Figure 3.
Identification of the TH3Z cognate antigen by expression cloning. (A) A prokaryotic cDNA expression library from day 5 BMDCs derived from B6 mice was screened in 129/J-derived BMDCs using the lacZ-inducible TH3Z T cells as described in Fig. 2 B. The lacZ response of TH3Z hybridoma to the bacterial pools in one of the 96-well plates screened is shown indicating the higher than background response to pool 224. (B) TH3Z response to individual bacterial colonies isolated from pool 224. Clone 224–26 was selected for further analysis. (C and D) The TH3Z response to clone 224–26 is antigen-specific and Ab MHC class II restricted. Clone 224–26 bacteria were cultured in medium alone (−Trp) or in medium containing the transcriptional inducer tryptophan (+Trp). Varying number of bacteria were fed to 105 129/J BMDCs/well. 105 TH3Z or the OVA/Ab-specific OTIIZ T cells were added and their lacZ response was assayed after overnight culture. (D) The right panel shows the TH3Z response to 129/J BMDC that were fed 106 clone 224–26 bacteria/well in medium alone or in the presence of mAb specific for the Ab MHC II or the Db MHC I molecules.