Comparable resident populations of T cells exist in the gut and the lung [44]. over-expressing DLL, we showed that this antibodies used by Clark Mcl-1-PUMA Modulator-8 em et al. /em were unable to detect native DLL, but instead stained 7AAD+ cells. Therefore, it is unlikely that this observed T-lineage commitment from HSC is usually mediated by DLL expressed on keratinocytes. In addition, we did confirm expression of the Notch-ligand Jagged-1 by keratinocytes. Conclusions Currently, and unfortunately, it remains difficult to explain the development or growth of T-cells described by Clark em et al. /em , but for the fate of patients suffering from lymphopenia it is Mcl-1-PUMA Modulator-8 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. Background Lymphopenia results in high mortality and morbidity among cancer patients receiving a hematopoietic stem cell (HSC) transplantation, or suffering from HIV contamination [1-5]. Eradication of hematological cancers is very successful using haplo-identical HSC transplantation [6], but many patients succumb to opportunistic infections that Rabbit polyclonal to ZNF138 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 problems associated with slow recovery of T cell levels involve the thymus: slow thymic reconstitution by blood-borne progenitors and thymic involution [10-14]. Because it is usually still not possible to control and/or reverse either of these processes, there is an obvious need to establish methods that generate a em de novo /em T cell repertoire em in vitro /em . However, the development of such systems is usually hampered because most processes that occur in the thymus are still enigmatic, especially how the thymus is usually capable to enforce self versus nonself recognition on developing thymocytes [15]. Understanding the process of positive/unfavorable selection, and reproducing the process em in vitro /em , would potentially help to reduce lymphopenia, especially in older patients as the thymus involutes with age. In this context, the results on thymus-independent T cell development previously described by Clark em et al. /em are remarkable. This method involves a seemingly simple co-culture system consisting of skin keratinocytes and fibroblasts grown on a three-dimensional (3D) tantalum covered scaffold (Statamatrix?) that, after 4 weeks co-culture with allogenic HSC, results in a population made up of 3-5% T cells tolerant to the skin-donor [16]. Even though T cells were detected, only a limited fraction of the expanded HSC actually became T-lineage committed; many cells differentiated towards Class II+ APC, a convenient aspect since it was suggested to be important for CD4+ T cell development [16]. Additional explanations for the extra-thymic development of T cells were the co-incidental expression of various components known to be important for HSC differentiation and thymus-function, such as Delta-Like Ligand (DLL) [8] and AutoImmune Regulator (AIRE) [17] by keratinocytes and fibroblasts [16], respectively. Even though direct mechanistic explanations for the extra-thymic development of T cells are lacking, and despite limited numbers, it still remains the only published method potentially capable of generating functional, clinical-grade, mature T cells em ex vivo /em . Because of the clinical importance, we made an effort to establish the skin-cell system in our laboratory and to Mcl-1-PUMA Modulator-8 characterize it in more detail. We observed that keratinocytes do express the Notch ligand Jagged1, but we did not find the abundant expression of DLL protein previously reported by Clark em et al. /em on keratinocytes. Furthermore, due to the growth characteristics.