Immune tolerance via FCR001 cell therapy compared with maintenance immunosuppression for kidney transplantation: Real-world evidence analysis of safety and efficacy.

While kidney transplantation (KTx) has traditionally required lifelong immunosuppression, an investigational stem cell therapy, FCR001, has been demonstrated to induce tolerance and eliminate the need for immunosuppression through the establishment of persistent mixed chimerism in a phase 2 clinical study. Real-world evidence (RWE) methods were employed to compare the safety and efficacy of non-myeloablative conditioning with FCR001 with standard of care [SOC] immunosuppression in a retrospective single-center analysis of outcomes among propensity score matched living-donor KTx receiving SOC (n = 144) or FCR001 (n = 36). Among the FCR001 recipients, 26 (72%) developed persistent chimerism allowing durable elimination of all immunosuppression. There was no significant difference in the composite primary endpoint (biopsy-proven acute rejection [BPAR], graft loss, or death) at 60 months (FCR001 27.8%, n = 10 and SOC 28.5%, n = 41; p = .9). FCR001 recipients demonstrated superior kidney function at 5 years (estimated glomerular filtration rate [eGFR] [mean ± standard deviation]: 64.1 ± 15.3) compared to SOC (51.7 ± 18.8; p = .02). At 5 years, FCR001 recipients experienced fewer complications including new-onset diabetes post-transplant, although two patients developed graft versus host disease. In conclusion, RWE demonstrated that KTx combined with non-myeloablative conditioning and FCR001 resulting in superior kidney function without increasing the risk of rejection, graft loss, or death among patients off immunosuppression.

Fms-Like Tyrosine Kinase 3-Ligand Contributes to the Development and Function of the Subpopulation of CD8α+ Plasmacytoid Precursor Dendritic Cells in CD8+ /TCR- Facilitating Cells.

Facilitating cells (FC) are a CD8+ TCR- bone marrow subpopulation that enhance engraftment of purified hematopoietic stem cells (HSC) and induce antigen-specific CD4+ CD25+ FoxP3+ regulatory T cell (Treg) in vivo. The major subpopulation in FC resembles plasmacytoid precursor dendritic cells (p-preDC) both phenotypically and functionally. Here, we report that the number of FC was significantly reduced in Fms-like tyrosine kinase 3-ligand-knockout (Flt3-L-KO) mice. Specifically, there was a selective decrease in the B220+ CD11c+ CD11b- p-preDC FC subpopulation. The p-preDC FC subpopulation in FC total is restored after Flt3-L administration to Flt3-L-KO mice. FC from Flt3-L-KO donors exhibit impaired facilitation of allogeneic HSC engraftment in ablatively conditioned mice (B6 → NOD) as well as in mice conditioned with reduced intensity conditioning (B6 → BALB/c). In addition, the number of CD4+ CD25+ Foxp3+ Treg from Flt3-L-KO mice is significantly decreased. This was associated with the expression of chemokine receptor CXCR3+ or CCR5+ on Treg. Treg from the spleen of Flt3-L-KO mice showed impaired facilitation of engraftment of allogeneic HSC compared to wild-type Treg. Flt3-L treatment significantly expanded Treg, and restored their facilitating function. These results suggest that Flt3-L is an important growth factor in the development and homeostasis of p-preDC FC and in the role of FC inducing generation of Treg. Flt3-L provides potent immunoregulatory properties that may be clinically useful to improve tolerance induction and enhance the function of allogeneic cell therapies. Stem Cells 2018;36:1567-1577.

Intragraft Molecular Pathways Associated with Tolerance Induction in Renal Transplantation.

The modern immunosuppression regimen has greatly improved short-term allograft outcomes but not long-term allograft survival. Complications associated with immunosuppression, specifically nephrotoxicity and infection risk, significantly affect graft and patient survival. Inducing and understanding pathways underlying clinical tolerance after transplantation are, therefore, necessary. We previously showed full donor chimerism and immunosuppression withdrawal in highly mismatched allograft recipients using a bioengineered stem cell product (FCRx). Here, we evaluated the gene expression and microRNA expression profiles in renal biopsy samples from tolerance-induced FCRx recipients, paired donor organs before implant, and subjects under standard immunosuppression (SIS) without rejection and with acute rejection. Unlike allograft samples showing acute rejection, samples from FCRx recipients did not show upregulation of T cell- and B cell-mediated rejection pathways. Gene expression pathways differed slightly between FCRx samples and the paired preimplantation donor organ samples, but most of the functional gene networks overlapped. Notably, compared with SIS samples, FCRx samples showed upregulation of genes involved in pathways, like B cell receptor signaling. Additionally, prediction analysis showed inhibition of proinflammatory regulators and activation of anti-inflammatory pathways in FCRx samples. Furthermore, integrative analyses (microRNA and gene expression profiling from the same biopsy sample) identified the induction of regulators with demonstrated roles in the downregulation of inflammatory pathways and maintenance of tissue homeostasis in tolerance-induced FCRx samples compared with SIS samples. This pilot study highlights the utility of molecular intragraft evaluation of pathways related to FCRx-induced tolerance and the use of integrative analyses for identifying upstream regulators of the affected downstream molecular pathways.

Facilitating cells: role in inducing transplantation tolerance.

PURPOSE OF REVIEW: This review discusses the role and mechanisms by which facilitating cells promote stem cell engraftment and induce tolerance in HLA-disparate kidney transplant recipients. RECENT FINDING: Facilitating cells in both mice and human are heterogeneous, consisting of several subpopulations. They have been shown to enhance stem cell engraftment in allogeneic recipients. They also increase hematopoietic stem cells (HSC) clonogenicity, enhance migration and homing of stem cells via secretion of cytokines/chemokines/growth factors, prevent apoptosis of stem cells and induce regulatory cells. This review summarizes the findings that led to the development of chimerism-based induction of tolerance using FCRx (a mobilized blood product enriched in stem cells and facilitating cells) in allogenic kidney transplant patients. SUMMARY: A phase-2 clinical trial based on FCRx therapy has been successful in inducing tolerance to living donor kidney allografts, leading to withdrawal of immunosuppression in over 70% of patients transplanted. The ultimate goal of establishing tolerance in the absence of immunosuppresive drugs can be achieved using FCRx therapy.

Leukocyte iNOS is required for inflammation and pathological remodeling in ischemic heart failure.

In the failing heart, iNOS is expressed by both macrophages and cardiomyocytes. We hypothesized that inflammatory cell-localized iNOS exacerbates left ventricular (LV) remodeling. Wild-type (WT) C57BL/6 mice underwent total body irradiation and reconstitution with bone marrow from iNOS-/- mice (iNOS-/-c) or WT mice (WTc). Chimeric mice underwent coronary ligation to induce large infarction and ischemic heart failure (HF), or sham surgery. After 28 days, as compared with WTc sham mice, WTc HF mice exhibited significant (p < 0.05) mortality, LV dysfunction, hypertrophy, fibrosis, oxidative/nitrative stress, inflammatory activation, and iNOS upregulation. These mice also exhibited a ~twofold increase in circulating Ly6Chi pro-inflammatory monocytes, and ~sevenfold higher cardiac M1 macrophages, which were primarily CCR2- cells. In contrast, as compared with WTc HF mice, iNOS-/-c HF mice exhibited significantly improved survival, LV function, hypertrophy, fibrosis, oxidative/nitrative stress, and inflammatory activation, without differences in overall cardiac iNOS expression. Moreover, iNOS-/-c HF mice exhibited lower circulating Ly6Chi monocytes, and augmented cardiac M2 macrophages, but with greater infiltrating monocyte-derived CCR2+ macrophages vs. WTc HF mice. Lastly, upon cell-to-cell contact with naïve cardiomyocytes, peritoneal macrophages from WT HF mice depressed contraction, and augmented cardiomyocyte oxygen free radicals and peroxynitrite. These effects were not observed upon contact with macrophages from iNOS-/- HF mice. We conclude that leukocyte iNOS is obligatory for local and systemic inflammatory activation and cardiac remodeling in ischemic HF. Activated macrophages in HF may directly induce cardiomyocyte contractile dysfunction and oxidant stress upon cell-to-cell contact; this juxtacrine response requires macrophage-localized iNOS.

DOCK2 is critical for CD8(+) TCR(-) graft facilitating cells to enhance engraftment of hematopoietic stem and progenitor cells.

CD8(+) TCR(-) graft facilitating cells (FCs) enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic and syngeneic recipients. The mechanisms by which FCs promote HSC engraftment and tolerance induction have not been fully elucidated. Here, we provide data to support a critical role for dedicator of cytokinesis 2 (DOCK2) in multiple aspects of FCs function. DOCK2(-/-) FCs exhibit compromised facilitative function in vivo as evidenced by the loss of engraftment-enhancing capability for c-Kit(+) Sca-1(+) lineage(-) (KSL) cells, and compromised ability to promote KSL cell homing and lodgment in hematopoietic niche. Deletion of DOCK2 abrogates the ability of FCs to induce differentiation of naïve CD4(+) CD25(-) T cells into FoxP3(+) regulatory T cells and interleukin-10-producing type 1 regulatory T cells in vitro. Moreover, DOCK2(-/-) FCs are unable to promote survival of KSL cells when cocultured with KSL cells. DOCK2(-/-) FCs also exhibit compromised migration to stroma-derived factor-1 in vitro and impaired homing to the bone marrow in vivo. In conclusion, our results demonstrate that DOCK2 is critical for FCs to maintain its immunomodulatory function and exert its trophic effects on KSL cells. These findings may have direct clinical relevance to promote HSC engraftment for treatment of autoimmunity, hemoglobinopathies, and to induce transplantation tolerance.

Facilitating cells in tolerance induction for kidney transplantation.

PURPOSE OF REVIEW: To describe the clinical outcomes and science behind a CD8/TCR facilitating cell-based hematopoietic stem cell transplant approach (termed FCRx) to induce tolerance to renal allografts without graft-versus-host disease (GVHD) and avoidance of long-term immunosuppressant drugs in living donor kidney transplant recipients. RECENT FINDINGS: Successful solid organ transplantation currently requires the life-long use of medications to suppress the immune system to prevent transplant rejection. Drug-based immunosuppression significantly increases the risk of infection and cancer, as well as being very costly. Development of new therapies to minimize or eliminate entirely the need for antirejection drugs is of great interest to the transplant community. Therapeutic cell transfer for the control of the human immune system represents a compelling approach to reduce or eliminate the need for antirejection drugs. SUMMARY: Establishment of durable hematopoietic macrochimerism under nonmyeloablative conditioning is achievable in mismatched recipients using facilitating cells and stem cells obtained from donor mobilized peripheral blood mononuclear cells. Persistently chimeric recipients developed donor-specific tolerance and were weaned off of immunosuppressive drugs over 12 months. They maintained stable renal function without development of acute or chronic GVHD.

HLA molecular mismatches and induced donor-specific tolerance in combined living donor kidney and hematopoietic stem cell transplantation.

Introduction: We investigated the potential role of HLA molecular mismatches (MM) in achieving stable chimerism, allowing for donor-specific tolerance in patients undergoing combined living donor kidney and hematopoietic stem cell transplantation (HSCT). Methods: All patients with available DNA samples (N=32) who participated in a phase 2 clinical trial (NCT00498160) where they received an HLA mismatched co-transplantation of living donor kidney and facilitating cell-enriched HSCT were included in this study. High-resolution HLA genotyping data were used to calculate HLA amino acid mismatches (AAMM), Eplet MM, three-dimensional electrostatic mismatch scores (EMS-3D), PIRCHE scores, HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence MM, and KIR ligands MM between the donor and recipient in both directions. HLA MM were analyzed to test for correlation with the development of chimerism, graft vs. host disease (GvHD), de novo DSA, and graft rejection. Results: Follow-up time of this cohort was 6-13.5 years. Of the 32 patients, 26 developed high-level donor or mixed stable chimerism, followed by complete withdrawal of immunosuppression (IS) in 25 patients. The remaining six of the 32 patients had transient chimerism or no engraftment and were maintained on IS (On-IS). In host versus graft direction, a trend toward higher median number of HLA-DRB1 MM scores was seen in patients On-IS compared to patients with high-level donor/mixed chimerism, using any of the HLA MM modalities; however, initial statistical significance was observed only for the EMS-3D score (0.45 [IQR, 0.30-0.61] vs. 0.24 [IQR, 0.18-0.36], respectively; p=0.036), which was lost when applying the Bonferroni correction. No statistically significant differences between the two groups were observed for AAMM, EMS-3D, Eplet MM, and PIRCHE-II scores calculated in graft versus host direction. No associations were found between development of chimerism and GvHD and non-permissive HLA-DPB1 T-cell epitope group MM, HLA-B leader sequence, and KIR ligands MM. Conclusion: Our results suggest an association between HLA-DRB1 molecular mismatches and achieving stable chimerism, particularly when electrostatic quality of the mismatch is considered. The non-permissive HLA-DPB1 T-cell epitope group, HLA-B leader sequence, and KIR ligands MM do not predict chimerism and GvHD in this combined kidney/HSCT transplant patient cohort. Further work is needed to validate our findings. Clinical trial registration: https://clinicaltrials.gov/study/NCT00498160, identifier NCT00498160.

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.

Evaluation of Immunocompetence and Biomarkers of Tolerance in Chimeric and Immunosuppression-free Kidney Allograft Recipients.

BACKGROUND: Thirty-seven patients have received a living-donor kidney transplant in a phase 2 study designed to induce tolerance with facilitated allogeneic hematopoietic stem cell transplant. The study protocol is based on tolerogenic CD8 + /T-cell receptor - facilitating cells (FCR001; also including hematopoietic stem cells and αß-T-cell receptor + T cells) and low-dose, nonmyeloablative conditioning. Persistent chimerism allowing full immunosuppression (IS) withdrawal was achieved in 26 patients (time off IS 36-123 mo). METHODS: We evaluated biomarkers of tolerance through urinary cell mRNA profiling and immunocompetence to respond to vaccination in these patients. We also assessed kidney function and metabolic parameters compared with standard-of-care patients on IS. RESULTS: Persistently chimeric patients retained chimerism after removal of IS and remained rejection free without donor HLA-specific antibody development. The presence of donor chimerism at >50% correlated with a signature of tolerance in urinary cell mRNA profiles, with a uniquely elevated increase in the ratio of cytotoxic T lymphocyte-associated protein 4 to granzyme B mRNA. Tolerance was associated with protection from recurrence of immune-mediated causes of kidney disease. Tolerant participants were safely vaccinated, developed protective immune responses, and did not lose chimerism after vaccination. When compared with kidney transplant recipients treated with standard IS, tolerant participants showed stable kidney function and reduced medication use for hypertension and hyperlipidemia. CONCLUSIONS: These results suggest that elimination of IS has distinct advantages in living-donor kidney allograft recipients.

Novel regulatory therapies for prevention of Graft-versus-host disease.

Graft-versus-host disease is one of the major transplant-related complications in allogeneic hematopoietic stem cell transplantation. Continued efforts have been made to prevent the occurrence of severe graft-versus-host disease by eliminating or suppressing donor-derived effector T cells. Conventional immunosuppression does not adequately prevent graft-versus-host disease, especially in mismatched transplants. Unfortunately, elimination of donor-derived T cells impairs stem cell engraftment, and delays immunologic reconstitution, rendering the recipient susceptible to post-transplant infections and disease relapse, with potentially lethal consequences. In this review, we discuss the role of dynamic immune regulation in controlling graft-versus-host disease, and how cell-based therapies are being developed using regulatory T cells and other tolerogenic cells for the prevention and treatment of graft-versus-host disease. In addition, advances in the design of cytoreductive conditioning regimens to selectively target graft-versus-host disease-inducing donor-derived T cells that have improved the safety of allogeneic stem cell transplantation are reviewed. Finally, we discuss advances in our understanding of the tolerogenic facilitating cell population, a phenotypically and functionally distinct population of bone marrow-derived cells which promote hematopoietic stem cell engraftment while reducing the risk of graft-versus-host disease.

Identification of inducible nitric oxide synthase in peripheral blood cells as a mediator of myocardial ischemia/reperfusion injury.

Although the late phase of ischemic preconditioning is known to be mediated by increased inducible nitric oxide synthase (iNOS) activity, controversy persists regarding the role of iNOS in ischemia/reperfusion (I/R) injury and, specifically, whether this protein is protective or detrimental. We hypothesized that iNOS is protective in myocytes but detrimental in inflammatory cells. To test this hypothesis, we created chimeric mice with iNOS-deficient peripheral blood cells by transplanting iNOS knockout (KO) bone marrow in wild-type (WT) mice after lethal irradiation. 2 months later, the mice underwent a 30-min coronary occlusion followed by 24 h of reperfusion. In WT naïve mice (iNOS(+/+) naïve; group I, n = 17), infarct size was 56.9 ± 2.8% of the risk region. In iNOS KO naïve mice with whole-body iNOS deletion (iNOS(-/-) naïve; group II, n = 10), infarct size was comparable to group I (53.4 ± 3.5%). When irradiated WT mice received marrow from WT mice (iNOS(+/+) chimera; group III, n = 10), infarct size was slightly reduced versus group I (44.3 ± 3.2%), indicating that irradiation and/or transplantation slightly decrease I/R injury. However, when WT mice received marrow from iNOS KO mice (iNOS(-/-) chimera; group IV, n = 14), infarct size was profoundly reduced (22.8 ± 2.1%, P < 0.05 vs. group III), indicating that selective deletion of iNOS from peripheral blood cells (with no change in myocardial iNOS content) induces protection against myocardial infarction. Together with our previous work showing the cardioprotective actions of NO donors, iNOS gene therapy, and cardiac-specific overexpression of iNOS, these data support a complex, dual role of iNOS in myocardial infarction (i.e., protective in myocytes but deleterious in blood cells). To our knowledge, this is the first study to identify a critical role of iNOS in peripheral blood cells as a mediator of myocardial I/R injury. The results support heretofore unknown differential actions of iNOS depending on cell source and have important translational implications.

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.

Plasmacytoid precursor dendritic cells facilitate allogeneic hematopoietic stem cell engraftment.

Bone marrow transplantation offers great promise for treating a number of disease states. However, the widespread application of this approach is dependent upon the development of less toxic methods to establish chimerism and avoid graft-versus-host disease (GVHD). CD8+/TCR- facilitating cells (FCs) have been shown to enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic recipients without causing GVHD. In the present studies, we have identified the main subpopulation of FCs as plasmacytoid precursor dendritic cells (p-preDCs). FCs and p-preDCs share many phenotypic, morphological, and functional features: both produce IFN-alpha and TNF-alpha, both are activated by toll-like receptor (TLR)-9 ligand (CpG ODN) stimulation, and both expand and mature after Flt3 ligand (FL) treatment. FL-mobilized FCs, most of which express a preDC phenotype, significantly enhance engraftment of HSCs and induce donor-specific tolerance to skin allografts. However, p-preDCs alone or p-preDCs from the FC population facilitate HSC engraftment less efficiently than total FCs. Moreover, FCs depleted of preDCs completely fail to facilitate HSC engraftment. These results are the first to define a direct functional role for p-preDCs in HSC engraftment, and also suggest that p-preDCs need to be in a certain state of maturation/activation to be fully functional.

Plasmacytoid dendritic cells regulate autoreactive B cell activation via soluble factors and in a cell-to-cell contact manner.

Plasmacytoid dendritic cells (pDCs) are specialized type I IFN producers, which play an important role in pathogenesis of autoimmune disorders. Dysregulated autoreactive B cell activation is a hallmark in most autoimmune diseases. This study was undertaken to investigate interactions between pDCs and autoreactive B cells. After coculture of autoreactive B cells that recognize self-Ag small nuclear ribonucleoprotein particles with activated pDCs, we found that pDCs significantly enhance autoreactive B cell proliferation, autoantibody production, and survival in response to TLR and BCR stimulation. Neutralization of IFN-alpha/beta and IL-6 abrogated partially pDC-mediated enhancement of autoreactive B cell activation. Transwell studies demonstrated that pDCs could provide activation signals to autoreactive B cells via a cell-to-cell contact manner. The involvement of the ICAM-1-LFA-1 pathway was revealed as contributing to this effect. This in vitro enhancement effect was further demonstrated by an in vivo B cell adoptive transfer experiment, which showed that autoreactive B cell proliferation and activation were significantly decreased in MyD88-deficient mice compared with wild-type mice. These data suggest the dynamic interplay between pDCs and B cells is required for full activation of autoreactive B cells upon TLR or BCR stimulation.

Strategic nonmyeloablative conditioning: CD154:CD40 costimulatory blockade at primary bone marrow transplantation promotes engraftment for secondary bone marrow transplantation after engraftment failure.

There is an increased risk of failure of engraftment following nonmyeloablative conditioning. Sensitization resulting from failed bone marrow transplantation (BMT) remains a major challenge for secondary BMT. Approaches to allow successful retransplantation would have significant benefits for BMT candidates living with chronic diseases. We used a mouse model to investigate the effect of preparative regimens at primary BMT on outcome for secondary BMT. We found that conditioning with TBI or recipient T cell lymphodepletion at primary BMT did not promote successful secondary BMT. In striking contrast, successful secondary BMT could be achieved in mice conditioned with anti-CD154 costimulatory molecule blockade at first BMT. Blockade of CD154 alone or combined with T cell depletion inhibits generation of the humoral immune response after primary BMT, as evidenced by abrogation of production of anti-donor Abs. The humoral barrier is dominant in sensitization resulting from failed BMT, because almost all CFSE-labeled donor cells were killed at 0.5 and 3 h in sensitized recipients in in vivo cytotoxicity assay, reflecting Ab-mediated cytotoxicity. CD154:CD40 costimulatory blockade used at primary BMT promotes allogeneic engraftment in secondary BMT after engraftment failure at first BMT. The prevention of generation of anti-donor Abs at primary BMT is critical for successful secondary BMT.

The beneficial effects of postinfarct cytokine combination therapy are sustained during long-term follow-up.

We have previously reported that administration of granulocyte colony-stimulating factor (G-CSF)+Flt-3 ligand (FL) or G-CSF+stem cell factor (SCF) improves left ventricular (LV) function and halts LV remodeling at 35 d after myocardial infarction (MI). In the current study, we investigated whether these beneficial effects are sustained in the long term - an issue of fundamental importance for clinical translation. Mice undergoing a 30-min coronary occlusion followed by reperfusion received vehicle (group I), G-CSF+FL (group II), G-CSF+SCF (group III), or G-CSF alone (group IV) starting 4 h after reperfusion and were euthanized 48 wk later. LV structure and function were assessed by serial echocardiography before and at 48 h and 4, 8, 16, 32, and 48 wk after MI. During follow-up, mice in group I exhibited worsening of LV function and progressive LV remodeling. Compared with group I, both groups II and III exhibited improved LV EF at 4 wk after MI; however, only in group II was this improvement sustained at 48 wk. Group II was also the only group in which the decrease in infarct wall thickening fraction, the LV dilatation, and the increase in LV mass were attenuated vs. group I. We conclude that the beneficial effect of G-CSF+FL on postinfarction LV dysfunction and remodeling is sustained for at least 11 months, and thus is likely to be permanent. In contrast, the effect of G-CSF+SCF was not sustained beyond the first few weeks, and G-CSF alone is ineffective. To our knowledge, this is the first long-term study of cytokines in postinfarction LV remodeling. The results reveal heretofore unknown differential actions of cytokines and have important translational implications.

FL/GCSF/AMD3100-mobilized Hematopoietic Stem Cells Induce Mixed Chimerism With Nonmyeloablative Conditioning and Transplantation Tolerance.

BACKGROUND: Mobilization of hematopoietic stem cells (HSCs) has become the preferred approach for HSC transplantation. AMD3100, a competitive inhibitor of C-X-C motif chemokine receptor-4, has been found to be a rapid mobilizing agent. The present study evaluated approaches to optimize the product collected. METHODS: Mobilized peripheral blood mononuclear cells (mPBMCs) from B6 mice were transplanted to recipient BALB/c mice conditioned with ablative or nonmyeloablative approaches. RESULTS: The optimal dose of AMD3100 was found to be 5.0 mg/kg. Optimal HSC mobilization was observed when AMD3100 (day 10) was coadministered with Flt3 ligand (FL) (days 1-10) and granulocyte colony-stimulating factor (GCSF) (days 4-10). There was a 228.8-fold increase of HSC with FL/GCSF/AMD3100 compared with AMD3100 treatment alone. When unmodified mPBMCs were transplanted into ablated allogeneic recipients, all recipients expired by day 40 from severe acute graft versus host disease (GVHD). When T cells were depleted from mPBMC, long-term survival and engraftment were achieved in majority of the recipients. When PBMC mobilized by FL/GCSF/AMD3100 were transplanted into recipients conditioned nonmyeloablatively with anti-CD154/rapamycin plus 100, 200, and 300 cGy of total body irradiation, 42.9%, 85.7%, and 100% of mice engrafted, respectively. Donor chimerism was durable, multilineage, and stable. Lymphocytes from mixed chimeras showed no response to host or donor antigens, suggesting functional bidirection T-cell tolerance in vitro. Most importantly, none of the engrafted mice exhibited clinical features of GVHD. CONCLUSIONS: FL/GCSF/AMD3100 is an efficient treatment to maximally mobilize HSC. Durable engraftment and donor-specific tolerance can be achieved with mPBMC in nonmyeloablative conditioning without GVHD.

Fms-related tyrosine kinase 3 expression discriminates hematopoietic stem cells subpopulations with differing engraftment-potential: identifying the most potent combination.

BACKGROUND: Fms-related tyrosine kinase 3 (Flt3)-ligand (FL) promotes the proliferation, differentiation, development, and mobilization of hematopoietic cells. We previously found that FL-mobilized hematopoietic stem cells (HSC) engraft efficiently, whereas FL-expanded bone marrow HSC do not. The function of FL-mobilized c-Kit(+) Sca-1(+)Lin(-)(KSL) subpopulations has not been systematically evaluated. A precise definition of the repopulating ability is needed to define which HSC subpopulations are critical for long-term chimerism and tolerance induction. FL significantly mobilized c-Kit(hi) and c-Kit(lo) Sca-1(+)Lin(-) cells into peripheral blood (PB). Here, we evaluated the influence of Flt3 expression on long-term repopulating ability of HSC subpopulations. METHODS: c-Kit(hi) or c-Kit(lo) KSL cells were sorted from PB of FL-treated green fluorescent protein-positive donors. The function of these cells was evaluated using competitive reconstitution assays, colony-forming units spleen, and colony forming cell assays. The function of c-Kit(hi) CD34(-)Flt3(-) KSL, c-Kit CD34(+)Flt3(-) KSL, c-Kit(hi) CD34(+)Flt3(+) KSL were investigated in an in vivo transplantation model. RESULTS: Only FL-mobilized PB c-Kit(hi) KSL cells exhibited high spleen colony-forming unit activity, generated high numbers of both lymphoid and myeloid colonies in vitro, and rescued ablated recipients. FL-mobilization expanded both c-Kit(hi) CD34(+)Flt3(-) cells (short-term HSC) and c-Kit(hi) CD34(-)Flt3(-) KSL cells (long-term HSC). There was a significant decrease in c-Kit CD34Flt3 KSL late multipotent progenitors in PB. A combination of c-Kit(hi) CD34Flt3 and c-Kit CD34(+)Flt3(-) KSL cells offered the most effective rescue of ablated recipients. CONCLUSIONS: These data suggest that engraftment of purified HSC is influenced by both short- and long-term repopulating populations and that Flt3 expression may be useful for selecting the most critical HSC subpopulations for transplantation.

Postinfarct cytokine therapy regenerates cardiac tissue and improves left ventricular function.

We systematically investigated the comparative efficacy of three different cytokine regimens, administered after a reperfused myocardial infarction, in regenerating cardiac tissue and improving left ventricular (LV) function. Wild-type (WT) mice underwent a 30-minute coronary occlusion followed by reperfusion and received vehicle, granulocyte colony-stimulating factor (G-CSF)+Flt-3 ligand (FL), G-CSF+stem cell factor (SCF), or G-CSF alone starting 4 hours after reperfusion. In separate experiments, chimeric mice generated by reconstitution of radioablated WT mice with bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice underwent identical protocols. Mice were euthanized 5 weeks later. Echocardiographically, LV function was improved in G-CSF+FL- and G-CSF+SCF-treated but not in G-CSF-treated mice, whereas LV end-diastolic dimensions were smaller in all three groups. Morphometrically, cytokine-treated hearts had smaller LV diameter and volume. Numerous EGFP-positive cardiomyocytes, capillaries, and arterioles were noted in the infarcted region in cytokine-treated chimeric mice treated with G-CSF+FL or G-CSF+SCF, but the numbers were much smaller in G-CSF-treated mice. G-CSF+FL therapy mobilized bone marrow-derived cells exhibiting increased expression of surface antigens (CD62L and CD11a) that facilitate homing. We conclude that postinfarct cytokine therapy with G-CSF+FL or G-CSF+SCF limits adverse LV remodeling and improves LV performance by promoting cardiac regeneration and probably also by exerting other beneficial actions unrelated to regeneration, and that G-CSF alone is less effective.