A Novel SNP-Based Method for Assessing Langerhans Cell Chimerism Following Hematopoietic Stem Cell Transplantation in Humans.
Authors: Kirkorian A. Yasmine , Collin Matthew P , Mielcarek Marco , Zhang Wandi , Neuberg Donna , Wu Catherine J , Merad Miriam 
Published: April 2020

Abstract
Graft-versus-host disease (GVHD) is a major cause of mortality following allogeneic hematopoietic cell transplantation (HCT). In a mouse model, we established that recipient Langerhans cells (LCs) survive transplantation and that their persistence is sufficient for cutaneous GVHD. To investigate whether this also applies to human transplantation we developed a molecular assay for assessing LC chimerism in patients undergoing HCT. Langerin (CD207), a C-type lectin type II transmembrane protein, is uniquely expressed by LCs in the epidermis and is highly polymorphic, containing numerous single nucleotide polymorphisms (SNPs). We therefore reasoned that quantification of Langerin-specific transcripts expressing a donor or host-specific Langerin polymorphism could enable measurement of LC-specific chimerism. Using the NCBI dbSNP database, we selected 5 SNPs located in the coding regions of the Langerin gene, with minor allele frequencies &gt; 0.200 (range: 0.248–0.480). Based on these frequencies, we applied binomial calculations and estimated the probability that this SNP panel could identify at least one informative polymorphism between any patient and donor pair to be 87%. These calculations were performed assuming each SNP an independent event, given the low levels of linkage disequilibrium between any two selected SNPs. To experimentally confirm that this panel of 5 SNPs would be informative for the majority of transplanted patients, PCR primers that would amplify the gene region around the SNP from genomic DNA (gDNA) were designed and optimized, and tested on samples of gDNA extracted from peripheral blood mononuclear cells (PBMC) of 7 patient/donor pairs. By using the quantitative sequencing platform, Pyrosequencing, we determined that one informative SNP could be identified in 3 of 5 HLA-matched related patient/donor pairs, and 2–3 informative SNPs in 2 of 2 HLA-matched unrelated patient/donor pairs. We next designed and optimized PCR primers that would amplify the gene region around the SNP from reverse transcribed total RNA extracted from skin biopsies. By gel electrophoresis, Langerin transcripts were successfully amplified from skin cDNA but not from PBMCs, confirming the restricted expression of this transcript in skin tissue. In addition, pyrosequencing of the Langerin transcripts demonstrated that all 5 SNPs can be readily detected. Ongoing studies now focus on applying this SNP panel to a cohort of 12 patients who have undergone HCT, and correlating results with clinical variables, such as conditioning regimen and occurrence and onset of GVHD. In addition, we are comparing this molecular assay with conventional methods for assessment of LC chimerism, which rely on fluorescence in-situ hybridization and immunohistochemistry in skin tissue, that are labor-intensive and feasible only in instances of sex-mismatched transplant. In conclusion, we demonstrate that sufficient genetic diversity exists within a LC-specific gene, such that a relatively small panel of 5 SNPs can frequently identify differences between donor and host. Among our samples, we were able to successfully distinguish donors from patients in 71% of cases, including 2 cases of HLA-matched related patient/donor pairs where the likelihood of having commonly inherited SNPs is increased. Our novel assay enables the analysis of a very rare and otherwise difficult to isolate immune cell population and may provide critical information for investigating the impact of Langerhans cell engraftment on immunological outcomes following hematopoietic stem cell transplantation.