Abstract

The heat shock protein gp96 forms non-covalent complexes with peptides. Gp96-peptide complexes, upon immunization, elicit peptide-specific MHC I-restricted CD8+ T lymphocyte responses. The high efficiency of this process (as little as I ng peptide complexed to 10 ug gp96 is sufficient) and the strigent requirement of professional antigen presenting cells (APCs) for the immunogenicity of gp96-peptide complexes led to the suggestion that APCs possess receptors for gp96 (gp96R). Gp96 bindings to the surface of APCs but not other cells in a saturable and competable manner and is internalized by APCs through receptor-mediated uptake by clathrin-coated pits. A 75kda gp96-binding molecule expressed on the surface of APCs but not other cells has been identified as a candidate gp96R. Uptake of gp96-bound peptides through gp96R leads to re-presentation of the peptides by MHC I of the APCs. The application aims to identify and characterize gp96R and its gene(s) and plans to carry out: 1. Identification and characterization of gp96-binding proteins on the surface of APCs. * Cross-linking of gp96 with gp96-binding proteins from APCs using reversible cross-linkers; * Chromatography of surface proteins of APCs over immobilized gp96 affinity columns; * Generation of monoclonal antibodies to gp96R using inhibition of re-presentation as assay; * Purification and characterization of gp96R. 2. Identification and characterization of transcripts specific for APCs which can re-present gp96-chaperoned peptides: * Subtraction of cDNA gp96R-cells from that of gp96R+cells; * Use of differential display to isolate gp96R transcripts; * Screening of expression library from gp96R+cells by gp96; 3. Determination if gp96,hsp90, hsp70 and Calreticulin share receptors on APCs: *Competition among the four HSPs for binding to APCs and re-presenting chaperoned peptides; 4. Characterization of the intracellular pathway through which gp96-peptide complexes traffic within the APC: *Ultrastructural analysis where gp96 and the peptide are tagged with different indicator moieties and the fate of each is followed through electron microscopy; *Analysis of epitope-flanking sequences which affect the processing and processing and presentation of gp96-chaperoned peptides in a manner that is different from that of un-chaperoned peptides.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA084479-02
Application #
6377711
Study Section
Experimental Immunology Study Section (EI)
Program Officer
Mccarthy, Susan A
Project Start
2000-07-01
Project End
2003-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
2
Fiscal Year
2001
Total Cost
$258,262
Indirect Cost

  Institution

Name
University of Connecticut
Department
Type
Other Domestic Higher Education
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030

  Publications

Srivastava, Pramod K (2015) Neoepitopes of Cancers: Looking Back, Looking Ahead. Cancer Immunol Res 3:969-77
Li, Changying; Buckwalter, Matthew R; Basu, Sreyashi et al. (2012) Dendritic cells sequester antigenic epitopes for prolonged periods in the absence of antigen-encoding genetic information. Proc Natl Acad Sci U S A 109:17543-8
Vatner, Ralph E; Srivastava, Pramod K (2010) The tailless complex polypeptide-1 ring complex of the heat shock protein 60 family facilitates cross-priming of CD8 responses specific for chaperoned peptides. J Immunol 185:6765-73
Testori, Alessandro; Richards, Jon; Whitman, Eric et al. (2008) Phase III comparison of vitespen, an autologous tumor-derived heat shock protein gp96 peptide complex vaccine, with physician's choice of treatment for stage IV melanoma: the C-100-21 Study Group. J Clin Oncol 26:955-62
Callahan, Margaret K; Garg, Manish; Srivastava, Pramod K (2008) Heat-shock protein 90 associates with N-terminal extended peptides and is required for direct and indirect antigen presentation. Proc Natl Acad Sci U S A 105:1662-7
Binder, Robert J; Kelly 3rd, John B; Vatner, Ralph E et al. (2007) Specific immunogenicity of heat shock protein gp96 derives from chaperoned antigenic peptides and not from contaminating proteins. J Immunol 179:7254-61
Binder, Robert J; Srivastava, Pramod K (2005) Peptides chaperoned by heat-shock proteins are a necessary and sufficient source of antigen in the cross-priming of CD8+ T cells. Nat Immunol 6:593-9
Li, Zihai; Qiao, Yi; Liu, Bei et al. (2005) Combination of imatinib mesylate with autologous leukocyte-derived heat shock protein and chronic myelogenous leukemia. Clin Cancer Res 11:4460-8
Binder, Robert J; Srivastava, Pramod K (2004) Essential role of CD91 in re-presentation of gp96-chaperoned peptides. Proc Natl Acad Sci U S A 101:6128-33
Makki, Amira; Weidt, Gunnar; Blachere, Nathalie E et al. (2002) Immunization against a dominant tumor antigen abrogates immunogenicity of the tumor. Cancer Immun 2:4

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