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Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes.

Wang RF, Appella E, Kawakami Y, Kang X, Rosenberg SA - J. Exp. Med. (1996)

Bottom Line: This epitope peptide was capable of sensitizing target cells for lysis by a CTL clone at 1 nM peptide concentration.Although some modified peptides could be recognized by the CTL clone, none were found to be better recognized by T cells than the parental peptide.Like other melamona differentiation antigens, TRP-2 was only expressed in melanoma, melanocytes, and retina, but not in other human tissues tested.

View Article: PubMed Central - PubMed

Affiliation: National Cancer Institute, Bethesda, Maryland 20892, USA.

ABSTRACT
The infusion of TIL586 along with interleukin-2 into the autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was previously isolated and shown to encode gp75 or TRP-1. Here we report that TRP-2 was identified as a second tumor antigen recognized by a HLA-A31-restricted CTL clone derived from the TIL586 cell line. The peptide LLPGGRPYR epitope was subsequently identified from the coding region of TRP-2 based on studies of the recognition of truncated TRP-2 cDNAs and the HLA-A31 binding motif. This epitope peptide was capable of sensitizing target cells for lysis by a CTL clone at 1 nM peptide concentration. Although some modified peptides could be recognized by the CTL clone, none were found to be better recognized by T cells than the parental peptide. Like other melamona differentiation antigens, TRP-2 was only expressed in melanoma, melanocytes, and retina, but not in other human tissues tested.

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Related in: MedlinePlus

Construction of deletions and subclones of the TRP-2 gene  and T cell recognition. The full-length cDNA of TRP-2 which comprises the 1,557-bp open reading frame is shown. Nucleotides are numbered from the first nucleotide from the 5′ untranslated region of TRP-2  cDNA. A series of deletion constructs and subcloning of DNA fragments  were made. T cell recognition of each construct was determined after  coculturing CTL clone 4 with COS-7 cotransfected with the DNA fragments shown above and the HLA-A31 gene.
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Figure 3: Construction of deletions and subclones of the TRP-2 gene and T cell recognition. The full-length cDNA of TRP-2 which comprises the 1,557-bp open reading frame is shown. Nucleotides are numbered from the first nucleotide from the 5′ untranslated region of TRP-2 cDNA. A series of deletion constructs and subcloning of DNA fragments were made. T cell recognition of each construct was determined after coculturing CTL clone 4 with COS-7 cotransfected with the DNA fragments shown above and the HLA-A31 gene.

Mentions: To determine the antigenic epitopes from TRP-2, a series of nested deletions of the TRP-2 gene from the 3′ end using ExoIII/S1 nuclease, as well as DNA fragments encoding the truncated form of TRP-2, were generated. These deletions and subcloned constructs were transfected into COS-7 cells along with the pBK-CMV plasmid containing the HLA-A31 cDNA. Recognition of the transfected COS cells was tested with the CTL clone 4 by measuring GM-CSF cytokine release from the CTL clone. Fig. 3 indicates that pTD1, 2, and 3 constructs retained the ability to stimulate cytokine release from the CTL clone 4, but pTD4 and pTD5 lost the stimulating activity to the CTL clone 4, indicating that the epitope(s) recognized by the CTL clone 4 was located in the region of nucleotides 836–1045. This was consistent with results obtained by the subcloning experiments. Although pTA and pTP lost the ability to stimulate cytokine release from CTL clone 4, pTK still remained positive in the cytokine release assay. Therefore, the epitopes resided in a DNA fragment flanked by the first PstI and KpnI sites as shown in Fig. 4.


Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes.

Wang RF, Appella E, Kawakami Y, Kang X, Rosenberg SA - J. Exp. Med. (1996)

Construction of deletions and subclones of the TRP-2 gene  and T cell recognition. The full-length cDNA of TRP-2 which comprises the 1,557-bp open reading frame is shown. Nucleotides are numbered from the first nucleotide from the 5′ untranslated region of TRP-2  cDNA. A series of deletion constructs and subcloning of DNA fragments  were made. T cell recognition of each construct was determined after  coculturing CTL clone 4 with COS-7 cotransfected with the DNA fragments shown above and the HLA-A31 gene.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211562&req=5

Figure 3: Construction of deletions and subclones of the TRP-2 gene and T cell recognition. The full-length cDNA of TRP-2 which comprises the 1,557-bp open reading frame is shown. Nucleotides are numbered from the first nucleotide from the 5′ untranslated region of TRP-2 cDNA. A series of deletion constructs and subcloning of DNA fragments were made. T cell recognition of each construct was determined after coculturing CTL clone 4 with COS-7 cotransfected with the DNA fragments shown above and the HLA-A31 gene.
Mentions: To determine the antigenic epitopes from TRP-2, a series of nested deletions of the TRP-2 gene from the 3′ end using ExoIII/S1 nuclease, as well as DNA fragments encoding the truncated form of TRP-2, were generated. These deletions and subcloned constructs were transfected into COS-7 cells along with the pBK-CMV plasmid containing the HLA-A31 cDNA. Recognition of the transfected COS cells was tested with the CTL clone 4 by measuring GM-CSF cytokine release from the CTL clone. Fig. 3 indicates that pTD1, 2, and 3 constructs retained the ability to stimulate cytokine release from the CTL clone 4, but pTD4 and pTD5 lost the stimulating activity to the CTL clone 4, indicating that the epitope(s) recognized by the CTL clone 4 was located in the region of nucleotides 836–1045. This was consistent with results obtained by the subcloning experiments. Although pTA and pTP lost the ability to stimulate cytokine release from CTL clone 4, pTK still remained positive in the cytokine release assay. Therefore, the epitopes resided in a DNA fragment flanked by the first PstI and KpnI sites as shown in Fig. 4.

Bottom Line: This epitope peptide was capable of sensitizing target cells for lysis by a CTL clone at 1 nM peptide concentration.Although some modified peptides could be recognized by the CTL clone, none were found to be better recognized by T cells than the parental peptide.Like other melamona differentiation antigens, TRP-2 was only expressed in melanoma, melanocytes, and retina, but not in other human tissues tested.

View Article: PubMed Central - PubMed

Affiliation: National Cancer Institute, Bethesda, Maryland 20892, USA.

ABSTRACT
The infusion of TIL586 along with interleukin-2 into the autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was previously isolated and shown to encode gp75 or TRP-1. Here we report that TRP-2 was identified as a second tumor antigen recognized by a HLA-A31-restricted CTL clone derived from the TIL586 cell line. The peptide LLPGGRPYR epitope was subsequently identified from the coding region of TRP-2 based on studies of the recognition of truncated TRP-2 cDNAs and the HLA-A31 binding motif. This epitope peptide was capable of sensitizing target cells for lysis by a CTL clone at 1 nM peptide concentration. Although some modified peptides could be recognized by the CTL clone, none were found to be better recognized by T cells than the parental peptide. Like other melamona differentiation antigens, TRP-2 was only expressed in melanoma, melanocytes, and retina, but not in other human tissues tested.

Show MeSH
Related in: MedlinePlus