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Figure 2. Diagram showing T-cell expression cloning strategy to identify critical virulence antigens associated with periodontal immunity in vivo. Genomic DNA of Aa was partially digested and size-fractionated (0.5–4.0 kb) before being ligated into the pTrcHis-C vector. Ligated plasmids were transformed into E. coli Top10 strain by electroporation and then distributed onto selective plates. The size of the genomic library was estimated at 
106 clones/µg DNA insert. Periodontal CD4+ T-cells were purified from Aa-HuPBL-NOD/SCID mice (Teng et al., 2000) and then expanded by rhIL-2 in vitro. Later, the CD4+ T-cells were transfected with a linearized DNA vector of an IL-2-inducible reporter construct with a lacZ sequence (NF-AT-lacZ). Hygromycin-resistant T-cells were then selected and expanded in hrIL-2. Thus, T-cell activation can be measured and visualized as lacZ expression in single cells or in bulk cultures. Controls included the sham- and lacZ-only transfected CD4+ T-cells, which did not yield any positive clones from screening. To screen bacterial antigens recognized by CD4+ T-cells, we plated E. coli clones from the Aa genomic library and added IPTG in vitro to induce protein expression. In parallel, autologous HuPBL-derived monocytes/macrophages were prepared and cultured overnight with rhIFN-
to up-regulate HLA. CD4+ T-cells from Aa-HuPBL-NOD/SCID mice, after being transfected with NF-AT-lacZ, described above, were then added to the co-cultures for overnight incubation. The lacZ(+) cells were visualized by being stained with buffered X-gal. The positive wells giving blue signals were sequentially subjected to further screening for confirmation by the use of fewer clones (i.e., 5–10) per well, until a single positive clone was identified (for details, see Teng and Hu, 2003).
