Hsiu-Ching Chang

Structural Basis of CD8 Coreceptor Function Revealed by Crystallographic Analysis of a Murine CD8αα Ectodomain Fragment in Complex with H-2Kb

Abstract The crystal structure of the two immunoglobulin variable–like domains of the murine CD8αα homodimer complexed to the class I MHC H-2K b molecule at 2.8 A resolution shows that CD8αα binds to the protruding MHC α3 domain loop in an antibody-like manner. Comparison of mouse CD8αα/H-2K b and human CD8αα/HLA-A2 complexes reveals shared as well as species-specific recognition features. In both species, coreceptor function apparently involves the participation of CD8 dimer in a bidentate attachment to an MHC class I molecule in conjunction with a T cell receptor without discernable conformational alteration of the peptide or MHC antigen-presenting platform.

Atomic structure of an alphabeta T cell receptor (TCR) heterodimer in complex with an anti-TCR fab fragment derived from a mitogenic antibody.

Each T cell receptor (TCR) recognizes a peptide antigen bound to a major histocompatibility complex (MHC) molecule via a clonotypic αβ heterodimeric structure (Ti) non‐covalently associated with the monomorphic CD3 signaling components. A crystal structure of an αβ TCR‐anti‐TCR Fab complex shows an Fab fragment derived from the H57 monoclonal antibody (mAb), interacting with the elongated FG loop of the Cβ domain, situated beneath the Vβ domain. This loop, along with the partially exposed ABED β sheet of Cβ, and glycans attached to both Cβ and Cα domains, forms a cavity of sufficient size to accommodate a single non‐glycosylated Ig domain such as the CD3ϵ ectodomain. That this asymmetrically localized site is embedded within the rigid constant domain module has implications for the mechanism of signal transduction in both TCR and pre‐TCR complexes. Furthermore, quaternary structures of TCRs vary significantly even when they bind the same MHC molecule, as manifested by a unique twisting of the V module relative to the C module.

The Structural Biology of CD2

The CD2 molecule is a 50-55KD transmembrane glycoprotein expressed on the vast majority of thymocytes and virtually all peripheral T lymphocytes. Its functions are two-fold: adhesion and activation. CD2 serves to facilitate conjugate formation between the T-lineage cell and its cognate partner via intermolecular interaction of CD2 and LFA-3 on the former and latter cells, respectively. Perturbation of the CD2 extracellular segment by certain combinations of anti-CD2 MAbs or LFA-3 and a single anti-CD2 MAb activate T-lineage function. These CD2-mediated activation events also synergize with signals mediated through the TCR to augment T-cell response. Based on microchemical analysis of immunoaffinity-purified human CD2 and cDNA and genomic cloning of mouse and human molecules, considerable structural information is now available. The mature surface human CD2 molecule consists of 327 amino acids: a 185 aa extracellular segment; a 25 aa hydrophobic transmembrane segment; and a 117 aa cytoplasmic domain rich in prolines and basic residues. The CD2 gene is comprised of five exons which span approximately 12 Kb on chromosome 1. A similar protein structure and gene exon organization is found for the mouse CD2 homologue. The CD2 adhesion domain is approximately 103 aa in length and is encoded by a single exon (exon 2). This domain is resistant to proteolysis, even though it lacks any intrachain disulfides and, like the entire extracellular segment protein expressed in a baculovirus system, binds to its cellular ligand, LFA-3. The latter occurs with a micromolar Kd. This relatively low affinity suggests that multivalent interactions among CD2 monomers on the T cells and individual LFA-3 structures on the cognate partner are important in enhancing the avidity of the T-cell interaction with its target or stimulator cell. The affinity of the CD2 extracellular segment for LFA-3 is not affected by truncations in the CD2 cytoplasmic domain, implying that ligand binding is not regulated by intracellular mechanisms. Given that CD2 mRNA expression and surface CD2 copy number are increased by more than one order of magnitude post-TCR stimulation, it is more likely that adhesion via CD2 is modulated by alteration in surface copy number. Analysis of early transduction events occurring via CD3-Ti (TCR) and CD2 including single channel Ca2+ patch-clamp recordings on living human T lymphocytes indicate a virtual identity of signals.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a common docking topology with substantial variation among different TCR–peptide–MHC complexes

Whether T-cell receptors (TCRs) recognize antigenic peptides bound to major histocompatability complex (MHC) molecules through common or distinct docking modes is currently uncertain. We report the crystal structure of a complex between the murine N15 TCR [1-4] and its peptide-MHC ligand, an octapeptide fragment representing amino acids 52-59 of the vesicular stomatitis virus nuclear capsid protein (VSV8) bound to the murine H-2Kb class I MHC molecule. Comparison of the structure of the N15 TCR-VSV8-H-2Kb complex with the murine 2C TCR-dEV8-H-2Kb [5] and the human A6 TCR-Tax-HLA-A2 [6] complexes revealed a common docking mode, regardless of TCR specificity or species origin, in which the TCR variable Valpha domain overlies the MHC alpha2 helix and the Vbeta domain overlies the MHC alpha1 helix. As a consequence, the complementary determining regions CDR1 and CDR3 of the TCR Valpha and Vbeta domains make the major contacts with the peptide, while the CDR2 loops interact primarily with the MHC. Nonetheless, in terms of the details of the relative orientation and disposition of binding, there is substantial variation in TCR parameters, which we term twist, tilt and shift, and which define the variation of the V module of the TCR relative to the MHC antigen-binding groove.

Expression, Purification, and Functional Analysis of Murine Ectodomain Fragments of CD8αα and CD8αβ Dimers

Soluble mouse CD8αα and CD8αβ dimers corresponding to the paired ectodomains (CD8f) or their respective component Ig-like domains (CD8) were expressed in Chinese hamster ovary cells or the glycosylation variant Lec3.2.8.1 cells as secreted proteins using a leucine zipper strategy. The affinity of CD8ααf for H-2Kb as measured by BIAcore revealed a ∼65 μm K d , similar to that of CD8αβf. Consistent with this result, CD8ααf as well as CD8αβf blocked the effector function of N15 T cell receptor transgenic cytolytic T cells in a comparable, dose-dependent fashion. Furthermore, both Lec3.2.8.1-produced and Chinese hamster ovary-produced CD8 homodimers and heterodimers were active in the inhibition assay. These results suggest that the Ig-like domains of CD8 molecules are themselves sufficient to block the requisite transmembrane CD8-pMHC interaction between cytolytic T lymphocytes and target cells. Moreover, given the similarities in co-receptor affinities for pMHC, the findings suggest that the greater efficiency of CD8αβ versus CD8αα co-receptor function on T cells is linked to differences within their membrane-bound stalk regions and/or intracellular segments. As recently shown for sCD8αα, the yield, purity and homogeneity of the deglycosylated protein resulting from this expression system is sufficient for crystallization and x-ray diffraction at atomic resolution.

Crystallization of a Deglycosylated T Cell Receptor (TCR) Complexed with an Anti-TCR Fab Fragment

A strategy to overexpress T cell receptors (TCRs) in Lec3.2.8.1 cells has been developed using the “Velcro” leucine zipper sequence to facilitate α-β pairing. Upon secretion in culture media, the VSV-8-specific/H2-Kb-restricted N15 TCR could be readily immunopurified using the anti-leucine zipper monoclonal antibody 2H11, with a yield of 5-10 mg/liter. Mass spectrometry analysis revealed that all attached glycans were GlcNAc2-Man5. Following Superdex 200 gel filtration to remove aggregates, wild-type N15 or N15s, a C183S variant lacking the unpaired cysteine at amino acid residue 183 in the Cβ domain, was thrombin-cleaved and endoglycosidase H-digested, and the two derivatives were termed iN15ΔH and N15sΔH, respectively, and sized by Superdex 75 chromatography to high purity. N-terminal and C-terminal microsequencing analysis showed the expected unique termini of N15 α and β subunits. Nevertheless, neither protein crystallized under a wide range of conditions. Subsequently, we produced a Fab fragment of the murine TCR Cβ-specific hamster monoclonal antibody H57 and complexed the Fab fragment with iN15ΔH and N15sΔH. Both N15sΔH-Fab[H57] and iN15ΔH-Fab[H57] complexes crystallize, with the former diffracting to 2.8-Å resolution. These findings show that neither intact glycans nor the conserved and partially exposed Cys-183 is required for protein stability. Furthermore, our results suggest that the H57 Fab fragment aids in the crystallization of TCRs by altering their molecular surface and/or stabilizing inherent conformational mobility.

The CD8α β co-receptor on double-positive thymocytes binds with differing affinities to the products of distinct class I MHC loci

The CD8 co‐receptor is essential for TCR‐dependent immune recognition and T cell development involving peptides bound to MHC class I (MHCI) molecules. The dominant interaction of CD8 α α and α β co‐receptors is with the α3 domain of an MHCI molecule. Whether this interaction is different for the products of various MHCI loci is currently unknown. Here we examine the interaction between H‐2Kb and H‐2Db, the two MHCI molecules in the C57BL / 6 mouse, and CD8 using H‐2Kb and H‐2Db tetramers. The MHCI molecules bind to the CD8α β co‐receptor on double‐positive thymocytes with different avidities (H‐2Kb > Db). The differences are linked to their respective α3 domains. Hence, an H‐2DbKb tetramer comprising Dbα1 – α2 and Kbα3 domains shows more binding than H‐2Db. We also quantitated the monomeric affinities of CD8α α and CD8α β for H‐2Kb and H‐2Db. The H‐2Kb interaction with CD8α α and CD8α β is stronger than that of H‐2Db. Given that T cell repertoire selection of DP thymocytes is a function of both TCR‐pMHCI and CD8α β‐pMHCI avidities, these differences may explain the dominant role of H‐2Kb as compared to H‐2Db in CD8 T cell development of C57BL / 6 mice. The influence of allelic and non‐allelic α3 polymorphisms on thymic selection processes are discussed.

Stalk Region of β-Chain Enhances the Coreceptor Function of CD8

CD8 glycoproteins are expressed as either αα homodimers or αβ heterodimers on the surface of T cells. CD8αβ is a more efficient coreceptor than the CD8αα for peptide Ag recognition by TCR. Each CD8 subunit is composed of four structural domains, namely, Ig-like domain, stalk region, transmembrane region, and cytoplasmic domain. In an attempt to understand why CD8αβ is a better coreceptor than CD8αα, we engineered, expressed, and functionally tested a chimeric CD8α protein whose stalk region is replaced with that of CD8β. We found that the β stalk region enhances the coreceptor function of chimeric CD8αα to a level similar to that of CD8αβ. Surprisingly, the β stalk region also restored functional activity to an inactive CD8α variant, carrying an Ala mutation at Arg8 (R8A), to a level similar to that of wild-type CD8αβ. Using the R8A variant of CD8α, a panel of anti-CD8α Abs, and three MHC class I (MHCI) variants differing in key residues known to be involved in CD8α interaction, we show that the introduction of the CD8β stalk leads to a different topology of the CD8α-MHCI complex without altering the overall structure of the Ig-like domain of CD8α or causing the MHCI to employ different residues to interact with the CD8α Ig domain. Our results show that the stalk region of CD8β is capable of fine-tuning the coreceptor function of CD8 proteins as a coreceptor, possibly due to its distinct protein structure, smaller physical size and the unique glycan adducts associated with this region.

Involvement of the TCR Cβ FG Loop in Thymic Selection and T Cell Function

The asymmetric disposition of T cell receptor (TCR) Cβ and Cα ectodomains creates a cavity with a side-wall formed by the rigid Cβ FG loop. To investigate the significance of this conserved structure, we generated loop deletion (βΔFG) and βwt transgenic (tg) mice using the TCR β subunit of the N15 CTL. N15βwt and N15βΔFG H-2b animals have comparable numbers of thymocytes in S phase and manifest developmental progression through the CD4−CD8− double-negative (DN) compartment. N15βΔFG facilitates transition from DN to CD4+8+ double-positive (DP) thymocytes in recombinase activating gene (RAG)-2−/− mice, showing that pre-TCR function remains. N15βΔFG animals possess ∼twofold more CD8+ single-positive (SP) thymocytes and lymph node T cells, consistent with enhanced positive selection. As an altered Vα repertoire observed in N15βΔFG mice may confound the deletions effect, we crossed N15αβ TCR tg RAG-2−/− with N15βΔFG tg RAG-2−/− H-2b mice to generate N15αβ RAG-2−/− and N15αβ.βΔFG RAG-2−/− littermates. N15αβ.βΔFG RAG-2−/− mice show an 8–10-fold increase in DP thymocytes due to reduced negative selection, as evidenced by diminished constitutive and cognate peptide-induced apoptosis. Compared with N15αβ, N15αβ.βΔFG T cells respond poorly to cognate antigens and weak agonists. Thus, the Cβ FG loop facilitates negative selection of thymocytes and activation of T cells.

Point mutations in the β chain CDR3 can alter the T cell receptor recognition pattern on an MHC class I\peptide complex over a broad interface area

To study how the T cell receptor interacts with its cognate ligand, the MHC/peptide complex, we used site directed mutagenesis to generate single point mutants that alter amino acids in the CDR3beta loop of a H-2Kb restricted TCR (N30.7) specific for an immunodominant peptide N52-N59 (VSV8) derived from the vesicular stomatitis virus nucleocapsid. The effect of each mutation on antigen recognition was analyzed using wild type H-2Kb and VSV8 peptide, as well as H-2Kb and VSV8 variants carrying single replacements at residues known to be exposed to the TCR. These analyses revealed that point mutations at some positions in the CDR3beta loop abrogated recognition entirely, while mutations at other CDR3beta positions caused an altered pattern of antigen recognition over a broad area on the MHC/peptide surface. This area included the N-terminus of the peptide, as well as residues of the MHC alpha1 and alpha2 helices flanking this region. Assuming that the N30 TCR docks on the MHC/peptide with an orientation similar to that recently observed in two different TCR-MHC/peptide crystal structures, our findings would suggest that single amino acid alterations within CDR3beta can affect the interaction of the TCR with an MHC surface region distal from the predicted CDR3beta-Kb/VSV8 interface. Such unique recognition capabilities are generated with minimal alterations in the CDR3 loops of the TCR. These observations suggest the hypothesis that extensive changes in the recognition pattern due to small perturbations in the CDR3 structure appears to be a structural strategy for generating a highly diversified TCR repertoire with specificity for a wide variety of antigens.