1. 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):
December 15, 2003
©2003 by American Society of Hematology

2. 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 HA 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 HA , 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 HA 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:
©2003 by American Society of Hematology

3. 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:
©2003 by American Society of Hematology