CD4+CD25+ regulatory T cells inhibit immune-mediated transgene rejection by David-Alexandre Gross, Marylène Leboeuf, Bernard Gjata, Olivier Danos, and Jean Davoust Blood Volume 102(13):4326-4328 December 15, 2003 ©2003 by American Society of Hematology

HA-specific CD4+CD25+ cells suppress anti-HA B- and T-cell immune responses. HA-specific CD4+CD25+ cells suppress anti-HA B- and T-cell immune responses. At day 0, anesthetized BALB/c mice were injected into the tibialis anterior with 25 μL AAV-HA (6 × 1011 physical particles (pp)/mL) and were either untreated (No cells) or intravenously injected twice at days 0 and 4 with 106 CD4+CD25+ cells from TCR-HA mice (HA-Tregs). Their splenocytes were tested at day 14 in a standard IFNγ-ELISPOT assay against the HA512-520 epitope, and spot-forming units (SFU) are represented after subtraction of background spots obtained with unpulsed splenocytes (A). To monitor the specificity of the response, AAV-HA-transduced mice were untreated (No cells), or injected twice with 106 CD4+CD25+ cells from TCR-HA mice (HA-Tregs), CD4+CD25-cells from TCR-HA mice (HA-Th), or CD4+CD25+ cells from BALB/c mice (Tregs). Splenocytes were tested at day 35 by IFNγ-ELISPOT assay (B), restimulated in vitro for 6 days with HA512-520, and tested in cytotoxic assay (C), and mouse sera were assayed for the presence of anti-HA IgG (D). For ELISPOT and ELISA assays, results represent the mean of 3 mice per group and are expressed as mean ± standard error of the mean (SEM). For cytotoxic assays, the percentage of specific lysis was calculated as the difference in lysis between HA512-520-pulsed (1 μM) and unpulsed P815 targets cells. Results from one representative mouse per group are shown. Comparison of SFU and IgG titers were performed using Mann-Whitney t test. Statistically significant P values less than .01 were found between HA-Tregs and each other group (**), and less than .05 between HA-Th and No cells (*). David-Alexandre Gross et al. Blood 2003;102:4326-4328 ©2003 by American Society of Hematology

HA-specific CD4+CD25+ cells allow transgene engraftment in transduced muscle. HA-specific CD4+CD25+ cells allow transgene engraftment in transduced muscle. BALB/c mice were injected intramuscularly at day 0 with AAV-HA, and were either untreated (A,D) or injected intravenously at days 0 and 4 with 106 CD4+CD25+ cells from BALB/c mice (B,E) or CD4+CD25+ cells from TCR-HA mice (C,F). At days 14 (A-C) and 35 (D-F), muscles were frozen and HA expression was assayed by immunohistochemistry using horseradish peroxidase (HRP)/diaminobenzidine (DAB) staining in brown on sections counterstained with methyl green, which stains the nuclei of muscle and infiltrating lymphoid cells in blue/green. Images are representative of 3 mice per group. Original magnification × 40. Quantification of HA expression was assessed by computer-assisted image analysis (Histolab; Microvision Instruments, Evry, France). For each mouse in each condition, 5 to 10 entire transverse sections of tibialis anterior were measured. The total section and HA-positive areas were determined by image texture and HRP/DAB color analysis, respectively. The reproducibility of image analysis was controlled using normal muscle sections as a standard on each slide. Results are expressed as an index of HA-positive area over the total tibialis anterior area and represent the mean ± SEM of 3 mice per group, at days 14 (G) and 35 (H). David-Alexandre Gross et al. Blood 2003;102:4326-4328 ©2003 by American Society of Hematology