2004;80(6):445C450. adhesion molecules, which prevents allogeneic T cell responses in mixed lymphocyte reactions.20 Cell death pathways are GPR120 modulator 2 complex and diverse, and the manner in which cells die can impact how they are perceived by the immune system, with apoptotic cells generally tolerated, and necrotic cells associated with inflammation.21 Apoptosis is a coordinated process of GPR120 modulator 2 cell death that involves regulated morphological and biochemical cellular change.22 In live cells, anionic phosphatidylserine (PS) is distributed around the cytoplasmic surface of the cell membrane.22 One of the earliest indicators of apoptosis is the translocation of PS from the internal to the external leaflet of the plasma membrane.21,22 Committed apoptotic cells also undergo caspase activation, chromatin condensation, and loss of cell membrane asymmetry, among other cellular changes.21,22 UVB light induces apoptosis in many different mouse and human cell types by various mechanisms.23C30 Different wavelengths and doses of UV light have different effects, with lower doses generally inducing apoptosis and higher doses inducing necrosis.29 The cell death pathway induced also depends on the cell type and is related to the extent of DNA damage. This paper focuses on WBCs, as contaminating lymphocytes in transfusion products are the most potent inducers of anti-MHC alloimmunization.30C32 UVB light can induce apoptosis in lymphocytes,24,25,29,33 but as doses and wavelengths of UV light can vary considerably, it is unclear if these findings will translate to blood products treated with commercially available PRT systems. There is some evidence to suggest that the Mirasol system may induce apoptosis. Yang et al. described PS exposure in human lymphocytes treated with riboflavin and an in-house source of UVB light,34 and Asano et al. reported GPR120 modulator 2 elevated PS exposure in rat WBCs following Mirasol treatment.19 Neither of these studies, however, evaluated indicators of apoptosis beyond PS exposure. UV treated cells have been shown to induce tolerance in humans and animal models. Extracorporeal photochemotherapy, in which autologous blood is usually treated with UVA light and psoralen before reinfusion, has been shown to regulate inflammatory immune responses in autoimmune diseases and graft-versus-host disease.35C38 Multiple infusions of allogeneic PBMCs treated with UVB light (lower doses than used in UV+R), can control donor specific humoral responses, and tolerance in this model can be conferred to na?ve mice by adoptive transfer of CD4+ CD25+ T cells.39,40 Treatment of PRP with UV+R reduces alloimmunization and provides WBC-dependent partial protection from subsequent exposure.15,16 As treatment induces rapid WBC death20 and apoptotic cells are suggested to have immune tolerant properties,41 we sought to determine the manner of death induced by UV+R treatment in both human and mouse WBCs. PS exposure, membrane asymmetry, caspase activity and chromatin condensation were evaluated in WBCs isolated from pathogen reduced PRP and compared CD300C with WBCs treated with known apoptosis or necrosis inducers. The immunogenicity of these cells was also compared to apoptotic and necrotic controls as described below. Apoptosis WBCs from non-leukoreduced PRP with and without UV+R treatment were evaluated for indicators of apoptosis. Empirically, we observed that human WBCs were more resistant to induction of PS exposure GPR120 modulator 2 than mouse cells when treated with the same concentration of ethanol (EtOH). Therefore, to generate experimental controls, an aliquot was treated with 5% total volume ethanol (mice) or 10% total volume ethanol (human) for 90 minutes at 37C for the induction of apoptosis.42C44 A separate aliquot was treated with a 10% total volume ethanol (mice), 20% total volume ethanol (human), or a cycle of freeze/thaw in dry ice and placed in 37C incubation for the induction of cell necrosis.42,45 Cells were washed prior to analysis. PS exposure was measured by.