Limits...
An alternative open reading frame of the human macrophage colony-stimulating factor gene is independently translated and codes for an antigenic peptide of 14 amino acids recognized by tumor-infiltrating CD8 T lymphocytes.

Probst-Kepper M, Stroobant V, Kridel R, Gaugler B, Landry C, Brasseur F, Cosyns JP, Weynand B, Boon T, Van Den Eynde BJ - J. Exp. Med. (2001)

Bottom Line: Such a dissociation of the translation of two overlapping ORFs from the same gene is unexpected.The antigenic peptide encoded by the alternative ORF is presented by human histocompatibility leukocyte antigen (HLA)-B*3501 and has a length of 14 residues.Binding studies of peptide analogues suggest that it binds by its two extremities and bulges out of the HLA groove to compensate for its length.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research and Cellular Genetics Unit, Université Catholique de Louvain, Brussels 1200, Belgium.

ABSTRACT
We show that cytotoxic T lymphocytes (CTLs) infiltrating a kidney tumor recognize a peptide encoded by an alternative open reading frame (ORF) of the macrophage colony-stimulating factor (M-CSF) gene. Remarkably, this alternative ORF, which is translated in many tumors concurrently with the major ORF, is also translated in some tissues that do not produce M-CSF, such as liver and kidney. Such a dissociation of the translation of two overlapping ORFs from the same gene is unexpected. The antigenic peptide encoded by the alternative ORF is presented by human histocompatibility leukocyte antigen (HLA)-B*3501 and has a length of 14 residues. Peptide elution indicated that tumor cells naturally present this 14 mer, which is the longest peptide known to be recognized by CTLs. Binding studies of peptide analogues suggest that it binds by its two extremities and bulges out of the HLA groove to compensate for its length.

Show MeSH

Related in: MedlinePlus

Identification of the peptide recognized by CTL 403A/9. (A) Partial sequence of the 5′ end of the M-CSF cDNA, showing the alternative ORF (boxed) coding for a 25-amino acid polypeptide (alt.M-CSF), and the initial part of the major ORF which encodes the leader sequence and the first residues of the mature M-CSF, which starts at position +1. The arrow indicates the boundary between exon 1 and 2 (reference 46). The antigenic peptide in the alt.M-CSF sequence is shown in bold. (B) CTL recognition of cells transfected with minigenes containing the alternative ORF encoding alt.M-CSF. 293-EBNA cells were transfected with plasmids containing HLA-B*3501 and minigenes encoding either the full-length alt.M-CSF product of 25 amino acids or a truncated product containing the first 20 amino acids. CTL 403A/9 was added after 24 h and production of IFN-γ was measured 24 h later. (C) Recognition of peptide LPAVVGLSPGEQEY by CTL 403A/9. Chromium-labeled LB1047-EBV B cells were incubated 30 min at 37°C with the indicated peptides at various concentrations. CTL 403A/9 was added at an effector/target ratio of 10, and chromium release was measured after 4 h.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2193327&req=5

Figure 3: Identification of the peptide recognized by CTL 403A/9. (A) Partial sequence of the 5′ end of the M-CSF cDNA, showing the alternative ORF (boxed) coding for a 25-amino acid polypeptide (alt.M-CSF), and the initial part of the major ORF which encodes the leader sequence and the first residues of the mature M-CSF, which starts at position +1. The arrow indicates the boundary between exon 1 and 2 (reference 46). The antigenic peptide in the alt.M-CSF sequence is shown in bold. (B) CTL recognition of cells transfected with minigenes containing the alternative ORF encoding alt.M-CSF. 293-EBNA cells were transfected with plasmids containing HLA-B*3501 and minigenes encoding either the full-length alt.M-CSF product of 25 amino acids or a truncated product containing the first 20 amino acids. CTL 403A/9 was added after 24 h and production of IFN-γ was measured 24 h later. (C) Recognition of peptide LPAVVGLSPGEQEY by CTL 403A/9. Chromium-labeled LB1047-EBV B cells were incubated 30 min at 37°C with the indicated peptides at various concentrations. CTL 403A/9 was added at an effector/target ratio of 10, and chromium release was measured after 4 h.

Mentions: This cDNA was 4 kb long, and its sequence corresponded to the full-length transcript of the gene encoding M-CSF, a cytokine also known as CSF-1, which promotes growth, differentiation, and activation of monocytes, macrophages, and osteoclasts 2223. To identify the region of the M-CSF transcript that encodes the antigenic peptide, we transfected truncated variants of the cDNA, and found that fragments containing only the 5′-terminal 335 nucleotides were able to confer antigenic expression (data not shown). We tested a series of synthetic peptides derived from this region of the M-CSF protein sequence for recognition by CTL 403A/9, but they were all negative. We then analyzed the 5′ nucleotidic sequence in the two other reading phases and identified an alternative ORF encoding a putative polypeptide of 25 amino acids (alt.M-CSF; Fig. 3 A). To determine whether the antigenic peptide was derived from this polypeptide, we constructed minigenes encoding either the full length or the first 20 amino acids of alt.M-CSF, and transfected them with the HLA-B*3501 cDNA into 293 cells. We observed that CTL 403A/9 recognized cells transfected with both constructs, suggesting that the antigenic peptide derived from the first 20 amino acids of this alternative translation product of the M-CSF cDNA (Fig. 3 B).


An alternative open reading frame of the human macrophage colony-stimulating factor gene is independently translated and codes for an antigenic peptide of 14 amino acids recognized by tumor-infiltrating CD8 T lymphocytes.

Probst-Kepper M, Stroobant V, Kridel R, Gaugler B, Landry C, Brasseur F, Cosyns JP, Weynand B, Boon T, Van Den Eynde BJ - J. Exp. Med. (2001)

Identification of the peptide recognized by CTL 403A/9. (A) Partial sequence of the 5′ end of the M-CSF cDNA, showing the alternative ORF (boxed) coding for a 25-amino acid polypeptide (alt.M-CSF), and the initial part of the major ORF which encodes the leader sequence and the first residues of the mature M-CSF, which starts at position +1. The arrow indicates the boundary between exon 1 and 2 (reference 46). The antigenic peptide in the alt.M-CSF sequence is shown in bold. (B) CTL recognition of cells transfected with minigenes containing the alternative ORF encoding alt.M-CSF. 293-EBNA cells were transfected with plasmids containing HLA-B*3501 and minigenes encoding either the full-length alt.M-CSF product of 25 amino acids or a truncated product containing the first 20 amino acids. CTL 403A/9 was added after 24 h and production of IFN-γ was measured 24 h later. (C) Recognition of peptide LPAVVGLSPGEQEY by CTL 403A/9. Chromium-labeled LB1047-EBV B cells were incubated 30 min at 37°C with the indicated peptides at various concentrations. CTL 403A/9 was added at an effector/target ratio of 10, and chromium release was measured after 4 h.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193327&req=5

Figure 3: Identification of the peptide recognized by CTL 403A/9. (A) Partial sequence of the 5′ end of the M-CSF cDNA, showing the alternative ORF (boxed) coding for a 25-amino acid polypeptide (alt.M-CSF), and the initial part of the major ORF which encodes the leader sequence and the first residues of the mature M-CSF, which starts at position +1. The arrow indicates the boundary between exon 1 and 2 (reference 46). The antigenic peptide in the alt.M-CSF sequence is shown in bold. (B) CTL recognition of cells transfected with minigenes containing the alternative ORF encoding alt.M-CSF. 293-EBNA cells were transfected with plasmids containing HLA-B*3501 and minigenes encoding either the full-length alt.M-CSF product of 25 amino acids or a truncated product containing the first 20 amino acids. CTL 403A/9 was added after 24 h and production of IFN-γ was measured 24 h later. (C) Recognition of peptide LPAVVGLSPGEQEY by CTL 403A/9. Chromium-labeled LB1047-EBV B cells were incubated 30 min at 37°C with the indicated peptides at various concentrations. CTL 403A/9 was added at an effector/target ratio of 10, and chromium release was measured after 4 h.
Mentions: This cDNA was 4 kb long, and its sequence corresponded to the full-length transcript of the gene encoding M-CSF, a cytokine also known as CSF-1, which promotes growth, differentiation, and activation of monocytes, macrophages, and osteoclasts 2223. To identify the region of the M-CSF transcript that encodes the antigenic peptide, we transfected truncated variants of the cDNA, and found that fragments containing only the 5′-terminal 335 nucleotides were able to confer antigenic expression (data not shown). We tested a series of synthetic peptides derived from this region of the M-CSF protein sequence for recognition by CTL 403A/9, but they were all negative. We then analyzed the 5′ nucleotidic sequence in the two other reading phases and identified an alternative ORF encoding a putative polypeptide of 25 amino acids (alt.M-CSF; Fig. 3 A). To determine whether the antigenic peptide was derived from this polypeptide, we constructed minigenes encoding either the full length or the first 20 amino acids of alt.M-CSF, and transfected them with the HLA-B*3501 cDNA into 293 cells. We observed that CTL 403A/9 recognized cells transfected with both constructs, suggesting that the antigenic peptide derived from the first 20 amino acids of this alternative translation product of the M-CSF cDNA (Fig. 3 B).

Bottom Line: Such a dissociation of the translation of two overlapping ORFs from the same gene is unexpected.The antigenic peptide encoded by the alternative ORF is presented by human histocompatibility leukocyte antigen (HLA)-B*3501 and has a length of 14 residues.Binding studies of peptide analogues suggest that it binds by its two extremities and bulges out of the HLA groove to compensate for its length.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research and Cellular Genetics Unit, Université Catholique de Louvain, Brussels 1200, Belgium.

ABSTRACT
We show that cytotoxic T lymphocytes (CTLs) infiltrating a kidney tumor recognize a peptide encoded by an alternative open reading frame (ORF) of the macrophage colony-stimulating factor (M-CSF) gene. Remarkably, this alternative ORF, which is translated in many tumors concurrently with the major ORF, is also translated in some tissues that do not produce M-CSF, such as liver and kidney. Such a dissociation of the translation of two overlapping ORFs from the same gene is unexpected. The antigenic peptide encoded by the alternative ORF is presented by human histocompatibility leukocyte antigen (HLA)-B*3501 and has a length of 14 residues. Peptide elution indicated that tumor cells naturally present this 14 mer, which is the longest peptide known to be recognized by CTLs. Binding studies of peptide analogues suggest that it binds by its two extremities and bulges out of the HLA groove to compensate for its length.

Show MeSH
Related in: MedlinePlus