Abstract
The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness.Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells.The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.
Highlights
Solid organ transplantation has transformed the care of patients with end-stage diseases of the kidney, liver, heart, and lungs
Culture the cells that pass through the column for 6 days in RPMI 1640 or DMEM supplemented with 2 ng/mL recombinant mouse granulocyte macrophage colony-stimulating factor (GM-CSF) and 2 ng/mL of recombinant human transforming growth factor β1 (TGFβ1)
This protocol results in long-term allograft survival that is dependent on Foxp3+ regulatory T cells[36,37]
Summary
Solid organ transplantation has transformed the care of patients with end-stage diseases of the kidney, liver, heart, and lungs. We showed that imaging flow cytometry can produce a very comparable measure of alloreactive T-cell frequency[23] and that changes in synapse frequency within a polyclonal T-cell population are predictive of graft outcome[23]. This approach has been optimized to measure the direct alloreactivity of CD4+ T cells, but, in principle, it could be developed to examine CD8+ T cells and the indirect pathway. In the future, the overall approach may be useful for the functional evaluation of T-cell responses in transplant recipients before transplant; immediately after transplant; and in the long-term, when drug minimization becomes an important goal
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