Allogeneic Chimerism Research Articles

Leveraging the lymphohematopoietic graft-versus-host reaction (LGVHR) to achieve allograft tolerance and restore self tolerance with minimal toxicity.

Mixed allogeneic chimerism has considerable potential to advance the achievement of immune tolerance to alloantigens for transplantation and the restoration of self-tolerance in patients with autoimmune disease. In this article, I review evidence that graft-versus-host (GVH) alloreactivity without graft-vs-host disease (GVHD), termed a lymphohematopoietic graft-vs-host reaction (LGVHR), can promote the induction of mixed chimerism with minimal toxicity. LGVHR was originally shown to occur in an animal model when non-tolerant donor lymphocytes were administered to mixed chimeras in the absence of inflammatory stimuli and was found to mediate powerful graft-vs-leukemia/lymphoma effects without GVHD. Recent large animal studies suggest a role for LGVHR in promoting durable mixed chimerism and the demonstration that LGVHR promotes chimerism in human intestinal allograft recipients has led to a pilot study aiming to achieve durable mixed chimerism.

Central tolerance promoted by cell chimerism

Historically, successful allotransplantation was only achieved by utilizing powerful immunosuppressive drugs that were exposing the patient to severe opportunistic infections. The thymus of the transplant recipient renders such therapy obligatory as it constitutively blocks self-reactive T cells while allowing alloreactive T cells to mature and populate the periphery. In 1992, a follow-up study revealed the presence of donor leukocytes in long-term transplant survivors. The stable persistence of recipient and donor leukocytes in the transplanted patient, referred to as “chimerism”, was considered the reason why in some cases it was even possible to stop immunosuppressive treatment without damaging the transplanted organ. Unfortunately, it quickly became evident that stable, persistent allogeneic chimerism was not easily achievable by design. Recently, a novel approach has been identified to help address this clinical gap in knowledge: Cotransplantation of a donor graft with a thymic organoid populated with donor precursor cells generates stable, long-term chimerism in the recipient. In humanized mice, the implantation of thymic organoids, populated with human donor inducible pluripotent stem cell (iPSC)-derived thymic epithelial cells (TECs) and the same donor CD34+ bone marrow precursors, induces tolerance to human leukocyte antigen (HLA)-matched donor tissues/organs. This technology will allow successful allotransplantation of cells/organs even between Major Histocompatibility Complex (MHC)-noncompatible individuals and allow getting rid of immunosuppressive treatments reducing recipient morbidity.

Mixed Chimerism after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with β Thalassemia: Impact on Outcome and Risk Factors

Background: Hematopoietic stem cell transplantation (HSCT) is the most effective curative option for patients with thalassemia major(TM). Early post-transplant mixed chimerism (MC) has known to be a predictor of secondary graft rejection. However, the impact of the persisting mixed chimerism on transplant outcome remains controversial. Recently Thiotepa(TT) has been decreased to reduce toxicity.There was no data available on reduced intensity conditioning on mixed chimersim in a large group of patients with thalassemia to date. Aims: To assess risk factors of mixed chimerism and to evaluate possible correlations between mixed chimerism and the development of complications after HSCT, in terms of graft versus host disease (GVHD) development, OS, TFS and GR. Patients and methods: From December2008 until December 2019, 618 patients with TM underwent HSCT at our center with median age of 6 (1-23) and median follow up time of 70 months(1-135), from HLA-identical sibling donors (SD) (n=212; 34.3%), unrelated donors (UD, n=313; 50.6%),sibling Cord blood(CB, n=49,7.9%)and parent donors (PD, n=44,7.1%). Source of graft were 538 (87.1%) from peripheral stem cells, 32(5.2%) from bone marrow and 48(7.8%)from sibling cord blood. Cy+Bu+Flu+TT+ATG conditioning regimen was used in 416 pts while a reduced conditioning regimen Cy+Bu+Flu+ATG was used for 167pts and Cy+Bu+Flu for the other 35 pts. Results Overall Survival (OS), Thalassemia-Free survival (TFS), Transplantation-related mortality (TRM) and graft rejection (GR) for the entire group were 94.8%, 92.7% ,5.2% and 2.4% respectively. MC was presented in 70 pts with 52 male and 18 female, defined as the presence of >5% residual recipient cells. Cumulative incidence of MC was 12.4% (Figure1). 5 pts had secondary graft failure and another 5 patients persisting MC post-transplant refractory to DLI following a second HSCT. There were two deaths ineffective to DLI and one die of idiopathic pneumonia and one of hemolytic uremic syndrome. The median MC present time was 4 months (1-59 months).No significant difference was observed in OS and Thalassemia-Free survival (TFS) between MC and PC pts. The cumulative probability of OS, TFS between MC pts and FC pts was 97.1% vs. 94.4% (P=0.343) and 89.6% vs. 93.2% (P=0.272). However, the incidence of MC was significantly associated with the development of graft failure/rejection. Graft failure occurred in 5 pts in the MC group vs.7 pts in the full donor chimerism (PC) group, the corresponding incidence were 7.6% vs. 1.6 % respectively. (P =0.001, Figure 2). Acute GVHD incidence was much lower in MC pts than in PC pts (2.9% vs. 12.7%, P=0.022, Figure 3 A), whereas MC pts had significantly higher chronic GVHD(16.8% vs. 4.2%, P=0.000 Figure 3 B). 2 of 70 MC pts developed I-II aGVHD. No MC pts develop II-IV aGVHD. MC patients were unlikely to have severe acute GVHD. MC was associated with an increased risk of chronic GVHD, mostly were DLI derived Patients receiving stem cells from cord blood of matched sibling donors had a high risk of MC.MC incidence of stem cell source from peripheral blood(PB), bone marrow(BM), cord blood (CB)were 10.6%, 10%, 34.2% respectively.(P=0.000, Figure 4). Unrelated donors had a lower risk of MC than sibling donors. Incidence of MC from unrelated donors was 9.5% vs. 16.1% from sibling donors (P=0.007, Figure 5) Intravenous Thiotepa (TT) in combination with Busulfan (Bu) , cyclophosphamide (Cy) anti-thymocyte globulin(ATG) on HSCT could reduce the risk of MC. The corresponding incidence of MC in Cy+Bu+Flu+TT+ATG, Cy+Bu+Flu+ATG, Cy+Bu+Flu conditioning regimen were 9.2%,15.9%,40.9% resepectively(P=0.000, Figure 6). There was also a significant difference in chimerism based on donor-recipient gender pairing (P = 0.041): male grafts into male patients having the greatest risk of MC and female grafts into female patients having the least. Patients who under 9 years old had a higher risk of MC than that of pts>9 years old (P=0.018, Figure 7), Summary: Our results show that patients who presented mixed chimerism showed a similar post-transplant outcome in OS and TFS but was associated with higher risk of graft failue.Patients receiving stem cells from CB had a high risk of MC than from PBSC. Reduced nonmyeloablative approach capable of achieving allogeneic mixed hematopoietic chimerism. Our data suggests that Cy+Bu+Flu+TT+ATG is a preferred conditioning regimenfor TM patients could reduce the risk of mixed chimerism. Disclosures No relevant conflicts of interest to declare.

Single Agent CD45-Targeted Antibody Drug Conjugate Enables Full Mismatch Allogeneic Hematopoietic Stem Cell Transplantation in a Murine HSCT Model

Introduction Allogeneic hematopoietic stem cell transplant (Allo-HSCT) is a potentially curative treatment for malignant and non-malignant blood disorders. However, current conditioning regimens limit the use of this curative procedure in many eligible patients due to regimen-related mortality and morbidities, including organ toxicity, infertility, and secondary malignancies. We are developing novel antibody drug conjugates (ADC) as conditioning agents that can achieve full myeloablation as a single agent that may reduce toxicity associated with current conditioning regimens. We have generated an anti-murine ADC targeting CD45 and assessed its effectiveness as single agent conditioning regimen in a fully allogeneic murine HSCT model. Methods Our tool CD45 ADC is engineered for rapid clearance (t1/2=1.7hr) to enable HSCT after conditioning. A single dose of 3 mg/kg is fully myeloablative in C57BL/6 mice. To determine if the tool CD45-ADC could successfully condition recipients for fully mismatched allo-HSCT, we evaluated the ability of a single dose of 5 mg/kg of the tool CD45-ADC to condition C57BL/6 hosts (H-2b, CD45.2+) for transplant with cells from CByJ.SJL(B6) donors (H-2d, CD45.1+). A matched dose of an isotype ADC (Iso-ADC) was used as a negative control, while 9 Gy TBI was used as a conventional conditioning positive control. Conditioned mice were transplanted with 4x107 whole BM cells, and peripheral blood chimerism was assessed over 22 weeks. At 22 weeks, donor hematopoietic cell chimerism was evaluated in the spleen, bone marrow, and thymus of recipients. Results In the fully mismatched Balb/c → C57Bl/6 allo-HSCT model, conditioning with a single dose of 5 mg/kg of CD45-ADC as a single agent was well tolerated and enabled full allogeneic donor chimerism (n=2 separate experiments). Peripheral blood chimerism was observed in mice conditioned with CD45-ADC at week 4 and maintained through week 22 (Figure 1). Multilineage reconstitution was observed in the T-, B-, and myeloid cell compartments with >90% donor chimerism seen in each compartment, indicative of HSC engraftment. These results were comparable to chimerism seen in the 9 Gy TBI positive control. Treatment with a non-targeting isotype ADC at a matched dose was not effective (Figure 1). For all groups, stem cell chimerism in the bone marrow matched that in the periphery. Splenic and thymic donor immune cell reconstitution was similar between CD45-ADC and TBI conditioning at week 22 (Figure 1), demonstrating that CD45-ADC efficiently depletes host lymphocytes in secondary lymphoid organs while preserving the capacity of the host thymus to support de novo generation of donor-derived T cells after transplantation. Conclusion Conditioning with CD45-ADC was well-tolerated, fully myeloablative, and enabled complete chimerism in a full mismatch allo-HSCT model as a single agent. This targeted, readily translatable approach for safer conditioning could improve the risk-benefit profile for allogenic and haploidentical HSCT and may extend the curative potential of HSCT to more patients suffering from blood cancers and other diseases that may benefit from HSCT. Disclosures Hyzy: Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Proctor:Magenta Therapeutics: Current Employment. Gillard:Magenta Therapeutics: Current Employment. Hammond:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sarma:Magenta Therapeutics: Ended employment in the past 24 months. Clark:Magenta Therapeutics: Current Employment. Bhat:Magenta Therapeutics: Current Employment. Lamothe:Magenta Therapeutics: Current Employment. Palchaudhuri:Magenta Therapeutics: Current Employment. Pearse:Magenta Therapeutics: Ended employment in the past 24 months. McDonagh:Magenta Therapeutics: Ended employment in the past 24 months. Kiem:Magenta Therapeutics: Consultancy; CSL: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees; Umoja: Membership on an entity's Board of Directors or advisory committees; Enochian: Membership on an entity's Board of Directors or advisory committees; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees. Wagner:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding; Magenta Therapeutics: Consultancy, Research Funding; BlueRock: Research Funding; Gadeta: Membership on an entity's Board of Directors or advisory committees. Blazar:Magenta Therapeutics: Consultancy; Fate Therapeutics Inc.: Research Funding; BlueRock Therapeutics: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-founder; BlueRock Therapeuetic: Consultancy. Boitano:Magenta Therapeutics: Ended employment in the past 24 months, Patents & Royalties. Cooke:Magenta Therapeutics: Ended employment in the past 24 months, Patents & Royalties. Davis:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Genetic profile considerations for induction of allogeneic chimerism as a therapeutic approach for type 1 diabetes mellitus

The major therapeutic modality for type 1 diabetes mellitus (T1DM) remains sustaining euglycemia by exogenous administration of insulin. Based on a new understanding of bone marrow structural and functional dynamics, a conditioning-free bone marrow transplantation (BMT), with reduced adverse effects, opens the possibility for evaluating β cell regeneration and restoration of euglycemia by induction of allogeneic chimerism in patients T1DM, as shown in a mouse model. With this therapeutic modality, donor bone marrow (BM) selection based on T1DM-predisposing and preventive phenotypes will improve treatment outcomes by limiting the risk of exacerbating the autoimmune processes in the BM recipient.

Stability of Chimerism in Non-Obese Diabetic Mice Achieved By Rapid T Cell Depletion Is Associated With High Levels of Donor Cells Very Early After Transplant.

Stable mixed hematopoietic chimerism is a robust method for inducing donor-specific tolerance with the potential to prevent rejection of donor islets in recipients with autoimmune type-1 diabetes. However, with reduced intensity conditioning, fully allogeneic chimerism in a tolerance resistant autoimmune-prone non-obese diabetic (NOD) recipient has rarely been successful. In this setting, successful multilineage chimerism has required either partial major histocompatability complex matching, mega doses of bone marrow, or conditioning approaches that are not currently clinically feasible. Irradiation free protocols with moderate bone marrow doses have not generated full tolerance; donor skin grafts were rejected. We tested whether more efficient recipient T cell depletion would generate a more robust tolerance. We show that a combination of donor-specific transfusion-cyclophosphamide and multiple T cell depleting antibodies could induce stable high levels of fully allogeneic chimerism in NOD recipients. Less effective T cell depletion was associated with instability of chimerism. Stable chimeras appeared fully donor-specific tolerant, with clonal deletion of allospecific T cells and acceptance of donor skin grafts, while recovering substantial immunocompetence. The loss of chimerism months after transplant was significantly associated with a lower level of chimerism and donor T cells within the first 2 weeks after transplant. Thus, rapid and robust recipient T cell depletion allows for stable high levels of fully allogeneic chimerism and robust donor-specific tolerance in the stringent NOD model while using a clinically feasible protocol. In addition, these findings open the possibility of identifying recipients whose chimerism will later fail, stratifying patients for early intervention.

Effect of Ex Vivo-Expanded Recipient Regulatory T Cells on Hematopoietic Chimerism and Kidney Allograft Tolerance Across MHC Barriers in Cynomolgus Macaques.

Infusion of recipient regulatory T (Treg) cells promotes durable mixed hematopoietic chimerism and allograft tolerance in mice receiving allogeneic bone marrow transplant (BMT) with minimal conditioning. We applied this strategy in a Cynomolgus macaque model. CD4 CD25 Treg cells that were polyclonally expanded in culture were highly suppressive in vitro and maintained high expression of FoxP3. Eight monkeys underwent nonmyeloablative conditioning and major histocompatibility complex mismatched BMT with or without Treg cell infusion. Renal transplantation (from the same BMT donor) was performed 4 months post-BMT without immunosuppression to assess for robust donor-specific tolerance. Transient mixed chimerism, without significant T cell chimerism, was achieved in the animals that received BMT without Treg cells (N = 3). In contrast, 2 of 5 recipients of Treg cell BMT that were evaluable displayed chimerism in all lineages, including T cells, for up to 335 days post-BMT. Importantly, in the animal that survived long-term, greater than 90% of donor T cells were CD45RA CD31, suggesting they were new thymic emigrants. In this animal, the delayed (to 4 months) donor kidney graft was accepted more than 294 days without immunosuppression, whereas non-Treg cell BMT recipients rejected delayed donor kidneys within 3 to 4 weeks. Early CMV reactivation and treatment was associated with early failure of chimerism, regardless of Treg cell administration. Our studies provide proof-of-principle that, in the absence of early CMV reactivation (and BM-toxic antiviral therapy), cotransplantation of host Treg cell can promote prolonged and high levels of multilineage allogeneic chimerism and robust tolerance to the donor.

Enhanced Allogeneic Engraftment Achieved Via Prenatal Tolerance Induction and Postnatal Anti-CD45 Immunotoxin Conditioned Hematopoietic Cell Transplantation

INTRODUCTION: In utero hematopoietic cell transplantation (IUHCT) is a non-myeloablative, non-immunosuppressive transplant approach that takes advantage of the immunologic immaturity of the fetus to engraft donor cells across immune barriers and induce donor specific immune tolerance (DST). Though it has the potential to treat many congenital hematologic disorders, IUHCT is limited by levels of engraftment below those anticipated to be therapeutic for most target diseases, including sickle cell anemia. Thus, a likely initial clinical application of IUHCT is to induce DST to allow for non-myeloablative, non-immunosuppressive postnatal "booster" transplants to increase engraftment. We have previously validated this approach in murine sickle cell, beta-thalassemia, and non-diseased models using low dose total body irradiation and busulfan prior to postnatal bone marrow transplantation (BMT). Although encouraging, less toxic approaches that specifically target the hematopoietic system would make IUHCT + postnatal BMT more applicable. CD45 is a membrane glycoprotein expressed by all leukocytes and hematopoietic stem cells. Recent studies have demonstrated enhanced engraftment in a congenic postnatal transplant mouse model following pre-transplant conditioning with an anti-CD45-saporin (SAP) immunotoxin (Palchaudhuri et al., Nat Biotech 2016; 34:738-45). We hypothesized that mixed allogeneic chimerism achieved by IUHCT can be enhanced after birth by conditioning with an anti-CD45-SAP immunotoxin followed by a same-donor allogeneic BMT.METHODS: 5x106 BM mononuclear cells from C57Bl/6 mice (H2Kb, CD45.2+) were injected intravenously into gestational day 14 (E14) Balb/c fetuses (H2Kd, CD45.2+). Donor cell peripheral blood (PB) chimerism was confirmed at 4 weeks of age, at which time chimeric mice were injected via tail vein with PBS (control) or CD45.2-SAP. Six days following treatment, chimeric animals were intravenously injected with 30x106 B6Pep3b (H2Kb, CD45.1+) BM cells. Additional control groups consisted of recipients of IUHCT alone without a postnatal BMT and naïve 5 week-old Balb/c mice which did not undergo IUHCT and only received CD45.2-SAP + postnatal BMT. Following postnatal BMT, PB was assessed bi-weekly by flow cytometry for total donor chimerism and the contribution of prenatal and postnatal donor cells to engraftment. Mice were weighed before each bleeding and evaluated for signs of GVHD. Statistical comparison was performed using the Student’s t-test for 2 samples at each time point assuming unequal variances.RESULTS: There was no significant difference in initial donor cell chimerism levels at 4 weeks of age between treatment (IUHCT+CD45.2-SAP+BMT, 12.4±2.2%, n=5) and control (IUHCT alone, 9.3±2.7%, n=18; IUHCT+BMT, 10.0±2.6%, n=7) groups prior to therapy. In contrast to naïve non-chimeric Balb/c mice undergoing a postnatal BMT (n=10) which demonstrated no donor cell engraftment, recipients of IUHCT and a postnatal BMT (both PBS and CD45.2-SAP treated groups) demonstrated postnatal donor cell engraftment, supporting DST achieved by IUHCT. While postnatal BMT after IUHCT failed to demonstrate a significant enhancement of engraftment compared to IUHCT alone (4.4±1.3% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p = 0.7), low-level allogeneic chimerism following IUHCT was significantly enhanced to high-level chimerism following conditioning with CD45.2-SAP+BMT (55.7±9.8% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p=0.0001) (Fig. 1). The majority of donor cell chimerism resulted from the postnatal donor population in recipients of CD45.2-SAP, supporting a mechanism of enhanced engraftment via a competitive advantage following recipient CD45.2-SAP treatment (Fig. 2). Chimerism was stable and multilineage at 22 weeks indicating enhanced stem cell/early progenitor engraftment. All mice demonstrated appropriate weight gain without GVHD.CONCLUSIONS: Prenatal tolerance induction achieved by IUHCT followed by postnatal CD45-SAP conditioning and same-donor BMT results in enhanced stable multilineage allogeneic engraftment at therapeutic levels for many target diseases. This work supports a potentially less toxic approach for the strategy of prenatal tolerance induction to facilitate non-myeloablative, non-immunosuppressive postnatal BMT and has the potential to significantly expand the applicability of IUHCT. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Enhanced in utero allogeneic engraftment in mice after mobilizing fetal HSCs by α4β1/7 inhibition

AbstractIn utero hematopoietic cell transplantation (IUHCT) is a novel nonmyeloablative approach that results in donor-specific tolerance and mixed allogeneic chimerism. Clinical application is limited by low levels of donor cell engraftment. Competition from endogenous hematopoietic stem cells (HSCs) for limited “space” in fetal hematopoietic organs remains a significant barrier to successful IUHCT. AMD3100, a CXCR4 inhibitor, and firategrast, an α4β1 and α4β7 integrin inhibitor (α4β1/7), have been shown to disrupt HSC retention in the postnatal hematopoietic niche. We hypothesized that maternal administration of AMD3100 and/or firategrast prior to IUHCT would mobilize endogenous HSCs from the fetal liver (FL) and result in preferential FL homing of donor HSCs and enhanced long-term engraftment following IUHCT in an allogeneic mouse model. We demonstrate that (1) both agents cross the placenta with rapidly detectable fetal serum concentrations following maternal administration; (2) firategrast treatment alone or with AMD3100 mobilizes endogenous HSCs from the FL and results in increased FL homing of donor HSCs following IUHCT; and (3) enhanced donor HSC homing following firategrast treatment translates into increased long-term multilineage donor cell engraftment. This approach highlights the potential of mobilization strategies to overcome barriers to successful engraftment and increase the clinical promise of IUHCT.

Impact of Mixed Xenogeneic Porcine Hematopoietic Chimerism on Human NK Cell Recognition in a Humanized Mouse Model.

Mixed chimerism is a promising approach to inducing allograft and xenograft tolerance. Mixed allogeneic and xenogeneic chimerism in mouse models induced specific tolerance and global hyporesponsiveness, respectively, of host mouse natural killer (NK) cells. In this study, we investigated whether pig/human mixed chimerism could tolerize human NK cells in a humanized mouse model. Our results showed no impact of induced human NK cell reconstitution on porcine chimerism. NK cells from most pig/human mixed chimeric mice showed either specifically decreased cytotoxicity to pig cells or global hyporesponsiveness in an in vitro cytotoxicity assay. Mixed xenogeneic chimerism did not hamper the maturation of human NK cells but was associated with an alteration in NK cell subset distribution and interferon gamma (IFN-γ) production in the bone marrow. In summary, we demonstrate that mixed xenogeneic chimerism induces human NK cell hyporesponsiveness to pig cells. Our results support the use of this approach to inducing xenogeneic tolerance in the clinical setting. However, additional approaches are required to improve the efficacy of tolerance induction while ensuring adequate NK cell functions.

Correction of murine hemoglobinopathies by prenatal tolerance induction and postnatal nonmyeloablative allogeneic BM transplants

AbstractSickle cell disease (SCD) and thalassemias (Thal) are common congenital disorders, which can be diagnosed early in gestation and result in significant morbidity and mortality. Hematopoietic stem cell transplantation, the only curative therapy for SCD and Thal, is limited by the absence of matched donors and treatment-related toxicities. In utero hematopoietic stem cell transplantation (IUHCT) is a novel nonmyeloablative transplant approach that takes advantage of the immunologic immaturity and normal developmental properties of the fetus to achieve mixed allogeneic chimerism and donor-specific tolerance (DST). We hypothesized that a combined strategy of IUHCT to induce DST, followed by postnatal nonmyeloablative same donor “booster” bone marrow (BM) transplants in murine models of SCD and Thal would result in high levels of allogeneic engraftment and donor hemoglobin (Hb) expression with subsequent phenotypic correction of SCD and Thal. Our results show that: (1) IUHCT is associated with DST and low levels of allogeneic engraftment in the murine SCD and Thal models; (2) low-level chimerism following IUHCT can be enhanced to high-level chimerism and near complete Hb replacement with normal donor Hb with this postnatal “boosting” strategy; and (3) high-level chimerism following IUHCT and postnatal “boosting” results in phenotypic correction in the murine Thal and SCD models. This study supports the potential of IUHCT, combined with a postnatal nonmyelablative “boosting” strategy, to cure Thal and SCD without the toxic conditioning currently required for postnatal transplant regimens while expanding the eligible transplant patient population due to the lack of a restricted donor pool.

Repeated Injections of IL-2 Break Renal Allograft Tolerance Induced via Mixed Hematopoietic Chimerism in Monkeys.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function.

Immune Reconstitution Kinetics following Intentionally Induced Mixed Chimerism by Nonmyeloablative Transplantation.

Establishing mixed chimerism is a promising approach for inducing donor-specific transplant tolerance. The establishment and maintenance of mixed chimerism may enable long-term engraftment of organ transplants while minimizing the use of immunosuppressants. Several protocols for inducing mixed chimerism have been reported; however, the exact mechanism underlying the development of immune tolerance remains to be elucidated. Therefore, understanding the kinetics of engraftment during early post-transplant period may provide insight into establishing long-term mixed chimerism and permanent transplant tolerance. In this study, we intentionally induced allogeneic mixed chimerism using a nonmyeloablative regimen by host natural killer (NK) cell depletion and T cell-depleted bone marrow (BM) grafts in a major histocompatibility complex (MHC)-mismatched murine model and analyzed the kinetics of donor (C57BL/6) and recipient (BALB/c) engraftment in the weeks following transplantation. Donor BM cells were well engrafted and stabilized without graft-versus-host disease (GVHD) as early as one week post-bone marrow transplantation (BMT). Donor-derived thymic T cells were reconstituted four weeks after BMT; however, the emergence of newly developed T cells was more obvious at the periphery as early as two weeks after BMT. Also, the emergence and changes in ratio of recipient- and donor-derived NKT cells and antigen presenting cells (APCs) including dendritic cells (DCs) and B cells were noted after BMT. Here, we report a longitudinal analysis of the development of donor- and recipient-originated hematopoietic cells in various lymphatic tissues of intentionally induced mixed chimerism mouse model during early post-transplant period. Through the understanding of immune reconstitution at early time points after nonmyeloablative BMT, we suggest guidelines on intentionally inducing durable mixed chimerism.

Suppression of lentivirus-mediated transgenic dendritic cells in graft-versus-host disease after allogeneic bone marrow transplantation in mice.

We determined whether genetically engineered immature dendritic cells (imDCs) mediated by lentiviral vectors alleviate acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT) in mice. We introduced the mouse chemokine receptor 7 (Ccr7) gene into the bone marrow-derived imDCs of C57BL/6 mice to construct genetically engineered imDCs. A 1:1 mixture of bone marrow and spleen cells from the donors was injected into the recipients, which were divided into four groups: radiation, transplantation, empty vector, and transgenic imDC groups. Symptoms, clinical scores, GVHD pathological changes, and survival times and rates of recipients were recorded; secretion of IFN-γ and IL-4, and allogeneic chimerism rates were detected. The survival time of the transgenic imDC group (27.5 ± 7.55 days) was significantly longer than in the other three groups (P < 0.01). The GVHD score of the imDC group mice was significantly lower than in the transplantation and empty vector groups (P < 0.05), which meant that mice in the transgenic imDC group had the lightest pathology damage in the target organs. In the transplantation group, IFN-γ increased while IL-4 decreased. In contrast, IFN-γ decreased and IL-4 increased in both empty vector and trans-imDC groups, and the difference was significant in the latter (P < 0.01). Thirty days or more following transplantation, the allogeneic chimerism rate was still 95-100%, suggesting complete donor type implantation. Ccr7 transfection into imDCs suppressed occurrence and severity of acute GVHD after allo-BMT in mice; the mechanism might be associated with IFN-γ decrease and IL-4 increase.

Differential Development of Donor Bone Marrow-Derived Thymocytes after Allogeneic in Utero Hematopoietic Cell Transplantation in the Murine Model

IntroductionIn Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been determined. In this study, we utilized the murine model of allogeneic IUHCT to analyze donor and host thymocyte development in the context of normal immune ontogeny.MethodsBone marrow (BM) cells (10x106) from C57/BL6 (B6, H2kb) mice were injected intravenously into Balb/c (H2kd) fetuses at embryonic day 14 (E14). E14 B6 fetuses injected with GFP B6 BM were used as congeneic controls. At indicated postnatal ages the thymocytes were delineated by multi-color flow cytometry. Cell apoptosis and proliferation were determined by Annexin V staining and in vivo BrdU incorporation, respectively. T cell alloreactivity was assessed by in vitro and in vivo MLR.ResultsOur findings demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocyte processing and from thymocyte development in normal control mice. While the phenotypic development of host thymocytes remained comparable with that of normal control Balb/c mice, the four major subsets of donor thymocytes showed altered distribution, with significantly higher proportions of single positive (SP) cells, and a dramatically lower proportion of CD4+CD8+ double positive (DP) cells, compared to their host-derived counterparts and B6 controls. The extent of the alteration is directly related to both BM chimerism levels and age. Higher levels of chimerism and/or older age are associated with more profound alterations in donor thymocyte distribution. Moreover, DP cells of donor origin show higher apoptosis and lower proliferation than those of the host. Donor TCR gamma/delta cells in DN cells which do not require positive selection based on MHC recognition are relatively increased compared to the host. Moreover, compared with the naive mice and congeneic chimeric mice, the donor BM-derived thymocytes in the allogeneic chimeric mice show increased proportion of DN3 and decreased proportion of DN4, but increased TCRβ+ proportions in both DN3 and DN4 cells, indicating that donor BM-derived thymocyte development is impeded during DN to DP transition, resulting from a MHC-restriction associated mechanism. In addition, in allogeneic chimeric mice, both host and donor BM-derived T cells are tolerant to allogeneic antigens in in vivo and in vitro MLR.ConclusionOur data suggests that in an allogeneic IUHCT system the immune reconstitution of the donor bone marrow-derived thymocytes differs from that of the host cells and that of normal mice. The data supports a mechanism of impaired MHC based positive selection of donor cells by the predominantly host thymic stroma resulting in lack of progression of a higher proportion of donor cells from the DN to DP stage of thymocyte development.Taken together, although donor BM-derived T cells undergo differential thymic development, permanent host-donor two-way tolerance could be achieved in the allogeneic IUHCT mouse model. These findings add to our understanding of the requirements for tolerance induction after IUHCT and have important clinical implications in choosing an optimal donor for IUHCT. DisclosuresNo relevant conflicts of interest to declare.

Quo vadis chimerism?

Although immunity in multicellular organisms is efficient in dealing with alien agents, it may fail for allogeneic chimerism. Natural chimerism is widely documented in nature, distributed in at least ten phyla of protists, invertebrates and plants, vertebrates and mammals, including humans; it is an important ecological/evolutionary tool manipulating metazoans' life history portraits. Instead of purging allogeneic nascent selfish cells, a 'double edged sword' chimerism emerges, displaying environmental dictated costs and benefits for the genotypes involved. Benefits include the development of synergistic complementation, the increase of genetic variability, the assurance of mate location, improved size-dependent ecological qualities (growth rates, reproduction, survivorship, competition, environmental tolerance) and more. Costs include the threat of somatic and germ cell parasitism, developmental instability, death, diseases, autoimmunity, sexual sterility and organ malformations, which develop as well in mammalian natural chimerism, including humans. Because of its importance, medical sciences should study and harness natural chimerism properties for clinical purposes.

Donor Specific Tolerance Of Renal Allografts Following Haploidentical In Utero Hematopoietic Cell Transplantation In The Canine Model

IntroductionIn utero hematopoietic cell transplantation (IUHCT) can result in allogeneic mixed hematopoietic chimerism and associated donor specific tolerance (DST). We have reported a 1-2% donor chimerism threshold for consistent DST in the murine model as defined by the ability to enhance engraftment postnatally with a same donor minimal conditioning BMT, and have confirmed this finding in the canine model of IUHCT. Recently, we have optimized the canine model and achieved higher levels of chimerism (average>10%). As prenatal tolerance induction for postnatal organ transplantation is one of the clinical goals of IUHCT, we hypothesized that the presence of donor chimerism (>2.0%) after haploidentical IUHCT in the canine model would be sufficient to allow same donor renal transplants without immunosuppression. MethodStable mixed hematopoietic macrochimerism was documented by VNTR after haploidentical IUHCT performed at 40 days gestation using maternal donor BM cells in 4 pups (chimerism levels 3-38%). One positive control canine with no detectable chimerism following IUHCT also underwent haploidentical renal transplantation. Renal transplantation was performed from the maternal donor at ages between 12 and 18 months, and the pups were serially followed by ultrasound of the graft, blood chemistry and urinalysis post transplant. At 60 days an open biopsy of the allograft was taken and at 6 months a graft nephrectomy was performed for histologic analysis. ResultsFollowing transplantation, all recipients demonstrated blood flow to the renal cortex and all laboratory values were within normal ranges. At 60 days and 6 months 3 of the 4 recipients demonstrated a graft without evidence of acute or chronic rejection. The recipient with the lowest level of chimerism (3%) demonstrated evidence of mild interstitial nephritis (Banff class 1 rejection) following transplantation at the time of renal biopsy which increased in severity to severe interstitial fibrosis and moderate tubular atrophy (Banff class 3 rejection) at the time of graft nephrectomy. The positive control canine without detectable chimerism demonstrated clinical and histologic evidence of acute rejection within one week of transplantation. ConclusionOur results support the ability of IUHCT to induce DST for haploidentical organ transplantation, in a large animal model, without the need for immunosuppression. In agreement with previous studies in the murine model there appears to be a threshold level of donor chimerism required for associated DST. Although our numbers are not adequate to establish absolute thresholds of chimerism predictive of DST for organ transplantation, it appears to be slightly higher than the 1-2% threshold established for cellular transplantation. Disclosures:No relevant conflicts of interest to declare.

Differential Development Of Donor Bone Marrow-Derived Thymocytes After Allogeneic In Utero Hematopoietic Cell Transplantation In The Murine Model

Introduction In Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been determined. In this study, we utilized the murine model of allogeneic IUHCT to analyze donor and host thymocyte development during postnatal ontogeny. Methods Bone marrow (BM) cells (10x106) from Foxp3GFP C57/BL6 (B6, H2kb) mice were injected intravenously into Foxp3GFP Balb/c (H2kd) fetuses at embryonic day 14 (E14). At indicated postnatal ages the thymocytes were delineated by multi-color flow cytometry. Cell apoptosis and proliferation were determined by Annexin V staining and in vivo BrdU incorporation, respectively. Results Our findings demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocyte processing and from thymocyte development in normal control mice. While the phenotypic development of host thymocytes remained comparable with that of normal control Balb/c mice, the four major subsets of donor thymocytes showed altered distribution, with significantly higher proportions of immature TCRβ-CD4+CD8- and TCRβ-CD4-CD8+ single positive (SP) cells, a slightly higher proportion of CD4-CD8- (DN) cells, and a dramatically lower proportion of CD4+CD8+ double positive (DP) cells, compared to their host-derived counterparts and B6 controls. The extent of the alteration is directly related to both BM chimerism levels and age. Higher levels of chimerism and/or older age are associated with more profound alterations in donor thymocyte distribution. Moreover, DP cells of donor origin show higher apoptosis and lower proliferation than those of the host. Donor DN cells and TCR gamma/delta cells which do not require positive selection based on MHC recognition are increased compared to the host. Donor T-regulatory cells were present at a higher frequency in the thymus and secondary lymphoid organs of chimeric animals compared to T-regulatory cells in host and normal strain controls. Conclusion Our data suggests that in an allogeneic IUHCT system the immune reconstitution of the donor bone marrow-derived thymocytes differs from that of the host cells and that of normal mice. The data supports a mechanism of impaired MHC based positive selection of donor cells by the predominantly host thymic stroma resulting in lack of progression of a higher proportion of donor cells from the DN to DP stage of thymocyte development. In contrast, generation of donor T-regulatory cells was not impaired and appeared to be increased in chimeric animals. The alloreactivity, regulatory suppression, and MHC restriction of the donor derived T cells, and the potential importance of these observations for clinical IUHCT remain to be determined. Disclosures: No relevant conflicts of interest to declare.

Humoral Immunity Induced By Viral Infection Provides a Major Barrier To Hematopoietic Cell Transplantation

BackgroundSensitization to MHC antigens because of transfusion, pregnancy, and previous grafts is among the most critical challenges to clinical transplantation. The presence of preformed donor-specific antibody increases the risk for bone marrow and solid organ graft rejection. It is well documented that some microbial molecules and microorganisms can directly induce the proliferation and differentiation of antibody-secreting cells from different B cells, regardless of their antigen specificity. The presence of a strong polyclonal B cell response resulting in hypergammaglobulinemia secretion has been described in several animal models of infection with viruses, parasites, and bacteria. Recent experiments in our lab showed that pathogen infection in naïve animals induces activation of naïve allospecific B cell and formation of de novo alloantibody. This prompted us to analyze the effect of pathogen infection induced alloantibody on hematopoietic cell transplantation. Herein, these studies show that preformed donor specific antibody induced by pathogen infection in the absence of donor-antigen is a major barrier to bone marrow engraftment. Methods/results: LCMV infection is a model system of viral infection and also an established system to examine virus nonspecific, polyclonal hypergammaglobulinemia. Naïve C57BL/6 (H2b) mice were infected with acute and chronic strain of LCMV (Armstrong and clone 13, n=10 each). Virally infected mice were bled weekly to analyze total IgG and formation of de novo alloantibody in the serum. Viral infection induced both hypergammaglobulinemia and the formation of de novo alloantibody (anti-H2d). Peak levels of donor-specific antibody in acute and chronically infected mice were increased to approximate 3 fold when compared to uninfected controls as assessed by flow cytometric crossmatch. The results were consistent with formation of anti-H2d antibody when measured by anti-H2d ELISA with peak level of approximately 336.3 ± 51.84 units/ml when compared to 2.179 ± 0.4597 units/ml in uninfected mice (p<0.0001, Figure 1). Interestingly, the response generated by chronic infection was relatively sustained (up to 75 days) when compared to the transient response induced by acute infection.Next we analyzed if virally induced preformed de novo alloantibody would represent a barrier to bone marrow engraftment. Naïve C57Bl/6 mice were irradiated with 950 rads and received 15x106BALB/c bone marrow. BALB/c bone marrow cells were either incubated ex vivo with 1:6 diluted sera from naïve B6 mice or with the sera from clone 13 infected mice (d28 post-infection). After incubation, bone marrow was washed and complement inactivated prior to infusion. All of the 10 mice receiving bone marrow incubated with naïve mice serum engrafted and survived, while only 1/10 mice survived receiving bone marrow incubated with serum from infected mice (Mean survival = >100 vs 24.5 days respectively, p < 0.0067, figure 2).We also examined the effects of virally induced preformed de novo alloantibody in a costimulation blockade based regimen to induce mixed allogeneic chimerism and transplant tolerance. Naïve C57BL/6 mice received 30x106 BALB/c bone marrow infusion along with costimulation blockade treatment (250ug each of CTLA4-Ig and anti-CD40L (MR1) at 0,2,4,6 days post transplant) after nonirradiation-based conditioning with busulfan (-3,-2 days. 20mg/kg IP injection). BALB/c bone marrow cells were either incubated ex vivo with 1:6 diluted sera from naïve B6 mice or with sera from clone 13 infected mice (d28 post-infection) as described above. Consistent with the experiments using the irradiation based conditioning regimen, mice receiving bone marrow incubated with naïve mice serum engrafted and established stable donor chimerism while none of mice receiving bone marrow incubated with serum from infected mice showed any donor cell engraftment Discussion:Allogeneic hematopoetic cell is used as therapy for hematologic malignancies and for non-malignant hematologic diseases. Our results indicate that preformed donor specific antibody induced by viral infection in the absence of previous exposure to donor-antigen is a major barrier to bone marrow engraftment. These results suggest that an individual's prior immune history to pathogens may significantly impact subsequent humoral responses upon bone marrow transplantation. Disclosures:No relevant conflicts of interest to declare.

Toward minimal conditioning protocols for allogeneic chimerism in tolerance resistant recipients

Mixed chimerism is a promising approach toward generating donor-specific immunological tolerance. However, chimerism induction can be toxic; therefore, there is an effort to develop non-myeloablative, minimal intensity protocols that can generate chimerism without the toxic side effects. Recently, with the goal of creating a minimalistic chimerism induction protocol in the tolerance resistant non-obese diabetic (NOD) mouse model, we identified pre-existing T cells as cells that resist fully allogeneic chimerism. With monoclonals targeting NOD T cells, we showed that long-term chimerism and tolerance toward donor islets could be established. However, this promising new protocol relied on the administration of a single dose of anti-CD40 ligand, which is not clinically applicable. In refining protocols to move even closer to clinical utility, we report here initial success at generating fully allogeneic mixed chimerism in NOD mice by adding cyclophosphamide to the conditioning regimen in place of anti-CD40 ligand antibodies.