Primed Bone Marrow Research Articles

Abstract 102: Trained Immunity Impairs Inflammation Resolution And Dampens Efferocytosis

Chronic inflammation is a major driver of atherosclerotic cardiovascular disease, and therapeutics that target inflammation reduce clinical cardiac events beyond levels seen with conventional strategies targeting cholesterol alone. Recent findings suggest innate immune cells maintain ‘memory’ of prior exposure to inflammatory stimuli, a phenomenon known as ‘trained immunity’. In response to inflammatory stimuli, macrophages undergo metabolic and epigenetic rewiring that primes them to mount an augmented response upon a second exposure. Oxidized low-density lipoproteins (oxLDL) have recently been shown to be potent triggers of trained immunity. While trained immunity has been shown to promote inflammation, little is known about how immune training impacts efferocytosis. Therefore, we hypothesize that trained immunity in macrophages promotes inflammation by impairing efferocytosis. We treated murine bone marrow progenitors with oxLDL for 24 hours, then washed and differentiated them into macrophages (BMDMs). Upon assessing efferocytosis, trained BMDMs were able to ingest a first apoptotic cell (AC) better than untrained BMDMs yet had an impaired ability to take up additional ACs, reflecting a defect in continual efferocytosis. Using an in vivo approach, we transplanted donor bone marrow from Ldlr -/- mice fed a chow or Western diet into naïve C57BL/6 recipients. After recovery, we elicited peritoneal macrophages to assess efferocytosis and found that recipients receiving marrow from Western diet fed Ldlr -/- mice not only displayed impaired efferocytosis, but also significantly upregulated PGE 2 production, suggesting impaired resolution. To determine whether PGE 2 is a mediator of trained immunity, we primed bone marrow progenitors with PGE 2 and differentiated them into BMDMs. We found that macrophages primed with PGE 2 elaborated higher levels of inflammatory cytokines in response to LPS stimulation than controls. Overall, these findings demonstrate that oxLDL/Western diet-training impinges on the resolution program by impairing efferocytosis and are durable effects that demonstrate heritability. Future directions include determining the impact of these findings on the development of atherosclerosis.

Abstract WP317: Phagocytic Potentials of Purinergic Receptor P2x4 Blockade After Ischemic Stroke

Backgrounds: We previously showed that genetic deletion as well as short term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides neuroprotection by reducing acute excessive inflammation and thus can be potential drug targets to treat ischemic stroke. However, the mechanism how P2X4R blockade provide long term benefits are not known. Here we hypothesize that P2X4R blockade increase phagocytic uptake of damaged or dead tissue and facilitate swift recovery. Methods: We used both 1μm size fluorescent phagocytic bead (Polyscience Inc) and pHrodo Red zymosan A Bioparticles conjugate (Thermofisher) to study in vitro and ex-vivo phagocytic uptake by LPS (200 μg/ml for 2 hours) primed Bone Marrow Derived Macrophages (BMDMs) from P2X4R KO and littermate WT mice. We also used flow sorted Ly6C hi and low monocytes for phagocytic uptake study obtained from perilesional ipsilateral mouse brain tissue of P2X4R KO and littermate WT mice after 3 or 7 days of stroke. The later mice were subjected to 60 mins of transient middle cerebral artery occlusion. Results: Both phagocytic beads and pHrodo bioparticle uptake study suggest significantly higher uptake (1.5 and 2-fold P<0.05 vs WT student t test n=4 mice/group x 2 technical replication) by BMDMs obtained from P2X4R KO mice. FACS sorted infiltrated Ly6Chi inflammatory monocytes from global P2X4R KO mice brain after 3 days of stroke show significantly (*P>0.05 vs WT) high phagocytic bead uptake as compared to WT control. Further these monocytes also showed elevated expression of CD36, a marker for scavenger receptor at 7 days after stroke. Conclusions: This study suggest that P2X4R blockade provide swift and sustained neuroprotection by increasing phagocytic uptake of damage tissue after ischemic stroke.

Bile acids induce IL-1α and drive NLRP3 inflammasome-independent production of IL-1β in murine dendritic cells.

Bile acids are amphipathic molecules that are synthesized from cholesterol in the liver and facilitate intestinal absorption of lipids and nutrients. They are released into the small intestine upon ingestion of a meal where intestinal bacteria can modify primary into secondary bile acids. Bile acids are cytotoxic at high concentrations and have been associated with inflammatory diseases such as liver inflammation and Barrett's Oesophagus. Although bile acids induce pro-inflammatory signalling, their role in inducing innate immune cytokines and inflammation has not been fully explored to date. Here we demonstrate that the bile acids, deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) induce IL-1α and IL-1β secretion in vitro in primed bone marrow derived dendritic cells (BMDCs). The secretion of IL-1β was found not to require expression of NLRP3, ASC or caspase-1 activity; we can't rule out all inflammasomes. Furthermore, DCA and CDCA were shown to induce the recruitment of neutrophils and monocytes to the site of injection an intraperitoneal model of inflammation. This study further underlines a mechanistic role for bile acids in the pathogenesis of inflammatory diseases through stimulating the production of pro-inflammatory cytokines and recruitment of innate immune cells.

Bone Marrow Quality Index: A Predictor of Acute Graft-versus-Host Disease in Hematopoietic Stem Cell Transplantation for Thalassemia

Bone marrow continues to be the preferred source of stem cells in allogenic transplantations for non-malignant disorders. Granulocyte-colony stimulating factor (G-CSF)-primed bone marrow (BM) is associated with low rates of acute GVHD and allows reduced collection volumes while ensuring speedy engraftment. However, variability in bone marrow harvest quality is a concern. This report analyses the utility of a novel indicator - Bone Marrow Quality Index (BMQI), which is a ratio of marrow cell count to peripheral blood cell counts, to predict acute GVHD. We analysed 184 consecutive first matched related donor bone marrow transplants for thalassemia using G-CSF-primed bone marrow over 6 years from March-2017 to April-2023 across 2 centres in India. Bone Marrow Quality Index (BMQI) was defined as the ratio of WBC count of G-CSF primed bone marrow to Peripheral WBC count within 24 hours of harvest. EBMT criteria were used for grading GVHD. Log-rank test was used to assess the impact of BMQI on GVHD. Chi-squared test was used to compare categorical data and Wilcoxon test was used to compare the numeric data. Cox proportional-hazards model was used to investigate the association of BMQI vis-à-vis other factors on acute GVHD. Of the 184 patients, 19 had a BMQI <0.9 and 18 between 0.9 and 1. The remaining 147 had BMQI >1. The proportion of patients with acute GVHD grade II-IV was 37%, 22% and 12% respectively for patients who received BMQI <0.9, 0.9-1 and >1 (p= 0.018). Patients who received BMQI <0.9 had a 3.1 times higher chance (95% CI being 0.9 to 10.6) of developing aGVHD II-IV and in those with BMQI 0.9-1 this was 2 times (95% CI being 0.5 to 6.6) higher. BMQI was the significant predictor associated with GVHD hazard (p=0.014). BMQI seems to be the most relevant and controllable predictor of acute GVHD. BMQI is a novel, informative, and very simple indicator which could influence GVHD prophylaxis decision-making. Our indicator is accurately measurable, inexpensive, precise, and timely; further it does not involve any sophisticated equipment, thus may be widely applicable. Prior knowledge of poor marrow quality may help intensify prophylaxis and monitoring for acute GVHD besides triggering review of collection procedures.

Efficient manufacturing and engraftment of CCR5 gene-edited HSPCs following busulfan conditioning in nonhuman primates

Hematopoietic stem cell gene therapy has been successfully used for a number of genetic diseases and is also being explored for HIV. However, toxicity of the conditioning regimens has been a major concern. Here we compared current conditioning approaches in a clinically relevant nonhuman primate model. We first customized various aspects of the therapeutic approach, including mobilization and cell collection protocols, conditioning regimens that support engraftment with minimal collateral damage, and cell manufacturing and infusing schema that reflect and build on current clinical approaches. Through a series of iterative invivo experiments in two macaque species, we show that busulfan conditioning significantly spares lymphocytes and maintains a superior immune response to mucosal challenge with simian/human immunodeficiency virus, compared to total body irradiation and melphalan regimens. Comparative mobilization experiments demonstrate higher cell yield relative to our historical standard, primed bone marrow and engraftment of CRISPR-edited hematopoietic stem and progenitor cells (HSPCs) after busulfan conditioning. Our findings establish a detailed workflow for preclinical HSPC gene therapy studies in the nonhuman primate model, which in turn will support testing of novel conditioning regimens and more advanced HSPC gene editing techniques tailored to any disease of interest.

231 - Safety and Graft Characteristics of G-CSF Primed Bone Marrow in Donors with Sickle Cell Trait

231 - Safety and Graft Characteristics of G-CSF Primed Bone Marrow in Donors with Sickle Cell Trait

Gremlin-1 Suppresses Hypertrophy of Engineered Cartilage In Vitro but Not Bone Formation In Vivo.

Current repair of articular cartilage (AC) often leads to a lower quality tissue with an unstable hypertrophic phenotype, susceptible to endochondral ossification and development of osteoarthritis. Engineering phenotypically stable AC remains a significant challenge in the cartilage engineering field. This motivates new strategies inspired from the extracellular matrix proteins unique to phenotypically stable AC. We have previously shown that the bone morphogenetic protein antagonist gremlin-1 (GREM1) protein, present in permanent but not transient cartilage, suppresses the hypertrophy of chondrogenically primed bone marrow stem cells (BMSCs) in pellet culture. The goal of this study was to assess the effect of GREM1 on the in vitro and in vivo phenotypic stability of porcine BMSC-derived cartilage engineered within chondro-permissive scaffolds. In addition, we explored whether GREM1 would synergize with physioxia, a potent chondrogenesis regulator, when engineering cartilage grafts. GREM1 did not influence the expression of chondrogenic markers (SOX-9, COL2A1), but did suppress the expression of hypertrophic markers (MMP13, COL10A1) in vitro. Cartilage engineered with GREM1 contained higher levels of residual cartilage after 4 weeks in vivo, but endochondral bone formation was not prevented. Higher GREM1 levels did not significantly alter the fate of engineered tissues in vitro or in vivo. The combination of physioxia and GREM1 resulted in a higher sulfated glycosaminoglycan deposition in vitro and a greater retention of cartilage matrix in vivo than physioxia alone, but again did not suppress endochondral ossification. Therefore, while physioxia and GREM1 regulate BMSC chondrogenesis in vitro and reduce cartilage loss in vivo, their use does not guarantee the development of stable cartilage. Impact Statement A major challenge associated with bone marrow stem cell (BMSC)-derived cartilage is that the chondrocyte-like cells have a tendency to undergo hypertrophic differentiation, yielding tissue unsuitable for use in hyaline articular cartilage (AC) repair. This is motivating the development of new tissue engineering strategies to generate phenotypically stable chondrocyte-like cells from BMSCs. In this study, we aimed to engineer phenotypically stable cartilage grafts using BMSCs seeded onto solubilized AC extracellular matrix-derived scaffolds and treated with the bone morphogenetic protein antagonist gremlin-1. This article describes the effects of potential therapeutic strategies that could improve the phenotypic stability of chondrogenically differentiated BMSCs, and examined the use of this strategy both in vitro and in vivo.

Inflamm-Aging-Related Cytokines of IL-17 and IFN-γ Accelerate Osteoclastogenesis and Periodontal Destruction

Periodontal disease (PD), as an age-related disease, prevalent in middle-aged and elderly population, is characterized as inflammatory periodontal tissue loss, including gingival inflammation and alveolar bone resorption. However, the definite mechanism of aging-related inflammation in PD pathology needs further investigation. Our study is aimed at exploring the effect of inflamm-aging-related cytokines of interleukin-17 (IL-17) and interferon-γ (IFN-γ) on osteoclastogenesis in vitro and periodontal destruction in vivo. For receptor activator of nuclear factor-κB ligand- (RANKL-) primed bone marrow macrophages (BMMs), IL-17 and IFN-γ enhanced osteoclastogenesis, with the expression of osteoclastogenic mRNA (TRAP, c-Fos, MMP-9, Ctsk, and NFATc1) and protein (c-Fos and MMP-9) upregulated. Ligament-induced rat models were established to investigate the role of IL-17 and IFN-γ on experimental periodontitis. Both IL-17 and IFN-γ could enhance the local inflammation in gingival tissues. Although there might be an antagonistic interaction between IL-17 and IFN-γ, IL-17 and IFN-γ could facilitate alveolar bone loss and osteoclast differentiation.

Dichotomic Potency of IFNγ Licensed Allogeneic Mesenchymal Stromal Cells in Animal Models of Acute Radiation Syndrome and Graft Versus Host Disease.

Mesenchymal stromal cells (MSCs) are being tested as a cell therapy in clinical trials for dozens of inflammatory disorders, with varying levels of efficacy reported. Suitable and robust preclinical animal models for testing the safety and efficacy of different types of MSC products before use in clinical trials are rare. We here introduce two highly robust animal models of immune pathology: 1) acute radiation syndrome (ARS) and 2) graft versus host disease (GvHD), in conjunction with studying the immunomodulatory effect of well-characterized Interferon gamma (IFNγ) primed bone marrow derived MSCs. The animal model of ARS is based on clinical grade dosimetry precision and bioluminescence imaging. We found that allogeneic MSCs exhibit lower persistence in naïve compared to irradiated animals, and that intraperitoneal infusion of IFNγ prelicensed allogeneic MSCs protected animals from radiation induced lethality by day 30. In direct comparison, we also investigated the effect of IFNγ prelicensed allogeneic MSCs in modulating acute GvHD in an animal model of MHC major mismatched bone marrow transplantation. Infusion of IFNγ prelicensed allogeneic MSCs failed to mitigate acute GvHD. Altogether our results demonstrate that infused IFNγ prelicensed allogeneic MSCs protect against lethality from ARS, but not GvHD, thus providing important insights on the dichotomy of IFNγ prelicensed allogenic MSCs in well characterized and robust animal models of acute tissue injury.

First-Line Choice for Severe Aplastic Anemia in Children: Transplantation from a Haplo-Identical Donor Versus Immunosuppressive Therapy

Background: Severe aplastic anemia (SAA) is a fatal disorder in children if treated inappropriately. According to the current algorithm, transplantation from a matched related donor (MRD) is recommended as the first-line option in children with SAA, if a MRD is unavailable, IST with a combination of anti-thymocyte globulin (ATG) and cyclosporine (CsA) represents the first-line choice. However, numerous limitations of immunosuppressive therapy (IST) exists. IST children may have incomplete recovery, and appear to be associated with an increased risk of clonal evolution and subsequent relapse. Additionally, the unavailability of horse-ATG in China also make the response of IST at a discount greatly. At the same time, great progresses in recent years in haploidentical transplantation might also promote changes in the algorithm of SAA treatment.Methods: We retrospectively compared the outcomes of children with SAA who received IST or who underwent hematopoietic stem cell transplantation (HSCT) from a haplo-identical donor (HID) as first-line choice between 2007 and 2016. The conditioning regimen for SCT from a haploidentical donor consisted of intravenous busulfan, cyclophosphamide and rabbit ATG. All of children were administered with granulocyte colonystimulating factor (G-CSF)-primed bone marrow (BM) combined with G-CSF-primed peripheral blood (PB) hematopoietic stem cells.Results: A total of 52 SAA children under the age of 17 years were initially treated with IST (n=24) or haplo-identical HSCT (n=28) as first-line choice. The last follow-up for all surviving patients was June 1, 2017. In IST group, by 6 months, 15 of the 21 subjects (71.4%) improved with first-line IST and achieved a partial response (n=10) or complete response (n=5). In SCT group, 27 patients (96.4%) achieved primary engraftment at a median of 12 (range, 10-21) days except one suffered from primary graft failure. The cumulative incidences (CI) of grade II-IV and of grade III-IV acute graft versus host disease (GVHD) were 48.1±1.0% and 11.1±0.4%, respectively. The 3-year CI of extensive chronic GVHD was 3.7±0.1%. The estimated 10-year overall survival were 73.4±12.6% and 89.3±5.8% in patients treated with IST or HID-HSCT (P=0.806). However, the failure-free survival was significantly inferior in patients receiving IST than in those undergoing transplantation from a HID [52.6±10.5% versus 89.3±5.8, P=0.008]. In univariate analysis, the choice of first-line immunosuppressive therapy was the only adverse predictor for failure-free survival. At the last follow-up, completely normal blood count was observed in 11 of 20 (55.0%) and 24 of 25 (96.0%) alive cases in IST and HID-HSCT cohort (P=0.003).Conclusions: Based on the superior FFS, high rate of engraftment and acceptable incidence of GVHD, the choice of HID HSCT for SAA children without a matched related donor as a first choice option seems reasonable. These suggest that SCT from a haplo-identical donor could be considered as first-line choice in children who lack a matched related donor, especially in experienced transplantation centers. DisclosuresNo relevant conflicts of interest to declare.

Graft Failure in Patients With Hematological Malignancies: A Successful Salvage With a Second Transplantation From a Different Haploidentical Donor.

Graft failure (GF) is a fatal complication of allogeneic stem cell transplantation, especially after haploidentical transplantation. The mortality of GF is nearly 100% without an effective salvage method. A second transplantation is usually necessary to save the patient's life. However, there is no standardized regimen, and the outcome is usually disappointing. We report on a prospective single-center study using a reduced-intensity conditioning regimen with different haploidentical donors (HIDs). Patients with GF after the first transplantation were enrolled in a prospective single-arm clinical trial (ClinicalTrials.Gov ID: NCT03717545) at the Peking University Institute of Hematology. The conditioning regimen consisted of fludarabine (30 mg/m2) (days−6 to−2) and cyclophosphamide (1,000 mg/m2/day) (days−5 to−4). Patients underwent a second transplant from a different HID using a granulocyte colony-stimulating factor primed bone marrow and peripheral blood stem cells. The primary outcome was neutrophil engraftment at day 28. The secondary outcomes included platelet engraftment at day 100, transplant-related mortality (TRM) at day 30, TRM at day 100, and overall survival (OS) at 1 year. From March 2018 to June 2020, 13 patients were enrolled in this clinical trial. Of the 13 patients, five had acute myeloid leukemia, five had acute lymphoblastic leukemia, two had myelodysplastic syndromes, and one had a non-Hodgkin lymphoma. The median age at first transplantation was 38 years (range, 8–55 years). As for the first transplantation, 11 patients underwent haploidentical transplantations and two underwent unrelated donor transplantations. At the time of GF, three patients had complete donor chimerism, five had mixed chimerism, and five had complete recipient chimerism. The median time from the first transplantation to the second transplantation was 49 (range 35–120) days. The medians of infused cell doses were as follows: mononuclear cells 7.93 (5.95–12.51) × 108/kg and CD34 + cells 2.28 (0.75–5.57) × 106/kg. All 13 patients achieved neutrophil engraftment after the second transplantation, with a median engraftment time of 11 (range 10–20) days after transplantation. The platelet engraftment rate on day 100 after transplantation was 76.9%. The TRMs at day 30, day 100, and 1-year were 0, 0, and 23.1%, respectively. The OS and disease-free survival at 1-year were 56.6 and 48.4%, respectively. For patients with GF after first transplantation, a second transplantation using a fludarabine/cyclophosphamide regimen from a different HID was a promising salvage option. Further investigation is needed to confirm the suitability of this method.

A Novel TBI Free Conditioning Protocol for Haploidentical Transplant in Acquired Aplastic Anemia: (FluCAB-Prime)

Introduction Allogeneic hematopoietic stem cell transplant (HSCT) is the standard treatment for patients younger than 40 years with Severe and Very Severe Aplastic Anemia (AA) who have a Matched Related Donor (MRD). For patients lacking a MRD, treatment options include immunosuppressive therapy (IST), matched unrelated donor (MUD) or alternate donor (haploidentical/cord blood) transplant. Pakistan has a population of around 23 million but there is no donor registry for MUD transplants and horse antithyomcyte globulin (hATG) is not available. Over past decade, results of haploidentical transplants have improved remarkably with use of post-transplant cyclophosphamide. However, most of these protocols incorporate total body irradiation (TBI) to improve engraftment and reduce graft failure. TBI is not available in most of the transplant centres across Pakistan. We have developed a novel TBI free conditioning regimen (NCT03955601) for haploidentical HSCT in acquired AA patients lacking a MRD. Materials and Methods We conducted a prospective, single centre, interventional trial (NCT03955601) using novel TBI free conditioning at AF bone marrow transplant centre (AFBMTC)/ National institute of blood and marrow transplant (NIBMT) for patients with acquired severe and very severe AA. Between July 2018 and March 2020, a total of 10 patients received related haploidentical transplant.Study inclusion criteria was diagnosis of severe and very severe AA, patients of both genders, age 2-60 years, Karnofsky performance status&amp;gt;70%. Exclusion criteria was presence of donor specific antibodies (DSA), inherited bone marrow failure syndrome, prior HSCT and severe sepsis. Conditioning regimen used was Fludarabine (Flu) 30 mg/m2 IV daily from day -7 to -3, Cyclophosphamide (Cy) 14.5 mg/kg IV daily on day -6 and -5 , rabbit Antithymocyte globulin (rATG) 5 mg /kg/day from day -6 to day-3; Busulphan (Bu) IV 3.2 mg per kg/day in 04 divided doses on day -3 and day-2, Granulocyte Colony Stimulating factor (GCSF) primed Bone marrow harvest (BMH) and/or PBSC graft on day 0 and day +1 respectively. Graft versus host disease (GVHD) prophylaxis used was post-transplant cyclophosphamide (PTCy) administered at a dose of 50mg/kg/day given daily on days +3 and +5 post-transplant, cyclosporine (CsA) from day +5 and mycophenolate mofetil (MMF) from day+5 to +35. Primary outcome measure was overall survival (OS) while secondary outcome measures included graft failure, treatment related mortality (TRM), disease free survival (DFS), GVHD free relapse free survival (GRFS), acute and chronic GVHD. Results: Ten patients were transplanted, 5 (50%) female and 5 (50%) male (table 1). Median age was 15.5 years (range 5-41 years). Median duration from diagnosis to transplant was 14 months (range 4-51 months). One patient received ATG prior to transplant. Median number of red cell concentrate (RCC) transfusions before transplant were 27 (8-90) and platelet transfusions 100(6-150). Median donor age was 23 years (10-41 years). Donor-recipient major ABO mismatch was present in 2(20%) while four (40%) had minor ABO mismatch. Primed bone marrow harvest (BMH) was used in 3(30%) while primed BM+PBSC was given in 7(70%) patients. Median CD34 dose given was 8 x 106/kg (range 5.1-16). Seven patients (70%) achieved sustained neutrophil engraftment. One patient had primary graft failure, one patient secondary graft failure at day 35 due to Cytomegalovirus infection and one patient (currently day +118) is having poor graft function. Acute skin GVHD stage-2 developed in 1 patient which settled with topical steroids. None of the patient developed chronic GVHD. Cytomegalovirus reactivation was documented in 8 (80%) patients. All donors and recipients were CMV seropositive pre-transplant. One patient (female, 20 years) had primary graft failure and died on day +31 with sepsis and multiorgan dysfunction syndrome (MODS). One patient (female, 14 years) had secondary graft failure due to CMV infection and died on day +44 with intracranial hemorrhage. Conditioning regimen was well tolerated without any treatment related morbidly and mortality. Median follow-up of study was 13 months (range 4-22 months). Overall survival of study cohort is 80%, DFS 70% and GRFS 70%. Conclusion: Our study shows that for countries lacking TBI, use of FluCAB-Prime protocol is feasible and is associated with low rates of acute and chronic GVHD. Disclosures No relevant conflicts of interest to declare.

Key Aspects of the Immunobiology of Haploidentical Hematopoietic Cell Transplantation.

Hematopoietic stem cell transplantation from a haploidentical donor is increasingly used and has become a standard donor option for patients lacking an appropriately matched sibling or unrelated donor. Historically, prohibitive immunological barriers resulting from the high degree of HLA-mismatch included graft-vs.-host disease (GVHD) and graft failure. These were overcome with increasingly sophisticated strategies to manipulate the sensitive balance between donor and recipient immune cells. Three different approaches are currently in clinical use: (a) ex vivo T-cell depletion resulting in grafts with defined immune cell content (b) extensive immunosuppression with a T-cell replete graft consisting of G-CSF primed bone marrow and PBSC (GIAC) (c) T-cell replete grafts with post-transplant cyclophosphamide (PTCy). Intriguing studies have recently elucidated the immunologic mechanisms by which PTCy prevents GVHD. Each approach uniquely affects post-transplant immune reconstitution which is critical for the control of post-transplant infections and relapse. NK-cells play a key role in haplo-HCT since they do not mediate GVHD but can successfully mediate a graft-vs.-leukemia effect. This effect is in part regulated by KIR receptors that inhibit NK cell cytotoxic function when binding to the appropriate HLA-class I ligands. In the context of an HLA-class I mismatch in haplo-HCT, lack of inhibition can contribute to NK-cell alloreactivity leading to enhanced anti-leukemic effect. Emerging work reveals immune evasion phenomena such as copy-neutral loss of heterozygosity of the incompatible HLA alleles as one of the major mechanisms of relapse. Relapse and infectious complications remain the leading causes impacting overall survival and are central to scientific advances seeking to improve haplo-HCT. Given that haploidentical donors can typically be readily approached to collect additional stem- or immune cells for the recipient, haplo-HCT represents a unique platform for cell- and immune-based therapies aimed at further reducing relapse and infections. The rapid advancements in our understanding of the immunobiology of haplo-HCT are therefore poised to lead to iterative innovations resulting in further improvement of outcomes with this compelling transplant modality.

Long-Term Follow-up of Hematopoietic Stem Cell Transplantation with the Modified Peking Regimen for the Treatment of Acute Leukemia

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment strategy for patients with acute leukemia. The ATG-based transplantation system initiated by Peking University people's hospital, known as the Peking regimen, has become a mainstream transplant system worldwide. Here, based on the Peking regimen, we report a modified protocol:(1)add Fludarabine and replace ATG with ATG-F in the conditioning regimen; (2)transfuse higher-dose cell collections from granulocyte-colony stimulating factor(G-CSF) primed bone marrow and peripheral blood samples; (3) add Basiliximab (a CD25-antibody) on day +3 for acute GVHD prophylaxis. In this study, 265 patients (158 patients with haplo-SCT and 107 patients with sibling-SCT) underwent allo-HSCT with our modified protocols. All patients achieved sustained full-donor chimerism. The incidence of grade II-IV and III-IV acute GVHD in haplo-SCT comparing with sibling-SCT was 36.1%(57/158) vs 17.8%(19/107)(P=0.001) and 13.3% (21/158) vs 9.3%(10/107)(P&gt;0.05) respectively. The 2-year cumulative incidence of total chronic GVHD and extensive chronic GVHD in haplo-SCT was 41% (65/158) and 15% (24/158) respectively. The 3-year cumulative incidence of non-relapse mortality (NRM) in haplo-SCT and sibling-SCT was 6.3% (10/158) and 4.7%(5/107) respectively(P&gt;0.05). The 100-day cumulative incidence of CMV viremia in haplo-SCT and sibling-SCT was 35.5% (56/158) and 23.4%(25/107) respectively(P=0.036). A total of 36 patients in haplo-SCT group and 24 patients in haplo-SCT group had recurrent disease, reaching a cumulative incidence of relapse of 20.8% in haplo-SCT and 23.4% in sibling-SCT at 3 years respectively(P&gt;0.05). The relapse ratio of haplo-SCT and sibing-SCT in the 1st year, between the 1st and the 2nd year and after 2 years was 21.5% vs 14.1%(P&gt;0.05), 1.3%(2/158) vs 0%(P&gt;0.05) and 0% vs 6.5%(P=0.009) respectively. The 3-year overall survival(OS) and leukemia-free survival(LFS) rates in haplo-SCT and sibling-SCT was 78.8% vs 74.2% and 76.8% vs 75.04% respectively(P&gt;0.05) by the Kaplan-Meier estimate. The 3-year GVHD-free and leukemia-free survival rates (GRFS) in haplo-SCT and sibling-SCT were 43.4% vs 69.5%(P=0.045) respectively. Lower OS in haplo-SCT was associated with III-IV aucte GVHD and lower MNC(&lt;19×10^8/L) in grafts by Cox regression analysis. In a word, the results from our experience showed that the modified protocol based on the Peking Regimen is safe and reliable for acute leukemia patients and brings on a long-stage survival post transplantation. Disclosures Zheng: Pfizer: Research Funding.

Relapsed Follicular Lymphoma and Haploidentical Transplantation Using Double Source of Hematopoietic Stem Cells and Post-Transplant Cyclophosphamide: Case Report

Follicular lymphoma (FL) is a heterogeneous disease with varying prognosis owing to differences in clinical, laboratory, and disease parameters. Although FL is considered incurable disease with standard chemotherapy, advances in treatment have improved disease its management and clinical outcomes. Treatment for relapsed or refractory patients is influenced by initial first-line therapy and the duration and quality of the response. Presently, there is no consensus for treatment of patients with early or multiple relapsed disease; however, numerous drugs, combination regimens, and transplant options have demonstrated efficacy. Considering that there is no consensus to treat such patients, we are reporting this case. A 37-yo woman presented in Sep 2005 with abdominal masses, constitutional symptoms and two years of recurrence of urinary and pelvic infections, with previous lymph nodes biopsies revealing hiperplasia. At this time grade I FL, stage IVB, with bone marrow involved was done. There was no response to four cycles of Rituximab and three of COP. In Jan 2006 a histological review confirmed the diagnosis. Then she underwent to six cycles of R-CHOP 21, resulting in complete remission (CR) was ruled out. Consolidation therapy consisted to IL-2 monthly four cycles, and an eight quarterly Rituximab for two years. Supportive care with polyclonal immunoglobuline infusion monthly was demanded to overcome frequent urinary and respiratory infections. After nine years, Dec 2015, constitutional symptoms returned and PET/CT was positive in mediastinum and there was bone marrow involvement. From Jan to Jun 2016, 6 cycles of Rituximab and Chlorambucil was done, and after the second one she complicated with pulmonary aspergillosis and there underwent voriconazole for 18 months. In Jul and Nov 2016 PET/CT revealed a second CR. Just four months later, BMB and pulmonary infiltrated disclosed a new relapse. Four cycles of Fludarabine 30 mg/m², Mitoxantrone 8 mg/m², Rituximab 375 mg/m² and dexamethasone were attempted, since Apr to Jul 2017. A fosfomycin therapy was done to prevent a recurrence of Escherichia coli multi-resistant urinary tract infections. The patient achieved a third CR at PET/CT in Aug 2017. Based on the last rapidly progression of the disease, and bone marrow involvement in all relapses, we proposed an allogeneic stem cell transplantation (allo-SCT). But she didn't have a match related donor. So, her twelve years old son was considered as an haploidentical donor. A reduced intensity conditioning (RIC) was performed with Cyclophosphamide 14.5 mg/Kg D-6 and D-5, Fludarabine 30 mg/m² D-6 to D-3, TBI 200 cGy at D-1. A double source of stem cells: primed-bone marrow (TNC 4.49 x 10⁸/Kg) plus peripheral blood stem cell (4.48 x 10⁶ CD34+/Kg) was infused on Oct-10-2017. Immunosuppressive therapy consisted of Cyclophosphamide 50 mg/Kg at D+3 and D+4 (PT-Cy), Tacrolimus and MMF starting at D+5, and G-CSF from D+5 until neutrophil engraftment. The neutrophil engraftment reached at D+14. Cytokine Releasing Syndrome, febrile neutropenia, rectal prolapse and grade I/II acute graft versus host disease (aGvHD) were immediate complications. Discharge occurred at day +18. CMV PCR positive was preemptive treated from Nov to Dec 2017. Steroid therapy was given for grade II aGvHD, Nov 2017 till Dec 2018. She had an Orthostatic Tachycardia Syndrome from Feb to Oct 2018; Respiratory Syncytial Virus, from Jul to Aug 2018; Rhino and B Influenza upper airway infection on Mar 2019. A complete chimerism was achieve at +100, +180 and +365 days. PET/CT confirmed CR at D+180 and +365. At the present she is off treatment since Jan 2019. In this situation a double source haplo-SCT was a successfully therapy overcame the complicated comorbidities before transplantation, with a good quality of life nowadays. Disclosures No relevant conflicts of interest to declare.

Novel Improvement of Haploidentical Hematopoietic Stem Cell Transplantation for Advanced Refractory/Relapsed Acute Leukemia

Introduction The advanced refractory/relapsed acute leukemia has a very dismal prognosis even with the salvage allogeneic hematopoietic stem cell transplantation (allo-HSCT). To develop a novel transplant protocol to achieve a rapid engraftment and quick graft-versus leukemia (GVL) effect is crucial. Based on previous observation, the G-CSF primed bone marrow (BM) plus peripheral blood mononuclear cells (PBMNCs) could not only increase mononuclear cells and CD34 cells, but also change T-cell biology by down-regulating the expression of adhesion and CD28/B7 molecules and increase the absolute number of D2APCS, which promotes a T-cell shift from T1 to T2 subset to secrete a higher anti-inflammatory cytokines of IL-4 and IL-10. After going through a rigorous conditioning, the recipients' T-cells are incapable to attack host. We propose that reinfusion of G-CSF primed donor PBMNCs to recipient on transplant day + 6 may cross mismatched MHC barrier thus accelerating engraftment. Patients and Methods From April 2018 to June 2019, 30 advanced-stage patients with refractory/relapsed acute leukemia were enrolled and underwent a salvage haploidentical (haplo)-HSCT. The median age was 12 (2-63) years with M/F ratio of 17/13. There were 21 patients with acute myelocytic leukemia (AML, 70%), 4 -cell acute lymphoblastic leukemia (B-ALL, 13%), 2 biphenotypic acute leukemia (BAL, 7%), 1 juvenile myelomonocytic leukemia (JMML, 3%), and 2 T-lymphoblast leukemia (T-LBL, 7%). The median BM blasts were 40% (5%-90%). Twenty-eight patients received a conditioning regimen including fludarabine 30mg/m2/d×4 day, cytarabine 2.0g/m2/d×4 days, busulfan 3.2mg/kg/d Q6h×4days, cyclophosphamide 1.5g/m2/d×2 days and melphalan 110mg/m2/d×1 day. Two patients with T-LBL received BCNU+Etoposide+Cytarabine+Melphalan (BEAM) regimen. Graft-versus-host disease (GVHD) prophylaxis contained methotrexate (MTX), cyclosporine A (CsA) and mycophenolate mofetil (MMF), plus ATG (Rabbit anti-human thymocyte immunoglobulin, Sangstat) 1.5mg/kg/d iv daily from day -4 to -1 and additional 1.5mg/kg on day +7 (total dose of 7.5 mg/kg). In addition, anti-CD25 monoclonal antibody (basiliximab) 20 mg on day+1 and +4 was given iv. Donors were primed with G-CSF at 3-4 ug/kg/d sq d1-5. On day 4 of G-CSF, donor BM cells were harvested and were infused unmanipulated on the same day to patient on transplant day 01. On day 5 of G-CSF injection, donor primed PBMNCs were harvested, part of which were immediately infused unmanipulated to recipient on transplant day 02. Part of the cells were cryopreserved and stored in the liquid nitrogen, which were then thawed and reinfused unmanipulated to recipient on day +6 (Table 1). Results All patients achieved a trilineage engraftment with a median time of 13.5 (5-16) days. The median neutrophil and platelet engraftment were 13 (11-19) days and 10 (5-22) days respectively (Fig.1). Among 29 evaluable patients, acute GVHD occurred in 10 (34.5%, Fig. 2) with a median time of 32 (14-60) days, including 5 cases (17%) of grade Ⅱ, 3 (10%) of grade Ⅲ and 2 (7%) of grade Ⅳ. Among the 28 evaluable patients for chronic GVHD, only 6 (21.4%, Fig.3) developed limited chronic GVHD with a median time of 103 (90-180) days. Twenty seven out of thirty (90%) patients achieved disease-free survival (DFS) with a median follow-up time of 6 (5-14) months. Three patients died from transplant related complication, including infection for 2 and GVHD for 1 respectively. Conclusion In this study, with an intensive myeloablative condition and salvage haplo-HSCT, by reinfusing additional unmanipulated donor PBMNCs to recipients on day +6 during their transplantation, a short engraftment time without engraftment failure, low lethal GVHD incidence, and a lower infection rate were able to be achieved. More importantly, most patients remain DFS at a median follow-up time of 6 months. Despite short-term follow-up. the outcomes are particularly encouraging. Disclosures No relevant conflicts of interest to declare.

Haploidentical Transplantation Using Double Source of Hematopoietic Stem Cells and Post-Transplant Cyclophosphamide for Acute Leukemias

Allogeneic stem cell transplantation (allo-SCT) represents a curative option for intermediate- and high-risk acute leukemias (AL). The number of unmanipulated haploidentical allo-SCT (haplo-SCT) is increasingly used with favorable outcomes. Incidence of graft-versus-host disease (GvHD) in haploidentical bone marrow (BM) transplants using post-transplant cyclophosphamide (PTCy) is low, counterbalanced by an excess in disease recurrence; and acute GvHD using mobilized peripheral blood stem cells (PBSC) ranges between 30% and 40%. The ultimate choice of graft source depends on the design of the full transplantation package based on transplantation center experience. We conducted a retrospective analysis of 32 patients (59% male), who received an haplo-PTCy with double source of stem cells, G-CSF primed bone marrow plus G-CSF-mobilized PBSC, for high-risk or advanced acute leukemia in two Brazilian centers, Hospital Santa Marcelina (n=23) and Hospital São Paulo (n=9), from 2013 to 2019. The median age patients were 27.5 years (range 17-60 years). Median disease time before haplo-PTCy was 8.9 months (3.6-108). There were 13 acute myeloid leukemia (AML), 17 acute lymphoblastic leukemia (ALL), one mixed phenotype acute leukemia and one dendritic cell leukemia. 6/17 ALL were Ph1 positive. 34% of the patients received 2 treatment protocols to achieve CR and 12.5% had submitted to more than two treatments. So, at the time of transplant 75% (n=24) was in first CR (CR1) although one (3%) patient was minimal residual disease (MRD) positive and six (18.7%) there were no MRD available. The others patients (n=8, 25%) were on second or third CR. The HCT-CI comorbidities was ≥ 3 in two patients, and there were 15 patients (46%) with carbapenem-resistant gram-negative bacilli (CRGNB) colonization before transplant. Panel reactive antibody was positive in two patients. The donor was a sibling in 68.7% (n=22), father, mother and child in two (6%), three (9%) and five (15%) patients, respectively. The conditioning was reduced intensity (RIC) in 87.5% (n=28) patients, with fludarabine, cyclophosphamide and total body irradiation (TBI) 200 cGy. After conditioning, patients received G-CSF primed bone marrow grafts in combination with PBSC no ex-vivo T cells depleted, and cyclophosphamide 50 mg/Kg/day IV on days +3 and +4 post-transplant, as well as GVHD prophylaxis. Six (18.7%) patients died for sepsis before 60 days (10 to 58 days), all had had CRGNB before transplant, four those ones with no grafting, died from days +10 to +19. Acute GvHD grade III-IV was observed in two patients, who died at +48 and +95 days. High mortality related to transplant (TRM) was observed considering all patients. CRGNB was a determining factor in these early deaths. If we excluded all CRGNB patients of this study the mortality could be 11% (2 patients with GVHD in 17 patients transplanted). Four patients (16%) has severe cGvHD. Nine patients (37%) relapsed in two years. Two years OS and DFS were 40% and 37.5%, respectively. In conclusion, with a median follow-up of 2 years, haplo-PTCy with double source leads to 40% overall survival in 32 patients with high-risk advanced acute leukemia, with 16% (n=2) being in treatment for cGvHD. The causes of death were relapse 43% (n=7), early sepsis in patients with CRGNB colonization 37.5% (n=6), grade IV acute GvHD in 12,5% (n=2), and one patient died for pneumonia community at D+285. Our data suggests that haplo-PTCY double source is a feasible option in these cases. However, CRGNB colonization in aggressive disease is the main factor that should be considered as exclusion for haplo-SCT in development countries. Disclosures No relevant conflicts of interest to declare.

Reduced Intensity Conditioning for Haploidentical Transplantation Using Double Source of Hematopoietic Stem Cells and Post-Transplant Cyclophosphamide for Severe Aplastic Anemia

Significant improvements in haploidentical stem cell transplantation (haplo-SCT) have confirmed its therapeutic role in severe aplastic anemia (SAA) and led to the evolution of treatment algorithms. However, the optimal conditioning regimen for haplo-SCT for SAA remains undefined. A recent review demonstrated haplo-SCT using NMA and RIC, engraftment between 75 to100% and OS one year of 75-100%. In our Institutions we started a double source since Jan 2017 using a RIC protocol that is the objective of this presentation. There were nine haplo-SCT in eight patients in two Brazilian centers, Hospital Santa Marcelina (n=5) and Hospital São Paulo (n=3). All patients were male, median age 26 years old (14-39), nocturnal paroxysmal hemoglobinuria clonality in five (62,5%); treated with Cyclosporine, Thymoglobulin (n=4), Eltrombopag (n=1), Eculizumab (n=2) and/or Alemtuzumab (n=1). Median time disease to transplant was 15,7 months (3.8-192). All patients submitted more than 20 RBC transfusions, and median of ferritin was 2,283 ng/mL (753-3,486). One patient had carbapenem-resistant gram-negative bacilli (CRNGB: Escherichia coli, Klebsiella pneumoniae and/or Pseudomonas aeruginosa). Panel reactive antibody was negative in all patients. The donor was a sibling in 44% (n=4), father and mother 22% each (n=2) and one patient received cells from his cousin after an early failure of engraftment from mother first transplant. The cause of this failure was a JC virus and adenovirus acute infections. The second haplo-SCT conditioning was Fludarabine, Cyclophosphamide and 600 cGy TLI. There were seven male donors. RIC was consisted of Fludarabine, Cyclophosphamide and total body irradiation (TBI) 200 cGy. The source was G-CSF primed bone marrow (G-BM) grafts in combination with PBSC no ex-vivo T cells depleted, and Cyclophosphamide 50 mg/Kg/day IV on days +3 and +4 post-transplant (PTCy), as well as graft versus host disease (GvHD) prophylaxis. The median of G-BM MNCs was 5.4 x 10⁸/Kg (2.3-8.7), and PBSC was 10 x 10⁶CD34+/Kg (4-18.7). One (11%) patient died for sepsis at day +13, he was the only with a confirmed CRGNB colonization before transplant. The ANC recovery was at day +17 (13-24). No one had Sinusoidal Obstructive Syndrome. Acute GvHD grade II and moderate chronic GvHD was observed in two patients (25%). Two patients had engrafting failure: the first at day +30 and received a successfully new haplo-SCT; the second at day +60, with a partial donor reconstitution without dependence of transfusion after immunosuppressive therapy. These two patients had female donors. The OS and DFS at two years were 87.5% and 75%, respectively. In conclusion, with a median follow-up of 30 months, haplo-SCT, PTCy with double source keep the 87.5% overall survival, with no one severe chronic GvHD. Our data suggests an excellent results for haplo-SCT, PTCy, RIC, double source in the SAA treatment. However, prospective well-designed trials need to confirm these results. Disclosures No relevant conflicts of interest to declare.

Macrophage colony-stimulating factor pretreatment of bone marrow progenitor cells regulates osteoclast differentiation based upon the stage of myeloid development.

Osteoclasts (OCs) are large, multinucleated bone resorbing cells originating from the bone marrow myeloid lineage, and share a common progenitor with macrophages and dendritic cells. Bone marrow cells (BMCs) are a common source for in vitro osteoclastogenesis assays but are a highly heterogeneous mixture of cells. Protocols for in vitro osteoclastogenesis vary considerably thus hindering interpretation and comparison of results between studies. Macrophage colony-stimulating factor (M-CSF) pretreatment is commonly used to expand OC progenitors (OCPs) in BMC cultures before in vitro differentiation. However, the failure of osteoclastogenesis of M-CSF primed bone marrow myeloid blasts has been reported. In this study, we used a simple method of differential adherence to plastic to enrich OCP from mouse BMCs. We found that M-CSF pretreatment of plastic-adherent BMCs (adBMCs) increased the number of CD11b-F4/80+ macrophages and decreased the number of CD11b+ monocytes resulting in decreased OC formation. M-CSF pretreatment of purified c-Kit+ progenitors weakly inhibited OC formation, whereas M-CSF pretreatment of purified c-Kit-CD11b+ progenitors promoted the formation of large OC. M-CSF pretreatment increased the proliferation of both purified c-Kit+ and c-Kit-CD11b+ cells and increased the percentage of CD11b-F4/80+ cells from c-Kit+ progenitors. In addition, M-CSF pretreatment increased the percentage of CD11b+ F4/80- cells from purified c-Kit-CD11b+ cells. M-CSF pretreatment increased the percentage of CD14 + CD16 + intermediate monocytes and subsequent OC formation from human 2adBMCs, and increased OC formation of purified CD14 + cells. Together, these results indicate that in vitro OCP expansion in the presence of M-CSF and bone marrow stromal cells is dependent upon the developmental stage of myeloid cells, in which M-CSF favors macrophage differentiation of multipotent progenitors, promotes monocyte maturation and supports differentiation of late-stage OCP cells.

Ligature-induced periodontitis induces systemic inflammation but does not alter acute outcome after stroke in mice.

BackgroundStroke is a major cause of disability and mortality. Poorer outcome after stroke is associated with concomitant inflammatory and infectious disease. Periodontitis is a chronic inflammatory disease of the dental supporting structures and is a prominent risk factor for many systemic disorders, including cardiovascular disease and stroke. While epidemiological studies suggest that periodontitis increases the likelihood of stroke, its impact on stroke severity is poorly understood. Here, we sought to determine the contribution of periodontitis to acute stroke pathology.MethodsWe characterized a murine ligature model of periodontitis for inflammatory responses that could potentially impact stroke outcome. We applied this model and then subjected mice to either transient or permanent middle cerebral artery occlusion. We also enhanced the periodontitis model with repeated intravenous administration of a periodontal-specific lipopolysaccharide to better mimic the clinical condition.ResultsLigature-induced periodontitis caused bone loss, bacterial growth, and increased local inflammatory cell trafficking. Systemically, periodontitis increased circulating levels of pro-inflammatory cytokines, and primed bone marrow monocytes to produce elevated tumour necrosis factor-alpha (TNFα). Despite these changes, periodontitis alone or in tandem with repeated lipopolysaccharide challenge did not alter infarct volume, blood–brain barrier breakdown, or systemic inflammation after experimental stroke.ConclusionsOur data show that despite elevated systemic inflammation in periodontitis, oral inflammatory disease does not impact acute stroke pathology in terms of severity, determined primarily by infarct volume. This indicates that, at least in this experimental paradigm, periodontitis alone does not alter acute outcome after cerebral ischemia.