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T-Cell Recognition and MHC Class II Presentation of Antigen

L. Teyton, M. Bondad, H. Celia, C. Garcia, A. Kang, G. Luo, C. Scott, R. Stefanko, T. Strattman, V. Apostoulopoulos, M. Wallace

IN VITRO T-CELL RECOGNITION

T-cell activation is the result of a productive interaction between the T-cell receptor (TCR) at the surface of a T cell and an MHC molecule at the surface of an antigen-presenting cell. This interaction is governed by the rules of MHC restriction, which limit the recognition of a T cell to a single specific ligand. The structural basis of the TCR-MHC interaction and the way the interaction leads to T-cell activation are still unclear. We focus our efforts on determining the structural parameters that govern MHC restriction, specificity, and T-cell activation.

We expressed a large number of soluble TCRs and MHC molecules in both class I-- and class II--restricted systems and used purified molecules and plasmon resonance to measure the strength of TCR-MHC interactions. In most cases, the affinity of TCRs for MHC molecules was low, from 10 to 100 µM. To determine which parts of the interface are the most important in providing the energy of binding, we used 2 complementary routes.

First, in collaboration with D. Kranz, University of Illinois, surface plasmon resonance was used to determine the binding of alanine mutants of each of the complementarity-determining regions of both the - and the ß-chains of TCRs to the peptide-MHC complex. Second, in collaboration with I. Wilson's laboratory, Department of Molecular Biology, the high-resolution 3-dimensional structures of 2 MHC-TCR complexes were determined.

The results of both approaches indicate that the MHC-TCR interaction is dominated by recognition of the MHC -helices. This information is our guide to additional mutagenesis and to an attempt to rationally design TCRs of high affinity. The goal of this work is to determine the basis of T-cell specificity and MHC restriction.

To complement this project, we have characterized the role of accessory molecules in T-cell recognition. For instance, CD8 increases the affinity of TCRs for MHC molecules. For studies to determine the mechanisms of this effect, recombinant CD8 molecules were purified and used in crystallization trials with TCRs and MHC molecules.

Finally, in collaboration with E. Kubalek and R. Milligan, Department of Cell Biology, we are using a new technique to reconstitute a functional TCR complex in vitro from recombinant molecules. Recombinant TCR and accessory molecules (CD3, CD4/CD8) are captured by their C-terminal histidine tag by lipids retaining a chelating cage at the hydrophilic pole. The capture orients the extracytoplasmic part of the molecule to have the same topology as a transmembrane molecule. Liposomes made with this lipid can be fused to hydrophobic surfaces to reconstitute monolayers that mimic T-cell membranes. Surface plasmon resonance measurements are carried out on such surfaces. This new technology was used to crystallize H-2K^b on a lipid layer and to show that MHC class I molecules anchored into membranes are fully accessible to T cells (Fig. 1).

MHC CLASS II PRESENTATION

Cell-mediated autoimmunity is generally restricted to MHC class II molecules. Some MHC class II molecules linked to autoimmunity have peculiar sequence features such as position 57 of the ß-chain of the MHC molecule associated with diabetes. We are evaluating the functional impact of these structural differences. Murine MHC class II molecules (i.e., I-A molecules) have been expressed, purified, and crystallized in collaboration with I. Wilson, and the structures of 2 I-A^d peptide complexes have been determined. These structures have revealed an unusual peptide-binding mode for I-A molecules: a minimal interaction between peptide side chains and MHC pockets. Thus, I-A^d peptides bind to the MHC molecule through a network of bonds between the main carbon chain of the peptides and the groove. In collaborative investigations done with A. Kang, Department of Molecular Biology, the results of phage display studies with I-A^d molecules corroborated this finding and indicated a weak peptide-binding motif consisting of a single hydrophobic residue at position 4 of the peptide.

The same approach was used with I-A^g7, a close structural relative of I-A^d. I-A^g7 is the only MHC class II molecule expressed by nonobese diabetic mice. Soluble I-A^g7 molecules were expressed and characterized in terms of stability, peptide binding, and class II--associated invariant chain binding. In contrast to the findings with I-A^d, phage display showed no peptide-binding motif for I-A^g7. Using the same molecules to screen a peptide library made from the autoantigen GAD65, we detected 4 peptides that bind I-A^g7 molecules. Three of these peptides are immunogenic in nonobese diabetic mice. Studies of the biology and the structures of I-A^g7 and autoimmune TCR-MHC complexes should indicate if unusual biophysical or structural features can explain autoreactivity.

In addition, in collaboration with A. Brünmark and L. Karlsson, R.W. Johnson Pharmaceutical Research Institute, peptide binding to MHC class II molecules is being studied in vitro by using (1) complexes composed of recombinant purified MHC class II molecules and the class II--associated invariant chain and (2) accessory molecules such as HLA-DM and HLA-DO.

PUBLICATIONS

Aichinger, G., Karlsson, L., Jackson, M.R., Vestberg, M., Vaughan, J.H., Teyton, L., Lechler, R.I., Peterson, P.A. Major histocompatibility complex class II-dependent unfolding, transport, and degradation of endogenous proteins. J. Biol. Chem. 272:29127, 1997.

Garcia, K.C., Degano, M., Pease, L.R., Huang, M., Peterson, P.A., Teyton, L., Wilson, I.A. Structural basis of plasticity in T cell receptor recognition of a self-peptide-MHC antigen. Science 279:1166, 1998.

Garcia, K.C., Tallquist, M.D., Pease, L.R., Brunmark, A., Scott, C., Degano, M., Stura, E., Peterson, P.A., Wilson, I.A., Teyton, L. -ß T-cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization. Proc. Natl. Acad. Sci. U.S.A. 94:13838, 1997.

Garcia, K.C., Teyton, L. T cell receptor peptide-MHC interactions: Biological lessons from structural studies. Curr. Opin. Biotechnol., in press.

Liljedahl, M., Winqvist, O., Surh, C.D., Wong, P., Teyton, L., Peterson, P.A., Brunmark, A., Rudensky, A.Y., Fung-Leung, W.P., Karlsson, L. Altered antigen presentation in mice lacking H2-O. Immunity 8:233, 1998.

Luxembourg, A., Borrow, P., Teyton, L., Brunmark, A.B., Peterson, P.A., Jackson, M.R. Biomagnetic isolation of antigen-specific CD8⁺ T cells usable in immunotherapy. Nature Biotechnol. 16:281, 1998.

Manning, T.C., Schlueter, C.J., Brodnicki, T.C., Parke, E.A., Speir, J.A., Garcia, K.C., Teyton, L., Wilson, I.A., Kranz, D.M. Alanine scanning mutagenesis of an ß T cell receptor: Mapping the energy of antigen recognition. Immunity 8:413, 1998.

Scott, C.A., Garcia, K.C., Stura, E., Peterson, P.A., Wilson, I.A., Teyton, L. Engineering protein for x-ray crystallography: The murine major histocompatibility complex class II molecule I-A^d. Protein Sci. 7:413, 1998.

Scott, C.A., Peterson, P.A., Teyton, L., Wilson, I.A. Crystal structures of two I-A^d-peptide complexes reveal high affinity can be achieved without large anchor residues. Immunity 8:319, 1998.