Nineteen subjects have more than 18 months' follow-up in a phase IIb tolerance protocol in HLA-mismatched recipients of living donor kidney plus facilitating cell enriched hematopoietic stem cell allografts (FCRx). Reduced intensity conditioning preceded a kidney allograft, followed the next day by FCRx. Twelve have achieved stable donor chimerism and have been successfully taken off immunosuppression (IS). We prospectively evaluated immune reconstitution and immunocompetence. Return of CD4 and CD8 T central and effector memory cell populations was rapid. T-cell receptor (TCR) Excision Circle analysis showed a significant proportion of chimeric cells produced were being produced de novo. The TCR repertoires posttransplant in chimeric subjects were nearly as diverse as pretransplant donors and recipients, and were comparable to subjects with transient chimerism who underwent autologous reconstitution. Subjects with persistent chimerism developed few serious infections when off IS. The majority of infectious complications occurred while subjects were still on conventional IS. BK viruria and viremia resolved after cessation of IS and no tissue-invasive cytomegalovirus infections occurred. Notably, although 2 of 4 transiently or nonchimeric subjects experienced recurrence of their underlying autoimmune disorders, none of the chimeric subjects have, suggesting that self-tolerance is induced in addition to tolerance to alloantigen. No persistently chimeric subject has developed donor-specific antibody, and renal function has remained within normal limits. Patients were successfully vaccinated per The American Society for Blood and Marrow Transplantation guidelines without loss of chimerism or rejection. Memory for hepatitis vaccination persisted after transplantation. Chimeric subjects generated immune responses to pneumococcal vaccine. These data suggest that immune reconstitution and immunocompetence are maintained in persistently chimeric subjects.

CD8-positive/T-cell receptor-negative (CD8 ؉ /TCR ؊ ) graft facilitating cells (FCs) are a novel cell population in bone marrow that potently enhance engraftment of hemopoietic stem cells (HSCs). Previously, we showed that the CD11c ؉ /B220 ؉ / CD11b ؊ plasmacytoid-precursor dendritic cell (p-preDC) FC subpopulation plays a critical but nonredundant role in facilitation. In the present study, we investigated the mechanism of FC function. We report that FCs induce antigen-specific CD4 ؉ / CD25 ؉ /FoxP3 ؉ regulatory T cells ( IntroductionRecently, a great deal of interest has focused on the therapeutic potential of cell-based therapies to induce tolerance. Of greatest interest is the subpopulation of bone marrow-derived plasmacytoidprecursor dendritic cells (p-preDCs) and the regulatory T cells (Tregs) that they induce. A major limitation to the use of p-preDCs and Tregs in vivo has been the failure to identify an approach to prevent them from losing their tolerogenic properties and becoming immunogenic after transplantation. We recently demonstrated that CD8␣-positive/T-cell receptor-negative (CD8␣ ϩ /TCR Ϫ ) graft facilitating cells (FCs) enhance the engraftment of hematopoietic stem cells (HSCs) and tolerance induction in allogeneic recipients. [1][2][3] FCs suppress graft-versus-host disease (GVHD) in vivo by producing CD4 ϩ /CD25 ϩ /FoxP3 ϩ Tregs 4 and induce Tregs in vitro in the presence of CpG. 5 The majority of CD8␣ ϩ /TCR Ϫ FCs share the B220 ϩ /CD11c ϩ /CD11b Ϫ p-preDC phenotype, and we have demonstrated the first in vivo engraftment-enhancing and tolerancepromoting effect of the p-preDC FC subpopulation. 2 Although removal of p-preDC FCs from total FCs completely abrogates facilitation, p-preDC FCs alone do not replace FCs to provide the full in vivo biologic effect of facilitation. The mechanism of FC function has yet to be precisely characterized.CD4 ϩ /CD25 ϩ /FoxP3 ϩ Tregs play a critical role in the maintenance of self-tolerance. 6 Defects in Treg development or homeostasis result in systemic autoimmunity, 7 whereas adoptive transfer of Tregs as a therapeutic method can control ongoing autoimmune diseases. [8][9][10] Recently, several studies have demonstrated an important role for Tregs in mediating transplantation tolerance in animal models, 11-14 but little is known about the mechanism of Treg development and homeostasis. [15][16][17] p-preDCs may be important in the generation of Tregs, as evidenced by their potential to facilitate engraftment of HSCs 2,18 and to prolong heart allograft survival. 19,20 In addition, in vitro activation of p-preDCs with CpGoligodeoxynucleotides (CpG-ODNs) induces the production of Tregs in vitro. 5,21 We therefore evaluated whether the mechanism of FC function in vivo is to induce Tregs.In the present study, we first evaluated whether FCs enhance allogeneic HSC engraftment in diabetes-prone nonobese diabetic (NOD) mice. Second, we investigated whether FCs induce the production of Tregs and examined their function using in vivo transplantation models and in vitr...

Background We recently reported that durable chimerism can be safely established in mismatched kidney recipients through nonmyeloablative conditioning followed by infusion of a facilitating cell (FC)-based hematopoietic stem cell transplant termed FCRx. Here we provide intermediate-term follow-up on this phase 2 trial. Methods Fifteen HLA mismatched living donor renal transplant recipients underwent low intensity conditioning (fludarabine, cyclophosphamide, 200cGyTBI), received a living donor kidney transplant on day 0, then infusion of cryopreserved FCRx on day +1. Maintenance immunosuppression(IS),consisting of tacrolimus and mycophenolate, was weaned over one year. Results All but one patient demonstrated peripheral blood macrochimerism post-transplantation. Engraftment failure occurred in a highly sensitized (PRA of52%) recipient. Chimerism was lost in 3patients at 2, 3, and 6 months post transplantation. Two of these subjects had received either a reduced cell dose or incomplete conditioning; the other 2 had PRA >20%. All demonstrated donor-specific hyporesponsiveness and were weaned from full dose immunosuppression. Complete immunosuppression withdrawal at one year post-transplant was successful in all patients with durable chimerism. There has been no GVHD or engraftment syndrome. Renal transplant loss occurred in 1 patient who developed sepsis following an atypical viral infection. Two subjects with only transient chimerism demonstrated subclinical rejection on protocol biopsy despite donor-specific hyporesponsiveness. Conclusions Low intensity conditioning plus FCRx safely achieved durable chimerism in mismatched allograft recipients. Sensitization represents an obstacle to successful induction of chimerism. Sustained T cell chimerism is a more robust biomarker of tolerance than donor-specific hyporeactivity.

Na,K-ATPase is essential for the regulation of cytoplasmic Na+ and K+ levels in lens cells. Studies on the intact lens suggest activation of tyrosine kinases may inhibit Na,K-ATPase function. Here, we tested the influence of Lyn kinase, a Src-family member, on tyrosine phosphorylation and Na,K-ATPase activity in membrane material isolated from porcine lens epithelium. Western blot studies indicated the expression of Lyn in lens cells. When membrane material was incubated in ATP-containing solution containing partially purified Lyn kinase, Na,K-ATPase activity was reduced by approximately 38%. Lyn caused tyrosine phosphorylation of multiple protein bands. Immunoprecipitation and Western blot analysis showed Lyn treatment causes an increase in density of a 100-kDa phosphotyrosine band immunopositive for Na,K-ATPase alpha1 polypeptide. Incubation with protein tyrosine phosphatase 1B (PTP-1B) reversed the Lyn-dependent tyrosine phosphorylation increase and the change of Na,K-ATPase activity. The results suggest that Lyn kinase treatment of a lens epithelium membrane preparation is able to bring about partial inhibition of Na,K-ATPase activity associated with tyrosine phosphorylation of multiple membrane proteins, including the Na,K-ATPase alpha1 catalytic subunit.

Composite tissue allotransplantation (CTA) is emerging as a potential treatment for complex tissue defects. It is currently being performed with increasing frequency in the clinic. The feasibility of the procedure has been confirmed through 30 hand transplantation, 3 facial reconstructions, and vascularized knee, esophageal, and tracheal allografts. A major drawback for CTA is the requirement for lifelong immunosuppression. The toxicity of these agents has limited the widespread application of CTA. Methods to reduce or eliminate the requirement for immunosuppression and promote CTA acceptance would represent a significant step forward in the field. Multiple studies suggest that mixed chimerism established by bone marrow transplantation promotes tolerance resulting in allograft acceptance. This overview focuses on the history and the exponentially expanding applications of the new frontier in CTA transplantation: immunology associated with CTA; preclinical animal models of CTA; clinical experience with CTA; and advances in mixed chimerism-induced tolerance in CTA. Additionally, some important hurdles that must be overcome in using bone marrow chimerism to induce tolerance to CTA are also discussed.