Analyses of cell populations from lymphoid organs of DO11.10-tg RAG-2^−/− mice by flow cytometry 60 days after AAV-EF1α-ova gene transfer. (A) Quantitation of CD25⁺ of TCR⁺CD4⁺, CD25⁺GITR⁺ and CD25⁺CTLA4⁺ of CD4⁺ cells from spleen in naive or vector-treated mice. Percentages of dual-positive cells (top-right quadrant) are indicated. Note that AAV-EF1α-ova–transduced mice showed substantial increase in CD25⁺, CD25⁺GITR⁺, and CD25⁺CTLA4⁺ of total CD4⁺ cells compared with control animals. Antibody stain was FITC-conjugated KJ1-26 or CD4, PE-conjugated GITR or CTLA4, and APC-conjugated CD25. Representative examples of FACS density plots are shown for individual samples of splenocytes from DO11.10-tg RAG-2^−/− mice. Also graphed are percent GITR⁺ (B) and CTLA4⁺ (C) of CD4⁺CD25⁺ cells derived from spleen, thymus, portal nodes, and inguinal nodes 60 days after AAV-EF1α-ova gene delivery (B,C).