Detailed Kinetics of the Direct Allo-Response in Human Liver Transplant Recipients: New Insights from an Optimized Assay

Abstract

Conventional assays for quantification of allo-reactive T-cell precursor frequencies (PF) are relatively insensitive. We present a robust assay for quantification of PF of T-cells with direct donor-specificity, and establish the kinetics of circulating donor-specific T cells after liver transplantation (LTx). B cells from donor splenocytes were differentiated into professional antigen-presenting cells by CD40-engagement (CD40-B cells). CFSE-labelled PBMC from LTx-recipients obtained before and at several time points after LTx, were stimulated with donor-derived or 3rd party CD40-B cells. PF of donor-specific T cells were calculated from CFSE-dilution patterns, and intracellular IFN-γ was determined after re-stimulation with CD40-B cells. Compared to splenocytes, stimulations with CD40-B cells resulted in 3 to 5-fold higher responding T-cell PF. Memory and naïve T-cell subsets responded equally to allogeneic CD40-B cell stimulation. Donor-specific CD4+ and CD8+ T-cell PF ranged from 0.5 to 19% (median: 5.2%). One week after LTx, PF of circulating donor-specific CD4+ and CD8+ T cells increased significantly, while only a minor increase in numbers of T cells reacting to 3rd party allo-antigens was observed. One year after LTx numbers of CD4+ and CD8+ T cells reacting to donor antigens, as well as those reacting to 3rd party allo-antigens, were slightly lower compared to pre-transplant values. Moreover, CD4+ and CD8+ T cells responding to donor-derived, as well as those reacting to 3rd party CD40-B cells, produced less IFN-γ. In conclusion, our alternative approach enables detection of allo-reactive human T cells at high frequencies, and after application we conclude that donor-specific T-cell PF increase immediately after LTx. However, no evidence for a specific loss of circulating T-cells recognizing donor allo-antigens via the direct pathway up to 1 year after LTx was obtained, underscoring the relative insensitiveness of previous assays.

Cytomegalovirus-Induced Expression of CD244 after Liver Transplantation Is Associated with CD8+ T Cell Hyporesponsiveness to Alloantigen

“…Cryopreserved PBMCs collected before LTx (pre-LTx), which were available in our bio bank, were used for baseline measurements. Cryopreserved splenocytes, isolated according to standard procedures (27) from splenic tissue of liver transplant donors, also were available in our bio bank. CD40-activated B cells were expanded from donor splenocytes, as described previously (27), and used as stimulator cells in allogeneic T cell-stimulation assays.…”

“…Third-party-derived CD40-activated B cells were expanded from splenocytes of an individual having the same number of HLA mismatches with the patient as the number of mismatches between patient and donor, but completely mismatched with the donor on HLA-A, HLA-B, and HLA-DR (27 (27). In addition, to determine responses to polyclonal stimulation, PBMCs were stimulated with PHA (5 mg/ml; Murex, Paris, France).…”

“…We used FACSDiva software (BD Biosciences) for analysis of phenotypic markers. Precursor frequencies (PFs), which are the proportions of the cells that respond to the stimulus, of alloreactive CD4 + and CD8 + T cells were calculated using ModFit LT software (Verity Software House), as previously described (27). Average PFs were calculated from duplicate assays.…”

Monocyte‐derived dendritic cells can induce autoreactive CD4+ T cells showing myeloid lineage directed reactivity in healthy individuals

“…For each sample, the absolute numbers of specific cell populations in the quantitative analyses were calculated with the following formula: ((numbers of beads added/numbers of beads acquired) × numbers of cells acquired). The frequency of autoDC-reactive T cells was calculated using the proliferation wizard in the FlowJo software following the approach described by Tapirdamaz et al [40]. Serum-free cell culture in X-VIVO15 medium (Lonza) was used in specific experiments to exclude reactivities against serum antigens.…”

Activation-induced CD137 is a fast assay for identification and multi-parameter flow cytometric analysis of alloreactive T cells

“…The disadvantages of these assays include its high labour-intensive character and complexity with respect to data analysis. The newer techniques have characterized T cell responses at the single-cell level by enzyme-linked immunospot (ELISPOT) [5,6] assay, carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled T cell flow cytometric analysis of proliferation [7,8] or cytokine flow cytometry [9,10]. ELISPOT has been used successfully to identify alloreactive cytokine-producing T cells with a lower limit of detection of 1:10·000 in the peripheral blood monuclear cell (PBMC) population [11].…”