T cells fail to develop in the human skin-cell explants system; an inconvenient truth

• Published in: BMC immunology Vol. 12; no. 1; p. 17
• Main Authors: Meek, Bob, Van Elssen, Catharina H M J, Huijskens, Mirelle J A J, van der Stegen, Sjoukje J C, Tonnaer, Siebe, Lumeij, Stijn B J, Vanderlocht, Joris, Kirkland, Mark A, Hesselink, Reinout, Germeraad, Wilfred T V, Bos, Gerard M J
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 18.02.2011; BioMed Central; BMC
• Subjects: Animals; Cell Culture Techniques; Cell development (Biology); Cell Differentiation; Cell Line; Cells, Cultured; Coculture Techniques; Correspondence; Fibroblasts - cytology; Fibroblasts - metabolism; Flow Cytometry; Fluorescent Antibody Technique; Hematopoietic stem cells; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - metabolism; HIV; Human immunodeficiency virus; Humans; Intercellular Signaling Peptides and Proteins - genetics; Intercellular Signaling Peptides and Proteins - metabolism; Intracellular Signaling Peptides and Proteins; Keratinocytes - cytology; Keratinocytes - metabolism; Ligands; Membrane Proteins - genetics; Membrane Proteins - metabolism; Methods; Mortality; Physiological aspects; Reverse Transcriptase Polymerase Chain Reaction; Skin - cytology; Stem cells; T cells; T-Lymphocytes - cytology; T-Lymphocytes - metabolism; Transplantation
• ISSN: 1471-2172; 1471-2172
• DOI: 10.1186/1471-2172-12-17

Haplo-identical hematopoietic stem cell (HSC) transplantation is very successful in eradicating haematological tumours, but the long post-transplant T-lymphopenic phase is responsible for high morbidity and mortality rates. Clark et al. have described a skin-explant system capable of producing host-tolerant donor-HSC derived T-cells. Because this T-cell production platform has the potential to replenish the T-cell levels following transplantation, we set out to validate the skin-explant system. Following the published procedures, while using the same commercial components, it was impossible to reproduce the skin-explant conditions required for HSC differentiation towards mature T-cells. The keratinocyte maturation procedure resulted in fragile cells with minimum expression of delta-like ligand (DLL). In most experiments the generated cells failed to adhere to carriers or were quickly outcompeted by fibroblasts. Consequently it was not possible to reproduce cell-culture conditions required for HSC differentiation into functional T-cells. Using cell-lines over-expressing DLL, we showed that the antibodies used by Clark et al. were unable to detect native DLL, but instead stained 7AAD+ cells. Therefore, it is unlikely that the observed T-lineage commitment from HSC is mediated by DLL expressed on keratinocytes. In addition, we did confirm expression of the Notch-ligand Jagged-1 by keratinocytes. Currently, and unfortunately, it remains difficult to explain the development or growth of T-cells described by Clark et al., but for the fate of patients suffering from lymphopenia it is essential to both reproduce and understand how these co-cultures really "work". Fortunately, alternative procedures to speed-up T-cell reconstitution are being established and validated and may become available for patients in the near future.