Kinetics Of Cell Activation Research Articles

Cytometry‐acquired calcium‐flux data analysis in activated lymphocytes

Flow cytometry enables the sequential determination of calcium levels in millions of stimulated lymphocytes over a short period of time. Current algorithms available are not suitable for the statistical analysis of this large amount of data. The authors aimed to develop a robust algorithm that fits a function to median values of measured data and provides an opportunity for statistical comparison between different calcium-flux measurements. The alteration of calcium signal was monitored in CD4+ cells loaded with calcium binding fluorescent dyes and stimulated with phytohemagglutinin; the alteration of calcium signal was monitored for 10 minutes. The authors also reanalyzed published calcium-flux data of CD3+ cells and Jurkat cells stimulated with different concentrations of anti-CD3 and thapsigargin. The authors fitted different functions to the medians of data per time unit and identified hormesis function as the best fitting one. On the basis of the optimally fitting function, the authors calculated the most relevant biological descriptors such as starting value, peak, time to reach the maximum, and time to reach 50% of maximum before and after the peak. Statistically significant differences in cell activation kinetics at different stimulatory concentrations were also demonstrated. This approach enables us to characterize the kinetics and distribution of calcium-flux data derived by flow cytometry and may be a reliable tool for the characterization of lymphocyte activation (for details see: http://calciumflux.intralab.eu).

Human T lymphocyte activation kinetics for identifying and targeting alloreactive T cells

Abstract Selective depletion of alloreactive T cells from a stem cell graft has the potential of reducing graft versus host disease (GvHD) while preserving graft versus malignancy and third party responses. For this purpose several techniques generate and deplete alloreactive cells, which are donor-derived T cells activated by recipient tissue. The kinetics of T cell activation in donor-recipient co-culture systems is critical in optimizing the timing of depletion of alloreactive T cells. We present the T cell activation kinetics in our preclinical system. Peripheral blood mononuclear cells (PBMCs) were derived from several pairs of unrelated healthy human volunteers. 2500 cGy irradiated cells (stimulators) were co-cultured with PBMCs (responders) in a 1:1 ratio and a concentration of 5 x 106/ml in serum free medium. Stimulator cells were labeled with PKH67 and the co-cultures were, analyzed for CD3, CD4, and CD25, expression by flow cytometry on days 0 through 7, using Topro-3 to exclude dead cells. We also added low dose Interleukin-2 (IL2) for augmenting the generation of alloreactive cells, to see if this made a difference. Our results show that alloreactive CD8 cells (CD3+, CD4−, CD25+) increased from ≤1 % on day 0, to 6.5 percent by day 5; the addition of IL2 amplified this to nearly 10% by day 5. CD4 + activated T cells (CD3+, CD4+, CD25+dim or total) did not appreciably increase over time and ranged between 2 to 5%; the addition of IL2 did not have any effect. T regulatory cells (CD3+, CD4+, CD25+bright) increased from ≤1 percent at baseline to 5% by day 5 and these were unaffected by the addition of IL2. The proportion of non-activated T lymphocytes, decreased with time of co-culture progression. Our results show that T lymphocyte activation, defined by CD25 expression, progressively increases through the first week of co-culture. This important observation will help in establishing the timing of allograft manipulation, for the selective depletion of alloreactive T cells in clinical hematopoietic stem cell transplantation. Download : Download high-res image (85KB) Download : Download full-size image

Human T Lymphocyte Activation Kinetics for Identifying and Targeting Alloreactive T Cells.

Selective depletion of alloreactive T cells from a stem cell graft has the potential of reducing graft versus host disease (GvHD) while preserving graft versus malignancy and third party responses. For this purpose several techniques generate and deplete alloreactive cells, which are donor-derived T cells activated by recipient tissue. The kinetics of T cell activation in donor-recipient co-culture systems is critical in optimizing the timing of depletion of alloreactive T cells. We present the T cell activation kinetics in our preclinical system. Peripheral blood mononuclear cells (PBMCs) were derived from several pairs of unrelated healthy human volunteers. 2500 cGy irradiated cells (stimulators) were co-cultured with PBMCs (responders) in a 1:1 ratio and a concentration of 5 x 106/ml in serum free medium. Stimulator cells were labeled with PKH67 and the co-cultures were, analyzed for CD3, CD4, and CD25, expression by flow cytometry on days 0 through 7, using Topro-3 to exclude dead cells. We also added low dose Interleukin-2 (IL2) for augmenting the generation of alloreactive cells, to see if this made a difference. Our results show that alloreactive CD8 cells (CD3+, CD4−, CD25+) increased from ≤1 % on day 0, to 6.5 percent by day 5; the addition of IL2 amplified this to nearly 10% by day 5. CD4 + activated T cells (CD3+, CD4+, CD25+dim or total) did not appreciably increase over time and ranged between 2 to 5%; the addition of IL2 did not have any effect. T regulatory cells (CD3+, CD4+, CD25+bright) increased from ≤1 percent at baseline to 5% by day 5 and these were unaffected by the addition of IL2. The proportion of non-activated T lymphocytes, decreased with time of co-culture progression. Our results show that T lymphocyte activation, defined by CD25 expression, progressively increases through the first week of co-culture. This important observation will help in establishing the timing of allograft manipulation, for the selective depletion of alloreactive T cells in clinical hematopoietic stem cell transplantation. [Display omitted]

Differentiation of immune cells challenged by bacteria in the common European starfish, Asterias rubens (Echinodermata)

Amoebocytes are the main effector cells of the echinoderm immune system. In starfishes, a taxon in which bacterial diseases have been rarely reported, amoebocytes are considered to be the only circulating and immune cell type. The present paper addresses the question of amoebocyte differentiation in the starfish Asterias rubens when challenged by bacteria. Starfishes were injected with FITC-coupled bacteria (Micrococcus luteus). Amoebocytes were collected at regular time intervals for 24 h. The cytometric characteristics and the phagocytic activity were studied by flow cytometry. Three amoebocyte groups of different size were identified. The cell concentrations of the two largest and more numerous of these groups (G2 and G3) were modulated by immune stimulation while the group of smallest, less numerous, cells (G1) was unaffected. All of these cell groups were phagocytic but their kinetics of cell activation and bacteria ingestion differed. G1 cells showed the lowest phagocytic activity while G3 cells had the highest and fastest phagocytic activity. Starfish amoebocytes appear to be segregated in three groups, two of them (G2 and G3) being immunomodulated and one of them presenting a very fast reaction to bacteria. It is suggested that the high efficiency of the immune system in starfishes is related to this fast reaction.

Mouse T-lymphocyte activation by Urtica dioica agglutinin : I. — Delineation of two lymphocyte subsets

Urtica dioica agglutinin (UDA) is a mouse T-lymphocyte-specific mitogen endowed with proliferative characteristics different from ConA, the prototypic T-lymphocyte mitogen. In particular, UDA induces 2-3-fold-reduced thymidine incorporation as compared to ConA. In an attempt to define the basis of this reduced proliferation, we analysed whether UDA binds to a unique subset of T lymphocytes or whether it activates only a T-cell subset. Cytofluorimetric analysis showed that this lectin binds uniformly to all T lymphocytes and does not, on this criterion, distinguish a particular T-cell subset. We next analysed whether UDA provokes the activation of all T lymphocytes. This was carried out by measuring the increase in cell size and the induction of the p55 chain of the IL2 receptor. The analysis showed that, throughout the kinetics of cell activation, only one subset of T lymphocytes increased in size and expressed the p55 chain of the IL2 receptor, suggesting that UDA activates only a subpopulation of T cells. This conclusion was strengthened by the analysis of 5-bromo-2-deoxyuridine (BrdU) incorporation into the DNA of UDA-activated cells. Two populations were easily identifiable: a BrdU-negative subset consisting of all the small p55-negative lymphocytes, and a BrdU-labelled subset including all the large p55-positive cells. BrdU was incorporated in both CD4+ and CD8+ cells, indicating that UDA did not distinguish helper from cytotoxic T lymphocytes. In addition to the p55 chain of the IL2R, all cycling cells expressed the Pgp-1 activation marker. The T lymphocytes, which bound UDA but did not proliferate, remained fully susceptible to subsequent stimulation by ConA. In conclusion, the capacity to proliferate upon UDA binding differentiates a UDA-sensitive from a UDA-refractory subset among splenic mouse T lymphocytes.