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Volumn 278, Issue 5335, 1997, Pages 108-109

Negative regulation by HLA-DO of MHC class II-restricted an tigen processing

Author keywords

[No Author keywords available]

Indexed keywords

HLA ANTIGEN; MAJOR HISTOCOMPATIBILITY ANTIGEN CLASS 2; B LYMPHOCYTE ANTIGEN; H 2O ANTIGEN; H-2O ANTIGEN; H2 M ANTIGENS; H2-M ANTIGENS; HLA ANTIGEN CLASS 2; HLA D ANTIGEN; HLA DMB; HLA DO ANTIGENS; HLA DR3 ANTIGEN; HLA-DMB; HLA-DO ANTIGENS; MHC CLASS II TRANSACTIVATOR PROTEIN; NUCLEAR PROTEIN; TRANSACTIVATOR PROTEIN;

EID: 0031551492     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: None     Document Type: Article
Times cited : (208)

References (52)
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    • 10 Raji cells was purified with a MaP.DMB-c affinity column (4). The peak fractions were pooled, and the DM-DO and DM complexes were fractionated by gel filtration with a Superose6 FPLC column (Pharmacia) equilibrated in 50 mM sodium acetate, 100 mM NaCl, 0.6% CHAPS (Pierce) (pH 5.0) at a flow rate of 0.4 ml/min. The presence of DM and DM-DO complexes in the fractions was determined by ELISA, and the peak fractions containing DM-DO complexes (19 to 21) were pooled and used in the subsequent experiments as DM-DO complexes. For the ELISA, polystyrene wells (Nunc-Immuno Plate MaxiSorb) were coated overnight at 4°C with 50 μl of the DM-specific mAb, MaP.DM2 (C. Hammond, in preparation), washed with phosphate-buffered saline (PBS) containing 0.01% Tween-20 (PBS-T), and masked for 30 min with PBS-T plus 1% bovine serum albumin (BSA). Samples containing DM or DM-DO complexes were incubated for 1 hour at room temperature, the wells were washed, and R.DOB/c or R.DMB/c (10 μg/ml) was added. Complexes were detected with horseradish peroxidase-labeled mouse anti-rabbit immunoglobulin G and Turbo-TMB (Pierce). We determined the percentage of remaining free DM in the DM-DO pool by first removing the DO (and DM-DO) by multiple rounds of immunoprecipitation with either R.DOB/c or a control antiserum and then determining the amount of free DM remaining in the immunoprecipitation supernatants by ELISA.
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    • DOA was amplified by PCR from a Raji cDNA library with primers specific for the 5′ end (5′-TCCCCC-GGGTACCGCCACCATGGCCCTCAGAGCAGGGCTG-3′) and 3′ end (5′-TCCAGTGTCCCCAGGTAATGATCGATGG-3′). The resulting PCR product was cloned into pCR2.1 (Invitrogen), sequenced, and subcloned into the eukaryotic expression vector pMCFR-PAC (13) to generate pMCFR-PAC/ DOA. CEM.CIITA cells were transfected with pM-CFR-PAC/DOA and pDOB#163 (10) at a ratio of 1:10, PMCFR-PAC/DOA alone, or empty vector alone as described (13). Clones resistant to puromycin were screened for DO expression by immunofluorescence with R.DOB/c. Two of the positive cell lines (CEM.CIITA.DO-1 and CEM.CIITA.DO-2) were chosen and used for subsequent experiments. OEM.CIITA-DOA cells were screened for DOA expression by RT-PCR analysis and CEM. CIITA. Vector-only cells were isolated by resistance to puromycin.
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    • CEM.CIITA.DO-1 cells were mixed at a ratio of 3:1 with untransfected CEM.CIITA cells and processed for immunofluorescence as described [N. F. Robbins, C. Hammond, L. Denzin, M. Pan, P. Cresswell, Hum. Immunol. 45, 13 (1996)] with the following modifications. After fixation of the cells on coverslips, they were treated with permeabilization solution [PBS containing 5% fetal bovine serum, 1% BSA, and saponin (15 μg/ml; Sigma)], 10 mM glycine, 10 mM Hepes (pH 7.4) for 15 min at room temperature, and the coverslips were placed in a 20-μl drop of a solution of R.DOB/c (50 μg/ml) in permeabilization solution for 30 min. The coverslips were then washed three times in permeabilization solution and treated with sequential rounds of Cer-CLIP.1 or MaP.DM1 (C. Hammond, in preparation) and secondary antibodies as described above, except that the saponin concentration was increased to 500 μg/ml. After extensive washing, the coverslips were mounted onto slides with Mowiol 4-88 solution (Calbiochem).
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    • C. Hammond, in preparation
    • CEM.CIITA.DO-1 cells were mixed at a ratio of 3:1 with untransfected CEM.CIITA cells and processed for immunofluorescence as described [N. F. Robbins, C. Hammond, L. Denzin, M. Pan, P. Cresswell, Hum. Immunol. 45, 13 (1996)] with the following modifications. After fixation of the cells on coverslips, they were treated with permeabilization solution [PBS containing 5% fetal bovine serum, 1% BSA, and saponin (15 μg/ml; Sigma)], 10 mM glycine, 10 mM Hepes (pH 7.4) for 15 min at room temperature, and the coverslips were placed in a 20-μl drop of a solution of R.DOB/c (50 μg/ml) in permeabilization solution for 30 min. The coverslips were then washed three times in permeabilization solution and treated with sequential rounds of Cer-CLIP.1 or MaP.DM1 (C. Hammond, in preparation) and secondary antibodies as described above, except that the saponin concentration was increased to 500 μg/ml. After extensive washing, the coverslips were mounted onto slides with Mowiol 4-88 solution (Calbiochem).
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    • note
    • Metabolic labeling of CEM.CIITA transfectants and affinity purifications were done as described (4, 13).
  • 52
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    • note
    • We thank C. Doyle, J. Brogdon, and J. Blum for cell lines; E. Long and J. Fontes for cDNA clones; and N. Dometios for preparation of the manuscript. We are grateful to B. Sadasivan for producing the CEM. CIITA cell line and C. Janeway for helpful discussions. Supported by a Donaghue Fellowship (L.K.D.), a fellowship from the Cancer Research Institute (C.H.), and by NIH grant Al14579 (D.B.S.). Funding for this research is from the Howard Hughes Medical Institute and NIH grant Al23081 (P.C.).


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