Administration of donor-derived transplant acceptance-inducing cells to the recipients of renal transplants from deceased donors is technically feasible.

Patient KW, a 36-yr-old male renal transplant recipient, received transplant acceptance-inducing cells (TAICs) as an adjunct immunosuppressive therapy. In the weeks post-transplantation, the patient's conventional immunosuppressive treatment was gradually minimized, such that, from the 21st wk post-transplantation onwards, the patient was stably maintained on tacrolimus monotherapy. Treatment with TAICs was without complication, both at the time of administration and in the four-yr follow-up period. It is concluded that the production and administration of TAICs to recipients of kidney transplants from deceased donors is technically feasible.

Preoperative treatment of a presensitized kidney transplant recipient with donor-derived transplant acceptance-inducing cells.

This report describes the case of patient FR, a 31-year-old recipient of a living-related kidney transplant from a donor against whom he was presensitized. Seventeen days prior to transplantation, a central venous infusion of transplant acceptance-inducing cells (TAICs) was administered to the patient. During the 27-month follow up, the patient experienced no acute rejection episodes under an immunosuppressive regime comprising anti-thymocyte globulin (ATG) induction, corticosteroids and tacrolimus. In a similar manner to the kidney transplant recipients treated preoperatively with TAICs in a previous study, patient FR achieved a state of donor-specific hypo-responsiveness. Most remarkably, the deliberate preoperative exposure of a sensitized patient to the sensitizing alloantigen did not heighten his response; on the contrary, after TAIC treatment and transplantation, HLA-specific antibodies were no longer detectable. The case of patient FR provides further evidence of the safety of pre-transplantation treatment with TAICs.

Transplant acceptance-inducing cells as an immune-conditioning therapy in renal transplantation.

The transplant acceptance-inducing cell (TAIC) is a type of immunoregulatory macrophage with the capacity to specifically dampen allogeneic rejection responses to a degree allowing safe minimization of conventional immunosuppressive therapy. In the first part of this report, the production and phenotype of the human TAIC is described. In the second part, an analysis is given of the TAIC-I clinical trial, in which 12 recipients of renal transplants from deceased donors were treated with donor-derived TAICs as an adjunct immune-conditioning therapy. Conventional immunosuppression was gradually withdrawn from 10 of these 12 patients over a period of 8 weeks, starting in the fourth week after transplantation. All but two patients tolerated cessation of steroid therapy, while the remaining eight patients were first weaned from sirolimus and then, in six cases, were also weaned to low-dose tacrolimus monotherapy. It is concluded that TAIC therapy is both safe and clinically practicable; however, the TAIC-I trial was unable to provide evidence that postoperative TAIC administration has a beneficial effect.

A cell-based approach to the minimization of immunosuppression in renal transplantation.

Five renal transplant recipients were preoperatively treated with transplant acceptance-inducing cells (TAICs) in a Phase-I safety study of TAICs as an adjunct immune-conditioning therapy in living-donor kidney transplantation. Initially, patients received anti-thymocyte globulin induction therapy in combination with tacrolimus and steroid immunosuppression. Over the course of 12 weeks, steroids were withdrawn and tacrolimus therapy was minimized. Three of the five patients were able to tolerate low-dose tacrolimus monotherapy and one patient was withdrawn from all immunosuppression for over 8 months. No acute or delayed adverse events were associated with the infusion of TAICs. Monitoring of the recipient anti-donor reactivity of TAIC-treated patients in mixed lymphocyte cultures demonstrated that, during periods of clinically stable graft function, recipient T-cell proliferation and cytokine secretion in response to stimulation with donor alloantigen was relatively suppressed. Therefore, although the TAIC-II trial did not provide conclusive evidence of a beneficial effect of preoperative TAIC treatment, the results were encouraging because they suggest that TAICs promote a state of alloantigen-specific unresponsiveness, which might allow safe minimization of pharmacological immunosuppression.

Macrophages driven to a novel state of activation have anti-inflammatory properties in mice.

Recurrent episodes of inflammation underlie numerous pathologies, notably those of inflammatory bowel diseases. In this study, we describe a population of macrophages in a novel state of activation that mitigates colitis in mice. The cells responsible for this effect, called IFN-gamma-stimulated monocyte-derived cells (IFNgamma-MdC), derive from mouse spleen, blood, and bone marrow monocytes and are distinguished from known macrophage populations by mode of generation, cell surface phenotype, and function. IFNgamma-MdC only arise when macrophages are cultivated in the presence of CD40L-expressing CD4+ T cells, M-CSF, and IFN-gamma. IFNgamma-MdC express markers including F4/80, CD11b/c, CD86, and CD274; they are negative for CD4, CD8, Gr1, CD19, CD80, and CD207. Functionally, IFNgamma-MdC are defined by their capacity to enrich cocultured T cell populations for CD4+CD25+Foxp3+ regulatory cells; this enrichment, constituting up to 60% or more of residual lymphocytes, is attributed to an expansion, but also to a cell contact and caspase-dependent depletion of activated T cells. In mice, IFNgamma-MdC delivered i.v. traffic to gut-associated peripheral lymphoid tissues, including the mesenteric lymph nodes, Peyer's patches, and colonic mucosa, and promote the clinical and histological resolution of chronic colitis. We conclude that IFNgamma-MdC represent macrophages in a novel state of activation, possessing multiple T cell-suppressive effects with therapeutic potential for the treatment of autoimmune inflammation.

WOFIE stimulates regulatory T cells: a 2-year follow-up of renal transplant recipients.

BACKGROUND: Initial interruption of immunosuppression for 72 hr was analyzed in renal transplant recipients according to Calne et al.'s "window of opportunity for immunologic engagement" (WOFIE) concept. METHODS: This pilot study was designed as a randomized, open-label, prospective trial of 40 recipients (20 in the WOFIE group, 20 in the control group) of cadaveric kidney transplants who were followed up for 2 years. Immunosuppression comprised tacrolimus (trough levels 5-8 ng/mL), daclizumab (1 mg per kilogram of body weight on day 0 and after 2, 4, 6, and 8 weeks), mycophenolate mofetil (1-2 g/day), and prednisolone (maintenance dose of 10 mg/day). After induction with daclizumab, prednisolone, and mycophenolate mofetil, immunosuppression was interrupted for 72 hr in the WOFIE group. Steroid withdrawal followed in both groups within 12 to 16 weeks posttransplant. RESULTS: Patient and graft survival did not differ significantly between the two cohorts. However, the WOFIE group experienced less acute rejection episodes and developed better graft function. Although all but one of the patients in the WOFIE group successfully discontinued steroid treatment, permanent steroid withdrawal was achieved in only 76.4% of the control group. After daclizumab discontinuation, the WOFIE group demonstrated an increase of CD4CD25 T cells in peripheral blood (P<0.05 vs. control group), which was stable over time and strongly correlated with a significantly higher expression level of Foxp3-mRNA. CONCLUSIONS: Initial interruption of immunosuppression for 72 hr correlates with the induction of regulatory immunologic mechanisms and allows early and reliable minimization of immunosuppressive treatment.

CD45 congenic bone marrow transplantation: evidence for T cell-mediated immunity.

CD45 congenic mice have been used to study stem cell engraftment in the absence of alloreactivity. Recently, impaired engraftment was reported in this model and attributed to weak immune reactivity. We have confirmed that there is indeed low-level reactivity mediated by CD8(+) cells and alpha beta-TCR(+) T cells. B6 (CD45.2) recipients were conditioned with total body irradiation (TBI) and transplanted with increasing doses of B6 (CD45.1) bone marrow cells (BMCs). Although chimerism was present at 1 month in all recipients, durable engraftment did not occur with <150 cGy of TBI, emphasizing the importance of long-term follow-up in evaluating nonmyeloablative conditioning approaches. A single dose of cyclophosphamide on day 2 also significantly enhanced engraftment. When B6 TCR beta(-/-), TCR delta(-/-), or TCR beta(-/-)/delta(-/-) (CD45.2) mice were transplanted with CD45.1 bone marrow, significantly enhanced engraftment occurred in recipients lacking alpha beta-TCR(+) T cells (p < .00005). Similarly, removal of alpha beta-TCR(+) host T cells in wild-type recipients resulted in enhanced engraftment. Engraftment was also significantly increased in CD8(-/-) and CD4(-/-)/8(-/-) recipients (p < .0005). Taken together, these results demonstrate that alpha beta-TCR(+) and CD8(+) T cells play a critical role in regulating engraftment of CD45 congenic marrow and suggest that these cells are the main effector cells in low-level alloreactivity to the CD45 disparity.

A delay in bone marrow transplantation after partial conditioning improves engraftment.

BACKGROUND: In the present study we examined the effect of the timing of marrow infusion on engraftment in nonmyeloablatively conditioned mice. METHODS: B10 mice were conditioned with decreasing doses of total body irradiation (TBI) and reconstituted with bone marrow cells (BMCs) from major histocompatibility complex-disparate donor B10.BR mice at 0 or 6 hr, or on days 1, 2, 3, 4, 5, 8, and 12 with respect to TBI. RESULTS: After undergoing conditioning with 700 cGy TBI and transplantation with 15 x 10(6) BMCs, 100% of recipients engrafted if the marrow was infused between 0 and 4 days after TBI. For lower doses of TBI, a delay in infusion of the marrow after TBI conditioning was associated with a significant increase in engraftment. Significantly less engraftment was achieved in animals conditioned with 600 cGy TBI if the marrow was infused at 0 or 6 hr compared with a 1- to 4-day delay. When the TBI was decreased to 500 cGy, engraftment occurred only when the transplant was performed between days 2 and 8. The highest proportion of recipients engrafted when the marrow was infused on day 4. This enhanced engraftment after a delay in marrow infusion is associated with a significant reduction in host mixed lymphocyte reaction reactivity and is correlated inversely with serum levels of interleukin-6 in the recipient. CONCLUSIONS: These data demonstrate for the first time that a delay between conditioning and marrow infusion significantly improves allogeneic engraftment in nonmyeloablatively conditioned recipients and reduces the total conditioning required.

WOFIE synergizes with calcineurin-inhibitor treatment and early steroid withdrawal in kidney transplantation.

BACKGROUND: According to the window of opportunity for immunologic engagement (WOFIE) concept, as designed by R. Calne, the authors prospectively analyzed the effect of a 72-hr immunosuppressive window on graft function in renal transplant recipients. METHODS: Immunosuppressive therapy comprised tacrolimus (trough levels, 5-8 ng/mL after day 8 posttransplantation), daclizumab (1 mg/kg body weight at day 0 and 2, 4, 6, and 8 weeks posttransplantation), mycophenolate mofetil (MMF) (1-2 g/day), and prednisolone. All patients received an induction therapy including daclizumab, prednisolone, and MMF. WOFIE patients were stopped from immunosuppression for 72 hr posttransplant. Steroids were withdrawn in both groups 12 to 16 weeks after transplantation and dual therapy with MMF and low-dose tacrolimus ensued. RESULTS: Thirty renal transplant recipients (16 in the WOFIE group and 14 in the control group) have been enrolled since May 2000. Patient and graft survival were 93.8% and 87.5%, respectively, in the WOFIE group and 100.0% and 92.9% in the control group, respectively. One patient in the WOFIE group died of cytomegalovirus infection with stable graft function. There were no grafts lost because of acute rejection or primary nonfunction in either group. Patients treated according to the WOFIE protocol revealed less acute rejection episodes during the time of observation (first biopsy-confirmed acute rejection rate 12.5% in the WOFIE group vs. 42.9% in the control cohort). Whereas 92.1% of the WOFIE patients were successfully discontinued from steroids, permanent steroid withdrawal was achieved in only 60% of the control cohort. CONCLUSIONS: Initial interruption of immunosuppression was associated with a decrease of acute graft rejections. Subsequently, the authors postulate a synergistic effect on the immunosuppressive efficiency of calcineurin inhibitors when combined with an initial drug-free window.

Hematopoietic stem cells from the marrow of mice treated with Flt3 ligand are significantly expanded but exhibit reduced engraftment potential.

BACKGROUND: Hematopoietic stem cells (HSC) can be significantly expanded by hematopoietic growth factors. Flt3 ligand (FL) is a hematopoietic growth factor that induces proliferation and mobilization of HSC into the peripheral blood. We previously reported that FL-mobilized HSC exhibit superior engraftment potential. The engraftment potential of FL-expanded HSC in the bone marrow compartment has not been evaluated. In this study, we investigated the effect of in vivo administration of FL on the engraftment potential of HSC expanded in the marrow. METHODS: B10.BR (H-2k) donor mice were treated for 10 days with 10 microg of FL per day. Partially conditioned allogeneic B10 (H-2b) recipients received whole bone marrow. Purified HSC (c-Kit+/Sca1+/lin-) from the marrow were also transplanted in ablated syngeneic B10.BR recipients. RESULTS: FL treatment significantly expanded HSC in the marrow compartment. The absolute number of T cells and granulocytes were unchanged whereas dendritic cells, facilitating cells, and HSC were significantly increased in the bone marrow of donor mice treated with FL compared with untreated mice. Mice conditioned with 700 cGy and transplanted with FL-treated allogeneic bone marrow showed a significantly lower rate of engraftment (14%) compared with recipients of bone marrow from untreated mice (100%). Syngeneic recipients transplanted with 500, 1000, 2000, or 3000 purified HSC from FL-treated donors also showed reduced long-term survival compared with mice transplanted with HSC from untreated donors. Cell cycle analysis revealed that significantly more bone marrow HSC were in cycle after FL treatment compared with unmanipulated controls. CONCLUSION: These data show that FL treatment for 10 days induces proliferation of HSC but reduces the engraftment potential of HSC harvested from the marrow. The reduced syngeneic engraftment of HSC indicates that FL treatment induces intrinsic changes in HSC, resulting in failure of long-term engraftment or self-renewal despite no change in characteristic phenotype of HSC.

CD8(+), alphabeta-TCR(+), and gammadelta-TCR(+) cells in the recipient hematopoietic environment mediate resistance to engraftment of allogeneic donor bone marrow.

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of alphabeta-TCR(+), gammadelta-TCR(+), alphabeta- plus gammadelta-TCR(+), CD8(+), or CD4(+) cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4(+) T cells. Mice lacking both alphabeta- and gammadelta-TCR(+) cells engrafted without conditioning, suggesting that both alphabeta- and gammadelta-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8(-) cell was targeted by the CyP conditioning. The CD8(+) cell effector function is mechanistically different from that for conventional T cells, and independent of CD4(+) T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.