Functional in vitro models composed of human cells will constitute an important platform in the next generation of system biology and drug discovery. This study reports a novel human-based in vitro Neuromuscular Junction (NMJ) system developed in a defined serum-free medium and on a patternable non-biological surface. The motoneurons and skeletal muscles were derived from fetal spinal stem cells and skeletal muscle stem cells. The motoneurons and skeletal myotubes were completely differentiated in the co-culture based on morphological analysis and electrophysiology. NMJ formation was demonstrated by phase contrast microscopy, immunocytochemistry and the observation of motoneuron-induced muscle contractions utilizing time lapse recordings and their subsequent quenching by D-Tubocurarine. Generally, functional human based systems would eliminate the issue of species variability during the drug development process and its derivation from stem cells bypasses the restrictions inherent with utilization of primary human tissue. This defined human-based NMJ system is one of the first steps in creating functional in vitro systems and will play an important role in understanding NMJ development, in developing high information content drug screens and as test beds in preclinical studies for spinal or muscular diseases/injuries such as muscular dystrophy, Amyotrophic lateral sclerosis and spinal cord repair.

The transfer of lymphocytes into severe combined immunodeficiency (SCID) mice induces a series of histological changes in the spleen, including the appearance of mature follicular dendritic cells (FDCs). Studies were undertaken to clarify the role of lymphotoxin (LT) in this process. The results show that SCID mice have a small and partially differentiated white pulp containing marginal zone and interdigitating dendritic cells, but lacking FDCs. Transferred spleen cells can segregate into T and B cell areas shortly after their injection to SCID mice. This ability is dependent on signaling through LT-β receptor (LT-βR), since blocking ligand–receptor interaction in recipient SCID mice ablates the capacity of the transferred cells to segregate. A week after lymphocyte transfer, host-derived FDCs appeared in the reconstituted SCID mice. This induction of FDCs is dependent on LT-βR signaling by B cells since LT-α−/− B cells are incapable of inducing development of FDCs in SCID mice, even after cotransfer of LT-α+/+ T cells. Therefore, LT plays at least two discrete roles in splenic organization. First, it appears that LT induces the differentiation of the white pulp to create sites for lymphocyte segregation. Second, LT expression by B cells drives the maturation of FDCs and the organization of B cell follicles.

Inflammatory mediators facilitate the maturation of dendritic cells (DC), enabling them to induce the activation, proliferation and differentiation of cognate T cells. The role of CD40 on DC and CD154 on T cells has been studied by the co‐adoptive transfer of antigen‐pulsed DC and TCR‐transgenic (Tg) T cells in vivo. It is shown that in the absence of CD40‐CD154 interactions, initial Tg T cell expansion occurs in vivo, but over time, T cell expansion cannot be sustained. The basis for the demise of the T cell population is likely due to the disappearance of the antigen‐pulsed DC in the draining lymph nodes when CD154‐CD40 interactions are interrupted. These findings show that both T cell and DC persistence in vivo is dependent on CD40‐CD154 interactions. In addition to the physical persistence of the DC, CD40 triggering of DC also greatly increases the period for which they can productively present antigen to Tg T cells. Hence DC persistence and antigen‐presenting cell capacity are both dependent on CD40 signaling. While TNF‐α can mature DC as measured by a variety of criteria, the unique capacity of CD40 signaling to sustain T cell responses and induce DC maturation is underscored by the inability of TNF‐α to rescue the immune deficiency of CD40–/– DC. Hence, the profound impact of CD154 deficiency on cell‐mediated immunity may be due to its ability to limit the duration of antigen presentation in vivo and cause the premature demise of antigen‐specific T cells.

Induction of transplantation tolerance to alloantigens without general immunosuppression remains an enduring challenge. Injecting a donor-specific transfusion (DST) of spleen cells together with blocking ␣CD154 antibody prior to graft transplantation is an effective way to induce long-lived graft acceptance. Using a novel T-cell receptor (TCR) transgenic (Tg) model of CD4 ؉ T-cell-mediated rejection, this study sheds new insights into the cellular basis for enhanced graft survival induced by DST and ␣CD154. The study shows that DST and ␣CD154 induce an early, robust, abortive expansion of the Tg T cells that results in profound anergy. This is contrasted with the more delayed, regional, productive response elicited by an allogeneic graft. Studies show that the induction of tolerance to the allograft induced by DST is mediated by indirect presentation by host antigen-presenting cells. Based on these observations, we conclude that DST and ␣CD154 preemptively tolerize the alloreactive T-cell compartment to prohibit subsequent responses to the immunogenic allograft.