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1842286224
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note
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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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1842400789
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note
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HLA-DR1 and -DR4 αβCLIP complexes were purified from T2.DR1 and T2.DR4 cells as described (4) with L243 (T2.DR4) or CerCLIP.1 (T2.DR1) affinity columns. For the DM-mediated peptide-exchange assay, DM or DM-DO preparations, matched for a DM concentration of 20 ng by ELISA, were incubated with ∼10,000 cpm of radiolabeled DR1 or DR4 αβCLIP complexes and biotinylated HAp peptide (KYVKQNTLKATK-biotin) in 20 mM tris, 130 mM NaCl (pH 7.4) containing 0.6% CHAPS (Pierce). The pH was adjusted to 5.0 by the addition of 1 M acetic acid, and the samples were incubated at 37°C for the times indicated in Fig. 3. After neutralization, the HAp-class II complexes were immunoprecipitated with streptavidin-agarose beads (Pierce) and the samples were separated by SDS-PAGE. The percentage of peptide-loaded class II molecules was quantitated by image analysis with a BioRad GS-525 Molecular imager.
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We cotransfected class II-negative CEM cells with CIITA-pcDNAI/Amp and pMCFR-Neo (13) at a ratio of 10:1 using previously described conditions [B. K. Sadasivan, A. Cariappa, G. L. Waneck, P. Cresswell, Cold Spring Harbor Symp. Quant. Biol. 55, 267 (1995)]. Clones resistant to geneticin were screened for expression of cell surface class II by flow cytometric analysis with the DR-specific mAb L243 [L. A. Lampson and R. Levy, J. Immunol. 125, 293 (1980)]. Positive clones were subcloned by limiting dilution, and the clone with the highest class II expression was used for subsequent DO transfection.
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0018937455
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We cotransfected class II-negative CEM cells with CIITA-pcDNAI/Amp and pMCFR-Neo (13) at a ratio of 10:1 using previously described conditions [B. K. Sadasivan, A. Cariappa, G. L. Waneck, P. Cresswell, Cold Spring Harbor Symp. Quant. Biol. 55, 267 (1995)]. Clones resistant to geneticin were screened for expression of cell surface class II by flow cytometric analysis with the DR-specific mAb L243 [L. A. Lampson and R. Levy, J. Immunol. 125, 293 (1980)]. Positive clones were subcloned by limiting dilution, and the clone with the highest class II expression was used for subsequent DO transfection.
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1842322506
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note
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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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0029979452
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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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Hum. Immunol.
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Robbins, N.F.1
Hammond, C.2
Denzin, L.3
Pan, M.4
Cresswell, P.5
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43
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0029979452
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C. Hammond, in preparation
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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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48
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0030920340
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Protein immunoblot analysis was done as described [P. J. Lehner, J. T. Karttunen, G. W. G. Wilkinson, P. Cresswell, Proc. Natl. Acad. Sci. U.S.A. 94, 6904 (1997)].
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Lehner, P.J.1
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Cresswell, P.4
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51
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1842274537
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note
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Metabolic labeling of CEM.CIITA transfectants and affinity purifications were done as described (4, 13).
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52
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1842269589
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note
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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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