Donor Bone Marrow Derived Macrophage Engraftment into the Central Nervous System of Allogeneic Transplant Patients

Introduction: Hematopoietic stem cell transplantation (HSCT) has had a major impact on the treatment of hematologic malignancies. Recent studies have shown the role HSCT can have in gene therapy by providing long-lived genetically modified cells to treat a variety of human diseases. It is well known that HSC and bone marrow-derived cells can differentiate into long-lived tissue macrophages and populate a wide spectrum of tissues including the brain. These cells are termed bone marrow derived macrophages and are akin to microglial cells in both morphology and function. There is an expanding literature of preclinical animal studies focused on the potential benefits of bone marrow derived-macrophage engraftment into the central nervous system (CNS). In this study we report the detection and characterization of donor bone marrow-derived macrophages in the cerebral cortex of allogeneic transplant patients. Methods: To determine the frequency of donor cell engraftment in post-transplant patients, we selected a cohort of 20 patients who had undergone a sex-mismatched transplant. Formalin fixed paraffin embedded cerebral cortex samples were obtained from the Fred Hutch tissue repository. Samples from male and female autologous transplants were used as controls. Tissue sections were stained by XY fluorescent in situ hybridization (FISH) to identify male and female cells. The XY FISH-stained slides were imaged at 40X magnification on a TissueFAX system. Scanned images were analyzed in blinded fashion using TissueQuest software. Male donor cells were defined by the presence of the Y chromosome within DAPI stained nuclei. Parameters were established using a small area and then applied to a larger area covering 10,000-15,000 cells. Identified donors were confirmed by manual inspection. Adjacent sections were used in Iba1 immunohistochemistry (IHC) studies to quantify the microglia/macrophage population. Select cases were used in double fluorescent Iba1 IHC (tyramide signal amplification)/XY-FISH studies to identify the donor cell type. Results: Intraparenchymal donor bone marrow derived cells were identified in all cerebral cortex sex mismatched samples. To determine the identity of donor cells, select cases were stained with fluorescent tyramide based Iba1 IHC, imaged, stained with XY FISH and re-imaged. The majority of donor cells (>80%) showed strong expression of Iba1, confirming them to be bone marrow-derived macrophages. In parallel Iba1 IHC studies we showed that microglial cells constitute ~12% of the scanned cell population. Thus, when computed as a percentage of the macrophage/microglial population, donor cells from myeloablative transplants range from 4.2-25%. The bone marrow derived cells are stable over time since length of the post-transplant period did not have a major impact on the number of donor cells. Prior animal studies have demonstrated the importance of conditioning (total body irradiation (TBI) or Busulfan) in providing access to the CNS and stimulating engraftment. Consistently, we found that the strength of the conditioning regimen had a significant impact on donor cell engraftment into the CNS. Donor cells in myeloablative cases (>1,000cGy) averaged 8.0% (4.2-14.9%) of microglial cells, while those in non-myeloablative cases (<300cGy) averaged 1.3% (1.2-1.3%). In agreement with preclinical studies, we also noted that myeloablative cases from Busulfan or Treosulfan based conditioning had similar levels of donor-derived cells as cases with TBI myeloablative conditioning, averaging 6.6% (4.4-8.3%) of microglial cells. Although only a limited number of samples were available for analysis, the highest level of donor engraftment was observed in patients who had received 2 separate transplants; on average they comprised 16.3% (12.2-25.1%) of microglial cells. Conclusion: This, the largest study of bone marrow-derived macrophages in post-transplant patients, shows that donor derived cells from myeloablative transplants account for 4.1-25.1% of microglial cells. Donor engraftment is highest following myeloablative conditioning or in patients receiving multiple transplants, and lowest in non-myeloablative cases. Our studies document the magnitude of donor-derived macrophages in the CNS following a bone marrow transplant and serve as a basis for future gene therapy studies targeting neurodegenerative disorders. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The Positive Impact of Donor Bone Marrow Cells Transplantation into Immunoprivileged Compartments on the Survival of Vascularized Skin Allografts

Using the vascularized skin allograft (VSA) model, we compared the tolerogenic effects of different allogeneic bone marrow transplantation (BMT) delivery routes into immunoprivileged compartments under a 7-day protocol immunosuppressive therapy. Twenty-eight fully MHC mismatched VSA transplants were performed between ACI (RT1a) donors and Lewis (RT11) recipients in four groups of seven animals each, under a 7-day protocol of alfa/beta TCRmAb/CsA (alpha/beta-TCR monoclonal antibodies/Cyclosporine A therapy). Donor bone marrow cells (BMC) (100 × 106 cells) were injected into three different immunoprivileged compartments: Group 1: Control, without cellular supportive therapy, Group 2: Intracapsular BMT, Group 3: Intragonadal BMT, Group 4: Intrathecal BMT. In Group 2, BMC were transplanted under the kidney capsule. In Group 3, BMC were transplanted into the right testis between tunica albuginea and seminiferous tubules, and in Group 4, cells were injected intrathecally. The assessment included: skin evaluation for signs and grade of rejection and immunohistochemistry for donor cells engraftment into host lymphoid compartments. Donor-specific chimerism for MHC class I (RT1a) antigens and the presence of CD4+/CD25+ T cells were assessed in the peripheral blood of recipients. The most extended allograft survival, 50–78 days, was observed in Group 4 after intrathecal BMT. The T cells CD4+/CD25+ in the peripheral blood were higher after intrathecal BMC injection than other experimental groups at each post-transplant time point. Transplantation of BMC into immunoprivileged compartments delayed rejection of fully mismatched VSA and induction of robust, donor-specific chimerism.

Inducible Sbds Deletion Impairs Bone Marrow Niche Capacity to Engraft Donor Bone Marrow After Transplantation

Bone marrow (BM) niche-derived signals are critical for facilitating engraftment after hematopoietic stem cell (HSC) transplantation (HSCT). HSCT is required for restoration of hematopoiesis in patients with inherited bone marrow failure syndromes (iBMFS). Shwachman-Diamond syndrome (SDS) is a rare iBMFS associated with mutations in SBDS. Previous studies have demonstrated that SBDS deficiency in osteolineage niche cells causes bone marrow dysfunction that promotes leukemia development. However, it is unknown whether BM niche defects caused by SBDS deficiency also impair efficient engraftment of healthy donor HSC following HSCT, a hypothesis that could explain morbidity seen after clinical HSCT for patients with SDS. Here, we report a mouse model with inducible Sbds deletion in hematopoietic and osteolineage cells. Primary and secondary BM transplantation (BMT) studies demonstrated that SBDS deficiency within BM niches caused poor donor hematopoietic recovery and specifically poor HSC engraftment after myeloablative BMT. We have additionally identified multiple molecular and cellular defects within niche populations that are driven by SBDS deficiency and that are accentuated or develop specifically following myeloablative conditioning. These abnormalities include altered frequencies of multiple niche cell subsets including mesenchymal lineage cells, macrophages and endothelial cells; disruption of growth factor signaling, chemokine pathway activation, and adhesion molecule expression; and p53 pathway activation, and signals involved in cell cycle arrest. Taken together, this study demonstrates that SBDS deficiency profoundly impacts recipient hematopoietic niche function in the setting of HSCT, suggesting that novel therapeutic strategies targeting host niches could improve clinical HSCT outcomes for patients with SDS.

Donor bone marrow-derived macrophage MHC II drives neuroinflammation and altered behaviour during chronic GVHD in mice

Graft-versus-host disease (GVHD) remains the leading cause of non-relapse mortality after allogeneic stem cell transplantation for haematological malignancies. Manifestations of GVHD in the central nervous system (CNS) present as neurocognitive dysfunction in up to 60% of patients, however, the mechanisms driving chronic GVHD in the CNS are yet to be elucidated. Our studies of murine chronic GVHD revealed behavioural deficits associated with broad neuroinflammation and persistent Ifng upregulation. By flow cytometry, we observed a proportional shift in the donor-derived T-cell population in the chronic GVHD brain from early CD8 dominance to later CD4 sequestration. RNA sequencing of the hippocampus identified perturbations to structural and functional synapse-related gene expression, together with the upregulation of genes associated with IFN-γ responses and antigen presentation. Neuroinflammation in the cortex of mice and humans during acute GVHD was recently shown to be mediated by resident microglia-derived TNF. In contrast, infiltration of pro-inflammatory MHC class II+ donor bone marrow-derived macrophages (BMDM) was identified as a distinguishing feature of chronic CNS GVHD. Donor BMDM, which comprised up to 50% of the CNS myeloid population, exhibited a transcriptional signature distinct from resident microglia. Recipients of MHC class II knockout bone marrow grafts exhibited attenuated neuroinflammation and behaviour comparable to controls, suggestive of a critical role of donor BMDM MHC class II expression in CNS chronic GVHD. Our identification of disease mediators distinct from those in the acute phase indicates the necessity to pursue alternative therapeutic targets for late-stage neurological manifestations.

A Prospective Controlled Trial to Evaluate Safety and Efficacy of in vitro Expanded Recipient Regulatory T Cell Therapy and Tocilizumab Together With Donor Bone Marrow Infusion in HLA-Mismatched Living Donor Kidney Transplant Recipients (Trex001)

Background: The induction of donor-specific immunological tolerance could improve outcome after kidney transplantation. However, no tolerance protocol is available for routine clinical use. Chimerism-based regimens hold promise, but their widespread application is impeded in part by unresolved safety issues. This study tests the hypothesis that therapy with polyclonal recipient regulatory T cells (Tregs) and anti-IL6R (tocilizumab) leads to transient chimerism and achieves pro-tolerogenic immunomodulation in kidney transplant recipients also receiving donor bone marrow (BM) without myelosuppressive conditioning of the recipient.Methods/design: A prospective, open-label, controlled, single-center, phase I/IIa academic study is performed in HLA-mismatched living donor kidney transplant recipients.Study group: Recipients of the study group receive in vitro expanded recipient Tregs and a donor bone marrow cell infusion within 3 days after transplantation and tocilizumab for the first 3 weeks post-transplant. In addition they are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Starting 6 months post-transplant, sirolimus and steroids are withdrawn in a step-wise manner in stable patients.Control group: Recipients of the control group are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Co-primary endpoints of safety (impaired graft function [eGFR <35 mL/min/1.73 m2], graft-vs.-host disease or patient death by 12 months) and efficacy (total leukocyte donor chimerism within 28 days post-transplant) are assessed. Secondary endpoints include frequency of biopsy-proven acute rejection episodes and subclinical rejection episodes on surveillance biopsies, assessment of kidney graft function, and the evaluation whether the study protocol leads to detectable changes in the immune system indicative of pro-tolerogenic immune modulation.Discussion: The results of this trial will provide evidence whether treatment with recipient Tregs and donor BM is feasible, safe and efficacious in leading to transient chimerism. If successful, this combination cell therapy has the potential to become a novel treatment option for immunomodulation in organ transplantation without the toxicities associated with myelosuppressive recipient conditioning.Trial registration: European Clinical Trials Database EudraCT Nr 2018-003142-16 and clinicaltrials.gov NCT03867617.