No abstract

Since its introduction, the propidium iodide (PI) flow cytometric assay has been widely used for the evaluation of apoptosis in different experimental models. It is based on the principle that apoptotic cells, among other typical features, are characterized by DNA fragmentation and, consequently, loss of nuclear DNA content. Use of a fluorochrome, such as PI, that is capable of binding and labeling DNA makes it possible to obtain a rapid (the protocol can be completed in about 2 h) and precise evaluation of cellular DNA content by flow cytometric analysis, and subsequent identification of hypodiploid cells. The original protocol enhanced the capacity for a rapid, quantitative measure of cell apoptosis. For this reason, since its publication, the PI assay has been widely used, as demonstrated by the large number of citations of the original paper and/or the continuous use of the method in many laboratories.

By comparing mRNA species expressed in dexamethasone (DEX)-treated and untreated murine thymocytes, we have identified a gene, glucocorticoid-induced leucine zipper (GILZ), encoding a new member of the leucine zipper family. GILZ was found expressed in normal lymphocytes from thymus, spleen, and lymph nodes, whereas low or no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. In thymocytes and peripheral T cells, GILZ gene expression is induced by DEX. Furthermore, GILZ expression selectively protects T cells from apoptosis induced by treatment with anti-CD3 monoclonal antibody but not by treatment with other apoptotic stimuli. This antiapoptotic effect correlates with inhibition of Fas and Fas ligand expression. Thus, GILZ is a candidate transcription factor involved in the regulation of apoptosis of T cells.

GITR (glucocorticoid-induced TNFR family related gene) is a member of the TNFR superfamily (TNFRSF) that is expressed in different cell types, including T lymphocytes. Because of a high homology in its cytoplasmic region with other known costimulatory members of the TNFRSF, we investigated whether GITR played a costimulatory role in T lymphocyte subpopulations. Our results show that the proliferation response of CD8 + and CD4 + peripheral T cell subpopulations was potentiated when a GITR costimulus was added to an anti-CD3 stimulus. Furthermore, expression of the main activation-induced receptor (IL-2R § ) and production of IL-2 and IFN-+ were increased more with a GITR costimulus than with anti-CD3 alone. GITR stimulation also enhanced anti-CD3-induced ERK phosphorylation, suggesting that GITR is involved in MAPK-pathway activation. Interestingly, CD4 + CD25 + regulatory T cell (Treg cell) proliferation was triggered by the GITR costimulus; Treg cell proliferation was paralleled by the loss of the anergic phenotype and suppressor activity. Nevertheless, unstimulated GITR -/-CD4 + CD25 + and GITR +/+ CD4 + CD25 + cells were equally able to exert suppressor activity on CD4 + CD25 -responder cells. These results indicate a novel function for GITR as costimulatory molecule of T cell subsets.

By comparing untreated and dexamethasone-treated murine T cell hybridoma (3DO) cells by the differential display technique, we have cloned a new gene, GITR (glucocorticoid-induced tumor necrosis factor receptor family-related gene) encoding a new member of the tumor necrosis factor͞nerve growth factor receptor family. GITR is a 228-amino acids type I transmembrane protein characterized by three cysteine pseudorepeats in the extracellular domain and similar to CD27 and 4-1BB in the intracellular domain. GITR resulted to be expressed in normal T lymphocytes from thymus, spleen, and lymph nodes, although no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. Furthermore, GITR expression was induced in T lymphocytes upon activation by anti-CD3 mAb, Con A, or phorbol 12-myristate 13-acetate plus Caionophore treatment. The constitutive expression of a transfected GITR gene induced resistance to anti-CD3 mAbinduced apoptosis, whereas antisense GITR mRNA expression lead to increased sensitivity. The protection toward T cell receptor-induced apoptosis was specific, because other apoptotic signals (Fas triggering, dexamethasone treatment, or UV irradiation) were not modulated by GITR transfection. Thus, GITR is a new member of tumor necrosis factor͞nerve growth factor receptor family involved in the regulation of T cell receptor-mediated cell death.