Fig. 1
B cell expression, epitope recognition, and direct antigenic presentation of retrovirally synthesized peptide-IgG
(A) Structure and proviral integration of murine Moloney leukemia retroviral construct MBAE.BAK. Ten micrograms of genomic DNA from transduced, G418-resistant (+) or control (−) A20 cells was digested with Sac I, fractionated on 0.8% agarose. Southern-blotted, and probed with 32P-labeled DNA probe containing three tandem copies of 12–26 sequence. Sac I digestion releases an ∼5.1 kb proviral genome. (B) Surface IgG1 expression on transduced, G418-resistant (+) or control (−) A20 (sIgG2b+) or CH31 (sIgM+) B cell lines. Cells were probed with either goat anti-mouse IgG1 (adsorbed)-PE (Caltag) or biotinylated anti-mouse IgG1b (Pharmingen) plus streptavidin-FITC. Dashed lines show isotype-matched or secondary reagent controls. (C) Detection of 12–26 peptide on secreted Ig-peptide H chain. Culture supernatants from transduced, G418-resistant (+) or control (−) J558L myelomas were immunoprecipitated with either goat anti-mouse IgG1 agarose or NIP-sepharose beads. Samples were resolved on 10% SDS-PAGE, transferred onto nitrocellulose, blocked with 2% BSA, and probed with biotinylated mAb B3.11 (anti-12-26 epitope) plus streptavidin-AP as a secondary reagent. (D) Tissue expression of 12–26 mRNA in long-term (∼3 months) recipients of gene-transferred (+) or mocktransduced (−) BM progenitors. RNA from bone marrow (B), thymus (T), or spleen (S) was assayed by 12–26 sequence RT-PCR (25). One-tenth of each PCR reaction (except for A20 controls: 1/100th) was Southern blotted and probed with a noncomplementary 32P-labeled 12–26-specific oligonucleotide. For simplicity, only a subset of representative tissues are shown. (E) Direct presentation of endogenously synthesized peptide to 12–26-specific T cell hybrid, 9C127. Microcultures (200 µl) of transduced or control A20 cells were plated at various dilutions with 2 × 104 9C127 per well for 24 hr. T cell stimulation in the absence or presence of various blocking antibodies (lower right) was assayed via CTLL assay with serially diluted supernatants and recombinant IL-2 standards.