Gene transfer into BALB/c mice that had received adoptive transfer of CFSE-labeled CD4⁺ splenocytes from 4 DO11.10 TCR mice. (A) Experimental strategy. Mice were injected intramuscularly with equal doses of AAV-CMV-OVA and AAV-null vector into contralateral legs 24 hours after adoptive T-cell transfer. (B) Flow cytometry to detect CFSE-stained cells (histograms, left column) and CFSE and KJ1-26 (DO11.10 TCR expression) dual-stained cells (scatter graphs, right column) in spleens (top row) and nondraining LNs (ND-LN, ie, LNs of AAV-null injected leg; bottom row). Note that about 70% of CFSE⁺ cells were TCR⁺, and about 30% of CFSE⁺ cells were TCR^-. Analysis shown here was done 5 days after injection of 1 × 10¹³ vg/leg. Each graph represents pooled cells from experimental animals. (C) Flow cytometry to detect CFSE-stained cells (histograms, left column) and CFSE and KJ1-26 (DO11.10 TCR expression) dual-stained cells (scatter graphs, right column) in LNs draining AAV-CMV-OVA–injected leg (D-LN) as a function of vector dose 5 days after gene transfer. Note a vector dose-dependent increase in cells with decreased CFSE fluorescence intensity indicating in vivo proliferation of TCR⁺ cells. (D) Estimate of percentage TCR⁺ cells that had undergone cell division in the draining LNs as a function of vector dose. (E) Hematoxylin and eosin stain of muscle tissue 10 days after AAV-CMV-OVA (left column) or AAV-null (right column) administration. Shown are representative sections for the 3 different vector doses. AAV-CMV-OVA–transduced muscle was also analyzed (day 10) for OVA expression and CD8⁺ cellular infiltrate (F), for infiltrate of IFN-γ–expressing cells (G), and for CD4⁺ and DO11.10 TCR⁺ cellular infiltrate (H). Arrows in panel H depict examples of CD4⁺TCR⁺ dual positive cells (yellow to orange color). Original magnification, × 100 (E-F), × 200 (G), and × 400 (H).