Simple conditioning with monospecific CD4+CD25+ regulatory T cells for bone marrow engraftment and tolerance to multiple gene products by David-Alexandre Gross, Pascal Chappert, Marylene Leboeuf, Virginie Monteilhet, Laetitia Van Wittenberghe, Olivier Danos, and Jean Davoust Blood Volume 108(6):1841-1848 September 15, 2006 ©2006 by American Society of Hematology

Antigen-specific in vivo expansion of CD4+CD25+ T cells from Marilyn mice. Antigen-specific in vivo expansion of CD4+CD25+ T cells from Marilyn mice. (A) FACS analysis of lymph node cells from female Marilyn mice labeled with anti-CD4, Vβ6, and CD25. (B) FoxP3 mRNA levels of purified CD25+ and CD25– cells from female B6 and Marilyn mice were determined by real-time PCR analysis on fresh splenocytes. (C-D) In vivo expansion of DBY-Tregs: B6- or DBY-Tregs labeled with 5 μM CFSE were transferred together with 10 × 106 female or male 45.1 splenocytes into recipient female 45.1 mice; n = 3 mice per group. At day 6, splenocytes were labeled with CD25, CD4, and CD45.2. Dot plots are gated on CD4+CD45.2+ cells. Statistical analysis was performed using the Mann-Whitney t test; *P ≤ .05. Panel A is representative of 3 experiments, and panels B-D are each representative of 2 experiments. Error bars in panels B and C show standard error of the mean. David-Alexandre Gross et al. Blood 2006;108:1841-1848 ©2006 by American Society of Hematology

Short-term engraftment of male BM and transient expansion of DBY-Tregs. Short-term engraftment of male BM and transient expansion of DBY-Tregs. (A-B) Short-term engraftment (day 28) of congenic male 45.1 BM (5 × 106 cells) transferred into B6 female 45.2 mice either untreated (-) or conditioned with one intravenous injection of a various number of DBY-Tregs or 1 × 106 B6-Tregs. Male B6 mice were engrafted as a positive control (CTRL). Percentages of CD45.1+ donor cells were analyzed in PBMCs. Results represent the mean of 3 to 6 mice per group ± standard error of the mean (SEM). (C) Transient expansion of DBY-Tregs. DBY-Tregs (1 × 105 cells) were transferred into B6 female mice with or without male BM (107 cells). At each time point, mice were killed and their splenocytes stained with CD45.1 and CD4. Graph represents the percentage of CD45.1+ cells in CD4+ 7-AAD– cells. Results represent the mean of 2 mice killed at each time point from groups of 8 mice per condition. Bars represent the 2 values of single-animal results. David-Alexandre Gross et al. Blood 2006;108:1841-1848 ©2006 by American Society of Hematology

Tolerance to male antigens occurs mainly through peripheral mechanisms. Tolerance to male antigens occurs mainly through peripheral mechanisms. (A) Congenic male 45.1 BM (15 × 106 cells) was transferred into intact (n = 5) or thymectomized female 45.2 B6 mice (n = 5) conditioned with single intravenous injections of 1 × 105 DBY-Tregs. (B) Male 45.2 BM from wild-type mice or from CD3null mice (15 × 106 cells) was transferred into congenic female 45.1 mice (n = 5 for each group) conditioned with single intravenous injections of 1 × 105 DBY-Tregs. In panels A and B, donor chimerism and percentage of T cells, expressed as a percentage of CD45.1+ or CD45.2+ cells, were analyzed in PBMCs at various time points after bone marrow transplantation (BMT). FACS stainings depicted at day 60 are shown. Panels A and B are each representative of 2 experiments. David-Alexandre Gross et al. Blood 2006;108:1841-1848 ©2006 by American Society of Hematology

Development of long-term, multilineage mixed chimerism. Development of long-term, multilineage mixed chimerism. (A) A total of 15 × 106 male 45.2 BM cells were transferred into congenic female 45.1 mice untreated (▴, n = 3) or conditioned with either 5 weekly intravenous injections of 2 × 105 to 5 × 105 DBY-Tregs (•, n = 5) or a single injection of 1 × 105 DBY-Tregs (○, n = 5). As a positive control, female 45.2 BM cells were transferred into congenic female 45.1 mice (X, n = 3). Donor chimerism expressed as a percentage of CD45.2+ cells was analyzed in PBMCs at various time points after BMT (A). Results represent the mean per group ± SEM. (B-C) Mice chimerized for more than 300 days (5 DBY-Treg injections) were killed, and cells from various organs were analyzed by FACS. (B) Splenocytes were stained with CD45.2-biotin/APC-streptavidin, PE-conjugated anti-CD8, anti-CD4, anti-B220, anti-CD11c, and anti–7-AAD. (C) Thymocytes were stained with CD4, CD3, CD45.2, and CD8 (no gate) or with CD3, CD45.2, and CD11c (FACS gated on CD3– 7-AAD– is shown). David-Alexandre Gross et al. Blood 2006;108:1841-1848 ©2006 by American Society of Hematology

Strong tolerance to male antigen in chimeric mice. Strong tolerance to male antigen in chimeric mice. (A-B) The absence of antimale CTL activity in chimeric mice. Male and female splenocytes labeled with 0.5 μM and 5 μM CFSE, respectively, were transferred into female mice, male mice, or chimeric female mice. PBMCs were labeled with PE anti-B220 and 7-AAD at various time points, and percentages of specific lysis of male over female splenocytes were calculated as detailed in “Materials and methods.” Results represent the mean of 2 mice per group assayed at 5 time points. Bars represent the 2 values of single-animal results. (C) The absence of antimale T-cell responses. Female, male, or chimeric mice were challenged subcutaneously with 50 μg UTY peptide emulsified in IFA. Splenocytes were tested on day 10 in a standard IFNγ ELISPOT assay against various doses of the UTY peptide. Results represent the mean of 3 mice per group ± SEM. David-Alexandre Gross et al. Blood 2006;108:1841-1848 ©2006 by American Society of Hematology

Hematopoietic chimerism does not impair immune response to third-party antigens. Hematopoietic chimerism does not impair immune response to third-party antigens. (A) Chimeric or naive female mice were challenged subcutaneously with 100 μg OVA protein emulsified in IFA. Splenocytes were tested at day 10 in a standard IFNγ ELISPOT assay against the OVA257 epitope. Serum was tested by ELISA for anti-OVA antibody. Results represent the mean of 3 mice per group ± SEM. (B-C) Susceptibility of male cells to immune cytolytic activity. Chimeric mice were challenged subcutaneously with OVA protein in IFA as in panel A and were infused at day 8 with male splenocytes (n = 2) or female splenocytes (n = 2) either pulsed with OVA257 (0.5 μM CFSE) or left unpulsed (5 μM CFSE). PBMCs were analyzed at day 0, 1, and 2. The percentage of specific lysis of pulsed over unpulsed cells was calculated as detailed in “Materials and methods,” similarly to Figure 5 with male over female cells. Panels B and C represent 1 of 2 similar results. David-Alexandre Gross et al. Blood 2006;108:1841-1848 ©2006 by American Society of Hematology

Secondary engraftment of tissues expressing EGFP transgene in the absence of DBY antigen. Secondary engraftment of tissues expressing EGFP transgene in the absence of DBY antigen. (A) A total of 7 × 106 male 45.2 BM cells from transgenic EGFP mice were transferred into 45.2 male hosts, 45.2 female hosts, or 45.2 female hosts conditioned with 105 DBY-Tregs. One representative FACS analysis of 3 experiments is shown (day 150). (B) Donor chimerism expressed as a percentage of EGFP-positive cells analyzed in PBMCs 5 months after EGFP BM transfer as in panel A. Results represent the mean of 3 mice per group ± SEM. (C-D) BM from EGFP × CD45.1 male or female mice was transferred into 45.2 EGFP chimeric or naive female mice. Representative FACS analysis (C) and percentage of EGFPhigh and CD45.1+ cells (D) in PBMCs at 5 months are shown. Results represent the mean of 3 mice per group ± SEM. Statistical analysis was performed using the Mann-Whitney t test; *P ≤ .05. (E) Long-term engraftment of EGFP female skin grafts in EGFP male-female chimeric mice (n = 8, ▪). In the controls, 5 of 6 chimeric mice lacking EGFP (♦) and 4 of 4 female B6 mice (▴) rejected the EGFP female skin graft between day 12 and day 16. The results presented are pooled from 2 independent experiments. David-Alexandre Gross et al. Blood 2006;108:1841-1848 ©2006 by American Society of Hematology