Chimerism Studies Research Articles

Preclinical Analysis of Candidate Anti-Human CD79 Therapeutic Antibodies Using a Humanized CD79 Mouse Model.

The BCR comprises a membrane-bound Ig that is noncovalently associated with a heterodimer of CD79A and CD79B. While the BCR Ig component functions to sense extracellular Ag, CD79 subunits contain cytoplasmic ITAMs that mediate intracellular propagation of BCR signals critical for B cell development, survival, and Ag-induced activation. CD79 is therefore an attractive target for Ab and chimeric Ag receptor T cell therapies for autoimmunity and B cell neoplasia. Although the mouse is an attractive model for preclinical testing, due to its well-defined immune system, an obstacle is the lack of cross-reactivity of candidate therapeutic anti-human mAbs with mouse CD79. To overcome this problem, we generated knockin mice in which the extracellular Ig-like domains of CD79A and CD79B were replaced with human equivalents. In this study, we describe the generation and characterization of mice expressing chimeric CD79 and report studies that demonstrate their utility in preclinical analysis of anti-human CD79 therapy. We demonstrate that human and mouse CD79 extracellular domains are functionally interchangeable, and that anti-human CD79 lacking Fc region effector function does not cause significant B cell depletion, but induces 1) decreased expression of plasma membrane-associated IgM and IgD, 2) uncoupling of BCR-induced tyrosine phosphorylation and calcium mobilization, and 3) increased expression of PTEN, consistent with the levels observed in anergic B cells. Finally, anti-human CD79 treatment prevents disease development in two mouse models of autoimmunity. We also present evidence that anti-human CD79 treatment may inhibit Ab secretion by terminally differentiated plasmablasts and plasma cells in vitro.

Application of flow cytometry in transfusion medicine: The Sanjay Gandhi Post Graduate Institute of Medical Sciences, India experience.

The application of flow cytometry (FC) is diverse and this powerful tool in used in multiple disciplines such as molecular biology, immunology, cancer biology, virology, and infectious disease screening. FC analyzes a single cell or a particle very rapidly as they flow past single or multiple lasers while suspended in buffered solution. FC has a great impact in the field of transfusion medicine (TM) due to its ability to analyze individual cell population and cell epitopes by sensitive, reproducible, and objective methodologies. The main uses of FC in TM are detection of fetomaternal hemorrhage, diagnosis of paroxysmal nocturnal hemoglobinuria, quantification of D antigen, detection of platelet antibody, quality control of blood components, for example, residual leukocyte counts and evaluation of CD34-positive hematopoietic progenitor cells in stem cell grafts. In recent years, FC has been implemented as an alternative method for the detection and characterization of red cell autoantibodies in autoimmune hemolytic anemia. Many workers considered FC as a very good complement when aberrant expression of various erythrocyte antigens needs to be elucidated. It has been extensively used in the resolution of ABO discrepancies and chimerism study. FC has also been used successfully in various platelet immunological studies. In the recent past, FC has been used in several studies to assess the platelet storage lesions and elucidate granulocyte/monocyte integrity and immunology. FC analysis of CD34+ stem cells is now the method of choice to determine the dosage of the collected progenitor cells. The technique is vastly used to evaluate residual leukocytes in leukodepleted blood components. We conclude that flow cytometers are becoming smaller, cheaper, and more user-friendly and are available in many routine laboratories. FC represents a highly innovative technique for many common diagnostic and scientific fields in TM. Finally, it is the tool of choice to develop and optimize new cellular and immunotherapeutic trials.

Crystal structure of Aedes aegypti trypsin inhibitor in complex with μ-plasmin reveals role for scaffold stability in Kazal-type serine protease inhibitor.

Kazal-type protease inhibitor specificity is believed to be determined by sequence of the reactive-site loop that make most, if not all, contacts with the serine protease. Here, we determined the complex crystal structure of Aedes aegypti trypsin inhibitor (AaTI) with μ-plasmin, and compared its reactivities with other Kazal-type inhibitors, infestin-1 and infestin-4. We show that the shortened 99-loop of plasmin creates an S2 pocket, which is filled by phenylalanine at the P2 position of the reactive-site loop of infestin-4. In contrast, AaTI and infestin-1 retain a proline at P2, rendering the S2 pocket unfilled, which leads to lower plasmin inhibitions. Furthermore, the protein scaffold of AaTI is unstable, due to an elongated Cys-V to Cys-VI region leading to a less compact hydrophobic core. Chimeric study shows that the stability of the scaffold can be modified by swapping of this Cys-V to Cys-VI region between AaTI and infestin-4. The scaffold instability causes steric clashing of the bulky P2 residue, leading to significantly reduced inhibition of plasmin by AaTI or infestin-4 chimera. Our findings suggest that surface loops of protease and scaffold stability of Kazal-type inhibitor are both necessary for specific protease inhibition, in addition to reactive site loop sequence. PDB ID code: 7E50.

A Pilot Phase I Trial of IL-21 Expanded Ideal-Donor Natural Killer (NK) Cells in Combination with Mogamulizumab in Patients with Cutaneous T-Cell Lymphomas (CTCL) or Adult T-Cell Leukemia/Lymphomas (ATLL)

A Pilot Phase I Trial of IL-21 Expanded Ideal-Donor Natural Killer (NK) Cells in Combination with Mogamulizumab in Patients with Cutaneous T-Cell Lymphomas (CTCL) or Adult T-Cell Leukemia/Lymphomas (ATLL)

Analysis of Hematopoietic Niche Components Post Allogeneic Transplantation

Timely and effective reconstitution of the hematopoietic niche is imperative post-allogeneic stem cell transplantation (alloHCT) for prevention of life-threatening opportunistic infections and relapses. Efficient engraftment is dependent on the ability of donor hematopoietic stem cells (HSCs) to expand and provide long-term multi-lineage progenitors in the recipient. Mesenchymal stem cells (MSCs) are essential in providing a conducive environment for HSC reconstitution and maintenance. Conditioning chemotherapy with or without total body irradiation is required to confer a competitive advantage to donor HSCs. However, myeloablative conditioning (MA) regimens also destroy the specialized stromal niches which support HSC self-renewal and maintenance (Acar et al., 2015), providing a plausible explanation for the long term cytopenias observed post allogeneic transplantation (Domenech et al., 1995). Previous studies in humans showed CD146-expressing stromal cells support the establishment of the hematopoietic cell microenvironment (Sacchetti et al., 2007). Expression of markers of stromal/progenitor cells (CD146, CD31 and CD34) was evaluated pre and post alloHCT at day 30 in bone marrow biopsies obtained from patients treated with either MA or reduced intensity conditioning (RIC) approaches. CD146 and CD31 expression was evaluated by immunohistochemistry performed on formalin-fixed, paraffin-embedded bone marrow core biopsies. Whole slide imaging (Aperio) was used to digitize the histology and positive cells were scored using image analysis software (strong positive pixel count; ST PPC). In bone marrow, CD146 is expressed on vascular endothelial cells, pericytes on sinusoids, adipose and bone marrow stromal cells. CD31 positive cells include vascular endothelial cells, sinusoidal cells, megakaryocytes, and myeloid cells (Fig. 1). The percent of CD34 positive stem cells was determined by flow cytometry in concurrent bone marrow aspirate cells.The indications for an alloHCT were AML (5/10), ALL (2/10) or MDS (3/10). Median age was 62 (59-69) years in the RIC group and 34 (25-46) years in the MA group. Average HCT CI score was higher in the RIC group (2.5 versus 1.2) (Table 1). 3/10 patients received total body irradiation (TBI) based conditioning. All patients received peripheral blood stem cell alloHCT and 7/10 were MUDs. Chronic GVHD was present (grade 2 or 3) in 3/10 patients in the RIC groups and 1 patient (grade 1) in the MA cohort. Chimerism studies showed optimum engraftment of donor cells in 2 patients in the RIC group and 1 patient in the MA (Table 1).On average, pretransplant bone marrow cellularity was lower in the RIC group. The average overall cellularity on D30 was similar in the two groups (Fig.2). The average percent of CD34 positive cells was similar pretransplant and lower at D30 in the RIC cohort compared to the MA group (0.96% CD34 pre alloHCT versus 0.30% D30 post alloHCT RIC; pre alloHCT 0.96% CD34 versus D30 post alloHCT 0.92% MA). The mean number of CD31 positive cells was higher in the RIC group compared to the MA cohort both pre-transplant and at D30 (Fig 3). On average, an increase in CD146 positive cells on D30 was noted in the alloHCT RIC group compared to the MA cohort (Fig. 4).In conclusion, the RIC cohort showed a pre-post-transplant increase in average overall bone marrow cellularity, more numerous CD31 and CD146 positive cells on average and an increase in CD146 positive cells at D30 post alloHCT compared to the MA group. Despite the modest number of patients, this is the first study to date comparing pre and post alloHCT niche components which may provide important insights in the reconstitution process and impact of conditioning. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Allogeneic mesenchymal stromal cells for cartilage regeneration: A review of in vitro evaluation, clinical experience, and translational opportunities.

The paracrine signaling, immunogenic properties and possible applications of mesenchymal stromal cells (MSCs) for cartilage tissue engineering and regenerative medicine therapies have been investigated through numerous in vitro, animal model and clinical studies. The emerging knowledge largely supports the concept of MSCs as signaling and modulatory cells, exerting their influence through trophic and immune mediation rather than as a cell replacement therapy. The virtues of allogeneic cells as a ready‐to‐use product with well‐defined characteristics of cell surface marker expression, proliferative ability, and differentiation capacity are well established. With clinical applications in mind, a greater focus on allogeneic cell sources is evident, and this review summarizes the latest published and upcoming clinical trials focused on cartilage regeneration adopting allogeneic and autologous cell sources. Moreover, we review the current understanding of immune modulatory mechanisms and the role of trophic factors in articular chondrocyte‐MSC interactions that offer feasible targets for evaluating MSC activity in vivo within the intra‐articular environment. Furthermore, bringing labeling and tracking techniques to the clinical setting, while inherently challenging, will be extremely informative as clinicians and researchers seek to bolster the case for the safety and efficacy of allogeneic MSCs. We therefore review multiple promising approaches for cell tracking and labeling, including both chimerism studies and imaging‐based techniques, that have been widely explored in vitro and in animal models. Understanding the distribution and persistence of transplanted MSCs is necessary to fully realize their potential in cartilage regeneration techniques and tissue engineering applications.

Anti-CD45 Radioimmunotherapy Followed By Haploidentical Allogeneic Hematopoietic Cell Transplantation for Advanced Acute Leukemia or High-Risk MDS

Background: Although hematopoietic cell transplantation (HCT) may offer the best chance for cure for patients with relapsed/refractory hematologic malignancies, many of these patients will relapse after HCT. Escalating doses of total body irradiation (TBI) or chemotherapy prior to HCT can decrease relapse, but incurs higher toxicity, resulting in no survival benefit. Therefore, there is a need to reduce relapse without increasing the toxicity of the conditioning regimen prior to HCT. In addition, many potentially curable candidates for HCT do not have a fully human leukocyte antigen (HLA) matched donor. This is an especially challenging hurdle for many ethnic minority patients, yet nearly all patients have a related donor identical for one HLA haplotype (i.e., haploidentical). Radioiodinated anti-CD45 radioimmunotherapy (RIT) has been shown to deliver high-doses of radiation specifically to hematolymphoid organs, while sparing normal tissues. We report a phase I/II study using iodine-131 (131I)-anti-CD45 antibody (BC8) combined with pre- and post-transplant cyclophosphamide, and reduced intensity conditioning prior to haploidentical HCT.Methods and Patients: Eligible patients over the age of 18 had relapsed or refractory acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), or high-risk (>5% active marrow blasts) myelodysplastic syndrome (MDS). Patients received a 131I-anti-CD45 dosimetry dose, followed by serial gamma-camera imaging for organ dosimetry estimates. One to two weeks later a therapeutic dose delivering escalating doses (12 to 26 Gy) to the liver (the normal organ that received the highest dose) was administered, followed by fludarabine (FLU; 30 mg/m2 on day -6 to -2), cyclophosphamide (CY; 14.5 mg/m2 on day -6 and -5), 200 cGy TBI on day -1, and subsequent haploidentical marrow HCT on day 0. For GVHD prophylaxis, patients received CY (50 mg/kg) on day+3 plus standard tacrolimus and mycophenolate mofetil.Results: A total of 25 patients have been treated (21 with advanced AML, 3 with ALL and 1 with high-risk MDS). Ten patients were in florid refractory relapse with 6-20% abnormal marrow blasts while 9 were in remission with minimal residual disease (MRD) 0.03-5.4% by flow cytometry, and only 6 were in remission and MRD negative by flow cytometry. All 24 patients who had marrow aspiration on day 28 after HCT achieved a morphologic remission, with only two patients showing MRD by flow cytometry (0.002 -1.8% abnormal blasts). There were no graft failures, and all patients with chimerism studies (n=15) had 100% donor CD3+ and CD33+ cell engraftment at day 28 after haploidentical HCT. Median time to neutrophil engraftment (ANC>500/ml for 3 consecutive days) was 15 (range, 12-19) days. Median time to platelet engraftment (platelets >20,000/ml for 7 consecutive days without transfusion support) was 23 (19-44) days. The maximum tolerated dose was not determined and there were no unexpected adverse events, as the most frequent grade ≥3 adverse events were infections/neutropenic fevers (32), gastrointestinal (22), and laboratory abnormalities (cytopenias 18; metabolic 16). Five patients had grades 3-4 acute GVHD. Twelve patients relapsed with median time to relapse of 2.4 months. Most deaths have been from relapse (11), and infections (2). Eight (32%) patients were alive at a median follow up of 4 years after HCT (range 1 to 9 years). Overall survival at one and three year was 42% and 26%, and progression-free survival was 40% and 25%, respectively (Figure).Conclusions: Although patients with active disease are not traditionally referred for HCT, the use of 131I-anti-CD45 RIT prior to a haploidentical HCT may facilitate improved survival for these patients. Given these results, anti-CD45 RIT prior to HCT using haploidentical donors warrants further investigation for patients with advanced leukemia and high-risk MDS. [Display omitted] DisclosuresOrozco:Actinium Pharmaceuticals: Other: Research Funding to Institution for sponsored Clinical Trials. Shadman:PLEXXIKON: Research Funding; AbbVie: Other: advisory board; Genentech: Consultancy, Research Funding; Emergent: Research Funding; TG Therapeutics: Research Funding; Gilead: Research Funding; Acerta Pharma: Research Funding; Celgene: Research Funding; Pharmacyclics: Other: advisory board, Research Funding; Merck: Research Funding. Gopal:Seattle Genetics: Consultancy, Research Funding. Pagel:Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

T-Cell Replete Peripheral Blood Haploidentical Transplant Using Immunogenic Dose of Cyclophosphamide with Fludarabine and Melphalan Results in Enhanced Cytokine Release Syndrome (CRS) with Robust Immune Recovery and Low Relapse Rates

The immune suppressive properties of high-dose Cyclophosphamide are well known; however, a growing literature has described its immune stimulatory properties when applied in low dose, mediated through proliferation of effector T cells and abrogation of regulatory T cells. In order to exploit the immunogenic property of low dose Cyclophosphamide we hypothesized that its addition to Melphalan and Fludarabine in the absence of the immune suppressive effect of radiation would result in robust effector T cells proliferation through enhanced cytokine release and antigen alloreactivity. (1)We examined the clinical outcomes and immune recovery of 14 consecutive haploidentical transplant patients with high risk hematological malignancies who received their first allogeneic transplant between 7/8/2015 and 1/10/2017. The immune recovery markers were assessed at day 60, 120 and 180 post-transplant and included absolute lymphocyte count (ALC), CD3+ CD4+, CD8+, activated T cells (CD3+HLA Dr+), CD19+ and NK cells (CD56/16+). Patients and graft characteristics are as shown in Table-1. The median duration of follow up for survivors is 15 months (range 6-25); and all but 3 patients have completed 1 year of follow up. The conditioning regimen was low dose Cyclophosphamide 14.5 mg/m2 on day -6 & day -5, Fludarabine 30 mg/m2 on days -6 to Days -2 and Melphalan 70 mg/m2 on day -3 & -2. Graft versus host disease (GVHD) prophylaxis was post-transplant Cyclophosphamide 50 mg/Kg on day +3 and day +4 along with Tacrolimus and Mycophenolate starting at day +5. Tacrolimus continued until day +180 (Target level 5-8) and Mycophenolate continued until Day+30. Chimerism studies were performed at day +30 and +100 post-transplant by variable number tandem repeat PCR analysis of peripheral blood & bone marrow. All patients engrafted except one patient who died at day 25 prior to engraftment. Median time to neutrophil engraftment was 21 days (range 12-57). All of the evaluable 13 patients for engraftment achieved 100% donor chimerism in bone marrow (unsorted) and peripheral blood (CD3, CD33 & CD 56) by day 100. The day100 and one year overall survival were 92.8% and 85.7% respectively. Only 1/14 patients has relapsed, and the cumulative incidence of relapse is 7.74% at 1 year post- transplant. Similarly, the cumulative incidence of treatment related mortality (TRM) is 7% at 1 year post transplant. Overall 3 patients have died (2 Patients TRM and 1 Patient Relapse related mortality). TRM was related to severe CRS grade 4 in one patient at day +25 and pneumonia at day +540 in another patient. Acute GVHD grade 2-4 developed in 4 patients of whom 2 had grade 3-4. Chronic GVHD developed in 8 patients (57%) but was extensive in only 3 patients. All patients developed CRS grade as previously proposed by Lee et al.Grade 4 CRS developed in 4/14 patients requiring ventilator support and dialysis, which led to one death during the first year post-transplant at day 25. Grade 1 CRS developed in 7 patients while 3 patients developed grade 2 CRS. Two of 4 patients with grade 4 CRS had delayed neutrophilic engraftment at day +34 and +57, and both achieved 100% donor chimerism by day 100 in bone marrow and peripheral blood. Post-Transplant immune recovery is shown in Fig-1. There was significant early recovery at day +60 in all T cell subsets. This recovery was most pronounced in activated T cells, giving support to our hypothesis. While we have observed a progressive reduction in the median number of early activated T cells at day +120 & +180, the number of helper T cells (CD4) and NK cells (CD56/16) maintained its early immune recovery. Additionally, we found more robust NK cell recovery in patients with grade 2-4 CRS (#6) compared to patients with less severe CRS grade 1 (#7). In contrast, CD4 recovery was more robust in patients with less severe CRS (Table 2)Conclusion:Fludarabine/Melphalan/Cyclophosphamide as a conditioning regimen prior to T-cell replete peripheral blood haploidentical transplant results in early robust immune recovery and may have the potential to decrease the risk of relapse in high risk hematological malignancies. The observed robust NK recovery in particular in patients with more severe CRS may be attributed to the lower risk of relapse. Further confirmation in a larger cohort of patients is needed to confirm these results.(1) Madondo MT, Quinn M, Plebanski M. Low dose cyclophosphamide: mechanisms of T cell modulation. Cancer Treat Rev 2016; 42: 3-9. [Display omitted] DisclosuresChandler:Vector Oncology: Consultancy; BMS: Consultancy; Janssen: Consultancy, Speakers Bureau.

Absence of Vasoactive Intestinal Peptide Synthesis in Recipients in Allogeneic Bone Marrow Transplant Increases Graft Versus Host Diseases

Introduction: Vasoactive intestinal polypeptide (VIP) is an immunosuppressive neuropeptide. Activation of donor T cells leading to graft-versus-host disease (GvHD) in allogeneic bone marrow transplant (allo-BMT) occurs via early post transplant interactions between donor T cells and host antigen presenting cells and leads to upregulation of immune co-inhibitory pathways, including expression of VIP in T cells and dendritic cells. We have previously shown VIP signaling favorably regulates the activation status and enhances the graft-versus-leukemia (GvL) activities of donor T cells (Li et al., Cancer Research 2016). The role of the VIP produced by host antigen presenting cells in regulating GvHD in allo-BMT is unknown. We tested the hypothesis that absence of VIP expression by host cells would augment donor T cell activation and increase GvHD.Methods and Results: We used the B10BR->B6 MHC fully mismatched murine model of allogeneic BMT. Recipient wildtype (WT) and VIP knockout B6 (VIP-KO) mice were lethally irradiated with 11Gy and injected i.v. with 5×106 T cell depleted (TCD) bone marrow cells (BM) plus 0, 1 or 3 ×106 splenocytes from WT B10BR donors. Fifteen mice (males and females) were assigned to each splenocytes dosage group. The survival of recipients after BMT was recorded daily and the average clinical GvHD scores were obtained by combining weight-loss, activity, posture, fur texture and skin integrity data following standard published procedures. All three groups of WT recipients had better survival than VIP-KO recipients. VIP-KO mice transplanted with TCD BM alone had only 33.3% survival at day 75 compared with 73.3% survival among the WT recipients. Chimerism studies on day 10 post-BMT showed equivalent level of donor chimerism: 80% donor in WT recipients vs 72% donor in VIP-KO recipients, indicating increased mortality in the VIP-KO recipients was not due to early graft rejection. The addition of donor splenocytes to the graft resulted in significantly more GvHD-related mortality in VIP-KO recipients compared with WT recipients (Figure 1), with 80% vs 100% day 75 survival among recipients of 1x106 splenocytes (p=NS) and 0% vs 40% survival (p<0.01) among recipients of 3x106 donor splenocytes, respectively.To determine how the absence of VIP production in KO recipients modulates immune effector mechanisms that contributes to the differential survival rate post allo-BMT, we analyzed the recipients' splenocytes on day 30 post transplant with flow cytometry and staining with CD3, CD4, CD8, CD62L, CD44 and PD1 antibodies. VIP-KO recipients transplanted with 5M TCD BM plus 1M splenocytes had more CD4+ central memory and effector T cells, and CD8+ central memory and effector T cells. VIP-KO recipients transplanted with 5×106 TCD BM alone in addition had significantly more CD4 and CD8 effector T cells (p<0.01). VIP-KO recipients transplanted with 5×106 TCD BM plus 1x106 splenocytes had more PD1 expression on donor CD4+ central memory and effector T cells, and CD8+ central memory effector T cells, consistent with enhanced antigen-mediated activation.To visualize VIP produced by the host cells, we transplanted C57/BL6 recipients expressing a GFP transgene under the control of the VIP promoter (n=10) with 5×106 TCD BM plus 3 ×106 splenocytes from WT B10BR donors, and visualized VIP gene promoter activity as GFP expression in recipient lymphoid tissues and GvHD target organs. Only small number of CD45+ VIP-GFP+ cells were detected from in host liver, spleen and lymph nodes on day 10 post transplant (Figure 2), suggesting residual host leukocytes, particularly dendritic cells, expressed VIP.Conclusion: VIP production by recipient antigen presenting cells is a key pathway in limiting the activation of donor T cells. Physiological expression of VIP by host leukocytes regulate the GvHD activity of donor T cells. Novel methods to enhance VIP production in transplant recipients or the parenteral administration of VIP receptor agonists may limit GvHD, and may be particularly important in allo-BMT where GvL is not necessarily critical for success, such as transplants for hemoglobinopathy. [Display omitted] DisclosuresWaller:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; PRA: Consultancy; AMGEN: Consultancy; National Institutes of Health: Research Funding; Coulter Foundation: Research Funding; Helocyte: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Katz Foundation: Research Funding; Chimerix: Equity Ownership; Cerus: Equity Ownership; Celldex: Consultancy.

Clinical Report on 6 Children with Hematopoietic Disease with Umbilical Cord Blood Transplantation.

To study the efficacy of hematopoietic stem cell transplant in children with umbilical cord blood 6 children received transplant in our hospital within these 3 years.Except for one patient suffered from acute severe aplastic anemia (SAA). All others had relapsed leukemia. Among them, 3 were relapsed ALL and another 2 were relapsed AML. The average age was 55 months (range, 38 months to 7 years old) and average weight was 17.8 kg (range, 13.5 ~28 kg) in this group. Only a 7 years old ALL-CR3 patient was conditioned with fraction total body irradiation, all the others were conditioned with non radiation regimen. Patients with ALL conditioned with Busulfan 16 mg + Cyclophosphomide 120 mg + VP1640 mg and patients with AML received Busulfan 16 mg + Cyclophosphomide 200 mg. Fludarabine 30 mg/m 2 × 5day +CTX 60 mg/kg × 2 day was chosen as the conditioning regimen for our SAA-I patient. 1 patient did not use ATG because the donor was matched related sibling, all the others received 6–30 mg/kg. day × 5 day ATG(Fresennius S). Only CSA was used for prophylaxis GVHD in this group.Two cord blood provided from Shanghai and Beijing cord blood bank separately and another 4 were from Guangzhou cord blood bank. The average total neuclear cell count was 10×107/kg (6.5~13×107/kg) and CD34+ cell was 0.81×106/kg (0.2~1.47×107/kg). 5 patients had hematopoitic reconstitution with the average time for absolute neutrophil over 0.1×109/L and 0.5×109/L 16 days (range, 14~19day) and 17days (range, 14~20day) respectively. The average time for transfusion independent platelet count recovered over 20×109/L, 50×109/L was 33.3 days (range, 27~39 days) and 49.3days (range, 48~51days) separately. 4 patients had grade II GVHD after transplantation. All patients infected with CMV and one died from CMV pneumonia on day +43, another one had encephalitis on day +124 and treated with anti-epilepsy drug till now. The average time for follow up was 29 months (13–41 months) in this group. One patient suffered from grade III hemorrhagic cystitis on day +60 and resolved on day +100 after being treated with hydration, alkalization and anti-virus drugs. One patient suffered from a great deal of hemorrhagic pericardial effusion on day +172, because of no response after several times of pericardial puncture, dissection of pericardium was received on day +182 and recovered since then. Another patient received transfusion with autologous back-up peripheral blood stem cells on day +40 because of graft failure confirmed by DNA chimerism study on day +34. This patient died of leukemia relapse on day +73. 4 patients have kept on disease free survival till now.These results further demonstrate umbilical cord blood transplant is a therapeutic option for children with very poor-prognosis leukemia and who lack an HLA-identical sibling or matched unrelated donor. But the decision of transplant for SAA patient with cord blood should be considered carefully.

Chimerism Monitoring Techniques after Hematopoietic Stem Cell Transplantation: An Overview of the Last 15 Years of Innovations.

Chimerism analysis is a well-established method for monitoring the state of hematopoietic stem cell transplantation (HSCT) over time by analyzing peripheral blood or bone marrow samples of the recipient in several malignant and non-malignant hematologic diseases. From a clinical point of view, a continuous monitoring is fundamental for an effective early therapeutic intervention. This paper provides a comparative overview of the main molecular biology techniques which can be used to study chimerism after bone marrow transplantation, focusing on their advantages and disadvantages. According to the examined literature, short tandem repeats (STR) analysis through simple PCR coupled with capillary electrophoresis (STR-PCR) is the most powerful method which guarantees a high power of differentiation between different individuals. However, other methods such as real-time quantitative PCR (qPCR), digital PCR (dPCR), and next-generation sequencing (NGS) technology were developed to overcome the technical limits of STR-PCR. In particular, these other techniques guarantee a higher sensitivity, which allows for the detection of chimerism at an earlier stage, hence expanding the window for therapeutic intervention. After a comparative evaluation of the various techniques, it seems clear that STR-PCR still remains the gold standard option for chimerism study, even if it is likely that both dPCR and NGS could supplement or even replace the common methods of STR analysis.

Passenger Lymphocyte Syndrome in a Pediatric Lung Transplant Recipient with Previous Bone Marrow Transplantation

Introduction Passenger lymphocyte syndrome (PLS) is a rare complication following solid organ transplantation (SOT). It consists of immune-mediated hemolysis due to antibodies produced by donor B-lymphocytes against recipient red blood cells [1]. Incidence across all SOT is as low as 0.5% in adults, with the highest rates being in lung and heart [2,3,4]. There have been no cases reported in children post-lung transplantation to our knowledge. Case Report A 12-year-old female received a double lung transplantation for bronchiolitis obliterans due to graft versus-host disease (GHVD). She had undergone allogeneic BMT 6 years prior for myelodysplastic syndrome (MDS). Her post-BMT course was complicated by lung, skin, and gastrointestinal GVHD. Lung transplant was uncomplicated: with donor blood group O+, recipient blood group A+, and crossmatch negative. On post-operative day 13, there was an acute drop in hemoglobin: from 131 g/L day 7, to 87 g/L day 13, to 66 g/L the same day. Septic workup was negative. Direct antiglobulin test was positive. Hemolysis markers were present with high LDH, high unconjugated bilirubin, and low haptoglobin. Eluate test was positive for anti-A1 antibody, consistent with a diagnosis of PLS. She received donor-type (O+) red cells, and was given IVIG 600 mg/kg for hypogammaglobulinemia. Hemoglobin recovered after day 21. Given her BMT history, concern around recurrent MDS was also raised. A chimerism study was sent on day 22 for this with the added benefit of examining for circulating lung-donor lymphocytes. No MDS was revealed. FISH revealed all cells were chromosomally XX (recipient had received BMT from a female donor), indicating no persistent circulating lung-donor lymphocytes since lung donor was male. Summary We report PLS following lung transplant in a 12-year-old post-BMT. Risk factors included minor ABO mismatch and organ type transplanted, with a high lymphocyte burden in lungs compared to other organs [3,4]. It is unclear whether prior allogeneic BMT and GVHD were added risk factors. Given higher PLS rates post-BMT [5], one could postulate that this may have also predisposed to PLS in this case. Albeit self-limiting, transfusion with donor blood group is necessary to prevent further hemolysis. Finally, a chimerism study for MDS has the added benefit of searching for lung-donor lymphocytes, if still present in circulation.

The beta-glucuronidase intracisternal A particle insertion model results in similar overall MPSVII phenotype as the single base deletion model when on the same C57BL/6J mouse background

Two unique gene mutations in the enzyme beta-glucuronidase (GUSB) that result in the lysosomal storage disease Mucopolysaccharidosis (MPS) type VII had previously been reported to have differing disease phenotype severities when compared on differing mouse strains. The MPSVII mouse has proven to be a highly efficacious model to study mucopolysaccharidoses and for evaluating potential gene or stem cell therapies for lysosomal storage diseases. We examined the single base pair deletion (MPSVII) and the intracisternal A particle element insertion (MPSVII2J) in GUSB compared with control animals by skeletal measures, electroretinography, auditory-evoked brainstem response and life span on a C57BL/6J background strain. In all measures, both mutations result in either a trend toward or significant changes from the background strain control. In all measures, there is no significant phenotypic difference between the two mutations. The 2J variant is a more easily genotyped and equally affected phenotype, which holds promise for further studies of chimerism and stem cell therapy approaches.

Donor Chimerism Study by Single Nucleotide Polymorphism using SYBR green based Real Time PCR.

Objective:To optimize and evaluate a real time PCR of Single Nucleotide Polymorphism by SYBR Green method for detection of donor chimerism after haematopoietic stem cell transplantation.Methods:This descriptive study was conducted at Genetic Resource Centre (GRC) Lab Rawalpindi from Oct 2017 - Dec 2019. A total of twenty patients of post haematopoietic stem cell transplant with various haematological disorders were studied to see the status of donor chimerism by using SNP real time PCR using SYBR Green method and short tandem repeat PCR. These patients had undergone allogeneic HSCT from HLA-matched sibling donors at Pakistan Institute of Medical Science and Armed Forces Bone Marrow Transplant Centre.Results:Real time PCR using SYBR Green was able to detect significant amount of chimerism in all 20 patients having undergone HSCT. Regarding precision of the real time PCR assay the mean value of donor chimerism was 94.1% (SD 3.96) and by STR PCR it was 95.1% (SD 1.41). The assay was found to be sensitive with a detection limit of <1%.Conclusion:Our results demonstrate that SNP analysis by SYBR Green real time PCR may be used for the evaluation of chimerism status in patients having undergone HSCT with a sensitivity of <1%. Hence donor chimerism by this sensitive method can be used in monitoring of chimerism in post-transplant patients with various haematological disorders.

Hematological Complications of Donor Origin Following Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplant (HSCT) offers curative therapy for some malignant hematological disease and marrow failure states. Post-transplant donor cell pathology, which might become more prevalent in long term survivors of allogeneic HSCT, warrants further study. We report five recipients of allogeneic HSCT from our institution who developed donor cell abnormalities, discuss potential mechanisms underlying these disorders and propose potential avenues for future study. Case 1 involves a 43-year-old female who developed leukopenia and adenopathy and was subsequently found to have diffuse large B-cell lymphoma. She received a matched unrelated donor (MUD) SCT from a 23-year-old male and subsequently developed a reactive lymphocytosis. In case 2, a 44-year-old male with myelodysplastic syndrome (MDS) received an allogenic peripheral blood (PB) SCT from his 50-year-old sister and later developed donor-cell derived acute myeloid leukemia (AML) with chimerism studies demonstrating 100% donor cells in CD3, CD33/66 and CD56. Case 3 involved a 72-year-old female with AML who received a PBSCT from her 65-year-old brother and subsequently developed MDS with peripheral blood chimerism studies demonstrating 100% donor cells in all three compartments. Case 4 involved a 40-year-old female with AML who achieved molecular remission following chemotherapy and underwent a double umbilical cord transplant, subsequently developing donor-cell derived AML with chimerism studies negative for host cells. Finally, case 5 highlighted a 54-year-old male with AML who underwent a MUDSCT from a 44-year-old male and passed away from progressive leukemia. Peripheral blood demonstrated only donor cells with near identity of phenotypic and molecular studies. It remains possible that no malignant cells exited the marrow. Nonetheless, this case in particular highlights the difficulty in precisely diagnosing donor cell leukemia (DCL). DCL correlates with increasing donor age, suggesting that age-related mutation in hematopoietic stem cells might be the cause. Notably, the average donor age in our case studies was 53 years old. Hematopoiesis is maintained by both hematopoietic stem and progenitor cells and during the normal aging process becomes clonally restricted. Clonal hematopoiesis of indeterminate potential (CHIP) describes the presence of a clonal blood cell population associated with a driver mutation at a VAF ≥ 2%, in the absence of severe cytopenia or a WHO defined disorder. These mutations are found in 5% of individuals greater than 50 years of age, with the most common gene mutations being found inDNMT3A, ASXL1,andTET2. The rate of development of overt neoplasia in patients with CHIP is 0.5% to 1% per year and these patients are 4 to 15 times more likely to develop a hematologic malignancy. CHIP mutations might confer a survival advantage allowing these clones to expand under the influence of a selection pressure such as chemotherapy or immunosuppression. These surviving clones might be the origin of DCL. DCL is estimated to account for approximately 1-5% of all post-transplant leukemia. When transplanted, expansion of these clones as required for repopulation of the hematopoietic stem cell niche might accelerate the acquisition of secondary mutations providing surviving mutated clones a survival advantage leading to malignancy. An open question remains particularly as it concerns older donors &amp;gt;55 years, as to whether they should be screened by next generation sequencing (NGS) for the presence of mutations known to be associated with CHIP or frank hematologic malignancy. If the incidence of DCL increases, consideration should be given to subjecting older donors to these screening methods. When multiple donors of different age are available preference can be assigned to younger donors to avoid possibly transmitting CHIP. Disclosures Fazal: BMS:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Jansen:Speakers Bureau;Novartis:Consultancy, Speakers Bureau;Celgene:Speakers Bureau;Gilead/Kite:Consultancy, Speakers Bureau;Glaxosmith Kline:Consultancy, Speakers Bureau;Agios:Consultancy, Speakers Bureau;Takeda:Consultancy, Speakers Bureau;Incyte Corporation:Consultancy, Honoraria, Speakers Bureau;Karyopham:Speakers Bureau;Amgen:Consultancy, Speakers Bureau;Stemline:Consultancy, Speakers Bureau;Jazz Pharma:Consultancy, Speakers Bureau.

Immune Hemolysis after a Hematopoietic Progenitor Cell Transplantation for Sickle Cell Disease: A Case Report

Background:The incidence of Delayed Hemolytic Transfusion Reaction (DHTR) is 0.04% but is higher in patients with sickle cell disease (SCD). Although it has been reported in hematopoietic progenitor cell (HPC) transplantation, its incidence in SCD following nonmyeloablative HPC transplantation remains unknown. We report a recipient with blood group O who received a nonmyeloablative HPC transplantation with blood group A three years ago. She is now typing as blood group A and developed a DHTR after receiving group A red blood cells (RBC). Case Report:A 42-year-old female was transferred from an outside hospital (OSH) for management of Acute T-Cell Lymphoblastic Leukemia. Three years prior, she received a group A+ matched sibling nonmyeloablative HPC transplantation for SCD. Her original blood group was O+, and she had a history of clinically significant RBC alloantibodies (anti-E, anti-C, anti-Goa, anti-Kell, and anti-Jkb). On admission to OSH (Day 1), the type and screen showed group A+ without ABO discrepancy. The antibody screen was negative. The OSH blood bank was unaware of her immunohematologic history, because it was not communicated to them by the OSH hematologists, and the only documented diagnosis was that of acute leukemia. Per SOP, OSH performed only immediate spin crossmatch. She was transfused three units of A+ RBC at OSH in preparation for her transfer to us on Day 3 (Fig. 1). At admission to our hospital, her laboratory parameters were suggestive of both tumor lysis syndrome and hemolysis. Her type and screen specimen was grossly hemolyzed. She typed as Group A+. The Direct Antiglobulin Test (DAT) was positive with polyspecific antisera, positive for IgG and negative for bound complement factors. Antibody screen was negative except for the anti-Goa. In the eluate, we identified anti-A or anti-A,B, which were not differentiated for lack of clinical implications. Per our request, OSH retrospectively performed a pre-transfusion DAT, which was negative, and an AHG crossmatch of the pre-transfusion sample. The results showed that the RBC transfused at OSH on Day 1 were incompatible (1+) and those transfused on Day 2 after O+ platelet transfusion were compatible (Fig. 1). This confirmed that the eluted antibodies were not passively transferred from the platelet transfusion but were rather isoagglutinins from the patient's own plasma of her original blood type O. Chimerism studies indicted the presence of only 25% donor CD3 and 30% donor myeloid cells. Further studies at our institution confirmed the hemolysis to be due to anti-A/A,B and not anti-Goa. Antibody titers of the patient's plasma with A1 and A2 cells were low (1) and negative, respectively. The titer of the eluate with A1 cells and B cells was 4 and 2, respectively. The crossmatch of the patient's plasma with A1 cells was negative at immediate spin and 37oC but positive at the IgG phase, which explains the negative crossmatch at immediate spin at OSH. We believe that the exposure of the 2 incompatible A+ RBC at OSH prompted an anamnestic response, causing the hemolysis of the transfused RBC. Subsequently, the patient required the transfusion of 3 additional RBC. Due to the presence of positive DAT developed after 24 hours of transfusion (on Day 3), the positive elution test, inadequate rise of post-transfusion Hb level and rapid fall in Hb back to the concentration pre-transfusion (Fig. 1), this reaction is best classified as a definitive DHTR in accordance with the CDC hemovigilance guidelines. Conclusion:This case is a warning for the perfect storm from the combination of HPC transplantation and SCD. Our patient had a history of transplantation for SCD and clinically significant alloantibodies. OSH blood bank was not aware of her immunohematologic history, and she received incompatible RBC units that were crossmatched at immediate spin only. She subsequently developed a DHTR which was clinically significant, requiring blood transfusion. This is a good reminder of the importance of good communication between clinicians and the transfusion services. The need for caution when using electronic crossmatch or immediate spin is also important, especially in this era of transplantation for SCD. The absence of RBC antibodies cannot be assumed when a transfusion history is lacking. Increasing awareness, prevention and early recognition and treatment are essential to avoid the potential fatal complication of hemolytic transfusion reactions. Disclosures No relevant conflicts of interest to declare.

CXXC5 variant in an immunodeficient patient with a progressive loss of hematopoietic cells

Partial loss of function variant in CXXC5 identified in an immunodeficient patient with progressive bone marrow failure, corrected by bone marrow transplant.

TLR2/6 signaling promotes the expansion of premalignant hematopoietic stem and progenitor cells in the NUP98–HOXD13 mouse model of MDS

Toll-like receptor 2 (TLR2) expression is increased on hematopoietic stem and progenitor cells (HSPCs) of patients with myelodysplastic syndromes (MDS), and enhanced TLR2 signaling is thought to contribute to MDS pathogenesis. Notably, TLR2 heterodimerizes with TLR1 or TLR6, and while high TLR2 is associated with lower-risk disease, high TLR6, but not TLR1, correlates with higher-risk disease. This raises the possibility of heterodimer-specific effects of TLR2 signaling in MDS, and in the work described here, we tested the effects of specific modulation of TLR1/2 versus TLR2/6 signaling on premalignant HSPCs. Indeed, chronic stimulation of TLR2/6, but not TLR1/2, accelerates leukemic transformation in the NHD13 mouse model of MDS, and conversely, loss of TLR6, but not TLR1, slows this process. TLR2/6 stimulation expands premalignant HSPCs, and chimeric mouse studies revealed that cell-autonomous signaling contributes to this expansion. Finally, TLR2/6 stimulation is associated with an enrichment of Myc and mTORC1 activities. While Myc inhibition partially suppressed the TLR2/6 agonist-mediated expansion of premalignant HSPCs, inhibition of mTORC1 exacerbated it, suggesting that these pathways play opposite roles in regulating the effects of TLR2/6 ligation on HSPCs. Together, these data reveal heterodimer-specific effects of TLR2 signaling on premalignant HSPCs, with TLR2/6 signaling promoting their expansion and leukemic transformation.

553 Clinical characteristics of acute graft-versus-host disease in small bowel and multi-visceral transplant recipients

Acute graft-versus-host disease (aGVHD) following stem cell transplantation is characterized by rash, liver dysfunction, and diarrhea. Although aGVHD is uncommon following solid organ transplantation, we find higher rates of aGVHD after intestinal (ITx) and multi-visceral transplantation (MVT). In these cases we find a unique presentation as the donor liver (if present) and intestine are spared, leaving skin as the main target. Between 2013 and 2019, we diagnosed 17 cases of aGVHD following ITx (n=9) and MVT including both intestine and liver (n=8). Median time to onset of aGVHD was 37 days. Erythema on the trunk, palms, or both was the most common presenting skin finding (15/17 patients). All patients had skin biopsies at the time of diagnosis. Patients presented with grade 1 (7/17), grade 2 (8/17), grade 3 (1/17), and grade 4 (1/17) aGVHD. Peripheral blood chimerism studies obtained in 11 patients revealed donor CD3+ lymphocytes in 5 (45%). Two patients had passenger lymphocyte syndrome, and partial donor stem cell engraftment was diagnosed in a third patient. Native GI tract involvement (usually rectosigmoid), was found in 7/17 patients; other sites included bone marrow, and native liver. In all, 6/17 patients had no evidence of extra-cutaneous aGVHD. Treatment strategies included systemic and topical corticosteroids, anti-thymocyte globulin, extracorporeal photopheresis, and/or JAK inhibitors. Complications of immunosuppression including opportunistic infections (aspergillosis and toxoplasmosis) and post-transplant lymphoproliferative disorder occurred in 7/17 patients. In summary, rash is typically the first, and may be the only, manifestation of aGVHD following luminal organ transplants. Diagnosis may be aided by time of onset of cutaneous manifestations, extra-cutaneous signs, and peripheral blood chimerism.

RPS28B mRNA acts as a scaffold promoting cis-translational interaction of proteins driving P-body assembly.

P-bodies (PBs) are cytoplasmic mRNA-protein (mRNP) granules conserved throughout eukaryotes which are implicated in the repression, storage and degradation of mRNAs. PB assembly is driven by proteins with self-interacting and low-complexity domains. Non-translating mRNA also stimulates PB assembly, however no studies to date have explored whether particular mRNA transcripts are more critical than others in facilitating PB assembly. Previous work revealed that rps28bΔ (small ribosomal subunit-28B) mutants do not form PBs under normal growth conditions. Here, we demonstrate that the RPS28B 3′UTR is important for PB assembly, consistent with it harboring a binding site for the PB assembly protein Edc3. However, expression of the RPS28B 3′UTR alone is insufficient to drive PB assembly. Intriguingly, chimeric mRNA studies revealed that Rps28 protein, translated in cis from an mRNA bearing the RPS28B 3′UTR, physically interacts more strongly with Edc3 than Rps28 protein synthesized in trans. This Edc3-Rps28 interaction in turn facilitates PB assembly. Our work indicates that PB assembly may be nucleated by specific RNA ‘scaffolds’. Furthermore, this is the first description in yeast to our knowledge of a cis-translated protein interacting with another protein in the 3′UTR of the mRNA which encoded it, which in turn stimulates assembly of cellular structures.