Immunological Monitoring of Renal Transplant Recipients to Predict Acute Allograft Rejection Following the Discontinuation of Tacrolimus

Contains fulltext : 69863.pdf (publisher's version ) (Open Access)BACKGROUND: Transplant patients would benefit from reduction of immunosuppression providing that graft rejection is prevented. We have evaluated a number of immunological markers in blood of patients in whom tacrolimus was withdrawn after renal transplantation. The alloreactive precursor frequency of CD4+ and CD8+ T cells, the frequency of T cell subsets and the functional capacity of CD4+CD25+FoxP3+ regulatory T cells (Treg) were analyzed before transplantation and before tacrolimus reduction. In a case-control design, the results were compared between patients with (n = 15) and without (n = 28) acute rejection after tacrolimus withdrawal. PRINCIPAL FINDINGS: Prior to tacrolimus reduction, the ratio between memory CD8+ T cells and Treg was higher in rejectors compared to non-rejectors. Rejectors also had a higher ratio between memory CD4+ T cells and Treg, and ratios <20 were only observed in non-rejectors. Between the time of transplantation and the start of tacrolimus withdrawal, an increase in naive T cell frequencies and a reciprocal decrease of effector T cell percentages was observed in rejectors. The proportion of Treg within the CD4+ T cells decreased after transplantation, but anti-donor regulatory capacity of Treg remained unaltered in rejectors and non-rejectors. CONCLUSIONS: Immunological monitoring revealed an association between acute rejection following the withdrawal of tacrolimus and 1) the ratio of memory T cells and Treg prior to the start of tacrolimus reduction, and 2) changes in the distribution of naive, effector and memory T cells over time. Combination of these two biomarkers allowed highly specific identification of patients in whom immunosuppression could be safely reduced

The presence of donor-specific human leukocyte antigen antibodies does not preclude successful withdrawal of tacrolimus in stable renal transplant recipients.

Item does not contain fulltextBACKGROUND: Because of the adverse events associated with the administration of immunosuppressive drugs, reduction of immunosuppression after solid-organ transplantation is highly desirable provided that graft rejection is prevented. In our transplant center, we used an immunosuppression reduction regimen in stable renal transplant patients. The presence of human leukocyte antigen (HLA) antibodies has been negatively associated with transplant outcome. Therefore, we evaluated the impact of HLA antibodies on the occurrence of acute rejection after immunosuppression reduction. METHODS: The presence and antigen specificity of HLA immunoglobulin G antibodies in serum samples were detected using enzyme-linked immunosorbent assay and single-antigen bead assays. Donor-specific cytotoxic potential was tested by standard CDC cross-match analysis. RESULTS: The presence of donor-specific or total HLA antibodies was not predictive for the occurrence of acute rejection after the reduction of immunosuppression. In addition, the presence of HLA antibodies did not preclude successful reduction of immunosuppression. After reduction of immunosuppression, newly formed HLA antibodies were seldom detected. Interestingly, evaluation of the cytotoxic potential of the detected HLA antibodies revealed that the one patient who developed donor-specific HLA antibodies and experienced a subsequent rejection episode was the only patient who carried cytotoxic HLA antibodies. This finding fueled the notion that the functional capacity, rather than the mere presence of donor-specific HLA antibodies, is indicative for transplant outcome. CONCLUSION: The presence of HLA antibodies does not preclude the successful reduction of immunosuppression in renal transplant patients with stable graft function

Differential cell viability of chondrocytes and progenitor cells in tissue-engineered constructs following implantation into osteochondral defects

Animal studies in cartilage tissue engineering usually include the transfer of cultured cells into chondral or osteochondral defects. Immediately at implantation, the cells are exposed to a dramatically changed environment. The aim of this study was to determine the viability of two cell types currently considered for cellular therapies of cartilage defects-chondrocytes and progenitor cells-shortiy after exposure to an osteochondral defect in rabbit knees. To that end, autogenic chondrocytes and periosteal cells were labeled with CM-DiI fluorochrome, seeded or cultured in PEGT/PBT scaffolds for periods up to 2 weeks, transferred into osteochondral defects, harvested 5 days postimplantation, and analyzed for cell viability. In order to further elucidate factors effecting cell viability within our model system, we investigated the effect of serum, 2) extracellular matrix surrounding implanted cells, 3) scaffold interconnectivity, and 4) hyaluronan, as a known cell protectant. Controls included scaffolds with devitalized cells and scaffolds analyzed at implantation. We found that the viability of periosteum cells (14%), but not of chondrocytes (65-95%), was significantly decreased after implantation. The addition of hyaluronan increased periostium cell viability to 44% (p < 0.05). Surprisingly, cell viability in less interconnected compression-molded scaffolds was higher compared to that of fully interconnected scaffolds produced by rapid prototyping. All other factors tested did not affect viability significantly. Our data suggest chondrocytes as a suitable cell source for cartilage repair in line with clinical data on several chondrocyte-based therapies. Although we did not test progenitor cells other the periosteum cells, tissue-engineering approaches using such cell types should take cell viability aspects into consideration

Changes in the functional capacity of T cells isolated from the peripheral blood of transplant patients between the time of transplantation (T0) and the start of tacrolimus withdrawal (T1).

<p>A positive value indicates an increase and a negative value indicates a decrease of the value in time. A CFSE MLR in combination with Modfit LT™ software was used to calculate the precursor frequency and the number of mitotic events of allo- and 3^rd-party-reactive CD4+ and CD8+ T cells. (A) The change (y-axis) in the precursor frequency of alloreactive CD4+ and CD8+ T cells. (B) The change in the number of mitotic events (ME) of CD4+ and CD8+ alloreactive T cells. (C) The functional capacity to suppress anti-donor responses of Treg isolated before transplantation (T0, white circles) and before the start of tacrolimus reduction (T1, black circles) in 5 rejectors (upper graphs) and 4 non-rejectors (lower graphs).</p

Patient characteristics.

*<p>deceased donors only</p

The frequency of effector, memory and regulatory T cell subsets in the peripheral blood of renal transplant patients immediately before the start of tacrolimus dose reduction (T1).

<p>(A) Representative dot plots of CD62L versus CD45RA and CD62L versus CD45RO. The analysis was performed on cells within the lymphocyte gate in the forward/side scatter plot. (B) The percentage of naïve (T_N, CD45RA+CD62L+), effector (T_E, CD45RA+CD62L-), central memory (T_CM, CD45RO+CD62L+) and effector memory (T_EM, CD45RO+CD62L-) T cells within the total CD4+ T cell subset. R vs. NR = not significant. (C) As described under A, for CD8+ T cells. R vs. NR = not significant. (D) Representative dot plot of CD25 versus FoxP3 in CD4+ lymphocytes. (E) The percentage of CD25+FoxP3+ regulatory T cells (Treg) within CD4+ lymphocytes. R vs. NR = not significant. (F) The ratio between the percentage of memory T cells and the percentage of Treg.</p

The functional capacity of allo- and 3^rd party-reactive CD4+ and CD8+ T cells and CD4+CD25^high regulatory T cells isolated from the peripheral blood of transplant patients immediately before the start of tacrolimus dose reduction (T1).

<p>A CFSE-MLR was used to determine the precursor frequency and mitotic events of alloreactive CD4+ (left) and CD8+ (right) T cells. CFSE labeled patient PBMC (1*10⁵) were stimulated with PKH labeled donor or 3^rd party cells (1*10⁵) for 6 days. Using flow cytometry, patient T cells were gated based on forward/side scatter and PKH exclusion and subsequent CD4 and CD8 staining. An example is shown in (A). The precursor frequency and mitotic events of alloreactive CD4+ and CD8+ T cells were calculated on the basis of the CFSE dilution pattern using Modfit LT™ software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002711#pone.0002711-Wells1" target="_blank">[22]</a>. (B) The precursor frequency of donor reactive CD4+ and CD8+ T cells. (C) The precursor frequency of 3^rd party reactive CD4+ and CD8+ T cells. (D) The number of mitotic events of alloreactive CD4+ and CD8+ T cells. (E) The number of mitotic events of 3^rd party reactive T cells. R vs. NR = not significant.</p

Changes in the frequency of naïve, effector, memory and regulatory T cell subsets in the peripheral blood of transplant patients between the time of transplantation (T0) and the start of tacrolimus withdrawal (T1).

<p>A positive value indicates an increase and a negative value indicates a decrease of the value in time. The change in the percentage of naïve (T_N, CD45RA+CD62L+), effector (T_E, CD45RA+CD62L-), central memory (T_CM, CD45RO+CD62L+) and effector memory (T_EM, CD45RO+CD62L-) T cell subsets within (A) the total CD4+ T cell population, and (B) the CD8+ T cell population. (C) The change in the percentage of peripheral blood CD4+CD25+FoxP3+ T cells within the total CD4+ T cell population.. R vs. NR = not significant.</p

The suppressive potential of Treg isolated from transplant patients immediately before the start of tacrolimus dose reduction.

<p>R = rejectors (n = 5), NR = non-rejectors (n = 4). CD4+CD25^high and CD4+CD25⁻ control cells were isolated from patient PBMC using fluorescence activated cell sorting resulting in >95 purity. The cells were expanded using anti-CD3/CD28 coated expander beads in combination with IL-2 and IL-15 for 14–21 days. After expansion, Treg were rested for 2–3 days after which their capacity to suppress anti-donor and anti-3^rd party responses was evaluated in a suppression assay, in which Treg were added at increasing ratios to a newly setup MLR (with donor or 3^rd party stimulator cells). Proliferation was measured at day 6 using ³H incorporation The percentage inhibition of proliferation (y-axis) of anti-donor (left) or anti-3^rd-party (right) responses by Treg, added at increasing ratios (x-axis) to the MLR, isolated from rejectors and non-rejectors. R vs. NR = not significant.</p