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 - PubMed (original) (raw)

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

M Probst-Kepper et al. J Exp Med. 2001.

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.

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Figures

Figure 1

Figure 1

Lytic activity of CTL clone 403A/9. Targets were: autologous tumor cells LB1047-RCC, allogeneic HLA-B35–positive renal carcinoma cells HA7-RCC and LE9211-RCC, and the corresponding EBV-transformed B cell lines LB1047-EBV, HA7-EBV, and LE9211-EBV. NK target K562 was also used as control. All target cells were treated with 100 U/ml IFN-γ 24 h before the test. Chromium release was measured after 4 h.

Figure 2

Figure 2

Isolation of a cDNA encoding the antigen recognized by CTL 403A/9. 293-EBNA cells were transfected transiently with cDNA 314 and with a plasmid construct encoding HLA-B*3501. CTL 403A/9 was added after 24 h and the amount of IFN-γ in the supernatant was measured 24 h later. LB1047-RCC cells were used as positive control.

Figure 4

Figure 4

Expression of the alt.M-CSF product in tubular kidney cells and in hepatocytes. Paraffin sections from: the renal tumor of patient LB1047 (A and B); a normal kidney biopsy from a graft donor (D and E); or a normal liver (G and H) were stained with either a rabbit control IgG (A, D, and G) or a rabbit antiserum raised against the alt.M-CSF polypeptide (B, E, and H). Cryosections from: the renal tumor of patient EB56 (C), a normal kidney sample from patient LB2043 (F), or a normal liver (I) were stained with a monoclonal antibody against M-CSF. Original magnifications: ×200 (A–C) and ×100 (D–I).

Figure 5

Figure 5

Recognition of normal kidney cells by CTL 403A/9. Short-term cell lines LB2046-PTEC and LB2043-PTEC (10,000 cells/well), which derive from proximal tubular epithelial cells (PTEC), were incubated with CTL 403A/9 (10,000/well), and the production of TNF in the supernatant was measured after 24 h. In the absence of CTL, LB2043-PTEC, and LB2046-PTEC produced 2 and 3 pg/ml TNF, respectively. As indicated, both lines were transiently transfected with HLA-B*3501, to ensure expression of the presenting molecule. Line LB2046-PTEC, which was already recognized by CTL 403A/9 in the absence of transfection, was later confirmed to express HLA-B35 naturally. The amount of M-CSF produced by the same cell lines was meas-ured by ELISA on culture supernatants and is indicated.

Figure 6

Figure 6

Elution of the natural peptide recognized by CTL 403A/9. (A) HPLC fractions of peptides eluted with acid from the HLA class I molecules of LB1047-RCC were tested for their ability to stimulate CTL 403A/9 after pulsing onto HLA-B35–positive target cells A110L-B35. The results of one representative experiment out of three are shown. (B) Synthetic peptide LPAVVGLSPGEQEY (150 pmol) was injected on the same HPLC column and its elution was monitored by UV absorption. (C) 1 pmol of the synthetic peptide was injected on the same column and the fractions were tested for CTL recognition as indicated in panel A.

Figure 3

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.

Figure 3

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.

Figure 3

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.

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