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A new antigen recognized by cytolytic T lymphocytes on a human kidney tumor results from reverse strand transcription.

Van Den Eynde BJ, Gaugler B, Probst-Kepper M, Michaux L, Devuyst O, Lorge F, Weynants P, Boon T - J. Exp. Med. (1999)

Bottom Line: It is encoded by an antisense transcript, RU2AS, which starts from a cryptic promoter located on the reverse strand of the first intron and ends up on the reverse strand of the RU2S promoter, which contains a polyadenylation signal.This mechanism of antigen expression is unprecedented and further illustrates the notion that many peptides recognized by T cells cannot be predicted from the primary structure of the major product of the encoding gene.It is absent in most normal tissues, but is expressed in testis and kidney, and, at lower levels, in urinary bladder and liver.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Brussels Branch, B-1200 Brussels, Belgium. vandeneynde@licr.ucl.ac.be

ABSTRACT
By stimulating blood lymphocytes from a renal cell carcinoma patient in vitro with the autologous tumor cells, we obtained cytolytic T lymphocyte (CTL) clones that killed several autologous and allogeneic histocompatibility leukocyte antigen (HLA)-B7 renal carcinoma cell lines. We identified the target antigen of these CTLs by screening COS cells transfected with the HLA-B7 cDNA and with a cDNA library prepared with RNA from the tumor cells. The antigenic peptide recognized by the CTLs has the sequence LPRWPPPQL and is encoded by a new gene, which we named RU2. This gene is transcribed in both directions. The antigenic peptide is not encoded by the sense transcript, RU2S, which is expressed ubiquitously. It is encoded by an antisense transcript, RU2AS, which starts from a cryptic promoter located on the reverse strand of the first intron and ends up on the reverse strand of the RU2S promoter, which contains a polyadenylation signal. This mechanism of antigen expression is unprecedented and further illustrates the notion that many peptides recognized by T cells cannot be predicted from the primary structure of the major product of the encoding gene. Antisense transcript RU2AS is expressed in a high proportion of tumors of various histological types. It is absent in most normal tissues, but is expressed in testis and kidney, and, at lower levels, in urinary bladder and liver. Short-term cultures of normal epithelial cells from the renal proximal tubule expressed significant levels of RU2AS message and were recognized by the CTLs. Therefore, this antigen is not tumor specific, but corresponds to a self-antigen with restricted tissue distribution.

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Isolation of a cDNA coding for the antigen recognized by CTL 361A/21. (A) MHC restriction of CTL 361A/21. CTL activation was followed by measuring the production of TNF after stimulation with autologous tumor cells in the presence of the indicated anti-HLA mAbs. The HLA typing of patient LE9211 is HLA-A3, -B7, -B35, -Cw4, and -Cw7. MZ2-MEL is an allogeneic melanoma line that was used as a negative control. Antibodies W6/32 (anti–HLA class I), 4E (anti–HLA-B/C), ME1 (anti–HLA-B7), and GAPA3 (anti–HLA-A3) were used as described (reference 13). 3,000 CTLs were mixed with 30,000 tumor cells and IL-2 (25 U/ml), and TNF production was measured 18 h later as described elsewhere (reference 11). (B) cDNA 4.1 cloned into plasmid pcDNAI/Amp was transfected into COS cells together with the HLA-B*0702 cDNA cloned into plasmid pcDSRalpha. CTL 361A/21 (3,000 cells) was added after 24 h, and TNF production was measured 18 h later.
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Figure 2: Isolation of a cDNA coding for the antigen recognized by CTL 361A/21. (A) MHC restriction of CTL 361A/21. CTL activation was followed by measuring the production of TNF after stimulation with autologous tumor cells in the presence of the indicated anti-HLA mAbs. The HLA typing of patient LE9211 is HLA-A3, -B7, -B35, -Cw4, and -Cw7. MZ2-MEL is an allogeneic melanoma line that was used as a negative control. Antibodies W6/32 (anti–HLA class I), 4E (anti–HLA-B/C), ME1 (anti–HLA-B7), and GAPA3 (anti–HLA-A3) were used as described (reference 13). 3,000 CTLs were mixed with 30,000 tumor cells and IL-2 (25 U/ml), and TNF production was measured 18 h later as described elsewhere (reference 11). (B) cDNA 4.1 cloned into plasmid pcDNAI/Amp was transfected into COS cells together with the HLA-B*0702 cDNA cloned into plasmid pcDSRalpha. CTL 361A/21 (3,000 cells) was added after 24 h, and TNF production was measured 18 h later.

Mentions: CTL clone 361/A21 was isolated by stimulating blood lymphocytes of a kidney cancer patient in vitro with autologous tumor cells LE9211-RCC, and by cloning the responding cells by limiting dilution 13. This clone was able to lyse both autologous and allogeneic tumor cells sharing the HLA-B7 specificity, but it did not lyse autologous EBV-transformed B cells or NK target K562 (Fig. 1). The HLA-B7 restriction of this CTL clone was confirmed by the observation that an anti–HLA-B7 antibody blocked the recognition of the tumor cells (Fig. 2 A). To identify the target antigen of CTL 361A/21, we prepared an oligo-dT–based unidirectional cDNA library with RNA from LE9211-RCC cells, and we transfected DNA from this library into COS cells together with DNA from an HLA-B7 cDNA plasmid construct. The transfected cells were screened by adding the CTLs to the microcultures and measuring the production of TNF. We isolated cDNA 4.1, which was able to stimulate the CTLs when transfected into COS cells together with the HLA-B7 cDNA (Fig. 2 B). This cDNA clone was 924 bp long. Its sequence was new and contained an open reading frame coding for a protein of 84 amino acids (Fig. 3). To exclude that recognition of the transfected COS cells was an artifact of high expression levels after transient transfection, we transfected an HLA-B7–positive sarcoma line with cDNA 4.1. A stable transfectant was obtained, and it was recognized by the CTLs (Fig. 4). To identify the antigenic epitope recognized by CTL 361A/21, we tested several synthetic peptides encoded by cDNA 4.1 and bearing the binding motif for HLA-B7, which is P in position 2 and L/F in position 9 14. We found that peptide LPRWPPPQL was able to sensitize autologous EBV-B cells to lysis by the CTLs (Fig. 5).


A new antigen recognized by cytolytic T lymphocytes on a human kidney tumor results from reverse strand transcription.

Van Den Eynde BJ, Gaugler B, Probst-Kepper M, Michaux L, Devuyst O, Lorge F, Weynants P, Boon T - J. Exp. Med. (1999)

Isolation of a cDNA coding for the antigen recognized by CTL 361A/21. (A) MHC restriction of CTL 361A/21. CTL activation was followed by measuring the production of TNF after stimulation with autologous tumor cells in the presence of the indicated anti-HLA mAbs. The HLA typing of patient LE9211 is HLA-A3, -B7, -B35, -Cw4, and -Cw7. MZ2-MEL is an allogeneic melanoma line that was used as a negative control. Antibodies W6/32 (anti–HLA class I), 4E (anti–HLA-B/C), ME1 (anti–HLA-B7), and GAPA3 (anti–HLA-A3) were used as described (reference 13). 3,000 CTLs were mixed with 30,000 tumor cells and IL-2 (25 U/ml), and TNF production was measured 18 h later as described elsewhere (reference 11). (B) cDNA 4.1 cloned into plasmid pcDNAI/Amp was transfected into COS cells together with the HLA-B*0702 cDNA cloned into plasmid pcDSRalpha. CTL 361A/21 (3,000 cells) was added after 24 h, and TNF production was measured 18 h later.
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Figure 2: Isolation of a cDNA coding for the antigen recognized by CTL 361A/21. (A) MHC restriction of CTL 361A/21. CTL activation was followed by measuring the production of TNF after stimulation with autologous tumor cells in the presence of the indicated anti-HLA mAbs. The HLA typing of patient LE9211 is HLA-A3, -B7, -B35, -Cw4, and -Cw7. MZ2-MEL is an allogeneic melanoma line that was used as a negative control. Antibodies W6/32 (anti–HLA class I), 4E (anti–HLA-B/C), ME1 (anti–HLA-B7), and GAPA3 (anti–HLA-A3) were used as described (reference 13). 3,000 CTLs were mixed with 30,000 tumor cells and IL-2 (25 U/ml), and TNF production was measured 18 h later as described elsewhere (reference 11). (B) cDNA 4.1 cloned into plasmid pcDNAI/Amp was transfected into COS cells together with the HLA-B*0702 cDNA cloned into plasmid pcDSRalpha. CTL 361A/21 (3,000 cells) was added after 24 h, and TNF production was measured 18 h later.
Mentions: CTL clone 361/A21 was isolated by stimulating blood lymphocytes of a kidney cancer patient in vitro with autologous tumor cells LE9211-RCC, and by cloning the responding cells by limiting dilution 13. This clone was able to lyse both autologous and allogeneic tumor cells sharing the HLA-B7 specificity, but it did not lyse autologous EBV-transformed B cells or NK target K562 (Fig. 1). The HLA-B7 restriction of this CTL clone was confirmed by the observation that an anti–HLA-B7 antibody blocked the recognition of the tumor cells (Fig. 2 A). To identify the target antigen of CTL 361A/21, we prepared an oligo-dT–based unidirectional cDNA library with RNA from LE9211-RCC cells, and we transfected DNA from this library into COS cells together with DNA from an HLA-B7 cDNA plasmid construct. The transfected cells were screened by adding the CTLs to the microcultures and measuring the production of TNF. We isolated cDNA 4.1, which was able to stimulate the CTLs when transfected into COS cells together with the HLA-B7 cDNA (Fig. 2 B). This cDNA clone was 924 bp long. Its sequence was new and contained an open reading frame coding for a protein of 84 amino acids (Fig. 3). To exclude that recognition of the transfected COS cells was an artifact of high expression levels after transient transfection, we transfected an HLA-B7–positive sarcoma line with cDNA 4.1. A stable transfectant was obtained, and it was recognized by the CTLs (Fig. 4). To identify the antigenic epitope recognized by CTL 361A/21, we tested several synthetic peptides encoded by cDNA 4.1 and bearing the binding motif for HLA-B7, which is P in position 2 and L/F in position 9 14. We found that peptide LPRWPPPQL was able to sensitize autologous EBV-B cells to lysis by the CTLs (Fig. 5).

Bottom Line: It is encoded by an antisense transcript, RU2AS, which starts from a cryptic promoter located on the reverse strand of the first intron and ends up on the reverse strand of the RU2S promoter, which contains a polyadenylation signal.This mechanism of antigen expression is unprecedented and further illustrates the notion that many peptides recognized by T cells cannot be predicted from the primary structure of the major product of the encoding gene.It is absent in most normal tissues, but is expressed in testis and kidney, and, at lower levels, in urinary bladder and liver.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Brussels Branch, B-1200 Brussels, Belgium. vandeneynde@licr.ucl.ac.be

ABSTRACT
By stimulating blood lymphocytes from a renal cell carcinoma patient in vitro with the autologous tumor cells, we obtained cytolytic T lymphocyte (CTL) clones that killed several autologous and allogeneic histocompatibility leukocyte antigen (HLA)-B7 renal carcinoma cell lines. We identified the target antigen of these CTLs by screening COS cells transfected with the HLA-B7 cDNA and with a cDNA library prepared with RNA from the tumor cells. The antigenic peptide recognized by the CTLs has the sequence LPRWPPPQL and is encoded by a new gene, which we named RU2. This gene is transcribed in both directions. The antigenic peptide is not encoded by the sense transcript, RU2S, which is expressed ubiquitously. It is encoded by an antisense transcript, RU2AS, which starts from a cryptic promoter located on the reverse strand of the first intron and ends up on the reverse strand of the RU2S promoter, which contains a polyadenylation signal. This mechanism of antigen expression is unprecedented and further illustrates the notion that many peptides recognized by T cells cannot be predicted from the primary structure of the major product of the encoding gene. Antisense transcript RU2AS is expressed in a high proportion of tumors of various histological types. It is absent in most normal tissues, but is expressed in testis and kidney, and, at lower levels, in urinary bladder and liver. Short-term cultures of normal epithelial cells from the renal proximal tubule expressed significant levels of RU2AS message and were recognized by the CTLs. Therefore, this antigen is not tumor specific, but corresponds to a self-antigen with restricted tissue distribution.

Show MeSH
Related in: MedlinePlus