In-Situ Proliferation May Explain the Persistence of Host Derived Langerhans Cells Following Allogeneic Hematopoietic Stem Cell Transplantation with Reduced Intensity Conditioning.

Langerhans cells (LCs) are antigen presenting cells found in the epidermis. They are thought to originate from bone marrow precursors although studies in mice suggest they have the ability to self renew in situ. Murine models have also shown that recipient LCs are required for the development of acute graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Engraftment of LCs following human allo-HSCT has previously been assessed using migration from isolated epidermal sheets, which may be inaccurate because of the potential for differential migration of donor and recipient cells. We therefore studied LC engraftment using fluorescence immunophenotyping and simultaneous in-situ hybridisation for X/Y chromosomes in sex mismatched transplants. Skin biopsies were performed at days 28, 56 and 100, 6 months and 1 year post transplant on 8 patients receiving alemtuzumab based RIC allo-HSCT regimens. Ten micron cryosections were taken, LCs labelled with anti-CD1a and X/Y chromosomes detected with the CEP X/Y probe kit. Slides were examined on a Zeiss LSM510 Meta confocal microscope and Z-stack images were collected to cover a volume of 100μm3. Chimerism of purified CD15+ and CD3+ peripheral blood cells was determined in parallel by polymerase chain reaction amplification of short tandem repeat sequences. Results are tabulated below and show that compared to CD15+ myeloid blood cells, donor LC engraftment is markedly delayed (day 28, 56 and 100 paired t-tests give P<0.0001, P=0.001 and P=0.0302 respectively). Of the 8 patients studied, 1 went on to develop biopsy proven GVHD. In all cases studied, recipient LCs could still be detected albeit in small numbers at the 1-year time point. Since these findings suggested that epidermal LCs are either very persistent or replicate in-situ, we next stained samples of normal and post transplant GVHD skin with both CD1a and the proliferation marker Ki67. In both cases a proportion of CD1a+ cells also expressed Ki67 (13.5% of normals, n=8; 18.6% of GVHD n=9 p=0.33). Both recipient and donor derived LCs appear to be capable of proliferating since both male and female Ki67+ CD1a+ cells were identified in 2 cases for which appropriate samples were available. In conclusion, our results provide independent confirmation that recipient LCs may persist for a prolonged period following allo-HSCT and show that at least in part, this may be due to self renewal within the epidermis.