Donor Cell Chimerism Research Articles

Efficacy and risk of donor-derived CAR-T treatment of relapsed B-cell acute lymphoblastic leukemia after hematopoietic stem cell transplantation

The one-year survival rate for patients experiencing a relapse of B-cell acute lymphocytic leukemia (B-ALL) following hematopoietic stem cell transplantation (HSCT) is approximately 30%. Patients experiencing a relapse after allogeneic HSCT frequently encounter difficulties in obtaining autologous CAR-T products. We conducted a study involving 14 patients who received donor-derived CAR-T therapy for relapsed B-ALL following HSCT between August 2019 and May 2023 in our center. The results revealed a CR/CRi rate of 78.6% (11/14), a GVHD rate of 21.4% (3/14), and a 1-year overall survival (OS) rate of 56%. Decreased bone marrow donor cell chimerism in 9 patients recovered after CAR-T therapy. The main causes of death were disease progression and infection. Further analysis showed that GVHD (HR 7.224, 95% CI 1.42–36.82, P = 0.017) and platelet recovery at 30 days (HR 6.807, 95% CI 1.61–28.83, P = 0.009) are significantly associated with OS after CAR-T therapy. Based on the findings, we conclude that donor-derived CAR-T cells are effective in treating relapsed B-ALL patients following HSCT. Additionally, GVHD and poor platelet recovery impact OS, but further verification with a larger sample size is needed.

Treatment of Mixed Chimerism After Hematopoietic Stem Cell Transplantation in Patients with Thalassemia Major

Background: Incomplete donor cell chimerism often occurs in thalassemia transplant due to host cells remain or reappear overtime, which is termed as mixed chimerism (MC). Objective: To compare the immunosuppression withdrawal (ISW) and donor lymphocyte infusion (DLI) in the correction of mixed chimerism (MC) after thalassemia transplantation. Methods: Eighty-seven patients with post-transplant MC admitted in our center from January 2010 to December 2019 were analyzed. Among them donor cells of 90%-95% and 75%-89% were classified as MC1 and MC2 respectively. MC3 donor cells <75%. The incidence and correction rate of MC, the occurrence rate of graft versus host disease (GVHD), timing of DLI were studied. Results: DLI was associated with higher correction rates and higher GVHD than ISW. In MC1 group, higher GVHD occurred in early and intermediate stage (<I>P</I> = 0.024/0.023) than ISW. In MC2 group, DLI in late stages had higher correction rates than ISW (<I>P</I> = 0.001). Conclusion: ISW was the primary strategy for MC1 patients. DLI should be given to the late-stage MC2 patients quickly. The earlier the treatment is provided, regardless of ISW or DLI, the more likely that patients develop GVHD.

Virus-specific TRM cells of both donor and recipient origin reside in human kidney transplants.

Tissue-resident lymphocytes (TRLs) are critical for local protection against viral pathogens in peripheral tissue. However, it is unclear if TRLs perform a similar role in transplanted organs under chronic immunosuppressed conditions. The present study aimed to characterize the TRL compartment in human kidney transplant nephrectomies and examine its potential role in antiviral immunity. The TRL compartment of kidney transplants contained diverse innate(-like) and adaptive TRL populations expressing the canonical residency markers CD69, CD103, and CD49a. Chimerism of donor and recipient cells was present in 43% of kidney transplants and occurred in all TRL subpopulations. Paired single-cell transcriptome and T-cell receptor (TCR) sequencing showed that donor and recipient tissue-resident memory T (TRM) cells exhibit striking similarities in their transcriptomic profiles and share numerous TCR clonotypes predicted to target viral pathogens. Virus dextramer staining further confirmed that CD8 TRM cells of both donor and recipient origin cexpress TCRs with specificities against common viruses, including CMV, EBV, BK polyomavirus, and influenza A. Overall, the study results demonstrate that a diverse population of TRLs resides in kidney transplants and offer compelling evidence that TRM cells of both donor and recipient origin reside within this TRL population and may contribute to local protection against viral pathogens.

A novel flow-cytometric based method to assess post-HSCT donorchimerism exploiting RNA hybridization.

Analysis of donor-recipient chimerism after hematopoietic stem cell transplantation (HSCT) is of pivotal importance for patient's clinical management, especially in the context of mixed chimerism. Patients are routinely monitored for chimerism in sorted subsets of peripheral blood cells. However, measurement of chimerism in sorted immune cell subsets is technically challenging and time consuming. We here propose a novel, flow cytometry-based approach to detect donor cell chimerism in sex-mismatched HSCT. We exploit RNA PrimeFlow™ system, based on RNA hybridization, to detect mRNA from a lysine demethylase encoded by Y chromosome, KDM5D. This approach allows to distinguish male and female derived cells with around 1% sensitivity. The procedure can be coupled with multiparametric immunophenotyping to assess chimerism in specific immune cell subsets without the need for prior FACS-sorting. We apply this method to a cohort of HSCT patients (n = 10) and we show that it is consistent with standard PCR-based method. We also show that different T lymphocyte subsets display variable degrees of donor chimerism, especially in CD8+ T cell compartment where we observe an enrichment for recipient chimerism in central memory T cells. This method can be exploited to advance current knowledge on immune reconstitution focusing on specific subsets avoiding prior FACS-sorting.

Updated Findings of a Phase II Study of Ruxolitinib Pre-, during- and Post-Hematopoietic Stem Cell Transplantation for Patients with Primary or Secondary Myelofibrosis

Background: Despite tremendous progress in the treatment of myelofibrosis in the last decade, the only curative therapy remains allogeneic hematopoietic cell transplantation (HCT). There are now three FDA-approved JAK inhibitors (JAKi) to treat symptomatic splenomegaly and constitutional symptoms in myelofibrosis, although these agents do not prevent disease progression. Discontinuation of JAKi before (pre) HCT is challenging as patients can experience return of symptoms. Thus, ruxolitinib is often continued during HCT in an off-label fashion; however, little is known about the safety and efficacy of this approach. Furthermore, there is increasing use of ruxolitinib after (post) HCT, following its approval in treating refractory graft-versus-host disease (GVHD). Methods: We previously reported the interim results of a phase II, multicenter, investigator-initiated trial investigating ruxolitinib given pre-, during- and for 1-year post-HCT for patients with primary or secondary MF (NCT03427866). Here we present the results of the study with full accrual of 43 patients. The primary endpoint was 1-year GVHD-free, relapse-free survival (GRFS). Secondary endpoints included overall (OS) and progression free survival (PFS), engraftment, and incidence of acute and chronic GVHD. Patients were treated with reduced intensity conditioning with fludarabine (30mg/m 2/d x 5 days) and melphalan (100 or 140mg/m 2 x 1). Patients received peripheral blood stem cell grafts from either 7/8 or 8/8 HLA-matched donors. GVHD prophylaxis consisted of standard tacrolimus and methotrexate. Results: 44 patients with myelofibrosis were enrolled and 43 underwent HCT between 9/2018 and 5/2023. Median age was 66 (range 46-75), 37% were female (n=16), and most patients had DIPSS intermediate-2/high risk (n=36), 60% (n=27) were receiving ruxolitinib prior to enrolling on the study. Most (n=35) received 8/8 matched unrelated transplants and had measurable splenomegaly by either ultrasound or CT (n=40) prior to HCT. At baseline, 25 patients had JAK2 mutation, 8 had CALR and 5 had MPL mutations, 13 patients had ASXL1 mutations (Figure 1). No differences in outcomes were observed by pre-HCT mutational status. At day 100, all but one patient had undetectable mutations by next generation sequencing. The most common grade 3/4 hematologic adverse events (AE) were anemia (n=12), thrombocytopenia (14), neutropenia (n=11). Non-hematologic grade 3/4 events included hypertriglyceridemia (n=3). Median time to neutrophil engraftment was 15 days (range 4-38), one patient did not engraft. Median time to platelet (>20x10 9) engraftment was 25 days (range 11-145). At day 30, 2 patients had a donor-cell chimerism below 90% (one ultimately relapsed) and at day 100, 2 patients had chimerism below 90 (one ultimately relapsed). With a median follow up among survivors of 13 months (range 0.7 - 26), 1-year GRFS was 74%. OS, PFS and cumulative incidence of NRM and disease relapse were, 86%, 79%, 10%, and 10%, respectively (Figure 2). The cumulative incidence of grade III-IV acute GVHD at 6 months was 2.4%. Only one patient had grade IV acute GVHD. One year moderate/severe chronic GVHD was 11%In univariable analysis, thrombocytopenia of <40 x 10 9/L pre-HCT was associated with worse OS (1-yr OS: 64% vs 92%, p=0.008). Splenomegaly (defined as >17 cm) was associated with worse PFS (1-yr PFS: 100% vs 64%, p=0.046). There was an improvement in OS in patients that received ruxolitinib and achieved response pre-HCT compared to those that did not but this did not achieve statistical significance (1-yr OS 100% vs 74%, p=0.054). Discussion: This study is the first, multicenter trial to test ruxolitinib use pre-, during- and post-HCT in patients with myelofibrosis. Our results demonstrate safety and feasibility of this approach. Favorable rates of engraftment were observed with ongoing ruxolitinib administration even through hematopoietic nadir. We continue to demonstrate favorable outcomes of PFS, OS and GRFS with a low incidence of severe acute and chronic GVHD. Our results strongly support the use of ruxolitinib in the peri- and post-transplant setting for patients with myelofibrosis undergoing HCT.

Decline in Cardiolipin in Hematopoietic Stem Cell during Aging Alters Their Regenerative Potential

Hematopoietic Stem Cells (HSCs) are known for their regenerative potential. This property allowed the use of HSC in bone marrow transplantation to treat hematological disorders such as leukemia. However, aging influences these characteristics of HSCs. The major hallmarks of aging are limited self-renewal and reconstitution capacity resulting in HSC exhaustion in aged individuals. Mitochondrial metabolism and activity are active drivers of HSC fate decisions and data from our lab shows that mitochondria in aged HSCs have increased sphericity with a polarized mitochondrial network and a lower mitochondrial membrane potential. An important aspect of mitochondrial functions is the lipid composition of their membranes. A lipid trafficking assay showed an atypical pattern of lipid incorporation by mitochondria in aged HSCs suggesting that mitochondrial lipids become abnormal upon aging. The overall content of Cardiolipin (CL) which is a signature mitochondrial lipid, found exclusively in the inner mitochondrial membrane was also reduced during aging. This was done with flow cytometry using a CL marker NAO (Non-Acridine Orange). Tafazzin encoded by the gene TAZ is crucial for remodeling CL into its mature form which is required for maintaining mitochondrial functions. Mutations in the TAZ gene have been known to cause a reduction in the synthesis of mature CL which is often associated with HSCs functional defect. Western Blot analysis showed a considerable decrease in Taz protein expression in aged stem and progenitor cells (HSPC) compared to young HSPC. To understand the effect of reduced Taz expression on HSC functions, we used a doxycycline inducible, sh-RNA mediated TAZ knock-down (KD) mouse model. Bone marrow analysis indicates that the frequency of HSC and multipotent progenitors decreases significantly in 6-month-old TAZ KD mice compared to wild type (WT) mice. Our data shows that following 5FU induction TAZ KD mice have a significant reduction in the multipotent progenitor and HSC population on day 10 and day 21 post 5FU, indicating TAZ KD decrease the regenerative potential of HSCs under stress conditions. Further pointing towards the significance of cardiolipin in HSC regeneration. TAZ KD mice also have abnormal peripheral blood recovery post 5FU mainly affecting the platelets. We next examined the repopulation potential of HSCs isolated from TAZ KD mice using serial competitive transplantation assays. While primary transplanted mice of TAZ KD HSCs exhibited similar donor-cell chimerism compared to mice transplanted with WT HSCs, about 50-60%, secondary recipient of TAZ KD cells had significantly lower donor-cell chimerism than secondary recipients of WT cells. Further, donor-derived HSC frequency in the bone marrow of recipients of TAZ KD HSCs 24 weeks following engraftment was also significantly lower than recipients of WT HSCs. Hence, Taz-deficiency causes a defect in HSC self-renewal under regenerative conditions. We next evaluated whether restoring cardiolipin levels in mid-aged HSCs could rescue their functions. For this, mid-aged HSC were incubated for 3 days under conditions that maintain HSC functions in vitro with Alcar (Acetyl-L-Carnitine), which promotes CL production. In lipid incorporation assay, Alcar supplementation restored mitochondrial membrane potential, mitochondrial organization and lipid incorporation of mid-aged HSCs. In competitive transplant assay, while mid-aged WT HSC treated with vehicle gave rise to a myeloid-bias graft, Alcar supplemented mid-aged WT HSC gave rise to a more balanced graft with increased ratio lymphoid to myeloid cells in the peripheral blood 16 weeks following transplantation, albeit the rescue was partial. This study indicates that HSC mitochondrial membrane lipids become abnormal with aging which causes a decline in HSC function and highlights the critical role of mitochondrial cardiolipin in HSC functions. It implies that metabolite supplementation is a viable option for restoring age-related HSC functional defects.

Outcome of first or second transplantation using unrelated umbilical cord blood without ATG conditioning regimen for pediatric bone marrow failure disorders

BackgroundUnrelated umbilical cord blood transplantation (UCBT) for bone marrow failure (BMF) disorders using conditioning regimens without Anti-Thymocyte Globulin (ATG) has been used as an alternative transplantation for emerging patients without matched-sibling donors. Experience with this transplant modality in children is limited, especially as a secondary treatment for transplant failure patients. ProcedureWe retrospectively reviewed 17 consecutive bone marrow failure patients who underwent unrelated umbilical cord blood transplantation in our center and received conditioning regimens of Total Body Irradiation (TBI) or Busulfan (BU) + Fludarabine (FLU) + Cyclophosphamide (CY). ResultsAmong the 17 BMF patients, 15 patients were treated with first cord blood transplantation and another 2 with secondary cord blood transplantation because of graft failure after first haploidentical stem cell transplantation at days +38 and +82.All patients engrafted with a median donor cell chimerism of 50 % at days +7 (range, 16 %–99.95 %) and finally rose to 100 % at days +30. Median time to neutrophil engraftment was 19 days (range, 12–30) and time to platelet engraftment was 32 days (range, 18–61). Pre-engraftment syndrome (PES) was found in 16 patients (94.11 %, 16/17). Cumulative incidence of grades II to IV acute GVHD was 58.8 % (95 % CI: 32.7–84.9 %), and 17.6 % (95 % CI: 2.6–37.9 %) of patients developed chronic GVHD. The 3-year overall survival (OS) and failure-free survival (FFS) rates were 92.86 ± 6.88 %. ConclusionUCBT is an effective alternative treatment for bone marrow failure pediatric patients. TBI/BU + FLU + CY regimen ensure a high engraftment rate for unrelated umbilical cord blood transplantation, which overcomes the difficulty of graft failure. Secondary salvage use of cord blood transplantation may still be useful for patients who have failed after other transplantation.

Maternal dendritic cells influence fetal allograft response following murine in-utero hematopoietic stem cell transplantation

BackgroundIntrauterine hematopoietic stem cell transplantation (IUT), potentially curative in congenital haematological disease, is often inhibited by deleterious immune responses to donor cells resulting in subtherapeutic donor cell chimerism (DCC). Microchimerism of maternal immune cells (MMc) trafficked into transplanted recipients across the placenta may directly influence donor-specific alloresponsiveness, limiting DCC. We hypothesized that dendritic cells (DC) among trafficked MMc influence the development of tolerogenic or immunogenic responses towards donor cells, and investigated if maternal DC-depletion reduced recipient alloresponsiveness and enhanced DCC.MethodsUsing transgenic CD11c.DTR (C57BL/6) female mice enabled transient maternal DC-depletion with a single dose of diphtheria toxin (DT). CD11c.DTR females and BALB/c males were cross-mated, producing hybrid pups. IUT was performed at E14 following maternal DT administration 24 h prior. Bone marrow-derived mononuclear cells were transplanted, obtained from semi-allogenic BALB/c (paternal-derived; pIUT), C57BL/6 (maternal-derived; mIUT), or fully allogenic (aIUT) C3H donor mice. Recipient F1 pups were analyzed for DCC, while maternal and IUT-recipient immune cell profile and reactivity were examined via mixed lymphocyte reactivity functional assays. T- and B-cell receptor repertoire diversity in maternal and recipient cells were examined following donor cell exposure.ResultsDCC was highest and MMc was lowest following pIUT. In contrast, aIUT recipients had the lowest DCC and the highest MMc. In groups that were not DC-depleted, maternal cells trafficked post-IUT displayed reduced TCR & BCR clonotype diversity, while clonotype diversity was restored when dams were DC-depleted. Additionally, recipients displayed increased expression of regulatory T-cells and immune-inhibitory proteins, with reduced proinflammatory cytokine and donor-specific antibody production. DC-depletion did not impact initial donor chimerism. Postnatal transplantation without immunosuppression of paternal donor cells did not increase DCC in pIUT recipients; however there were no donor-specific antibody production or immune cell changes.ConclusionsThough maternal DC depletion did not improve DCC, we show for the first time that MMc influences donor-specific alloresponsiveness, possibly by expanding alloreactive clonotypes, and depleting maternal DC promotes and maintains acquired tolerance to donor cells independent of DCC, presenting a novel approach to enhancing donor cell tolerance following IUT. This may have value when planning repeat HSC transplantations to treat haemoglobinopathies.

Local Delivery of Adipose Stem Cells Promotes Allograft Survival in a Rat Hind-Limb Model of Vascularized Composite Allotransplantation.

Adipose stem cells (ASCs) are a promising cell-based immunotherapy because of their minimally invasive harvest, high yield, and immunomodulatory capacity. In this study, the authors investigated the effects of local versus systemic ASC delivery on vascularized composite allotransplant survival and alloimmune regulation. Lewis rats received hind-limb transplants from Brown Norway rats and were administered donor-derived ASCs (passage 3 or 4, 1 × 10 6 cells/rat) locally in the allograft, or contralateral limb, or systemically at postoperative day 1. Recipients were treated intraperitoneally with rabbit anti-rat lymphocyte serum on postoperative days 1 and 4 and daily tacrolimus for 21 days. Limb allografts were monitored for clinical signs of rejection. Donor cell chimerism, immune cell differentiation, and cytokine expression in recipient lymphoid organs were measured by flow cytometric analysis. The immunomodulation function of ASCs was tested by mixed lymphocyte reaction assay and ASC stimulation studies. Local-ASC-treated recipients achieved significant prolonged allograft survival (85.7% survived >130 days; n = 6) compared with systemic-ASC and contralateral-ASC groups. Secondary donor skin allografts transplanted to the local-ASC long-term surviving recipients accepted permanently without additional immunosuppression. The increases in donor cell chimerism and regulatory T-cells were evident in blood and draining lymph nodes of the local-ASC group. Moreover, mixed lymphocyte reaction showed that ASCs inhibited donor-specific T-cell proliferation independent of direct ASC-T-cell contact. ASCs up-regulated antiinflammatory molecules in response to cytokine stimulation in vitro. Local delivery of ASCs promoted long-term survival and modulated alloimmune responses in a full major histocompatibility complex-mismatched vascularized composite allotransplantation model and was more effective than systemic administration. ASCs are a readily available and abundant source of therapeutic cells that could decrease the amount of systemic immunosuppression required to maintain limb and face allografts.

Detecting Donor-Derived DNA by Real-Time PCR in Recipients Suspected of Graft-Versus-Host-Diseases After Liver Transplantation: A Case Series and Literature Review.

BACKGROUND Graft-versus-host disease (GVHD) after liver transplantation (LT) is a rare but fatal complication. GVHD diagnosis is usually based on clinical symptoms and pathologic confirmation. However, it is often misdiagnosed due to its non-specific symptoms. Here, we report the detection of donor-cell chimerism using peripheral blood (PB) donor-derived deoxyribonucleic acid (ddDNA) for 3 cases with suspected GVHD after LT (GVHD-LT) through real-time quantitative polymerase chain reaction (qPCR) assay targeting 39 insertions and/or deletions of chromosomes. MATERIAL AND METHODS The qPCR assay for detecting donor-cell chimerism was performed for 3 post-LT patients with suspected GVHD using KMRtype® and KMRtrack® assays (GenDx, Netherlands). The mean recipient/donor-cell fraction of informative markers unique to each recipient or donor was calculated. RESULTS In Case 1, who received living donor LT (LDLT) from his daughter, initial sign was diarrhea at post-operative day (POD) #23. Case 2 received unrelated deceased donor LT and initial sign was cytopenia at POD #29. Case 3 received LDLT from her son and GVHD associated cytopenia was developed at POD #80. Average PB ddDNA fractions in post-transplant samples of cases 1, 2, and 3 were 39.68%, 78.38%, and 4.76%, respectively. Despite an active treatment including steroid and tumor necrosis factor-alpha inhibitor, 2 patients (cases 1 and 2) died due to multiple organ failures. CONCLUSIONS Early detection of donor-cell chimerism may help halt fatal progression of GVHD-LT. A qPCR test targeting INDEL of chromosomes would be a helpful procedure for timely diagnosis of GVHD.

Bodyweight and Absolute Lymphocyte Count Based Dosing of Rabbit Anti-Thymocyte Globulin Results in Early CD4+ Immune Reconstitution in Patients with Inborn Errors of Metabolism Undergoing Umbilical Cord Blood Transplantation

Background Rabbit anti-thymocyte globulin (rATG) is used in allogeneic hematopoietic cell transplantation (HCT) to prevent graft failure through depletion of host T cells and decrease the risk of graft-versus-host disease (GVHD) through partial depletion of donor T cells. However, it has highly variable pharmacokinetics. Previous reports suggest rATG dosing should be based on recipient bodyweight and absolute lymphocyte count (ALC) prior to initiating rATG. This new dosing nomogram reduces rATG overexposure, potentially leading to faster immune reconstitution and subsequently superior overall outcomes. Methods Since January 12, 2017, we use a previously described rATG dosing nomogram based on bodyweight and ALC before administering rATG in patients with inborn errors of metabolism receiving an umbilical cord blood transplant at the University of Minnesota Masonic Children's Hospital. The proposed rATG dosing varied from 2 to 10 mg/kg based on bodyweight and recipient ALC prior to initiating rATG, starting 8 or 9 days before HCT. The myeloablative conditioning regimen was Busulfan based, with addition of Fludarabine and Cyclophosphamide for lymphodepletion, as well as Rituximab and/ or Bortezomib for prevention of immune-mediated cytopenia. For GVHD prophylaxis cyclosporin A or tacrolimus were used with methylprednisolone. The primary endpoint was CD4+ immune reconstitution (>50 CD4+ T-cells/mL) within 100 days after transplantation. Secondary endpoints included overall survival, graft failure, acute and chronic GVHD, myeloid and T cell donor chimerism at one year post transplant, time to neutrophil engraftment and viral reactivations of cytomegalovirus, adenovirus and Epstein Barr virus. The study protocol was approved by the Institutional Review Board of University of Minnesota. Results Between February 20, 2017, and June 28, 2021, 27 patients were included in the study. Median follow-up time was 31 months (IQR 22.0 to 38.5) and median age was 1.45 years (IQR 0.7 to 3.9). Ten (37%) of 27 patients were female. The baseline characteristics are presented in Table 1. rATG dose was adjusted below 10 mg/kg recipient body weight in 7 (26%) of 27 patients on average by 29% (SD 14.9%). The CD4+ recovery at 100 days post HCT was reached in 22 (85%) of 26 patients (Figure 1A). Overall, four (15%) patients died resulting in overall survival of 83% (95%CI 67% to 100%). Causes of death were intracranial bleeding due to immune-mediated thrombocytopenia (this patient was transplanted before introduction of bortezomib and rituximab for immune-mediated cytopenia prevention), diffuse alveolar hemorrhage, respiratory syncytial virus infection and disease progression. Median time to neutrophil engraftment was 14 days (IQR 12.5 to 18.0), and no graft failure was observed. All surviving patients had full myeloid chimerism and 14 of 23 (61%) of patients had full T cell donor chimerism at one year post HCT. Two (7%) patients developed acute GVHD grade II to IV (Figure 1B), and no patients had chronic GVHD. Six patients (22%) had cytomegalovirus viremia, five of them were seropositive before transplant, one patient was seronegative as was the mother of the donor. One patient, who was seronegative before transplant, had Epstein-Barr virus reactivation requiring treatment, no patient had adenovirus reactivation. Conclusion In pediatric patients with inborn errors of metabolism individualized dosing of rATG was associated with a robust and early CD4+ immune reconstitution, with low therapy related mortality, low GVHD incidence compared to historical findings and stable full myeloid chimerism. These results are in line to the findings of the PARACHUTE trial previously published. However, the PARACHUTE trial included only small number of patients with inborn errors of metabolism. Table 1. Baseline characteristicsFigure 1A. Cumulative incidence of CD4+ immune reconstitutionFigure 1B. Cumulative incidence of acute Graft-versus-host disease Grade 2 to 4Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Donor T and NK Cells with a Special Tissue-Resident Memory Phenotype Migrate into the Periphery of Lung Transplant Recipients - A Potential Feature for Tolerance Development

<h3>Purpose</h3> After lung transplantation (LuTx), a transient chimerism of donor cells can be observed in recipient blood due to the migration of lymphocytes from the transplanted lung into the periphery. We aimed to characterize the phenotype of donor T and NK cells and to investigate their potential origin from tissue-resident memory (TRM) T and NK cells. <h3>Methods</h3> Lymphocyte dynamics in recipient blood were determined in n=97 LuTx patients directly (T0), 24 hours (T24) and 3 (wks) weeks after LuTx using flow cytometry. Donor T and NK cells were analyzed by HLA class I allele-specific mAb in n=44 LuTx recipients. T and NK cell markers were used to discriminate circulating from TMR subsets in blood, organ storage solutions (perfusates, n=97), explanted lung parenchyma (n=28) and donor trachea (n=17). Single cell mRNA sequencing of explant lung parenchyma was conducted (n=16). <h3>Results</h3> In peripheral blood of all recipients, donor-derived T and NK cells were detected at T0, T24 and 3 wks after LuTx, showed higher CD69 expression compared to recipient cells (p=0.01 to 0.04) and were mostly CCR7⁻ memory cells. This phenotype was similar to T and NK cells in corresponding perfusates. In recipient parenchyma and donor trachea, most CD69⁺ T and NK cells showed coexpression of TRM markers (CD103, CD49a, PD-1) with significant enrichment in trachea (p<0.05). These other TRM markers were not found in circulating donor T and NK cells or in perfusates, arguing for different memory T and NK cell subsets. Sc-mRNA sequencing confirmed distinct TRM and TRM-like T cell subsets in lung parenchyma according to separate expression profiles. The presence of TRM T and NK cells did not have an impact on primary graft dysfunction (PGD) 24h after LuTx. Yet, patients with high frequencies of donor T cells showed a trend towards CLAD-free survival 2 years post LuTx, although no statistical significance was reached (p=0.15). <h3>Conclusion</h3> Our results demonstrate that donor T and NK cells found in recipient blood immediately after LuTx are separate subsets from circulating as well as pulmonary TRM T and NK cells, since they express CD69 but lack expression of other classical TRM markers. Donor T cells may be clinically relevant for tolerance induction and long-term survival after transplantation due to their unique features.

Real-World Experience of Cryopreserved Allogeneic Hematopoietic Grafts during the COVID-19 Pandemic: A Single-Center Report

In response to the widespread COVID-19 pandemic, cryopreservation of allogeneic donor apheresis products was implemented to mitigate the challenges of donor availability and product transport. Although logistically beneficial, the impact of cryopreservation on clinical outcomes and graft composition remains unclear. In this study, we compared outcomes and graft composition with cryopreserved versus fresh allografts in the setting of allogeneic hematopoietic cell transplantation (allo-HCT). We retrospectively analyzed the clinical outcomes of 30 consecutive patients who received cryopreserved allografts between March and August 2020 and 60 consecutive patients who received fresh allografts before the COVID-19 pandemic. Primary endpoints were hematopoietic engraftment and graft failure (GF), and secondary outcomes were overall survival (OS), relapse-free survival (RFS) and nonrelapse mortality (NRM). In addition, extended immunophenotype analysis was performed on cryopreserved and prospectively collected fresh apheresis samples. Compared with recipients of fresh allografts, both neutrophil and platelet recovery were delayed in recipients of cryopreserved reduced-intensity conditioning (RIC) allo-HCT, with a median time to engraftment of 24 days versus 18 days (P = .01) for neutrophils and 27 days versus 18 days (P = .069) for platelets. We observed primary GF in 4 of 30 patients in the cryopreserved cohort (13.3%) versus only 1 of 60 patients (1.7 %) in the fresh cohort (P = .03). Cryopreserved RIC allo-HCT was associated with significantly lower median total, myeloid, and T cell donor chimerism at 1 month. OS and RFS were inferior for cryopreserved graft recipients (hazard ratio [HR], 2.16; 95% confidence interval [CI], 1.00 to 4.67) and HR, 1.90; 95% CI, 0.95 to 3.79, respectively. Using an extended immunophenotype analysis, we compared 14 samples from the cryopreserved cohort to 6 prospectively collected fresh apheresis donor samples. These analyses showed both a decrease in total cell viability and a significantly reduced absolute number of natural killer cells (CD3−CD56+) in the cryopreserved apheresis samples. In this single-institution study, we found delayed engraftment and a trend toward clinical inferiority of cryopreserved allografts compared with fresh allografts. Further evaluation of the use of cryopreserved allografts and their impact on clinical and laboratory outcomes is warranted.

Donor CD3 Chimerism After Allogeneic Transplantation Predicts Probability Of Relapse For Patients Treated For Acute Myelogenous Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), and Non-Hodkgin's Lymphoma (NHL)

Introduction The ability to predict relapse in patients after HSCT is crucial for tailoring therapy including pre transplant conditioning regimens and post transplant interventions. Donor bone marrow CD34+ cell chimerism analyses has previously been reported to be predictive for post transplant relapse for acute leukemia. Rapid immune reconstitution after HSCT may be important in the development of a robust graft versus leukemia response. Methods We performed for a sequential series of patients a retrospective analysis of donor cell chimerism (DCC) of peripheral blood CD3+ cells at 4 week intervals starting at day 28 after transplantation. Donor bone marrow CD34+ chimerism was evaluated at day 84 after transplantation. Percent chimerism was measured by PCR based amplification of short tandem repeats (STR). From 2009-2012, percent DCC data were collected for patients with ALL (n=39), NHL (n=26), and AML (n=68). Median age at diagnosis was 45 (range 25-69), 51 (range 23-66), and 57 (range 38-75), respectively. Patients received either PBSC or bone marrow cells from related (n=81) or unrelated donors (n=52). At transplantation, patients with ALL were in CR1 (n=27), CR2 (n=4), CR3 (n=1), PIF (n=3), relapse (n=2), PR (n=5); patients with NHL were in PR (n=9), CRU1 (n=4), CRU2 (n=3), Cr1 (n=4), CR2 (n=3), CR3 (n=4); patients with AML were in CR1 (n=38), CR2 (n=6), relapse (n=8), PIF (n=13). Conditioning regimens for patients with ALL were cyclophosphamide (cy) and fractionated total body irradiation (TBI) (n=27) or fludarabine (flu) and melphalan (mel) (n=11). Patients with NHL were conditioned with flu/cy and rituximab (n=15), or flu/mel (n=8). Patients with AML were conditioned with either a myeloablative regimen of flu/busulfan+TBI (n=51), or a reduced intensity regimens of flu/mel or flu/cy/TBI (n=16). Those who had NHL or AML and received unrelated donor cells were given rituximab. Post transplant immunosuppression was tacrolimus and methotrexate for most patients. Patients were censored for relapse or death. For analysis of DCC, patients who relapsed or did not relapse were divided into quartiles by diagnosis. The probability of relapse was tested for quartile groups using chi square analysis. Adjustments were not made for competing risks. Results ALL patients in the upper three quartiles of CD3+ DCCs (1 of 34 patients, median=100%, range=84-100%, [Table 1][1]) compared to patients in the bottom quartile were less likely to relapse (3 of 7 patients, median=94%, range=82-95%, p<0.002). Similarly, the upper 2 quartiles of NHL patients who had at least 95% day 28 CD3+ DCC were less likely to relapse (2 of 16 patients, median 95%, range 63-100%, p<0.007, [Table 1][1]). 7 of 48 patients with AML in the upper 3 quartiles who had day 28 CD3+ DCC of at least 90% (median=100%, range 60-100%) relapsed, trending lower compared to those in the lower quartile (p<0.06). CD3+ DCCs for day 56 and day 84 were not statistically significant for predicting future relapse. View this table: Table 1 CD3+ DCC at 28 days Day 84 CD34+ DCCs were also analyzed as described. The upper quartile of patients with ALL who did not relapse achieved a 100% DCC (1 of 23 patients relapsed, median 100%, range 82-100%, [Table 2][2]). Those that did not achieve 100% DCC for CD34+ cells by day 84 more likely to relapse (p<0.009). For patients with NHL, the upper 3 quartiles of those who did achieve 83% CD34+ DCC (1 of 7, median=90%, range 83-100%, p<0.008) by day 84 were less likely to relapse. The same method was used for patients with AML and those that did achieve 90% CD34+ DCC for cells by day 84 were not likely to relapse (5 of 35, median=96%, range=63-100%, p<0.09). View this table: Table 2 CD34+ DCC at 84 days Conclusions These findings suggest that failure to achieve early donor cell engraftment predicts for relapse. Relapse remains a major cause for treatment failure. Improvements in transplant conditioning may improve early lymphoid (CD3+) and myeloid (CD34+) engraftment and decrease the risk of disease relapse. Early monitoring of CD3+ DCC may identify patients who would benefit from post transplant intervention. Disclosures: No relevant conflicts of interest to declare. [1]: #T1 [2]: #T2

Stable Engraftment with Haploidentical Peripheral Blood Stem Cell Transplantation in Adult Patients with Sickle Cell Disease

The widespread application of allogeneic hematopoietic stem cell transplantation (HSCT) to cure patients with clinically aggressive sickle cell disease (SCD) has been limited by a lack of HLA-matched sibling donors. HSCT from a haploidentical donor represents a feasible option to increase transplant availability to a majority of the patients. However, current published studies using haploidentical donors in adults with SCD have resulted in long-term event-free survival rates of only 50-57%.Here we describe 10 consecutive haploidentical HSCT (haplo-HSCT) performed in adult patients with clinically aggressive SCD lacking an HLA-matched sibling donor. The median age of recipients was 27 years (range, 20-52 years) and all 10 patients met at least two standard SCD eligibility criteria for HSCT (Table 1). Based on our successful experience in matched related peripheral blood stem cell (PBSC) HSCT using a nonmyeloablative conditioning regimen with alemtuzumab 1mg/kg and 3 Gy total body irradiation (TBI), we initially performed two haplo-HSCT using the same conditioning regimen followed by high dose post-transplant cyclophosphamide (PT-Cy) at 50mg/kg on day+3 and +4. Both SCD patients failed to engraft donor cells and recovered autologous neutrophils on day +39 and day +34, respectively.In the following 8 patients, transplanted between September 2015 and March 2017, we utilized a modified version of the Hopkins protocol with: fludarabine 30mg/m2, Cy 14.5mg/kg, 3 Gy TBI (increased from the original 2 Gy dose), antithymocyte globulin 0.5mg/kg, PT-Cy 50mg/kg, and infusing mobilized PBSC instead of marrow cells (Table 1). G-CSF at 5µg/kg was administered starting on day +12 post-HSCT in neutropenic patients with HbS <30% and the median time to neutrophil engraftment was 22 days (range, 18-23 days). All 8 patients initially engrafted donor cells and only 1 (12%) subsequently developed secondary graft failure. The remaining 7 (88%) currently maintain a stable donor cell engraftment. Transplant related toxicities included: neutropenic fever resolving after antibiotic therapy (n=8/8), ≥ grade II mucositis (n=3/8), CMV reactivation (n=2/8), and small intracranial hemorrhages (n=2/8) on (i) day +10 in a patient refractory to platelet transfusions that resolved after cross-matched platelets became available, and, (ii) on day +12 in a patient with prior stroke history that resolved after increasing the platelet threshold from 50 to 100 x103/µL. Acute GVHD ≥ grade 2 was seen in 2/8 patients (one non-compliant with immunosuppressive treatment), with complete resolution after steroid therapy in one case, and transformation into chronic GVHD (skin, eye and liver) in the other. No other cases of chronic GVHD have been observed to date. With a median follow up of 9 months (range, 5-22 months), all 8 patients are alive and 7 (88%) maintain >90% donor cell chimerism with an improved median hemoglobin concentration from 8.8g/dL (range, 6.4-12.8g/dL) to 13.3g/dL (range, 9.9-15.3g/dL).Our study demonstrates that modifying the Hopkins conditioning regimen by increasing the TBI dose from 2Gy to 3Gy and using PBSC instead of bone marrow grafts allows 88% of SCD patients to achieve stable donor engraftment without severe toxicity. We also demonstrate that G-CSF can be safely administered to adult patients with SCD after HSCT. Haploidentical HSCT is the only curative option for many adult patients with clinically advanced SCD and our study suggests a basis for larger, multicenter clinical studies. [Display omitted] DisclosuresPatel:Celgene: Consultancy, Honoraria. Khan:Takeda: Research Funding; Gilead: Consultancy; Teva: Speakers Bureau. Gordeuk:Emmaus Life Sciences: Consultancy.

Non-Myeloablative Transplant in Severe Sickle Cell Disease Is Safe & Effective. Single Center Experience from Saudi Arabia

Background:Sickle cell disease (SCD) is a severe monogenic disorder in the world with >300,000 newly born afflicted children per year. Prevalence of this disorder is high in sub-Saharan Africa, Mediterranean basin, Middle East and India. Patients with severe disease phenotype manifested with stroke or other complications may benefit from allogeneic hematopoietic stem cell transplant (SCT). Recent reports in pediatric patients using myeloablative regimen showed excellent outcomes but similar progress in adults has been hampered by higher rates of transplant related mortality (TRM) and infertility.Methods:We conducted a study of adult patients at our institution with SCD whom underwent a matched related SCT during the period of 2014-2017. Medical records of eligible patients were retrospectively reviewed and extracted. All patients received hydroxyurea (HU) at maximally tolerated doses with hyper-transfusion 2-3 months prior to SCT. Iron chelation was given for patients with ferritin > 1000 ug/L. The preperative regimen was non-myeloablative (NMA) consisting of alemtuzumab 1 mg/kg divided over 5 days (day -7 to -3) followed by Total Body Irradiation (TBI) 300 cGy on day -1. Stem cells were collected from peripheral blood via apheresis after 5 days of GCSF with target CD34 cells of 10×106/kg. Graft vs. host disease (GVHD) prophylaxis consisted of sirolimus starting from day -2 and continued for one year post SCT with dose modification based on chimerism. Event free survival (EFS) was defined from time to SCT to graft failure, development of GVHD or death from any cause. EFS and overall survival (OS) via time to end point analysis was computed using the Kaplan-Meir test.Results:A total of 18 adults patients were eligible and further analysed. Median age was 23.5 (14 - 39) years with 14 males and 4 females. All patients were on HU prior to SCT. Median ECOG performance status was 1 (0-1). Indications for transplant were recurrent vaso-occlusive crisis (VOC) refractory to standard therapy in 61%, stroke or other central nervous system (CNS) complication in 38%, recurrent avascular necrosis of joints in 38%, recurrent acute chest syndrome in 22% and severe disease requiring recurrent intensive care unit admission (ICU) in 17%. All donors were related siblings fully matched at 10/10 human leucocyte antigen (HLA) loci with median age of 23.6 years. A total of 11 (61%) of donors were sickle cell carriers with average Hgb S 33%. Stem cell mobilization was with 5 ug/kg GCSF in all donors and the average stem cell collected 11.6 x 106/kg. No significant complications resulted from GCSF use in donors with sickle cell trait.17 patients were conditioned with alemtuzumab / TBI regimen and GVHD prophylaxis with sirolmus. One patient who failed prior transplant received fludarabine, busulfan and thymoglobulin with GVHD prophylaxis as cyclosporine and methotrexate. Median duration of neutrophil engraftment was 20 days and platelet engraftment 2.6 days with half of patient's platelet nadir > 20. Average hemoglobin (Hb) pre- and post-SCT was 83 g/L and 142 g/L respectively. Average hemoglobin (Hb) S pre- and post-SCT was 77% and 13% respectively. Peri-SCT adverse events were 9 (50%) patients experienced neutropenic fever, 12 (67%) mucositis, 6 (33%) diarrhea. None of the patients developed acute or chronic GVHD. No SCD crises developed post SCT. One patient developed aplastic anemia while retaining 71% donor cell chimerism and underwent a second successful SCT from the same donor with fludarabine, cyclophosphamide and thymoglobulin. All patients have stable mixed chimerism resulting in full donor myeloid engraftment and normalization of blood count. With a median follow up of 9.1 months (2.3-32), all patients are alive and free of sickle cell crises. At last follow up, OS and EFS were 100% and 88.9%, respectively as shown in Figure 1.Conclusion:Herein, we report that SCT in adults with severe SCD using NMA regimen is safe, feasible and results in amelioration of disease severity. These excellent results are encouraging given the high prevalence and debilitating nature of SCD in our region. Follow up is on-going to assess the feasibility of sirolimus tapering and sustainability of donor chimerism status over time. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Efficacy of Donor Lymphocyte Infusion in CD34+ Selected Allogeneic Hematopoietic Stem Cell Transplant for Myeloid and Lymphoid Malignancies: A Single Center Experience

Introduction Donor lymphocyte infusion (DLI) has been used to prevent or treat relapse after allogeneic hematopoietic stem cell transplantation (alloHCT) since the 1990s. Retrospective series have reported variable efficacy (CML up to 60%, ALL 0% in some series) with rates of DLI-associated graft-versus host disease (GVHD) of 40-60%. DLI's ability to potentiate graft versus leukemia effects and GVHD are thought to be primarily T Cell mediated, and T cell chimerism, repertoire, and exhaustion may have important influence on outcomes.The use of ex-vivo T cell depleted CD34 selected alloHCT has been shown to provide similar survival to unmodified grafts but with lower acute and chronic GVHD, and is now being studied in a randomized trial exploring calcineurin inhibitor free GVHD prevention regimens (BMT CTN 1301). There is however limited data on DLI in this context. Here we describe safety and efficacy outcomes of DLI in recipients of CD34 selected alloHCT.Methods This retrospective study included all CD34 selected alloHCT recipients at our center who received DLI from January 2005 to January 2016 for treatment or prevention of relapse. We excluded patients with multiple myeloma as these results have been reported elsewhere. Primary outcomes were maintenance or achievement of disease remission, and change in whole bone marrow and peripheral T Cell chimerism over time. A loess smoothed curve was used to visually display changes in chimerism over time and to provide time point chimerism estimates.Results 58 patients received DLI for frank relapse (n=20), minimal residual disease (MRD, n=11), or for relapse prevention in the context of mixed marrow or peripheral blood chimerism (n=27). AML (40%) and MDS (29%) were the most common diagnoses, and most patients (74%) had intermediate disease risk index (Table 1). All patients received peripheral blood stem cell grafts and myeloablative conditioning. Median DLI dose was 0.5 x 106 CD3 cells/kg (0.01-10.0) with 86% of patients receiving doses between 0.1 and 1.0 x 106 CD3 cells/kg. DLI was most often given once (67%), with some patients having 2 (21%) or > 2 treatments (12%). Of the 20 patients who received DLI for frank relapse, 3 (15%) achieved remission, and of the 11 patients who received DLI for minimal residual disease, 8 (73%) achieved remission. Of the 27 patients with mixed chimerism treated with preventive DLI, remission was maintained in 22 (81%). Achieving or maintaining remission was not independently associated with underlying diagnosis, use of adjunctive hypomethylating agents or stem cell boost, or DLI dose or frequency in this cohort. DLI resulted in an average whole bone marrow donor chimerism increase after DLI, regardless of DLI indication (9% at 90 days; Fig 1). T Cell donor chimerism also increased after DLI, with a more pronounced increase in patients treated for relapse (50%) than those treated for relapse prevention (12%; Fig 1). Prior to DLI, rates of grade II-IV and III-IV acute GVHD were 10 and 3% respectively, and no patients had moderate or severe chronic GVHD. All GVHD had resolved prior to DLI administration. After DLI we observed 2 episodes (3%) of late acute grade II-IV GVHD (both grade III skin), and 4 episodes (7%) of moderate-severe chronic GVHD with median onset of 99 and 80 days post DLI, respectively (range 44-401).Conclusions DLI given after CD34+ selected alloHCT was associated with GVHD rates that were substantially lower than reported in other DLI contexts. Disease response to DLI was higher in MRD positive patients than for those with frank relapse (15 vs 73%), suggesting that regular MRD assessment and prompt DLI administration may be an effective relapse prevention strategy in MRD positive patients. The low relapse rate observed in patients with mixed chimerism receiving preventive DLI (19%) was not substantially different than historical relapse rates observed in CD34+ selected patients at our institution, so does not yet provide a convincing signal to explore DLI for relapse prevention in the absence of MRD. In summary, DLI given after T Cell depleted alloHCT was associated with low rates of GVHD and may be an effective strategy to prevent frank relapse in MRD positive patients after alloHCT. Acknowledging the inherent limitations of our small sample size, our observations suggest broader exploration of this safe and potentially effective strategy is warranted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reduction of Immunosuppression after Disease-Specific Donor Cell Chimerism Decrease Can Prevent Hematological Relapse Following Allogeneic Stem Cell Transplantation after Non-Myeloablative (NMA) Conditioning — Results of a Prospective Phase II Study. Eudract #: 2007-002420-15

Introduction: Relapse of the disease remains a major burden after stem cell transplantation (HCT) following myeloablative conditioning and even more after reduced intensity and non-myeloablative conditioning. Efforts to reduce relapse incidence involve early immunosuppression withdrawal and prophylactic buffy coat infusion in high risk patients. In this phase II study we analyzed the role of disease-specific chimerism driven immunosuppression.Methods: Between May 2008 and July 2012, 200 consecutive patients (pts) (130 male and 70 female) with a median age of 62.0 (range 26.2-74.8) years (y) with hematological malignancies were transplanted after NMA conditioning. The conditioning regimen consisted of fludarabine 30mg/m2 and total body irradiation (200 cGy) followed by immunosuppression with cyclosporine A (CSA) and mycophenolate mofetil (MMF). The primary objective of the study was to determine the incidence of relapse after a decrease of >10% disease-specific donor cell chimerism (DCC). Furthermore, the incidence of graft vs. host disease (GvHD) and the reconversion of DCC were investigated.Pts were transplanted for acute myeloid leukemia (AML; n=85; 42.5%), multiple myeloma (MM; n=31; 15.5%), myelodysplastic syndrome (MDS; n=26; 13%), chronic lymphocytic leukemia (CLL; n=22; 11%), B-Non-Hodgkin Lymphoma (B-NHL; n=11; 5.5%), T-NHL (n=6; 3.0%), acute lymphoblastic leukemia (ALL; n=7; 3.5%), myeloproliferative neoplasia (MPN; n=6; 3%), MDS/MPN (n=3; 1.5%) and chronic myeloid leukemia (CML; n=4; 2%). Donors were matched-related (MRD)(n=26;13%), matched unrelated (MUD) (n=129; 64.5%) and mismatched unrelated on allele or antigen level (n=45;22.5%).DCC analyses were performed on flow sorted cells on day (d)+ 28, d +56, d+ 84, d + 180 and every 6 months following. As a disease specific DCC line we used CD34 for AML, MDS and MPN pts, CD19 for CLL and B-NHL, CD138 for myeloma pts. The pts with T-NHL were monitored by CD3 chimerism. In addition, engraftment was monitored by CD3 chimerism.Results: Overall survival was 52.9 (95% Confidence Interval (CI) 46.4 - 60.3)% at 2y, and 43.9 (CI 37.3 - 51.6)% at 5y. Relapse incidence was median 28.2 (CI 22.1 - 34.6)% at 2 y and 30.3 (CI 23.9 - 36.9)% at 5 y. Non-relapse mortality (NRM) was 24.3 (CI 18.5-30.5)% at 2y and 30.4 (CI 23.9-37.1)% at 5y.DCC (≥90%) was observed in 116 of the 200 (58.0%) pts, 43 patients (21.5%) showed incomplete disease-specific DCC or reduction of DCC in association with hematological relapse: AML (n=17), MDS (n=4), MM (n=7), CLL (n=7), B-NHL (n=1), T-NHL (n=1), MPN (n=2), ALL (n=2), CML (n=1), MDS/MPN (n=1) and 41 patients (20%) had an isolated line specific DCC decrease after a median of 106 (28-2975) days following HCT.In the 41 pts with decrease in disease-specific DCC without hematological relapse, 1 was off immunosuppression and 3 pts had an active acute or chronic GvHD. In 37 pts CSA and MMF was reduced prematurely. 5 pts showed acute GvHD grade ≥III after reduction and 32 pts GvHD grade 0-II. Of the 37 pts, 27 (73.0%) showed no increase of DCC. 24 (88.9%) pts died, 18 due to relapse, 6 due to non-relapse causes. 3 pts are alive after salvage chemotherapy. 10 of the 37 patients (26.5%) reached full DCC after a median of 29 (21 - 267) days following immunosuppression taper and one recovered from the decrease of DCC and hematological relapse after discontinuing the immunosuppression.43 pts (15.5%) showed no full DCC with (n=12) or without hematological relapse (n=31). Following reduction of immunosuppression 32 pts relapsed, of which 4 are alive after salvage therapy. 2 pts rejected the HCT and are alive after 2nd HCT.85 AML pts were separately analysed, of which 43 experienced a complete DCC at any time point (27 are alive without relapse, 16 died due to non-relapse causes). 25 (29.4%) of which 24 pts were without active GvHD had isolated DCC decreased and were tapered. 7 converted to full DCC. All 17 pts showing no full disease specific DCC with or without simultaneous relapse relapsed. 15 pts died of relapse, 2 are alive after salvage therapy.Conclusion: Decrease of disease-specific DCC was observed in 20% of pts and in 29.4% of the AML pts. Of those 27.7 % in the total population and 29.2 % in the AML group showed recovery of DCC without hematological relapse after tapering of immunosuppression. We conclude that DCC is a useful tool to avoid relapse in the early phase of HCT while the patient is on immunosuppression. [Display omitted] DisclosuresFranke:Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Takeda: Consultancy. Lange:Novartis: Honoraria, Research Funding. Schwind:Novartis: Consultancy.

Regulatory T Cells Prevent Donor Cell Rejection in a Mouse Model of Delayed Gestational Age in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism without myeloablation/immunosuppression. It has the potential to treat a number of congenital hematologic disorders, including as Sickle cell disease. Central tolerance, through which donor-reactive host T cells and host-reactive donor T cells are deleted, is instrumental to the induction of donor-specific tolerance (DST) and the prevention of graft-versus-host-disease (GVHD). The window of central tolerance induction in humans occurs at a gestational age that will pose significant technical challenges and exclude from treatment all fetuses evaluated after 14 weeks gestation. We have previously demonstrated that IUHCT results in the peripheral induction of traditional and non-traditional regulatory T cells that contribute to the suppression of GVHD (Riley et al. ASH 2016 Abstract #4540). We hypothesized that regulatory T cells also contribute to DST and, furthermore, may be used to expand the window of central tolerance induction to a later point in gestation.Methods: A mouse model of delayed gestational age IUHCT was developed in which 9.7x10^6 C57/Bl6 (B6) GFP T cell-depleted (TCD) bone marrow (BM) cells were injected intravenously into day of life 0 (P0) naive Balb/c pups via the facial vein. The ability of IUHCT-induced regulatory T cells to prevent donor cell rejection was assessed by injecting 9.7x10^6 B6 GFP TCD BM cells plus 5x10^6 CD4+ splenocytes harvested from 2-4 week old chimeric mice. Fetuses injected with 9.7x10^6 B6 GFP TCD BM cells at gestational day 14 (E14) served as positive controls for tolerance and engraftment. Peripheral blood (PB) was analyzed at 4, 8, 12, and 24 weeks of age for donor cell chimerism (%CD45+H2kb+GFP+/CD45+H2kd+) and for engraftment (defined as chimerism >1% at 4 weeks). Weight gain and histology of the skin, liver, lungs, and small bowel were analyzed to confirm the absence of GVHD. PB was analyzed at 6 months of age to confirm multilineage engraftment. An in vivo mixed lymphocyte reaction (MLR) was performed at 6 months of age to confirm long-term DST. B6-sensitized Balb/c mice served as positive controls for alloreactivity. Comparison of continuous data was performed using Student's t test. Binary data were compared using Fischer's exact test. All tests were two-sided with an alpha level set at 0.05 for significance.Results: Our model of delayed gestational age IUHCT was associated with donor cell rejection in 52 of 56 (92.9%) pups injected with TCD BM only. Co-injection of CD4+ chimeric splenocytes significantly improved engraftment (100% vs. 7.1%, p<0.001) and chimerism (7.56% vs. 0.46% at 4 weeks, p=0.003) compared to TCD BM alone. No difference in engraftment (100% vs. 90%, p=0.501) or chimerism (7.56% vs. 7.66% at 4 weeks, p=0.961) was observed compared to fetuses injected with TCD BM at E14 (Figures 1A and 1B). These trends persisted at subsequent time points. Pups injected with TCD BM plus CD4+ chimeric splenocytes displayed normal weight gain and no histologic evidence of GVHD. Survival to adulthood was 100%. Donor cells of all hematopoietic lineages were present in the PB at 6 months of age. On in vivo MLR, pups injected with TCD BM plus CD4+ chimeric splenocytes displayed significantly reduced T cell proliferation compared to B6-sensitized Balb/c mice and P0 pups injected with TCD BM only. T cell proliferation did not differ significantly from fetuses injected with TCD BM at E14 (Figure 1C).Conclusion: The induction of tolerance following IUHCT is important for maintaining successful long-term engraftment and requires injection at an early gestational age to avoid donor cell rejection by the maturing fetal immune system. This study demonstrates an important role for regulatory T cell populations in the induction of DST following IUHCT. The adoptive transfer of regulatory T cells from a chimeric mouse to a naïve P0 pup inhibits donor cell rejection in our model of delayed gestational age IUHCT, allowing central tolerance induction and long-term engraftment to occur despite the increased maturity of the recipient immune system. These findings represent proof-of-principle that augmentation of regulatory T cell-dependent peripheral tolerance is one approach by which IUHCT could be delayed to a later point in gestation, decreasing the technical difficulty of the procedure and allowing fetuses evaluated after 14 weeks to be eligible for treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Generation of reconstituted hemato-lymphoid murine embryos by placental transplantation into embryos lacking HSCs

In order to increase the contribution of donor HSC cells, irradiation and DNA alkylating agents have been commonly used as experimental methods to eliminate HSCs for adult mice. But a technique of HSC deletion for mouse embryo for increase contribution of donor cells has not been published. Here, we established for the first time a procedure for placental HSC transplantation into E11.5 Runx1-deficient mice mated with G1-HRD-Runx1 transgenic mice (Runx1-/-::Tg mice) that have no HSCs in the fetal liver. Following the transplantation of fetal liver cells from mice (allogeneic) or rats (xenogeneic), high donor cell chimerism was observed in Runx1-/-::Tg embryos. Furthermore, chimerism analysis and colony assay data showed that donor fetal liver hematopoietic cells contributed to both white blood cells and red blood cells. Moreover, secondary transplantation into adult recipient mice indicated that the HSCs in rescued Runx1-/-::Tg embryos had normal abilities. These results suggest that mice lacking fetal liver HSCs are a powerful tool for hematopoiesis reconstruction during the embryonic stage and can potentially be used in basic research on HSCs or xenograft models.