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Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma.

Bloom MB, Perry-Lalley D, Robbins PF, Li Y, el-Gamil M, Rosenberg SA, Yang JC - J. Exp. Med. (1997)

Bottom Line: In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer.A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs.By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2.

View Article: PubMed Central - PubMed

Affiliation: Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Recently, major advances have been made in the identification of antigens from human melanoma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer. Progress in this area is likely to require accurate preclinical animal models, but the availability of such models has lagged behind developments in human tumor immunology. Whereas many of the identified human melanoma antigens are normal tissue differentiation proteins, analogous murine tumor antigens have not yet been identified. In this paper we identify a normal tissue differentiation antigen, tyrosinase-related protein 2 (TRP-2), expressed by the murine B16 melanoma which was found by screening a cDNA library from B16 with tumor-reactive cytotoxic T lymphocytes (CTL). A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs. By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2. It was further demonstrated that a CTL line raised from splenocytes by repeated stimulation in vitro with this peptide could recognize B16 tumor and was therapeutic against 3-d-old established pulmonary metastases. The use of TRP-2 in a preclinical model of tumor immunotherapy may be helpful in suggesting optimal vaccination strategies for cancer therapy in patients.

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Release of mIFN-γ by line A when incubated with MC-38  (H2b) and varying amounts of TRP-2181–188 peptide (circles). No significant  mIFN-γ release was seen when MC-38 was incubated with peptide without line A (square) or with line A without peptide (triangle).
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Figure 3: Release of mIFN-γ by line A when incubated with MC-38 (H2b) and varying amounts of TRP-2181–188 peptide (circles). No significant mIFN-γ release was seen when MC-38 was incubated with peptide without line A (square) or with line A without peptide (triangle).

Mentions: Peptides of eight amino acids from TRP-2 were then synthesized using the H2-Kb binding motif described by Rammensee (9, 10). 15 such peptides were identified in the normal reading frame of TRP-2. MC38 murine tumor cells expressing high levels of H2-Kb were incubated with each of these peptides and tested for their ability to stimulate line A T cells. Only one peptide, VYDFFVWL (TRP2181–188) mediated line A recognition of peptide-pulsed MC38. MC38 incubated with TRP-2181–188 caused a release of 6.0 U/ml/24 h of IFN-γ from line A versus ⩽1.0 U/ml/24 h for the other 15 peptides tested, and 0.6 U/ml/ 24 h for MC38 without peptide. The nucleotides coding for TRP-2181–188 are contained within the 622-bp sequence common to all five of the clones identified by cDNA library screening. Peptide concentrations of 100 ng/ml or higher were required for stimulation of cytokine release from line A T cells (Fig. 3).


Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma.

Bloom MB, Perry-Lalley D, Robbins PF, Li Y, el-Gamil M, Rosenberg SA, Yang JC - J. Exp. Med. (1997)

Release of mIFN-γ by line A when incubated with MC-38  (H2b) and varying amounts of TRP-2181–188 peptide (circles). No significant  mIFN-γ release was seen when MC-38 was incubated with peptide without line A (square) or with line A without peptide (triangle).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196033&req=5

Figure 3: Release of mIFN-γ by line A when incubated with MC-38 (H2b) and varying amounts of TRP-2181–188 peptide (circles). No significant mIFN-γ release was seen when MC-38 was incubated with peptide without line A (square) or with line A without peptide (triangle).
Mentions: Peptides of eight amino acids from TRP-2 were then synthesized using the H2-Kb binding motif described by Rammensee (9, 10). 15 such peptides were identified in the normal reading frame of TRP-2. MC38 murine tumor cells expressing high levels of H2-Kb were incubated with each of these peptides and tested for their ability to stimulate line A T cells. Only one peptide, VYDFFVWL (TRP2181–188) mediated line A recognition of peptide-pulsed MC38. MC38 incubated with TRP-2181–188 caused a release of 6.0 U/ml/24 h of IFN-γ from line A versus ⩽1.0 U/ml/24 h for the other 15 peptides tested, and 0.6 U/ml/ 24 h for MC38 without peptide. The nucleotides coding for TRP-2181–188 are contained within the 622-bp sequence common to all five of the clones identified by cDNA library screening. Peptide concentrations of 100 ng/ml or higher were required for stimulation of cytokine release from line A T cells (Fig. 3).

Bottom Line: In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer.A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs.By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2.

View Article: PubMed Central - PubMed

Affiliation: Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Recently, major advances have been made in the identification of antigens from human melanoma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer. Progress in this area is likely to require accurate preclinical animal models, but the availability of such models has lagged behind developments in human tumor immunology. Whereas many of the identified human melanoma antigens are normal tissue differentiation proteins, analogous murine tumor antigens have not yet been identified. In this paper we identify a normal tissue differentiation antigen, tyrosinase-related protein 2 (TRP-2), expressed by the murine B16 melanoma which was found by screening a cDNA library from B16 with tumor-reactive cytotoxic T lymphocytes (CTL). A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs. By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2. It was further demonstrated that a CTL line raised from splenocytes by repeated stimulation in vitro with this peptide could recognize B16 tumor and was therapeutic against 3-d-old established pulmonary metastases. The use of TRP-2 in a preclinical model of tumor immunotherapy may be helpful in suggesting optimal vaccination strategies for cancer therapy in patients.

Show MeSH
Related in: MedlinePlus