Mtb, we observed ∼1 log less of colony-forming units of Mtb in the lungs. Thus, after aerosol infection, T reg cells proliferate and accumulate at sites of infection, and have the capacity to suppress immune responses that contribute to the control of Mtb.

CD1d-restricted iNKT cells, the 'Swiss-Army knife' of the immune system

Natural Killer T cells are a distinct lymphocyte lineage that regulates a broad range of immune responses. NKT cells recognize glycolipids presented by the non-classical MHC molecule CD1d. Structural insight into the TCR/glycolipid/CD1d tri-complex has revealed an unusual and unexpected mode of recognition. Recent studies have also identified some of the signaling events during NKT cell development that give NKT cells their innate phenotype. Pathogen-derived glycolipid antigens continue to be found, and new mechanisms of NKT cell activation have been described. Finally, NKT cells have been shown to be remarkably versatile in function during various immune responses. Whether these extensive functional capacities can be attributed to a single population sensitive to environmental cues or if functionally distinct NKT cell subpopulations exist remains unresolved.

Evolutionarily Conserved Amino Acids That Control TCR-MHC Interaction

The rules for the conserved reaction of alphabeta T cell receptors (TCRs) with major histocompatibility complex (MHC) proteins plus peptides are poorly understood, probably because thymocytes bearing TCRs with the strongest MHC reactivity are lost by negative selection. Thus, only TCRs with an attenuated ability to react with MHC appear on mature T cells. Also, the interaction sites between TCRs and MHC may be inherently flexible and hence difficult to spot. We reevaluated contacts between TCRs and MHC in the solved structures of their complexes with these points in mind. Relatively conserved amino acids in the TCR complementarity-determining regions (CDR) 1 and CDR2 are often used to bind exposed areas of the MHC alpha-helices. These areas are exposed because of small amino acids that allow somewhat flexible binding of the TCRs. The TCR amino acids involved are specific to families of variable (V) regions and to some extent different rules may govern the recognition of MHCI versus MHCII.

Crossreactive T cells spotlight the germline rules for αβ T cell receptor interactions with MHC molecules

To test whether highly crossreactive alphabeta T cell receptors (TCRs) produced during limited negative selection best illustrate evolutionarily conserved interactions between TCR and major histocompatibility complex (MHC) molecules, we solved the structures of three TCRs bound to the same MHC II peptide (IAb-3K). The TCRs had similar affinities for IAb-3K but varied from noncrossreactive to extremely crossreactive with other peptides and MHCs. Crossreactivity correlated with a shrinking, increasingly hydrophobic TCR-ligand interface, involving fewer TCR amino acids. A few CDR1 and CDR2 amino acids dominated the most crossreactive TCR interface with MHC, including Vbeta8 48Y and 54E and Valpha4 29Y, arranged to impose the familiar diagonal orientation of TCR on MHC. These interactions contribute to MHC binding by other TCRs using related V regions, but not usually so dominantly. These data show that crossreactive TCRs can spotlight the evolutionarily conserved features of TCR-MHC interactions and that these interactions impose the diagonal docking of TCRs on MHC.

Germline-encoded amino acids in the αβ T cell receptor control thymic selection

An αβ T-cell response depends on the recognition of antigen plus major histocompatibility complex (MHC) proteins by its antigen receptor (TCR). The ability of peripheral αβ T cells to recognize MHC is at least partly determined by MHC-dependent thymic selection, by which an immature T cell survives only if its TCR can recognize self MHC. This process may allow MHC-reactive TCRs to be selected from a repertoire with completely random and unbiased specificities. However, analysis of thymocytes before positive selection indicated that TCR proteins might have a predetermined ability to bind MHC. Here we show that specific germline-encoded amino acids in the TCR promote ‘generic’ MHC recognition and control thymic selection. In mice expressing single, rearranged TCR β-chains, individual mutation of amino acids in the complementarity-determining region (CDR) 2β to Ala reduced development of the entire TCR repertoire. Altogether, these results show that thymic selection is controlled by germline-encoded MHC contact points in the αβ TCR and indicate that the diversity of the peripheral T-cell repertoire is enhanced by this ‘built-in’ specificity.

T Cell Receptor CDR2β and CDR3β Loops Collaborate Functionally to Shape the iNKT Cell Repertoire

Mouse type I natural killer T cell receptors (iNKT TCRs) use a single V alpha 14-J alpha 18 sequence and V beta s that are almost always V beta 8.2, V beta 7, or V beta 2, although the basis of this differential usage is unclear. We showed that the V beta bias occurred as a consequence of the CDR2 beta loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent V beta-CD1d affinities are further modulated by the hypervariable CDR3 beta loop, thereby defining a functional interplay between the two iNKT TCR CDR beta loops. These V beta biases revealed a broadly hierarchical response in which V beta 8.2 > V beta 7 > V beta 2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.

A minimal binding footprint on CD1d-glycolipid is a basis for selection of the unique human NKT TCR

Although it has been established how CD1 binds a variety of lipid antigens (Ag), data are only now emerging that show how αβ T cell receptors (TCRs) interact with CD1-Ag. Using the structure of the human semiinvariant NKT TCR–CD1d–α-galactosylceramide (α-GalCer) complex as a guide, we undertook an alanine scanning mutagenesis approach to define the energetic basis of this interaction between the NKT TCR and CD1d. Moreover, we explored how analogues of α-GalCer affected this interaction. The data revealed that an identical energetic footprint underpinned the human and mouse NKT TCR–CD1d–α-GalCer cross-reactivity. Some, but not all, of the contact residues within the Jα18-encoded invariant CDR3α loop and Vβ11-encoded CDR2β loop were critical for recognizing CD1d. The residues within the Vα24-encoded CDR1α and CDR3α loops that contacted the glycolipid Ag played a smaller energetic role compared with the NKT TCR residues that contacted CD1d. Collectively, our data reveal that the region distant to the protruding Ag and directly above the F′ pocket of CD1d was the principal factor in the interaction with the NKT TCR. Accordingly, although the structural footprint at the NKT TCR–CD1d–α-GalCer is small, the energetic footprint is smaller still, and reveals the minimal requirements for CD1d restriction.

Adaptability of the semi-invariant natural killer T-cell receptor towards structurally diverse CD1d-restricted ligands

The semi‐invariant natural killer (NK) T‐cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the α‐chain of the NKTcr is invariant, the β‐chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an α‐linked monosaccharide (α‐galactosylceramide and α‐galactosyldiacylglycerol) and the β‐linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their β‐chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including α‐galactosylceramide, the structurally similar α‐galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr β‐chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr β‐chain allows these cells to recognise unique aspects of structurally diverse CD1d‐restricted ligands.

T cells and their eons-old obsession with MHC

T cells bearing receptors made up of α and β chains (TCRs) usually react with peptides bound to major histocompatibility complex proteins (MHC). This bias could be imposed by positive selection, the phenomenon that selects thymocytes to mature into T cells only if the TCRs they bear react with low but appreciable affinity with MHC + peptide combinations in the thymus cortex. However, it is also possible that the polypeptides of TCRs themselves do not have random specificities but rather are biased toward reaction with MHC. Evolution would therefore have selected for a collection of TCR variable elements that are prone to react with MHC. If this were to be so, positive selection would act on thymocytes bearing a pre biased collection of TCRs to pick out those that react to some extent, but not too well, with self MHC + self‐peptides. A problem with studies of this evolutionary idea is the fact that there are many TCR variable elements and that these differ considerably in the amino acids with which they contact MHC. However, recent experiments by our group and others suggest that one group of TCR variable elements, those related to the mouse Vβ8 family, has amino acids in their CDR2 regions that consistently bind a particular site on an MHC α‐helix. Other groups of variable elements may use different patterns of amino acids to achieve the same goal. Mutation of these amino acids reduces the ability of T cells and thymocytes to react with MHC. These amino acids are present in the variable regions of distantly related species such as sharks and human. Overall the data indicate that TCR elements have indeed been selected by evolution to react with MHC proteins. Many mysteries about TCRs remain to be solved, including the nature of auto‐recognition, the basis of MHC allele specificity, and the very nature and complexity of TCRs on mature T cells.

MAIT Cell Recognition of MR1 on Bacterially Infected and Uninfected Cells

Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant αβ TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is resistant to proteinase K digestion and does not extract with common lipids, suggesting an unexpected class of antigen(s) might be recognized by this unique lymphocyte population.