CyTOF PBMCs were treated as described for circulation cytometry. were much less damaged by the treatment. The selective and effective killing effect RO4987655 on the activated cells was also seen after co-cultivating activated and resting T cells. Under our clinically relevant experimental conditions, ALA-PDT killed activated T cells more selectively and efficiently than 8-MOP/UV-A. Monocyte-derived dendritic cells (DCs) were not affected by the treatment. Incubation of ALA-PDT damaged T cells with autologous DCs induced a downregulation of the co-stimulatory molecules CD80/CD86 and also upregulation of interleukin 10 (IL-10) and indoleamine 2,3-dioxygenase expression, two immunosuppressive factors that may account for the generation of tolerogenic DCs. Overall, the data support the potential use of ALA-PDT strategy for improving ECP by selective and effective killing of activated T cells and induction of immune tolerance. < 0.05. Since the efficacy of ALA-PDT modality largely depends upon the cellular ability to produce PpIX during ALA incubation, the amounts of ALA-induced PpIX in resting and activated PBMCs were measured. As shown in Physique 1D, the histogram for the PpIX production shifts towards the right (higher PpIX amount) in the activated T cells incubated with ALA when compared to that of resting cells with or without ALA incubation (Physique 1D). The activated T cells without ALA also showed a small increase in PpIX (Physique 1D). This may be explained by the fact that this proliferative cells may use endogenous ALA more effectively to produce and accumulate some PpIX after being activated with anti-CD3/CD28 antibodies. The effects of different T cell activation protocols around the ALA-induced PpIX production were also examined in PBMCs. As shown in Physique 1E, the various activation protocols led to a 5- to 60-fold increase in ALA-PpIX production in activated T cells compared to resting T cells. Activation with anti-CD3/CD28 antibodies induced significantly more PpIX (< within the same doses of ALA than that with PHA or CSC in the CD3+ T cells. Circulation cytometry has a technical challenge when the broad fluorescence peak from PpIX is usually measured in combination with multi-staining procedures. In contrast, the CyTOF mass cytometer enables analysis of the expression of a large number of proteins simultaneously by using antibodies coupled to stable heavy metal isotopes using the Time-of-flight Inductively Coupled Plasma Mass Spectrometry (TOF ICPMS) technology. With more than RO4987655 120 detection channels in the CyTOF mass cytometer, the maximum per-cell information can be obtained from a single sample without the need for compensation. To check if PpIX signals interfere with the measurements of other wavelengths during circulation cytometry analyses, cells from FANCG your same PBMC sample were analyzed by both circulation cytometry and CyTOF mass cytometry for comparison. The results from the CyTOF analysis were comparable to those obtained by circulation cytometry for the different T cell subsets (Table A1). 2.2. Effects of the Parameters on ALA-Induced PpIX Production No significant cytotoxicity was observed in the resting and anti-CD3/CD28 activated CD3+ T cells incubated with ALA at a dose of 3 or RO4987655 10 mM in the dark for 1 h (Physique A3). Physique 2A shows the effects of ALA concentrations (1, 3, and 10 mM) and incubation occasions (1, 4, and 24 h) around the PpIX production in the CD3+ T cells. Incubation with 1 mM ALA for 24 h induced the highest PpIX production. Generally, a lower ALA dose for a longer incubation time led to a higher PpIX production in the ranges of ALA concentrations and incubation occasions studied. However, to be clinically feasible for ALA-ECP, 1-h ALA incubation was tested in this study. Open in a separate window Physique 2 Effects of the parameters affecting ALA-induced PpIX production. Healthy donor PBMCs were activated in vitro with anti-CD3/CD28 antibodies for 3 days. (A) effects of different ALA incubation time intervals on PpIX production in resting and anti-CD3/CD28 activated CD3+ T cells; (B) the effect of cell density on ALA-induced PpIX production; (C) the effect of heat (RT and 37 C) on ALA-PpIX production in resting, anti-CD3/IL-2 activated and anti-CD3/CD28 activated PBMCs. The cell density also.