Lonnie P. Lybarger

Assistant Professor, Cell Biology and Anatomy

Ph.D., Louisiana State University Health Sciences Center

Postdoctoral Research, Washington University School of Medicine

RESEARCH SUMMARY: MHC class I antigen presentation

The research interests of the lab center on the biology of MHC class I molecules, which play a central role in immune responses against both intracellular pathogens and tumors. Within the endoplasmic reticulum (ER), class I molecules bind peptides that are derived from the degradation of endogenously synthesized proteins. If cells are infected by a virus, for example, class I molecules may also bind unique virus-encoded peptides. The assembly of class I molecules requires the concerted activity of several accessory molecules in the ER, which are collectively referred to as the peptide-loading complex. Once peptides bind to class I heavy chains, the complexes are transported to the cell surface where the peptides are accessible for scanning by CD8+ T cells. If CD8 T cells detect the presence of a foreign (non-self) peptide amid the pool of peptides on class I, the T cells can mount the appropriate response against those cells that display the foreign peptide. Within this framework, there are two major efforts in the lab:

1) The first concerns virus-encoded molecules that inhibit MHC class I-restricted antigen presentation. These are employed by viruses to evade the host immune response. I particular, we study a family of related molecules that all possess ubiquitin ligase activity that is required for class I modulation. In the case of the mK3 protein of g-herpesvirus 68, class I molecules are degraded rapidly following synthesis in the ER. Our studies have revealed that mK3 requires class I-dedicated ER accessory molecules (peptide-loading complex) in order to degrade class I heavy chains. We continue to explore the molecular interactions that are required for mK3 substrate recognition and function. In addition, this system is being used as an entry point to understand ER degradation pathways under normal conditions.

2) The second project centers on the characterization and applications of single-chain versions of class I. We, in collaboration with the lab of Ted Hansen, have developed a unique approach for the generation of class I molecules that display uniform, high levels of single antigenic peptides. These molecules are formed by linking together all three components of a class I molecule (peptide, light chain, and heavy chain) in a single polypeptide. This technology appears to be widely applicable, since we have applied it successfully to several class I/peptide combinations. The ability to engineer class I to present high levels of a single peptide has many potential applications. Currently, we are investigating various applications, such as: i) Expression following DNA vaccination to prime CD8+ T cell responses against tumors and viruses. ii) Tetramer production with recombinant forms of these molecules to enumerate antigen-specific T cells. iii) Transgenic expression in mice to study CD8+ T cell selection and biology on a single peptide/MHC class I background.

RECENT PUBLICATIONS:

Myers, N. B., Harris, M. B., Connolly, J. M., Lybarger, L., Yu, Y. Y. L., and Hansen, T. H. 2000. Kb, Kd, and Ld molecules share common tapasin dependencies as determined using a novel epitope tag. Journal of Immunology. 165:5656.

Harris, M. R., Lybarger, L., Yu, Y. Y. L., Myers, N. B., and Hansen, T. H. 2001. Association of Erp57 with mouse MHC class I molecules is tapasin dependent and mimics that of calreticulin and not calnexin. Journal of Immunology. 166:6686.

Chun, T., Grandea, A. G., III., Lybarger, L., Forman, J., Van Kaer, L., and Wang, C-R. 2001. Functional roles of TAP and tapasin in the assembly of M3-N-formylated peptide complexes. Journal of Immunology. 167:1507.

Lybarger, L., Yu, Y. Y. L., Chun, T., Wang, C-R., Grandea, A. G., III., Van Kaer, L., and Hansen, T. H. 2001. Tapasin enhances peptide-induced expression of H2-M3, but is not required for the retention of open conformers. Journal of Immunology. 167:2097.

Harris, M. R., Lybarger, L., Myers, N. B., Hilbert, C., Solheim, J. C., Hansen, T. H., and Yu, Y. Y. L. 2001. Interactions of HLA-B27 with the peptide loading complex as revealed by heavy chain mutations. International Immunology. 13:1275.

Yu, Y.Y. L., Harris, M. R., Lybarger, L., Kimpler, L. A., Virgin, H.W., and Hansen, T. H. 2002. Physical association of the K3 protein of (g-herpesvirus-68 with MHC class I molecules with impaired peptide and $b2m assembly. Journal of Virology. 76:2796.

Yu, Y.Y.L., Netuschil, N., Lybarger, L., Connolly, J.M., and Hansen, T.H. 2002. Cutting Edge: Single-chain trimers of MHC class I molecules form stable structures that potently stimulate antigen-specific T cells and B cells. Journal of Immunology. 168:3145.

Lybarger, L., Wang, X., Harris, M.R., Virgin, H.W., and Hansen, T.H. 2003. Virus subversion of the MHC class I peptide-loading complex. Immunity. 18:121.

Miley, M.J., Truscott, S.M., Gilfillan, S., Yu, Y.Y.L., Fremont, D.H., Hansen, T.H., and Lybarger, L. 2003. Biochemical features of the non-classical MHC protein MR1 consistent with an immunological function. Journal of Immunology. 170:6090.

Norris, H.P., Lybarger, L., Martin, A.J., Andersen, H., Chervenak, D.C., and Chervenak, R. 2003. TCRb enhancer activation occurs in some but not all cells with T cell lineage developmental potential. Cellular Immunology. 222:164.

Lybarger, L., Yu, Y.Y.L., Miley, M.J., Fremont, D.H., Myers, N.M., Primeau, T., Truscott, S. M., Connolly, J.M., and Hansen, T.H. 2003. Enhanced immune presentation of a single-chain class I molecule engineered to optimize linkage of a C-terminal extended peptide. Journal of Biological Chemistry. 278:27105.

Norris, H.N., Andersen, H., Martin, A.J., Lybarger, L., Chervenak, D.C., and Chervenak, R. 2003. Activation of the TCRb chain enhancer in pluripotent hematopoietic precursors. Cellular Immunology. 222:164.

Wang, X., Lybarger, L., Connors, R., Harris, M.H., Virgin, H.W., and Hansen, T.H. 2004. A model for the interaction of mK3 protein of MHV68 with class I and TAP/tapasin. Journal of Virology. In press.

Jaramillo, A., Narayanan, K., Campbell, L.G., Benshoff, N.D., Lybarger, L., Hansen, T.H., Fleming, T.P., Dietz, J.R., Mohanakumar, T. 2004. Recognition of HLA-A*0201-Restricted Mammaglobin-A-Derived Epitopes by CD8+ Cytotoxic T lymphocytes from Breast Cancer Patients. Breast Cancer Research and Treatment. In press.

Wang, X., Connors, R., Harris, M.R., Hansen, T.H., and Lybarger, L. 2004. Requirements for the selective degradation of ER-resident MHC class I proteins by the viral immune evasion molecule, mK3. Manuscript submitted.