1. Immunomodulatory effect of 5-azacytidine (5-azaC): potential role in the transplantation setting
by Luis I. Sánchez-Abarca, Silvia Gutierrez-Cosio, Carlos Santamaría, Teresa Caballero-Velazquez, Belen Blanco, Carmen Herrero-Sánchez, Juan L. García, Soraya Carrancio, Pilar Hernández-Campo, Francisco J. González, Teresa Flores, Laura Ciudad, Esteban Ballestar, Consuelo del Cañizo, Jesus F. San Miguel, and Jose A. Pérez-Simon
Blood
Volume 115(1):
January 7, 2010
©2010 by American Society of Hematology

2. Effect of 5-azaC on the activation pattern of T cells.
Effect of 5-azaC on the activation pattern of T cells. (A) Dot plot showing the forward scatter (FSC) and side scatter (SSC) of resting versus anti-CD3 plus anti-CD28 stimulated T cells. The addition of 5-azaC significantly inhibited the activation of T cells as shown by a lower FSC/SSC among stimulated 5-azaC–treated T cells as well as a lower number of T cells expressing CD25 and intracellular IFN-γ and CD40L. A representative case of 9 experiments is shown. (B) Box-plot showing the dose-dependent effect of 5-azaC on the different activation parameters analyzed. (C) Supernatants CBA cytokine assays after 4 days of culture. A representative case of 5 cases analyzed is shown.
Luis I. Sánchez-Abarca et al. Blood 2010;115:
©2010 by American Society of Hematology

3. T-cell proliferation and cell-cycle analyses.
T-cell proliferation and cell-cycle analyses. (A) PKH staining showing the number of duplications of T cells under stimulation with either phytohemagglutinin A or anti-CD3 plus anti-CD28. A representative case of 8 cases is shown. (B) Box plot showing the dose response effect of 5-azaC on T-cell proliferation. (C) Cell-cycle analysis in stimulated T cells in the absence (i) or presence (ii) of 5-azaC at 1μM (P = .01). A representative case of 8 cases is shown.
Luis I. Sánchez-Abarca et al. Blood 2010;115:
©2010 by American Society of Hematology

4. T-cell viability assays.
T-cell viability assays. (A) Dot plot showing T-cell viability as assessed by Annexin V and 7-AAD staining among unstimulated or stimulated T cells in the absence or the presence of 1μM of the drug. A representative experiment of 8 is shown. (B) Box plot representation showing the effect of 5-azaC on T-cell viability. Significant differences were only observed at a dose of 10μM of the drug.
Luis I. Sánchez-Abarca et al. Blood 2010;115:
©2010 by American Society of Hematology

5. Effect of 5-azaC on the expression of genes involved in regulation of immune response and cell cycle.
Effect of 5-azaC on the expression of genes involved in regulation of immune response and cell cycle. (A) RNA expression of FOXP3, FOXO3a, IFN-γ, and IL-10 as evaluated by quantitative PCR. Exposure to 5-azaC significantly increased the expression of FOXP3 and FOXO3a and decreased the expression of IFN-γ and IL-10 at different time-points during the T-cell culture. (B) RNA expression of p16, p27, p53, and p73; a significant increase in the expression of all these genes was observed at different time points after exposure to the drug (n = 4 cases analyzed).
Luis I. Sánchez-Abarca et al. Blood 2010;115:
©2010 by American Society of Hematology

6. Luis I. Sánchez-Abarca et al. Blood 2010;115:107-121
©2010 by American Society of Hematology

7. Luis I. Sánchez-Abarca et al. Blood 2010;115:107-121
©2010 by American Society of Hematology

8. Luis I. Sánchez-Abarca et al. Blood 2010;115:107-121
©2010 by American Society of Hematology

9. Luis I. Sánchez-Abarca et al. Blood 2010;115:107-121
©2010 by American Society of Hematology

10. Effect of 5-azaC on the number of regulatory T cells in peripheral blood and spleen of mice after transplantation.
Effect of 5-azaC on the number of regulatory T cells in peripheral blood and spleen of mice after transplantation. Regulatory T cells in peripheral blood and spleen of (A) C57BL/6 mice and (B) Balb/c mice transplanted with C57BL/6 BM plus splenocytes and 5-azaC 60 and 84 hours after transplantation and on days 19, 21, and 23 after transplantation. Dot plot shows 1 representative case of 4 analyzed.
Luis I. Sánchez-Abarca et al. Blood 2010;115:
©2010 by American Society of Hematology