Assays for Alloreactive Responses by PCR

Abstract

In organ transplantation, major efforts are currently developed to minimize or even to withdraw immunosuppressive drugs. Different protocols have therefore been proposed to induce graft tolerance, that is, the survival of an allograft in the absence of continuing immunosuppression. They generally involve pre-transplant conditioning followed by hematopoietic stem cell infusion. Follow-up of immune status is mandatory in this kind of protocol, in order to investigate tolerance and consequently to reduce or withdraw immunosuppression without graft rejection. However, assessment of the alloreactive response using currently available techniques is labor-intensive and requires significant volume of patient's blood. The implementation of such tolerance protocols is thus difficult in routine practice. In this chapter, we describe a novel real-time polymerase chain reaction method based on interferon-gamma and interleukin-2 mRNAs quantification that requires only 10 ml of blood. Moreover, results can be obtained within 48 h. A modified mixed lymphocyte reaction (MLR) using whole blood T lymphocytes as responsive cells and CD3-depleted peripheral blood mononuclear cells (PBMCs) as stimulators is described. An alternative, that is, the use of PBMC as responding cells instead of whole blood, is also depicted. We successfully applied the technique in the monitoring of anti-donor reactivity in living donor liver graft recipients. We suggest that this rapid MLR assay could be valuable for the monitoring of patients undergoing solid organ or hematopoietic stem cell transplantation.

Whole blood assessment of antigen specific cellular immune response by real time quantitative PCR: a versatile monitoring and discovery tool

“…Indeed, similar techniques have already been used to monitor responsiveness to bacterial products, and, in particular, to LPS [ 14 , 24 ]. Furthermore, allospecific immune responses have also been assessed by qRT-PCR in whole blood [ 15 , 16 ] and responsiveness to allergen stimulation has been explored by testing IL-4 gene expression in whole blood [ 13 ], predominantly attributed to circulating basophils.…”