Research Activities

The primary function of CD8⁺ cytotoxic T lymphocytes (CTLs) is to recognize and eliminate infected or malignant cells. CTL specificity results from T cell antigen receptors that recognize pathogen-derived or tumor-specific peptides bound to MHC class I molecules. Many of the proteins from which class I binding peptides are derived are metabolically stable, with long half-lives. If these proteins are degraded only infrequently, how does an infected or malignant cell generate sufficient peptides for presentation to CTLs? This is especially relevant when one considers the relatively short time required by many intracellular pathogens to replicate and spread to neighboring cells. To determine how protein stability influences peptide-class I generation, we expressed stable and rapidly degraded forms of a chimeric protein in vaccinia virus infected cells and quantified surface peptide-class I complexes using a monoclonal antibody specific for Ova_257-264 associated with the K^b class I molecule. Our studies showed, irrespective of the half-life of the chimeric protein, that the vast majority of peptides are derived from a cohort of newly synthesized protein molecules that are targeted for almost immediate destruction by the proteasome. These rapidly degraded proteins appear to be defective ribosomal products (DRiPs), which result from errors in protein translation and/or folding. The efficiency of processing for these proteins is one surface peptide-class I complex generated for every 500-3000 proteins degraded. About 20-35% of these protein molecules are degraded during or immediately after they are synthesized. Significantly, values for processing efficiencies and DRiP formation are consistent whether murine cell lines or professional antigen presenting cells (pAPC), such as bone marrow dendritic cells and peritoneal macrophages, are assayed. These studies reveal an inefficiency in protein synthesis and folding that has as its byproduct a pool of rapidly presented peptides that can be recognized by CTLs early after pathogen infection.

One of the primary goals of this lab is to understand how the source of protein from which MHC class I binding peptides is derived influences the host immune response. More specifically, we would like to determine how presentation of a given peptide differs depending on whether the protein from which it was derived was synthesized by the presenting cells own biosynthetic machinery (viral infections, malignancies), another cell from the same organism (cross-presentation) or a prokaryotic pathogen?

Antigen Presentation From Intracellular Bacteria

The system examining presentation from viral proteins synthesized by host cell ribosomes is well established (see summary above). This system will be adapted to examine presentation in cells infected with the intracellular bacterial pathogen, Listeria monocytogenes. We will express identical chimeric proteins containing the Ova_257-264 peptide in recombinant vaccinia virus and L. monocytogenes and compare the kinetics of presentation in both professional and non-professional antigen presenting cells. The existence of mutant forms of L. monocytogenes will allow us to further expand the scope of these studies. For example, peptide generation can be compared between wild-type L. monocytogenes, which enters the host cell cytosol shortly after infection, and a mutant strain that is unable to escape the host cell phagosome. These studies will help determine whether the mode of entry of a protein into the degradative pathway affects the kinetics and/or efficiency of antigen presentation.

Antigen Cross-Presentation

T cells can respond to antigens presented on professional antigen presenting cells such as dendritic cells (DCs) that originated in other infected cells (a process known as cross-priming or cross-presentation). DCs acquire antigen for cross-presentation in the form of polypeptides or full-length proteins that must then be processed into antigenic peptides by the DC. Importantly, the peptides presented by cross-priming DCs must be the same as those that will be presented by infected cells in order to be targeted for elimination by effector CTLs. To this end, we will determine whether the kinetics of Ova_257-264 presentation differs between direct (biosynthesized) presentation in non-professional antigen presenting (APC) cells and cross-presentation in DCs. These studies will be performed using cells (e.g. bone marrow derived DC) isolated from both wild type mice as well as mice with specific mutations in components of their antigen processing machinery, such as the transporter associated with antigen processing (TAP) and the immunoproteasome.  It is expected that results from these studies will lead to experiments addressing the establishment of effective memory responses using in vivo mouse models of viral and bacterial infection.

Selected References

Qian, S.B., Princiotta, M.F., Bennink, J.R. and J.W. Yewdell. Characterization of rapidly degraded polypeptides in mammalian cells reveals a novel layer of nascent protein quality control. J. Biol. Chem. 2005. Jan 6;281(1):392-400.

Truckenmiller, M. E., Princiotta, M. F., Norbury, C. C. and R.Hl. Bonneau. Corticosterone impairs MHC class I antigen presentation by dendritic cells via reduction of peptide generation. J.  Neuroimmunol. 2005. Mar;160(1-2):48-60.

Norbury, C.C., Basta, S., Donohue, K.B., Tscharke, D.C., Princiotta, M.F., Berglund, P., Gibbs, J., Bennink, J.R. and J.W. Yewdell. CD8+ T cell cross-priming via transfer of proteasome substrates. Science. 2004 May 28;304(5675):1318-21.

Yewdell, J.W. and M. F. Princiotta. Proteasomes get by with lots of help from their friends. Immunity. 2004. Apr;20(4):362-63.

Houde, M., Bertholet, S., Gagnon, E., Brunet, S., Goyette, G., Laplante, A., Princiotta, M.F., Thibault, P., Sacks, D. and M. Desjardins. Phagosomes are competent organelles for antigen cross-presentation.

Nature. 2003 Sep 25;425(6956):402-6.

Princiotta, M.F., Finzi, D., Qian, S., Gibbs, J., Schuchmann, S., Buttgereit, F., Bennink, J.R. and J.W. Yewdell. Quantitating protein synthesis, degradation and endogenous antigen processing. Immunity. 2003 Mar;18(3):343-54.

Norbury, C.C., Princiotta, M.F., Bacik, I., Brutkiewicz, R.R., Wood, P., Elliott, T., Bennink, J.R. and J.W. Yewdell. Multiple antigen-specific processing pathways for activating naive CD8(+) T cells in vivo. J Immunol. 2001 Apr 1;166(7):4355-62.

Princiotta, M.F., Schubert, U., Chen, W.S., Bennink, J.R., Myung, J., Crews, C.M. and J.W. Yewdell. Cells adapted to the proteasome inhibitor 4-hydroxy- 5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone require enzymatically active proteasomes for continued survival. Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):513-8.

Berg, R.E., Princiotta, M.F., Irion, S., Moticka, J.A., Dahl, K.R. and U.D. Staerz

Positive selection of an H2-M3 restricted T cell receptor. Immunity. 1999 Jul;11(1):33-43.

Anton, L.C., Schubert, U., Bacik, I., Princiotta, M.F., Wearsch, P.A., Gibbs, J., Day, P.M., Realini, C., Rechsteiner, M.C., Bennink, J.R. and J.W. Yewdell. Intracellular localization of proteasomal degradation of a viral antigen. J Cell Biol. 1999 Jul 12;146(1):113-24.

Princiotta, M.F., Lenz, L.L., Bevan, M.J. and U.D. Staerz

H2-M3 restricted presentation of a Listeria-derived leader peptide.

J Exp Med. 1998 May 18;187(10):1711-9.