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.

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