Clinical Value of Flow Cytometric Assessment of Dendritic Cell Subsets in Peripheral Blood after Solid Organ Transplantation

Over the last years, the clinical value of flow cytometric monitoring of circulating dendritic cells (DCs) following solid organ transplantation attains more and more importance in transplantation medicine. The central role of DCs in transplantation immunology can be attributed to their function as professional antigen-presenting cells: firstly, DCs capture and present antigens and present these antigens to antigen-specific T cells. This process is enriched by bidirectional communication between DCs and T cells leading to T cell polarization into various effector and regulatory types 1. Secondly, within the scope of tolerance induction, DCs present self or foreign antigens to immature CD4+CD8+ thymocytes or induce anergy in peripheral T cells 2. This central role in the immunological processes makes DCs valuable to monitor immune function status after organ transplantation. Our study group recently published results in Cytometry B about the prognostic value of flow cytometric monitoring of the plasmacytoid DCs (pDCs) and myeloid DC (mDCs) subsets in peripheral whole blood of long-term heart transplanted patients 3. Patients were classified in two study groups regarding their calcineurin-inhibitor immunosuppressive drug, which is the cornerstone of an immunosuppressive drug therapy after transplantation. One group was treated with tacrolimus (TAC), and the other group with cyclosporine A (CsA)-based drug regimen, respectively. The TAC group had a higher expression of pDCs in peripheral blood compared to the CsA group. In general, TAC-treated patients had less biopsy proven acute rejection (BPAR) in their medical history compared to CsA-treated patients. Moreover, patients with higher values of pDCs had less events of BPAR as patients with low pDC values. Our results indicate that DC subsets could serve as useful biomarkers for detecting patients at risk of acute cellular rejection after heart transplantation. Interestingly, a small study in 20 heart transplanted (HTx) patients showed that expression of mDCs in peripheral blood was lower in patients with BPAR in contrast to patients without rejection. These results suggest that DC subsets may have the potential to serve as surrogate markers of rejection in the early period after HTx 4. In summary, this study results in organ transplanted patients revealed that analysis of circulating DCs enables (i) monitoring of an optimized immunosuppressive therapy following HTx 3, 5, (ii) analysing tolerance-inducing effects following transplantation 6, and (iii) drawing conclusions on the incidence of rejection 3. However, little is known about the development of immunological cell populations and especially about DCs within the first year post organ transplantation. Thus, we designed a study, which was approved by the local Ethic Committee of Leipzig, to monitor expression of pDCs and mDCs in peripheral blood of 46 heart transplanted patients, who gave written consent, over the first 12 months post-transplantation (Fig. 1). Peripheral whole blood samples of HTx patients were analysed for mDCs and pDCs in the first and the second quarter as well as in the second half-year following HTx (Fig. 2), while mDCs were defined as lin-1−/HLA-DR+/CD11chigh/CD123low cells, pDCs were defined as lin-1−/HLA-DR+/CD11 clow/CD123high cells (Fig. 2A). The initial immunosuppressive regimen consisted of TAC, mycophenolate mofetil (MMF) and steroids. After the first three months post-HTx the immunosuppressive triple drug therapy was changed in cases of drug-related side effects from TAC to CsA or as a consequence of clinical events other than rejections, for example CMV-infection. Overview about the study type, study design, the time course, and detailed information about the panel design. Flow cytometric analysis of blood DC subsets. An example of a 4-color flow cytometric gating strategy and analysis of DC subsets in patients with heart transplantation was shown (A): Lymphocytes and monocytes were gated (R1) using the forward- and side-scatter characteristics. Then, the DC population was identified by positive staining of HLA-DR and negative staining with lineage cocktail-1 (lin1) antibodies (antibodies against CD3, CD14, CD16, CD19, CD20, and CD56). CD123 and CD11c) (R2). mDCs and pDCs were identified by their expression profiles of CD11c and CD123, respectively. mDCs were defined as lin-1−/HLA-DR+/CD11chigh/CD123low cells, pDCs were defined as lin-1−/HLA-DR+/CD11 clow/CD123high cells. In the case of missing values, multiple imputation by chained equations was performed for construction of a combined set. The development of DC subsets at different time points (first, second, third, and fourth quarter) within the first year following heart transplantation: (B) percentages of pDCs; (C) percentages of mDCs and (D) the ratio of pDCs/mDCs. Statistically significant P values (>0.05) were documented in the box plots. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] We observed a significant (P < 0.001) and continuous increase of the percentage of pDCs over the first year post-HTx (Fig. 2B). Whereas no significant changes were observed for mDCs (P = 0.062) and subsequently for the pDC/mDC ratio (P = 0.240; Figs. 2C and 2D). Data of a previous study support the observation that healthy volunteers have a higher expression of pDCs in peripheral blood compared to long-term immunosuppressed HTx patients 5. Thus, it is hypothesized that the surgical trauma and the initial high-dose immunosuppression are reducing the numbers of mDCs and pDCs in peripheral blood followed by an increase of the DC numbers over the first year post-HTx. As a result of our recent observation we recommend to consider the time point of analyzing expression of pDCs and mDCs in peripheral blood post-transplantation. Changes of both DC subsets are described in 20 patients before, and at five different time points, until 38 weeks after HTx. The total numbers of DCs decreased significantly after 1-week post-HTx and remained lower than the pre-HTx condition until 38 weeks following transplantation 4. A similar decrease of circulating DC subsets was found in two studies observing the early postoperative period after kidney transplantation 7, 8. One study investigated total DC counts as well as the expression of mDCs and pDCs in peripheral blood at 0, 1, 7, and 28 days and 1 year in 33 patients 7. Blood DCs decreased dramatically at day 1 after transplantation and slowly increased by day 7. Until day 28 DC counts increased continuously, but did not reach the level before surgery. The other study included 24 kidney allograft recipients and pictured the kinetics of mDCs and pDCs before and at three different time points (7, 30, and 90 days) after transplantation 8. Similar to the results of Ma et al. this study by Hesselink et al. demonstrated a dramatic decrease of total DC counts and of DC subsets directly after transplantation (day 7) and a continuous increase in the following 12 weeks until the end of the study period. Furthermore, patients with additional immunosuppressive treatment because of acute rejection experienced an even more marked decrease in DC counts in the early postoperative period compared to patients without rejection 8. Until now it is unknown, how to explain the reduction of DC levels in the context of transplant-relevant immunological processes. However, both DC subsets, mDCs and pDCs, are known to promote tolerance to alloantigens; however, DCs are arguably better known to prime the immune response 9. The reason for low numbers of DC subsets post-HTx must be ascribed to the administered immunosuppressive regimen 4. The role of DCs in the field of tolerance after organ transplantation is still under investigation. It is known that tolerance-inducing effects are different for various solid organs 10. For instance, the ‘pDC hypothesis’ for kidney and liver allografts indicates that donor pDCs transferred with the kidney or liver allograft traffic to the host thymus and lymph nodes, where they facilitate the activation and expansion of donor-specific regulatory T cells (Tregs) and thus, promoting tolerance 10. Such processes have not been described after HTx or lung transplantation. Thus, it is not possible to directly transfer the knowledge about tolerance-inducing effects from one organ system to another. The immunological processes have to be investigated separately for each organ system in the future. For the same reason, the usage of DCs as a cellular therapy in transplantation holds controversial aspects. Beside the requested tolerance-inducing effects, there is a risk of sensitizing the recipients 9. Thus, future transplant research should focus on the identification of functionally different DC. A promising approach is the flow cytometric monitoring of the blood dendritic cell marker (BDCA)-1, -2, -3, and -4, which seem to distinguish between functional different DCs. While BDCA-1 (CD1c) and BDCA-3 (CD141) are present on subsets of mDCs, BDCA-2 (CD303) and BDCA-4 (CD304) are expressed on pDC subsets. Some of these promising DC subset markers were used in monitoring panels for diseases such as multiple myeloma or different types of leukaemia 11, 12. If a flow cytometric analysis of functional DCs in peripheral blood could shed light in current problems after organ transplantation needs to be explored in the future. The current results show the clinical value of flow cytometry as a diagnostic tool in monitoring DC subsets. Thus, flow cytometric experts should be encouraged to develop protocols to study DCs and their role in immunological processes after organ transplantation.