A PCR-Based Assay for the Detection of Langerhans-Cell Chimerism after Allogeneic Stem Cell Transplantation.

Abstract

Early acute GVHD of the skin frequently occurs in patients after allogeneic hematopoietic stem cell transplantation. Although T cell depletion reduces the incidence and severity, it does not completely prevent skin GVHD. This leads to a prolonged need for immunosuppressive medication in a significant number of patients. For the induction of acute GVHD, the stimulation of donor T cells by residing host antigen presenting cells such as Langerhans cells of the skin (LCs) plays a central role. The absence of donor T cells after depletion, however, seems to hamper an early switch of LCs from host to donor origin. Therefore, the monitoring of LC chimerism is of great interest. We and others have provided evidence for a delayed switch in LC chimerism after T cell depleted reduced intensity stem cell transplantation. However, the assays used so far either are imprecise when applying low numbers of isolated cells or they depend on the detection of the Y-chromosome in skin sections of sex-mismatched transplants. In an attempt to set up a more sensitive assay of general applicability, we combined the detection of donor chimerism and tissue specific markers in a single multiplex PCR. We established PCRs for 10 different cDNA regions of constitutively expressed genes containing single nucleotide polymorphisms (SNPs). The SNP-containing products of the multiplex PCR were subsequently analyzed by the primer extension method (minisequencing) and subsequently analyzed by capillary electrophoresis. All SNP-containing cDNAs were expressed in peripheral blood mononuclear cells (PBMCs) as well as in isolated CD4- and CD8-positive T-lymphocytes, in myeloid dendritic cells, LCs, and keratinocytes. When we tested this approach on PBMCs of 10 patients and their HLA-matched sibling donors, the assay distinguished all pairs in 1 to 6 out of 10 systems. In a subsequent step, the 10plex PCR was combined with the tissue specific markers langerin for LCs and cytokeratin 10 to distinguish LCs from keratinocytes. Their expression was detected using gene-specific probes in the same minisequencing reaction used for the detection of SNPS. The resulting 12plex assay distinguished sibling donors from the patients with the same specificity and, in the same reaction, detected Langerin as well as cytokeratin 10 in purified LCs and keratinocytes, respectively. In summary, we established a sensitive assay allowing the simultaneous detection of donor chimerism together with the tissue specificity of isolated LCs that is independent of sex-mismatched donors. The addition of further tissue specific markers might allow performing chimerism studies on other tissue resident antigen presenting cells.