Melanoma-Reactive Class I-Restricted Cytotoxic T Cell Clones Are Stimulated by Dendritic Cells Loaded with Synthetic Peptides, but Fail to Respond to Dendritic Cells Pulsed with Melanoma-Derived Heat Shock Proteins In Vitro

Abstract

Immunization with heat shock proteins (hsp) isolated from cancer cells has been shown to induce a protective antitumor response. The mechanism of hsp-dependent cellular immunity has been attributed to a variety of immunological activities mediated by hsp. Hsp have been shown to bind antigenic peptides, trim the bound peptides by intrinsic enzymatic activity, improve endocytosis of the chaperoned peptides by APCs, and enhance the ability of APCs to stimulate peptide-specific T cells. We have investigated the potential capacity of hsp70 and gp96 to function as a mediator for Ag-specific CTL stimulation in an in vitro model for human melanoma. Repetitive stimulation of PBLs by autologous DCs loaded with melanoma-derived hsp did not increase the frequency of T cells directed against immunodominant peptides of melanoma-associated Ags Melan-A and tyrosinase. In contrast, repeated T cell stimulation with peptide-pulsed DCs enhanced the number of peptide-specific T cells, allowing HLA/peptide multimer-guided T cell cloning. We succeeded in demonstrating that the established HLA-A2-restricted CTL clones recognized HLA-A2+ APCs exogenously loaded with the respective melanoma peptide as well as melanoma cells processing and presenting these peptides in the context of HLA-A2. We were not able to show that these melanoma-reactive CTL clones were stimulated by autologous dendritic cells pulsed with melanoma-derived hsp. These results are discussed with respect to various models for proving the role of hsp in T cell stimulation and to recent findings that part of the immunological antitumor activities reported for hsp are independent of the chaperoned peptides.

Generation of Murine CTL by a Hepatitis B Virus-Specific Peptide and Evaluation of the Adjuvant Effect of Heat Shock Protein Glycoprotein 96 and Its Terminal Fragments

“…Immunotherapies using HSPs to generate specific antitumor responses have been evaluated in clinical studies (16, 39 -41). In this report the gp96 -7/9-mer peptide complex and its terminal fragment-7/9-mer peptide complex could not elicit obvious peptide-specific CTL in mice, consistent with recent observations in vitro (42). However, when mixed with different amounts of peptides directly, gp96 and its N-terminal fragment (N333) showed adjuvant effects in enhancing the peptide-specific CTL response, and this response was peptide concentration dependent.…”

“…The findings implied that the N-terminal part of gp96 might contain the very motif to stimulate the innate immune system. However, additional work is necessary to completely rule out the possibility that proteins copurified with gp96 may contribute to its immunological effect, as suggested by several recent publications (42,43).…”

The messenger and the message: gp96 (GRP94)-peptide interactions in cellular immunity

“…These workers, too, conclude that stress protein-peptide complexes are unlikely to contribute to the phenomenon of cross-priming, rather, larger (poly)peptide proteasome substrates serve as the physiological message (Norbury et al 2004). Consistent with this view, Fleischer et al (2004) reported that though complexes of gp96 and synthetic melanoma peptide antigens can be efficiently processed by APC to yield stimulation of melanoma reactive CTL clones, no significant melanoma reactive CTL activity was obtained from APC pulsed with melanoma-derived gp96.…”

Designer T cells by T cell receptor replacement

“…J76 cells are TCRa and TCRb deficient but express all CD3 components. The human CMV-specific T cell clone T21 (Vb20) (submitted for publication) and the Melan-A-specific T cell clone InRi1 (Vb14) [28] were cultured with RPMI 1640 medium supplemented with 10% human serum (Valley Biomedical, Winchester, USA), 100 U/mL penicillin/streptomycin, 2 mM glutamine, 10 ng/ mL anti-CD3 mAb (Janssen-CILAG, Neuss, Germany), 50 U/mL IL-2, and 2 ng/mL IL-15. As feeder cells, 2.5 Â 10 7 allogeneic PBMC (irradiated with 30 Gy) and 5 Â 10 6 B lymphoblastoid cell lines (irradiated with 100 Gy) from healthy donors were added per T30 tissue culture flask (Greiner).…”

“…Cytolytic activity was analyzed in a standard 4-h chromiumrelease assay as described [28]. T2 cells were labeled with 100 lCi 51 Cr (ICN Biochemicals, Irvine, USA) and then loaded with 10 lg/mL peptide.…”