Chimerism Analysis of Langerhans Cells Early after T Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation.

Abstract

Skin is the most frequently affected organ in acute graft versus host disease (GVHD). Data from murine studies support the hypothesis that the interaction of residing host Langerhans cells of the epidermis (LC) and donor T cells is crucial for the initiation of acute GVHD. Donor T cells are also necessary to induce the switch of LC from host to donor origin after allogeneic stem cell transplantation (SCT). In an ongoing clinical protocol applying alemtuzumab-based T cell depleted (TCD) allogeneic SCT (Meyer, Blood 2007; 109:374), we observed acute skin GVHD occurring early after transplantation. We therefore intended to analyse the LC chimerism in patients undergoing this protocol. So far, LC-chimerism analysis in humans has been performed by the detection of the Y-chromosome restricting it to sex-mismatched donor/recipient pairs. Here we introduce a new method to isolate LC from small skin samples at high purity for a sensitive STR-based chimerism analysis of general applicability. Epidermal skin layers were obtained from 6 mm punch biopsies by dispase I digestion. A small slice of epidermis was used for immunofluorescent staining. The remaining sample was digested by trypsin, and CD1a/MHC-class II-positive LC were sorted by flow cytometry. This approach resulted in a mean purity of > 96% with skin of healthy individuals. However, the density of LC early after SCT following non-TCD myeloablative regimens had previously been shown to be much lower compared to healthy individuals. By CD1a-staining, we were able to show that this is also the case after TCD reduced intensity SCT. Nevertheless, LC could be purified in all of 8 analyzed patients. The isolated LC numbers ranged from 10 to more than 1000. In 4 patients we performed a re-analysis of the isolated cells by flow cytometry and confirmed a purity exceeding 97%. We obtained reliable results for LC chimerism in 6 of 8 patients. After the RNA-isolation protocol was further improved, we were able to detect signals even with 35 isolated LC in patient MZ-47. In two patients, the majority of isolated LC were of donor origin whereas the other 4 patients had predominantly host LC (patients' characteristics and chimerism results are summarized in Table 1). None of the patients developed spontaneous acute GVHD so far. For patients MZ-37 and MZ-43 LC-chimerism was also performed after day +50 post HSCT and showed a switch to >97% donor chimerism at that time. In summary, we have established a sensitive method that enables the chimerism analysis on highly purified LC independent of sex-mismatched donor/recipient pairs. Our results on a few patients' samples can not yet be related to clinical events. This assay, however, allows the comprehensive investigation of the chimerism of LC and potentially of other tissue-resident antigen presenting cells to study their impact on GVHD in humans.Table 1patientdonortypesex (patient/donor)LC (n)purity (%)donor chimerism (%)37MSDF/M112797,7<339MMUDF/M75098,6>9740MMUDM/M180098,31343MMUDM/M10n.a.n.a.44MSDM/M157n.a.n.a.45MSDM/F325> 983646MMUDM/M355n.a.4847MSDM/M35n.a.>90MSD, matched sibling donor; MMUD, mismatched unrelated donor; n.a., not applicable; M, male; F, female