Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleosidebased DNA methyltransferase inhibitor that induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in the G 0 to G 1 phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor-␣ and interferon-␥. This effect was not attributable to a proapoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes that induce cell-growth arrest, such as DCUN1D2, U2AF2, GADD45B, or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, overexpression of FOXP3, and expansion of regulatory T cells. Finally, the administration of 5-azaC after transplantation prevented the development of graft-versushost disease, leading to a significant increase in survival in a fully mismatched bone marrow transplantation mouse model. In conclusion, the current study shows the effect of 5-azaC in T lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways that must be explored to prevent graft-versus-host disease. (Blood.

The online version of this article contains a supplementary appendix. BackgroundIn vitro depletion of alloreactive T cells using the proteasome inhibitor bortezomib is a promising approach to prevent graft-versus-host disease after allogeneic stem cell transplantation. We have previously described the ability of bortezomib to selectively eliminate alloreactive T cells in a mixed leukocyte culture, preserving non-activated T cells. Due to the role of regulatory T cells in the control of graft versus host disease, in the current manuscript we have analyzed the effect of bortezomib in regulatory T cells. Design and Methods Conventional or regulatory CD4+ T cells were isolated with immunomagnetic microbeads based on the expression of CD4 and CD25. The effect of bortezomib on T-cell viability was analyzed by flow cytometry using 7-amino-actinomycin D staining. To investigate the possibility of obtaining an enriched regulatory T-cell population in vitro with the use of bortezomib, CD4 + T cells were cultured during four weeks in the presence of anti-CD3 and anti-CD28 antibodies, IL-2 and bortezomib. The phenotype of these long-term cultured cells was studied, analyzing the expression of CD25, CD127 and FOXP3 by flow cytometry, and mRNA levels were determined by RT-PCR. Their suppressive capacity was assessed in co-culture experiments, analyzing proliferation and IFN-γ and CD40L expression of stimulated responder T cells by flow cytometry. ResultsWe observed that naturally occurring CD4 + CD25+ regulatory T cells are resistant to the pro-apoptotic effect of bortezomib. Furthermore, we found that long-term culture of CD4 + T cells in the presence of bortezomib promotes the emergence of a regulatory T-cell population that significantly inhibits proliferation, IFN-γ production and CD40L expression among stimulated effector T cells. ConclusionsThese results reinforce the proposal of using bortezomib in the prevention of graft versus host disease and, moreover, in the generation of regulatory T-cell populations, that could be used in the treatment of multiple T-cell mediated diseases.Key words: bortezomib, regulatory T cells, graft-versus-host disease, prevention.Citation: Blanco B, Pérez-Simón JA, Sánchez-Abarca LI, Caballero-Velazquez T, Gutierrez-Cossío S, Hernández-Campo P, Díez-Campelo M, Herrero-Sanchez C, Rodriguez-Serrano C, Santamaría C, Sánchez-Guijo FM, del Cañizo C, and San Miguel JF. Treatment T-cell population. Haematologica 2009;94:975-983. doi:10.3324/haematol.2008 This is an open-access paper. with bortezomib of human CD4 + T cells preserves natural regulatory T cells and allows the emergence of a distinct suppressor

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Graft-versus-host disease (GVHD) is the major cause of morbidity and mortality among patients undergoing allogeneic hematopoietic stem cell transplantation. Although animal models have been clearly established for the study of skin, liver, and gut, currently there is no equivalent experiemental model for analyzing ocular involvement, which is rather common, especially among patients diagnosed with chronic GVHD. In the current study we have developed a murine model of ocular GVHD and, for the first time, we describe the histopathologic features involving cornea and limbus, which could play a role in the physiopathology of the disease at the ocular level. Our results represent a major finding that allows us to define a model for evaluating new therapeutic strategies for treating ocular GVHD prior to their use in clinical setting.

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BackgroundChronic graft-versus-host disease (cGVHD) is a major complication after allogeneic stem cell transplantation with an adverse effect on both mortality and morbidity. In 2005, the National Institute of Health proposed new criteria for diagnosis and classification of chronic graft-versus-host disease for clinical trials. New sub-categories were recognized such as late onset acute graft-versus-host disease and overlap syndrome. Design and MethodsWe evaluated the prognostic impact of the new sub-categories as well as the clinical scoring system proposed by the National Institute of Health in a retrospective, multicenter study of 820 patients undergoing allogeneic stem cell transplantation between 2000 and 2006 at 3 different institutions. Patients were retrospectively categorized according to the National Institute of Health criteria from patients' medical histories. ResultsAs far as the new sub-categories are concerned, in univariate analysis diagnosis of overlap syndrome adversely affected the outcome. Also, the number of organs involved for a cut-off value of 4 significantly influenced both cGVHD related mortality and survival. In multivariate analysis, in addition to NIH score, platelet count and performance score at the time of cGVHD diagnosis, plus gut involvement, significantly influenced outcome. These 3 variables allowed us to develop a simple score system which identifies 4 subgroups of patients with 84%, 64%, 43% and 0% overall survival at five years after cGVHD diagnosis (score 0: HR=15.96 (95% CI: 6.85-37.17), P<0.001; score 1: HR=5.47 (95% CI: 2.6-11.5), P<0.001; score 2: HR=2.8 (95% CI: 1.32-5.93), P=0.007). ConclusionsIn summary, we have identified a powerful and simple tool to discriminate different subgroups of patients in terms of chronic graft-versus-host disease related mortality and survival.Key words: cGVHD, prognostic factors, NIH classification, overlap syndrome, delayed acute GVHD.Citation: Pérez-Simón JA, Afram G, Martino R, Piñana JL, Caballero-Velazquez T, Ringden O, Valcarcel D, Caballero D, Remberger M, de Paz Y, Sierra J, San Miguel J, and Hagglund H. Evaluation of prognostic factors among patients with chronic graft-versus-host disease. Haematologica 2012;97(8):1187-1195. doi:10.3324/haematol.2011 This is an open-access paper. Evaluation of prognostic factors among patients with chronic graft-versus-host disease ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n

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