Immune reconstitution/immunocompetence in recipients of kidney plus hematopoietic stem/facilitating cell transplants.

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.

Tolerance induction in HLA disparate living donor kidney transplantation by donor stem cell infusion: durable chimerism predicts outcome.

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 transplantation termed FCRx. Here we provide intermediate-term follow-up on this phase II trial. METHODS: Fifteen human leukocyte antigen-mismatched living donor renal transplant recipients underwent low-intensity conditioning (fludarabine, cyclophosphamide, 200 cGy TBI), received a living donor kidney transplant on day 0, then infusion of cryopreserved FCRx on day +1. Maintenance immunosuppression, consisting of tacrolimus and mycophenolate, was weaned over 1 year. RESULTS: All but one patient demonstrated peripheral blood macrochimerism after transplantation. Engraftment failure occurred in a highly sensitized (panel reactive antibody [PRA] of 52%) recipient. Chimerism was lost in three patients at 2, 3, and 6 months after transplantation. Two of these subjects had received either a reduced cell dose or incomplete conditioning; the other two had PRA greater than 20%. All demonstrated donor-specific hyporesponsiveness and were weaned from full-dose immunosuppression. Complete immunosuppression withdrawal at 1 year after transplantation was successful in all patients with durable chimerism. There has been no graft-versus-host disease or engraftment syndrome. Renal transplantation loss occurred in one 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.

Simultaneous bone marrow and composite tissue transplantation in rats treated with nonmyeloablative conditioning promotes tolerance.

BACKGROUND: Approaches to safely induce tolerance in vascularized composite allotransplantation (VCA) with chimerism through bone marrow transplantation (BMT) are currently being pursued. However, VCA was historically performed sequentially after donor chimerism was established. Delayed VCA is not clinically applicable due to the time constraints associated with procurement from deceased donors. A more clinically relevant approach to perform both BMT and VCA simultaneously was evaluated. METHODS: Wistar Furth (RT1A) rats were treated with a short course of immunosuppressive therapy (anti-αß-TCR monoclonal antibody, FK-506, and anti-lymphocyte serum). One day before BMT, rats were treated with varying doses of total body irradiation (TBI) followed by transplantation of heterotopic osteomyocutaneous flaps from hindlimbs of August Copenhagen Irish (RT1A) rats. RESULTS: Eighty percent of rats conditioned with 300 cGy TBI and 40% of rats receiving 400 cGy TBI accepted the VCA. Mixed chimerism was detected in peripheral blood at 1 month after VCA, but chimerism was lost in all transplant recipients by 4 months. Most peripheral donor cells originated from the BMT and not from the VCA. Acceptors of VCA were tolerant of a donor skin graft challenge and no anti-donor antibodies were detectable, suggesting a central deletional mechanism for tolerance. Regulatory T cells (Treg) from spleens of acceptors more potently suppressed lymphocyte proliferation than Treg from rejectors in the presence of donor stimulator cells. CONCLUSIONS: These studies suggest that simultaneous BMT and VCA may establish indefinite allograft survival in rats through Treg-mediated suppression and thymic deletion of alloreactive T cells.

A critical role for the TLR4/TRIF pathway in allogeneic hematopoietic cell rejection by innate immune cells.

We show for the first time that signaling through the TLR4/TRIF pathway plays a critical role in allogeneic bone marrow cell (BMC) rejection. This appears to be unique to BMCs as organ allografts are rejected mainly via MyD88 signaling. Using T- or T-/B-cell-deficient mice, we found that BMC allorejection occurred early before T-cell activation and was T- and B-cell independent, suggesting an effector role for innate immune cells in BMC rejection. We further demonstrated the innate immune signaling in BMC allorejection by showing superior engraftment in mice deficient in TRIF or TLR4 but not in MyD88 or TLR3. The restored cytotoxicity in TRIF-deficient recipients transferred with wild-type F4/80(+) or NK1.1(+) cells suggests TRIF signaling dependence on macrophages or NK cells in early BMC rejection. Production of the proinflammatory cytokine IL-6 and TRIF relevant chemokine MCP-1 was significantly increased early after bone marrow transplantation. In vivo specific depletion of macrophages or NK innate immune cells in combination with anti-CD154/rapamycin resulted in additive-enhanced allogeneic engraftment. The requirement for irradiation was completely eliminated when both macrophages and NK cells were depleted in combination with anti-CD154/rapamycin to target T- and B-cells, supporting the hypothesis that two barriers involving innate and adaptive immunity exist in mediating the rejection of allogeneic BMCs. In summary, our results clearly demonstrate a previously unappreciated role for innate immunity in BMC allorejection via signaling through a unique MyD88-independent TLR4/TRIF mechanism. These findings may have direct clinical impact on strategies for conditioning recipients for stem cell transplantation.

The need for inducing tolerance in vascularized composite allotransplantation.

Successful hand and face transplantation in the last decade has firmly established the field of vascularized composite allotransplantation (VCA). The experience in VCA has thus far been very similar to solid organ transplantation in terms of the morbidity associated with long-term immunosuppression. The unique immunological features of VCA such as split tolerance and resistance to chronic rejection are being investigated. Simultaneously there has been laboratory work studying tolerogenic protocols in animal VCA models. In order to optimize VCA outcomes, translational studies are needed to develop less toxic immunosuppression and possibly achieve donor-specific tolerance. This article reviews the immunology, animal models, mixed chimerism & tolerance induction in VCA and the direction of future research to enable better understanding and wider application of VCA.

Innate and adaptive immune responses are tolerized in chimeras prepared with nonmyeloablative conditioning.

BACKGROUND: Mixed chimerism is an effective approach for tolerance induction in transplantation. Strategies to achieve mixed chimerism with relatively low toxicity have significantly expanded the clinical use of chimerism. METHODS: Allogeneic bone marrow transplants were performed between B6 (H2(b)) and BALB/c (H2(d)) mice. Recipient B6 mice were nonmyeloablatively conditioned with anti-αß-T-cell receptor, anti-CD154, or rapamycin alone or in different combinations. A total of 15 × 10(6) BALB/c bone marrow cells were transplanted after varying doses of cGy of total body irradiation. RESULTS: Pretreatment of recipients with anti-CD154 and rapamycin with or without T-cell lymphodepletion reduced the total body irradiation requirement to 100 cGy for establishing stable mixed chimerism. The mixed chimeras accepted donor islet allografts long term. Lymphocytes from mixed chimeras did not respond to host or donor antigens, yet were reactive to major histocompatibility complex-disparate third-party alloantigens, demonstrating functional donor-specific T-cell tolerance. No antibodies against donor and host were detected in mixed chimeras, suggesting humoral tolerance. Mixed chimeras showed no cytotoxicity to donor cells, but a similar rapid killing rate for major histocompatibility complex disparate third-party B10.BR cells compared with T-cell-deficient and wild-type controls in in vivo cytotoxicity assays, suggesting donor-specific tolerance in the innate immune cells was achieved in mixed chimeras. CONCLUSIONS: Mixed chimeras prepared with low-intensity nonmyeloablative conditioning exhibit systemic tolerance in innate immunity and tolerance in adaptive T- and B-cell immune responses.

Differential outcomes in prediabetic vs. overtly diabetic NOD mice nonmyeloablatively conditioned with costimulatory blockade.

OBJECTIVE: Autoimmune diabetes can be reversed with mixed chimerism. However, the myelotoxic agents currently required to establish chimerism have prevented the translation of this approach to the clinic. Here, we investigated whether multimodal costimulatory blockade would enhance chimerism and promote islet allograft tolerance in spontaneously diabetic nonobese diabetic (NOD) mice. MATERIALS AND METHODS: Prediabetic and spontaneously diabetic NOD mice were preconditioned with anti-CD8 monoclonal antibody before conditioning with 500 cGy total body irradiation and transplantation with 30 × 10(6) B10.BR bone marrow cells. Overtly diabetic animals were conditioned similarly and transplanted with 300 to 400 B10.BR islets. After irradiation, both groups of recipients were treated with anti-CD154, anti-OX40L, and anti-inducible T-cell costimulatory monoclonal antibodies. Urine, blood glucose levels, and chimerism were monitored. RESULTS: Conditioning of NOD mice with costimulatory blockade significantly enhanced engraftment, with 61% of mice engrafting at 1 month. Eleven of 12 chimeric animals with engraftment at 1 month remained diabetes-free over a 12-month follow-up, whereas nonchimeric animals progressed to diabetes. In contrast, similar conditioning prolonged islet allograft survival in only 2 of 11 overtly diabetic NOD recipients. Chimerism levels in the 9 islet rejector animals were 0%. CONCLUSIONS: Although nonmyeloablative conditioning reversed the autoimmune process in prediabetic NOD mice, the same regimen was significantly less effective in establishing chimerism and reversing autoimmune diabetes in spontaneously diabetic NOD mice.

Evidence that FoxP3+ regulatory T cells may play a role in promoting long-term acceptance of composite tissue allotransplants.

BACKGROUND: FoxP3/CD4/CD25 regulatory T cells (Treg) play an important role in maintaining peripheral tolerance and are potent suppressors of T-cell activation. In this study, we evaluated the role of Treg in peripheral tolerance to composite tissue allografts (CTA). METHODS: Mixed allogeneic chimeric rats were prepared by preconditioning recipients with anti-αß-T-cell receptor monoclonal antibody followed by total body irradiation. Animals received T-cell-depleted August Copenhagen Irish bone marrow cells followed by antilymphocyte serum and FK-506. A modified osteomyocutaneous hindlimb flap composed of bone and all limb tissue components was placed in animals with chimerism greater than or equal to 1% on day 28. Recipients with CTA surviving more than or equal to 6 months were evaluated for Treg. Skin samples from tolerant long-term allogeneic transplanted, syngeneic transplanted, rejected, and naïve animals were immunostained with fluorochrome-conjugated anti-FoxP3 and anti-CD4 monoclonal antibody and visualized under a laser confocal microscope. RESULTS: Significant CD4/FoxP3 Treg infiltrates were observed in tolerant donor-allograft skin samples. No graft infiltrating FoxP3 cells were observed in rejector, naïve, or skin from syngeneic CTA. In parallel experiments, mixed leukocyte reaction assays were performed to investigate the suppressor function of Treg cells. Splenocytes from tolerant, rejected, and naïve rats were sorted by flow cytometry for CD4/CD25 T cells. Treg demonstrated similar suppressive levels between the three groups. CONCLUSIONS: These data suggest that Treg may play an important role in maintenance of tolerance and promoting graft acceptance in long-term CTA acceptors and may explain the favorable outcomes observed in clinical CTA recipients.

CD8α+ plasmacytoid precursor DCs induce antigen-specific regulatory T cells that enhance HSC engraftment in vivo.

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 (Tregs) in vivo. The majority of chimeric Tregs were recipient derived. Chimeric Tregs harvested at ≥ 4 weeks after transplantation significantly enhanced engraftment of donor- and recipient-derived HSCs, but not third-party HSCs, in conditioned secondary recipients, demonstrating antigen specificity. Although Tregs were present 2 and 3 weeks after transplantation, they did not enhance engraftment. In contrast, week 5 and greater Tregs potently enhanced engraftment. The function of chimeric Tregs was directly correlated with the development of FoxP3 expression. Chimeric Tregs also induced significantly stronger suppression of T-cell proliferation to donor antigen in vitro. Removal of p-preDC FCs resulted in impaired engraftment of allogeneic HSCs and failure to produce chimeric Tregs, suggesting that the CD8α(+) p-preDC subpopulation is critical in the mechanism of facilitation. These data suggest that FCs induce the production of antigen-specific Tregs in vivo, which potently enhance engraftment of allogeneic HSCs. FCs hold clinical potential because of their ability to remain tolerogenic in vivo.

Plasmacytoid precursor dendritic cells from NOD mice exhibit impaired function: are they a component of diabetes pathogenesis?

OBJECTIVE: Plasmacytoid precursor dendritic cell facilitating cells (p-preDC FCs) play a critical role in facilitation of syngeneic and allogeneic hematopoietic stem cell (HSC) engraftment. Here, we evaluated the phenotype and function of CD8(+)/TCR(-) FCs from NOD mice. RESEARCH DESIGN AND METHODS: The phenotype of CD8(+)/TCR(-) FCs was analyzed by flow cytometry using sorted FCs from NOD, NOR, or B6 mice. The function of NOD FCs was evaluated by colony-forming cell (CFC) assay in vitro and syngeneic or allogeneic HSC transplantation in vivo. RESULTS: We report for the first time that NOD FCs are functionally impaired. They fail to facilitate engraftment of syngeneic and allogeneic HSCs in vivo and do not enhance HSC clonogenicity in vitro. NOD FCs contain subpopulations similar to those previously described in B6 FCs, including p-preDC, CD19(+), NK1.1(+)DX5(+), and myeloid cells. However, the CD19(+) and NK1.1(+)DX5(+) subpopulations are significantly decreased in number in NOD FCs compared with disease-resistant controls. Removal of the CD19(+) or NK1.1(+)DX5(+) subpopulations from FCs did not significantly affect facilitation. Notably, Flt3 ligand (FL) treatment of NOD donors expanded FC total in peripheral blood and restored facilitating function in vivo. CONCLUSIONS: These data demonstrate that NOD FCs exhibit significantly impaired function that is reversible, since FL restored production of functional FCs in NOD mice and suggest that FL plays an important role in the regulation and development of FC function. FCs may therefore be linked to diabetes pathogenesis and prevention.

Plasma membrane Ca(2+) -ATPase associates with CLP36, alpha-actinin and actin in human platelets.

The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca(2+) in resting platelets. Earlier studies demonstrated that the 4b isoform of PMCA interacts via its C-terminal end with the PDZ domains of membrane-associated guanylate kinase proteins. Activation of saponin-permeabilized platelets in the presence of a peptide composed of the last ten residues of the PMCA4b C-terminus leads to a significant decrease of PMCA associated with the cytoskeleton, suggesting that PDZ domain interactions play a role in tethering the pump to the cytoskeleton. Here we present experiments conducted to evaluate the mechanism of this association. Co-immunoprecipitation assays coupled with liquid chromatography/tandem mass spectrometry analysis and immunoblotting were used to identify proteins that interact with PMCA in the resting platelet. Our results indicate that the only PDZ domain-containing protein associated with PMCA is the LIM family protein, CLP36. Glutathione-S-transferase pull-down from a platelet extract using a fusion protein containing the C-terminal PDZ domain binding motif of PMCA confirmed binding of CLP36 to PMCA. Gel filtration chromatography of detergent-solubilized platelets demonstrated the existence of a 1,000-kDa complex containing PMCA and CLP36, and in addition, alpha-actinin and actin. Immunoflourescence microscopy confirmed the co-localization of PMCA with CLP36 in resting and activated platelets. Taken together these results suggest that PMCA is localized in non-filamentous actin complexes in resting platelets by means of PDZ domain interactions and then associates with the actin cytoskeleton during cytoskeletal rearrangement upon platelet activation. Thus, in addition to the reversible serine/threonine and tyrosine phosphorylation events previously described in human platelets, PMCA function may be regulated by interactions with anchoring and cytoskeletal proteins.

Past, present, and future prospects for inducing donor-specific transplantation tolerance for composite tissue allotransplantation.

Composite tissue allotransplantation (CTA) is among the most immunologically complex and newest transplant fields. Although the field has made considerable advances, there are still concerns that these procedures are performed to enhance quality-of-life issues and are not lifesaving procedures that restore physiologic function. Two challenges limit the widespread application of CTA; the first is chronic rejection, the most prevailing cause of organ allograft failure after transplantation; the second barrier is the numerous health complications associated with lifelong immunosuppressive therapy. Several tolerance-inducing strategies, including costimulatory blockade, T-cell depletion, mixed chimerism, and gene targeting of transplanted organs, have the potential to induce lifelong tolerance to organ allografts without chronic immunosuppression. Effective clinical tolerance protocols that improve CTA acceptance and offer an alternative to the requirement for chronic immunosuppressive therapy could be a major advance in the field. Tolerance would allow allotransplantation to provide a currently unmet need for reconstruction of large tissue defects. This article reviews the history of CTA, current challenges and complications, and offers future directions for CTA research in strategies to induce tolerance.

The influence of SRC-family tyrosine kinases on Na,K-ATPase activity in lens epithelium.

PURPOSE: Na,K-adenosine triphosphatase (ATPase) is essential for the regulation of cytoplasmic ion concentrations in lens cells. Earlier studies demonstrated that tyrosine phosphorylation by Lyn kinase, a Src-family member, inhibits Na,K-ATPase activity in porcine lens epithelium. In the present study, experiments were conducted to compare the ability of other Src-family kinases (Fyn, Src, and Lck) and Fes, a non-Src-family tyrosine kinase, to alter Na,K-ATPase activity. METHODS: Membranes prepared from porcine lens epithelium were incubated with partially purified tyrosine kinases in buffer containing 1 mM adenosine triphosphate (ATP). ATP hydrolysis in the presence and absence of ouabain was used to measure Na,K-ATPase activity. Western blot analysis was used to examine phosphotyrosine-containing proteins and tyrosine kinase expression. RESULTS: Fyn reduced Na,K-ATPase activity by approximately 30%. In contrast, Src caused a approximately 38% increase of Na,K-ATPase activity. Na,K-ATPase activity in membrane material treated with Lck or Fes was not significantly altered, even though Lck and Fes treatment induced robust tyrosine phosphorylation. Added exogenously, each tyrosine kinase induced a different pattern of membrane protein tyrosine phosphorylation. As judged by immunoprecipitation, Src, Fyn, Lyn, and Lck elicited tyrosine phosphorylation of the Na,K-ATPase alpha1 protein. Src, Fyn, Lyn, Lck, and Fes were each detectable in the epithelium by Western blot. CONCLUSIONS: The results indicate considerable variation in the Na,K-ATPase activity response of lens epithelium to different tyrosine kinases. This could perhaps explain why inhibition of Na,K-ATPase activity is reported to be caused by tyrosine phosphorylation in some tissues, whereas stimulation of Na,K-ATPase activity is observed in other tissues.

The influence of protein tyrosine phosphatase-1B on Na,K-ATPase activity in lens.

The abnormal sodium content of many cataracts suggests Na,K-ATPase is vital for maintenance of eye lens transparency. Since tyrosine phosphorylation is considered a possible regulatory mechanism for Na,K-ATPase, experiments were conducted to test the influence of protein tyrosine phosphatase-1B (PTP-1B) on Na,K-ATPase activity. Membrane material was isolated separately from porcine lens epithelium and fiber cells. Tyrosine phosphoproteins, Na,K-ATPase alpha1 polypeptide and PTP-1B were examined by Western blot. Na,K-ATPase activity was determined by measuring ATP hydrolysis in the presence or absence of ouabain. Western blot analysis revealed tyrosine phosphorylation of multiple membrane proteins in both lens cell types, the differentiated fiber cells and non-differentiated epithelium. When membrane material was subjected to immunoprecipitation using an antibody directed against Na,K-ATPase alpha1, a colocalized phosphotyrosine band was detected in lens fibers but not epithelium. Incubation with PTP-1B caused a approximately 50% increase of Na,K-ATPase activity in fiber membrane material. Na,K-ATPase activity in lens epithelium membrane material was not significantly altered by PTP-1B treatment even though PTP-1B was demonstrated to cause dephosphorylation of multiple membrane proteins in the epithelium as well as fibers. While endogenous PTP-1B was detected in both cell types, endogenous tyrosine phosphatase activity was low in both epithelium and fiber membrane material. The results illustrate endogenous tyrosine phosphorylation of Na,K-ATPase alpha1 polypeptide in fibers. Na,K-ATPase alpha1 in lens fibers may be a potential target for PTP-1B.

The influence of Lyn kinase on Na,K-ATPase in porcine lens epithelium.

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.