عزل الخلايا التائية المنظمة CD4(+)‎CD25(hi)‎ و تقييم وظيفتها الكابتة للمناعة في المختبر

الملخص EN

Background: The regulatory T cells subpopulation (Tregs) are professional suppressor lymphocytes that perform unique functions as modulators of almost all aspects of the immune response.

Isolation of Treg subpopulation in sufficient numbers and high purity is a precondition to gain insights into Treg cell suppressor mechanisms and exploring the possibility of manipulating their functionsboth positively or negatively in vitro and in vivo.

Objective: This study aimed at isolating highly purified CD4(+)CD25(high/bright) and evaluating their function measured by their suppressing the proliferation of activated CD4+CD25−and their production of Interleukin-2 (IL-2) in vitro.

Methods: CD4+ T cells were first isolated by negative selection.

This step was followed by Flow sorting of two subpopulation based on their expression levels of CD25.

In vitro suppression assay was performed using anti-CD3 and anti-CD28 beads as surrogate antigen presenting cells, and the suppression ability of CD4(+)CD25(high/bright) was determined via measuring their inhibition of responding cells (Tresp) proliferation and IL-2 production.

Results: We isolated polyclonal CD4(+)CD25(hi/bright) Tregs and effector helper T cells CD4(+)CD25(-) from peripheral blood of healthy subjects at purities exceeded 99%.

In vitro suppression assay clearly revealed that CD4(+)CD25(hi/bright) Tregs potently suppressed both the proliferation of activated CD4+CD25− and production of IL-2.

The observed suppression was dependent on numbers of Tregs added to the assay.

Conclusion: The protocol adapted in this study can be used to obtain highly-pure Tregs and effector T lymphocyte subpopulations needed to perform the in vitro suppression assay from the same donor/patient, and evade contamination with activated effector T cells CD4(+)CD25(lo).