Production and In Vitro Characterization of Altered Tag Transformed Morine Fibroblasts

CDS+ Cytotoxic T lymphocytes (CTL) have been known to play a significant role in tumor suppression through their role in adaptive cell-mediated immunity. CTL operate in conjunction with the Major Histocompatibility Complex Class I (MHC I) mediated presentation of intracellular proteins by all nucleated somatic cells. To be effective, the CDS+ T cell receptor (TCR) must be able to detect specific amino acids of intracellular processed peptides bound and presented by MHC I molecules. MHC I function in binding small polypeptide segments of 8-10 amino acids in length in the endoplasmic reticulum and transporting them to the cell surface to be observed by sentry CD g+ Lymphocytes. The MHC I binding peptides are produced by proteolytic processing of the transient pool of proteins actively translated on cellular ribosomes. Biochemically MHC I-mediated peptide presentation to CDS+ T killer cells (CTL) requires efficient binding of peptide anchor residues ( single amino acids with extruding side chains) to specific sites on the external face of the MHC I, spatially located between two a-helices. Additional amino acid side chains require projection up from the MHC I face to be available for TCR interaction. Each different allelic MHC I molecule cannot bind all possible peptides; thus, to present the broadest spectrum there must be a balance between high binding affinity and a wide specificity (Madden 1995). When bound, the MHC I complex containing the bound peptide is targeted for localization to the cell membrane, where the MHC I/bound peptide complex is extruded on the cell surface. In the presence of epitope-specific CTL, the bound peptide should trigger recognition by CTL via the physicochemical interaction of the TCR with spatially available amino acid side chains of the presented MHC bound peptide and the MHC molecule itself. Upon CTL recognition of foreign antigen, intracellular signal transduction events are initiated terminating with the observer activated CTL effecting a directly targeted cytolytic response against the peptide presenting target cell (Janeway and Travers 1994)

Identifying H-2b-restricted CD4+ T Lymphocyte Recognition Epitopes Within Simian Virus 40 Large Tumor Antigen

CD4+ T lymphocytes play a vital role in immune system function but little is known about their role in controlling SV40 T ag-induced tumors in C57BL/6 mice. In order to better investigate their role in the control of tumors, it is necessary to identify CD4 epitope(s) within the Tag. Four CDS epitopes have been identified but the CD4 epitopes have not been identified. To facilitate the identification of the epitopes, T cell hybridomas containing a Lacz reporter gene were used. CD4+ hybridoma clones capable of recognizing SV 40 Large Tumor Antigen (T ag) were obtained from heterogeneous populations through serial dilution and screening with a colorimetric (CPRG) assay in which bone marrow-derived dendritic cells were pulsed with purified SV 40 T ag protein. To map the location of the epitope target of each clone, four of the clones were chosen for use in a screening assay utilizing a library of overlapping synthetic 15mer peptides representing the entire SV 40 T ag amino acid sequence. The identification of such CD4+ epitopes within the T ag will make it possible to determine the immunogenic and regulatory properties of each epitope (in vivo) and determine whether differences in reactivity demonstrated by the various hybridoma clones against the purified SV 40 T ag protein are epitope or hybrid dependent

Comparing the Immunological Potencies of Two Viral Epitopes: LT529-543 from the Simian Virus 40 Large Tumor Antigen vs. LT678-690 from the Large Tumor Antigen of Murine Polyomavirus

The results of this study are part of our ongoing effort to understand factors which control the efficiency with which tumor epitope-specific CD8+ T lymphocytes can control the progression of SV40 Tag-induced tumors in murine models. We wish to explore the requirement and role of SV40 Tag-specific CD4+ T cells in establishing and maintaining tumor control and regulating tumor-induced CD8+ T cell tolerance. We have recently identified residues 529-543 of SV40 Tag as a CD4+ class II epitope. Splenocytes from mice immunized with B6/K-1,4,5 cells were used to search for additional epitopes within SV40 Tag. Peptide immunizations were also used to compare the immunogenicity of two epitopes that induce CD4+ T cells responses: LT529-543 from SV40 Tag and LT678-690 from murine Polyomavirus Large T antigen (mPyT). While the mPyT LT678-690 peptide was shown to be strongly immunogenic within the context of mPyT viral infections by others (Lin et al., 2010), synthetic mPyT LT678-690 and SV40 Tag LT529-543 peptides induced similar, but low levels of active CD4+ T cells. Cells immunized with each peptide in combination with the HBV helper peptide showed a weak response to the SV40 Tag peptide when compared to the mPyT peptide. These results suggest that the lack of virus-induced immunity may limit the immunological potency of the mPyT 678-690 epitope, but that in the presence of a potent helper peptide, the mPyT peptide is about twice as potent an immunogen as the SV40 epitope

Modification of a Tumor Antigen Determinant To Improve Peptide/Mhc Stability is Associated With Increased Immunogenicity and Cross-Priming a Larger Fraction of Cd8+ T Cells

Altered peptide ligands (APLs) with enhanced binding to MHC class I can increase the CD8+ T cell response to native Ags, including tumor Ags. In this study, we investigate the influence of peptide-MHC (pMHC) stability on recruitment of tumor Ag-specific CD8+ T cells through cross-priming. Among the four known H-2b-restricted CD8+ T cell determinants within SV40 large tumor Ag (TAg), the site V determinant ( 489QGINNLDNL497) forms relatively low-stability pMHC and is characteristically immunorecessive. Absence of detectable site V-specific CD8+ T cells following immunization with wild-type TAg is due in part to inefficient cross-priming. We mutated nonanchor residues within the TAg site V determinant that increased pMHC stability but preserved recognition by both TCR-transgenic and polyclonal endogenous T cells. Using a novel approach to quantify the fraction of naive T cells triggered through cross-priming in vivo, we show that immunization with TAg variants expressing higher-stability determinants increased the fraction of site V-specific T cells cross-primed and effectively overcame the immunorecessive phenotype. In addition, using MHC class I tetramer-based enrichment, we demonstrate for the first time, to our knowledge, that endogenous site V-specific T cells are primed following wild-type TAg immunization despite their low initial frequency, but that the magnitude of T cell accumulation is enhanced following immunization with a site V variant TAg. Our results demonstrate that site V APLs cross-prime a higher fraction of available T cells, providing a potential mechanism for high-stability APLs to enhance immunogenicity and accumulation of T cells specific for the native determinant. © 2012 The American Association of Immunologists, Inc

Inefficient Cross-Presentation Limits the Cd8+ T Cell Response to a Subdominant Tumor Antigen Epitope

CD8+ T lymphocytes (TCD8) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote TCD8 subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes TCD8 specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce TCD8 specific for the subdominant T Ag epitope V. Using adoptively transferred T CD8 from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic TCD8 in B6 mice required cross-presentation by host APC. However, robust expansion of these TCD8 required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific TCD8 contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting TCD8 responses to cancer. © 2005 The American Association of Immunologists, Inc