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

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.Lymphopenia results in high mortality and morbidity among cancer patients receiving a hematopoietic stem cell (HSC) transplantation, or suffering from HIV infection [1-5]. Eradication of hematological cancers is very successful using haplo-identical HSC transplantation [6], but many patients succumb to opportunistic infections that are the direct consequence of the lymphopenia, mainly involving the T cell pool [7]; it often takes more than 200 days before (mainly CD4+) T cell levels have normalized again. This underlines the need for, and explains the general interest in, methods capable of enhancing T cell reconstitution [8,9].Two important prob