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P052 Allograft inflammatory factor-1 (AIF-1) stimulates Th1 differentiation of human T-cells and protects them from apoptosis

D. Cano-Martínez*1, J. Monserrat2, B. Hernández-Breijo1, P. Sanmartín-Salinas1, I.D. Román1, M.D. Fernández-Moreno1, S. Marsal3, J.P. Gisbert4, L. Guijarro1

1Universidad de Alcalá and CIBERehd, Systems Biology, Alcalá de Henares, Spain, 2Universidad de Alcalá, Medicine and Medical Specialties, Alcalá de Henares, Spain, 3Hospital Universitari Vall d'Hebron, Gastroenterology Unit, Barcelona, Spain, 4Hospital Universitario de La Princesa, IIS-IP and CIBERehd, Gastroenterology Unit, Madrid, Spain

Background

In previous studies, we have demonstrated that AIF-1 is predominantly found in naïve CD4+ T cells. Activation with anti-CD3/CD28-coated magnetic beads of T-cells stimulated the AIF-1 expression and its secretion to extracellular medium (Cano-Martínez et al. Gastroenterology 2014. 146(5): s822).

The aim of this study was to determinate AIF-1 effect on differentiation of human PBMC (peripheral blood mononuclear cells) stimulated with anti-CD3/CD28-coated magnetic beads. Furthermore, the effect of AIF-1 on proliferation and apoptosis was evaluated in human PBMC.

Methods

Blood of healthy volunteers was used after the application of informed consent. T-cell differentiation was studied by qRT-PCR of Il-2 (interleukin-2), IFN- ϒ (interferon ϒ ), IL-4 (interleukin 4), IL-17 (interleukin 17), IL-10 (interleukin 10) and TGF β -1 (transforming growth factor-beta) of human PBMC in vitro. Four conditions were compared: a) basal conditions (incubation of PBMC during 24h in presence of FBS); b) clonal T-cell expansion (addition of anti-CD3/CD28-coated magnetic beads); c and d) Th1 differentiation (addition of anti-CD3/CD28-coated magnetic beads, IL-12 and anti-IL-4) in presence or absence of AIF-1 (6 nM). Proliferation of human PBMC was evaluated by cell counting and by propidium iodide-flow cytometry. Regulation of cell cycle was analyzed and Western blot analysis of Rb, pRb, E2-F and STAT-1 and pSTAT-1. Apoptosis of the different cell populations was studied by multiparametric flow cytometry using Annexin-V and by Western blot of caspase-3.

Results

After incubation of human PBMC during 24h with anti-CD3/CD28-coated beads, we observed an increased expression of IL-2 (p<0,001) without changes in CD25, the alpha chain of the IL-2 receptor; these data were in accordance with the increase in the number of cells and with the phosphorylation of Rb and STAT-1. The clonal expansion seems to affect the cells producing IFN- ϒ (Th1 response), IL-4 (Th2 response) and IL-10 (Treg response) (p<0,001). Not changes were observed in the levels of IL-17 (Th17 response). After incubation of PBMC with anti-CD3/CD28-coated beads, IL-12 and anti-IL-4 (Th1 differentiation), we observed an increase in IFN- ϒ (p<0,001) without changes in the other cytokines previously studied. The presence of AIF-1 in the medium during Th1 differentiation increased the levels of IL-2 and IFN- ϒ (p<0,001), but not IL-4, Il-17, Il-10 and TGF- β mRNA levels. Moreover, AIF-1 protected CD4+ T cells from the apoptosis observed during Th1 differentiation

Conclusion

Our Results demonstrate that exogenous AIF-1 amplified Th1 differentiation induced in vitro by increasing IL-2 and IFN- ϒ*** and by protecting CD4+ T from apoptosis