Detection of alloreactive T cells by flow cytometry: a new test compared with limiting dilution assay

BACKGROUND: Frequencies of alloreactive T cells determined by limiting dilution assays (LDA) may not adequately reflect the donor-reactive immune status in transplant recipients. To reevaluate LDA frequencies, we developed a flow cytometry test for direct determination of alloreactive T-cell frequencies and compared these frequencies with classical LDA estimates of frequencies. METHODS: For determination of frequencies by flow cytometry, peripheral blood lymphocytes (or lymphocytes taken from primary mixed lymphocyte culture) were stimulated with either Epstein-Barr virus-transformed lymphoblastoid cell lines or T cell-depleted spleen cells and stained for intracellular interferon (IFN)-gamma production and CD69. In lung transplant recipients, frequencies of IFN+ alloreactive T cells were compared with LDA frequencies, that is, cytotoxic T lymphocyte precursors and helper T lymphocyte precursors. RESULTS: With flow cytometry, alloreactive T cells were detected after overnight allostimulation as IFN-gamma CD69bright cells (range, 0.1-0.58% and 0.1-0.66% of total CD4 and CD8 cells, respectively). Frequencies increased 25-fold or more when lymphocytes were prestimulated in primary mixed lymphocyte culture before testing. After lung transplantation, mean donor-specific IFN+ CD8 T-cell frequencies did not decrease as mean donor-specific LDA cytotoxic T lymphocyte precursor frequencies, whereas no difference was seen in pretransplantation samples or third-party-specific frequencies at both time points. Mean frequencies of IFN+ CD4 did not differ from helper T lymphocyte precursors at both time points, but frequencies did not correlate. CONCLUSIONS: The flow cytometry test allows a direct measurement of alloreactive T-cell frequencies and demonstrates a discrepancy between donor-specific IFN+ CD8 T-cell frequencies and LDA CLTp after transplantation. This may be a result of the existence of "functional diverse" alloreactive T cells or of activation-induced cell death of donor-reactive T cells during long (LDA) culturing, which is avoided in the flow cytometry test.