Anti-A20 Id mAb can inhibit A20 tumor cell growth in vivo, but ultimately Id-negative tumor cells escape. (A) Escapee tumor cells from anti-A20 Id mAb-treated mice or A20 tumors derived from nontreated mice were stained for surface or intracellular IgG2a expression and analyzed by flow cytometry. Histograms are gated on live lymphocytes and are representative of 5 separate tumors. (B) Escapee tumor cells from anti-A20 Id mAb-treated mice or A20 tumors derived from nontreated mice were surface-stained with anti-κ light chain antibody or anti-A20 Id mAb. Graphs are gated on live lymphocytes and are representative of 5 separate tumors. (C) Escapee cells were plated for 4 days in the presence of either anti-A20 Id mAb or anti-38C13 Id mAb. Cells were then pulsed with [³H]thymidine for 12 hours and harvested. (D) Escapee tumor cells were cultured in vitro for 24 hours in complete media. Cells were then stimulated with anti-A20 Id mAb (S) or an isotype control mAb (I) for 1 hour at 37°C. The proteins were lysed, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotted, and probed for total tyrosine phosphorylation expression. (E) Wild-type mice were inoculated with escapee tumor cells subcutaneously and then treated intraperitoneally with saline, anti-A20 Id mAb, or an isotype control mAb (anti-38C13 Id) 3 hours later. Each line represents a group consisting of 10 mice.