Low Level Donor Research Articles

A Phase I Study to Determine the Maximum Tolerated Dose of ex Vivo Expanded Natural Killer Cells Derived from Unrelated, HLA-Disparate Adult Donors

The administration of allogeneic natural killer (NK) cells following a lymphodepleting chemotherapy regimen is emerging as a well-tolerated therapeutic approach in the management of various malignancies. Contrary to the expected complications of allogeneic T cell therapy, there remains no evidence of graft-versus-host disease (GVHD) mediated by NK cells in numerous clinical trials. On the contrary, preclinical and clinical studies suggest that NK cells do not induce GVHD and in fact may prevent its development following allogeneic hematopoietic cell transplantation (HCT). In this study, we sought to determine the maximum tolerated dose of non-HLA-matched donor NK cells derived from peripheral blood and ex vivo expanded using a novel feeder cell platform. In a single-center Phase I clinical trial using a 3×3 design, 9 subjects each received 2 infusions of NK cells 2 weeks apart following a preparative regimen of cyclophosphamide (60 mg/kg i.v.) and fludarabine (25 mg/m2/day i.v for 5 days). No exogenous cytokines were administered. NK cells were administered at 3 dose levels: 1×107/kg, 2.5×107/kg, and 5×107/kg. Three subjects had myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML), and the other 6 subjects had colorectal carcinoma. Recipients were monitored over a 4-week period for GVHD as well as other adverse events and for persistence of donor NK cells in systemic circulation. Disease assessment was started at 28 days following the first NK cell infusion and continued until postinfusion day 100 or disease progression. In all 9 study subjects, there was no occurrence of GVHD and no dose-limiting toxicities that would warrant cohort expansion at any of the 3 planned cell dose levels. Low-level donor NK cell persistence was observed up to 4 weeks after the first NK cell infusion at all dose levels. The best observed response was a complete response with incomplete platelet recovery in a MDS subject who experienced disease relapse after prior allogeneic HCT. Other responses were stable disease in 1 subject with MDS and 2 subjects with colorectal cancer up to postinfusion day 100. This off-the-shelf, third-party NK cell product can be administered safely without inducing GVHD and exhibits in vivo persistence promoted by preparative lymphodepletion alone. The observed clinical responses could be enhanced by administration of exogenous cytokine support, as well as complementary approaches that promote NK cell function in the tumor microenvironment.

Effect of N <sub>2</sub>O treatment on performance of back channel etched metal oxide thin film transistors

In this paper, the rare earth element praseodymium-doped indium tin zinc oxide semiconductor is used as the channel layer of the thin film transistor, and the aluminum oxide-based wet back channel etched thin film transistor is successfully prepared. The effect of N <sub>2</sub>O plasma treatment on the back-channel interface of thin film transistor is studied, and the effect of treatment power and time on device performance are studied in detail. The results show that the good device performance can be obtained under certain power and time treatment, and the prepared device has good thermal stability of positive bias and negative bias under light conditions. The results from high-resolution transmission electron microscopy show that the amorphous structure of the metal oxide semiconductor material can effectively resist the wet etchant, and that no obvious component segregation phenomenon is found. Further, X-ray photoelectric spectroscopy tests show that N <sub>2</sub>O plasma treatment can form an oxygen-rich, low-carrier-concentration interface layer at the interface. On the one hand, it can effectively resist the damage of the back channel caused by the plasma of plasma enhanced chemical vapor deposition (PECVD), and on the other hand, it acts as a passivation body of hydrogen from PECVD plasma, suppressing the generation of low-level donor state of hydrogen. This study provides an important reference for low-cost, high-efficiency thin film transistor performance optimization methods.

Lung transplantation outcomes after crossing low-level donor specific antibodies without planned augmented immunosuppression.

It is unknown whether some donor specific antibodies (DSA) can be crossed at the time of lung transplant without desensitization or augmented induction immunosuppression. This study assessed whether crossing low-level pre-transplant DSA (defined as mean fluorescence intensity [MFI] 1000-6000) without augmented immunosuppression is associated with worse retransplant-free or chronic lung allograft dysfunction (CLAD)-free survival. Of the 458 included recipients, low-level pre-transplant DSA was crossed in 39 (8.6%) patients. The median follow-up time was 2.2years. There were 15 (38.5%) patients with Class I DSA and 24 (61.5%) with Class II DSA. There was no difference in adjusted overall retransplant-free survival between recipients where pre-transplant DSA was and was not crossed (HR: .98 [95% CI=.49-1.99], P=.96). There was also no difference in CLAD-free survival (HR: .71 [95% CI=.38-1.33], P=.28). There was no difference in Grade 3 PGD at 72h (OR: 1.13 [95% CI=.52-2.48], P=.75) or definite or probable AMR (HR: 2.22 [95% CI=.64-7.61], P=.21). Lung transplantation in the presence of low-level DSA without planned augmented immunosuppression is not associated with worse overall or CLAD-free survival among recipients with intermediate-term follow-up.

5-Azacytidine depletes HSCs and synergizes with an anti-CD117 antibody to augment donor engraftment in immunocompetent mice

AbstractDepletion of hematopoietic stem cells (HSCs) is used therapeutically in many malignant and nonmalignant blood disorders in the setting of a hematopoietic cell transplantation (HCT) to eradicate diseased HSCs, thus allowing donor HSCs to engraft. Current treatments to eliminate HSCs rely on modalities that cause DNA strand breakage (ie, alkylators, radiation) resulting in multiple short-term and long-term toxicities and sometimes even death. These risks have severely limited the use of HCT to patients with few to no comorbidities and excluded many others with diseases that could be cured with an HCT. 5-Azacytidine (AZA) is a widely used hypomethylating agent that is thought to preferentially target leukemic cells in myeloid malignancies. Here, we reveal a previously unknown effect of AZA on HSCs. We show that AZA induces early HSC proliferation in vivo and exerts a direct cytotoxic effect on proliferating HSCs in vitro. When used to pretreat recipient mice for transplantation, AZA permitted low-level donor HSC engraftment. Moreover, by combining AZA with a monoclonal antibody (mAb) targeting CD117 (c-Kit) (a molecule expressed on HSCs), more robust HSC depletion and substantially higher levels of multilineage donor cell engraftment were achieved in immunocompetent mice. The enhanced effectiveness of this combined regimen correlated with increased apoptotic cell death in hematopoietic stem and progenitor cells. Together, these findings highlight a previously unknown therapeutic mechanism for AZA which may broaden its use in clinical practice. Moreover, the synergy we show between AZA and anti-CD117 mAb is a novel strategy to eradicate abnormal HSCs that can be rapidly tested in the clinical setting.

Lung Transplantation Outcomes after Crossing Low Level Donor Specific Antibodies without Augmented Immunosuppression

<h3>Purpose</h3> Lung transplant recipients with pre-transplant donor specific antibodies (DSA) are often treated with augmented immunosuppression. It is unknown whether some DSA can be safely crossed without additional therapies. We implemented a protocol allowing transplant when crossing select low level DSA (defined as mean fluorescence intensity (MFI) 1000-5000) without planned augmented immunosuppression. <h3>Methods</h3> This was a single center retrospective cohort study between 4/1/2015-8/31/2020. All recipients received solumedrol and basiliximab induction without desensitization. Presence of low level pre-transplant DSA was recorded. All post-transplant DSA was monitored within 14 days of transplant and then at routine intervals. The primary study outcomes were overall survival, definite CLAD≥1-free survival, and definite antibody mediated rejection (AMR), all defined according to ISHLT consensus guidelines. <h3>Results</h3> Of the 453 recipients, 36 (7.9%) had a low-level pre-transplant DSA crossed at transplant (Table 1). 13 had Class I antibodies and 26 had Class II. The median historical DSA MFI was 1800 (IQR 1300-2400) and the median most recent MFI was 1200 (IQR 950-2000). Among recipients where pre-transplant DSA was crossed, 17 (47.2%) had persistent post-transplant DSA with a median peak MFI of 7400. Class II antibodies were more likely to be detected post-transplant than Class I (57.7% vs 15.4%, p=0.02). There was no statistical difference in definite AMR in recipients where pre-transplant DSA was and was not crossed (8.3% vs 3.1%, p=0.11). With a median follow-up time of 2.4 years, there were no differences in adjusted overall survival (HR=1.14, 95% CI=0.57-2.32, p=0.71) or CLAD≥1-free survival (HR=0.82, 95% CI=0.44-1.54, p=0.54). <h3>Conclusion</h3> Lung transplantation in the presence of low level DSA without planned augmented immunosuppression was not associated with worse overall or CLAD-free intermediate-term survival but may be associated with increased AMR.

Low Level Donor Chimerism of CD19 CAR-T Cells Returned to Complete Donor Chimerism in Patients with Relapse After Allo-Hematopoietic Stem Cell Transplant.

PurposeTo investigate the donor chimerism changes and curative effects associated with the use of autologous anti-CD19 chimeric antigen receptor (CAR) T cells with B-cell acute lymphoblastic leukemia (B-ALL) presenting with a low donor chimerism level and relapse after allogeneic hematopoietic stem cell transplant (allo-HSCT).MethodsNine patients with B-ALL showing low donor chimerism level and relapse after allo-HSCT were enrolled. Patients 1–3 received CD19 CAR-T cell therapy using cells derived from autologous peripheral blood mononuclear cells (PBMCs) (comprising a mixture of patient and original donor cells) as their donors could not provide PBMCs. Samples from the other six patients (Patients A–F) were investigated only in vitro. The changes in the degree of donor chimerism, function of the CD19 CAR-T cells and T cells in all nine patients were analyzed in vitro. The therapeutic effects and adverse events (AEs) were also evaluated in Patients 1–3.ResultsThe CAR-T cells and T cells in all nine patients showed complete donor chimerism restoration following a 12-day culture period in vitro. These CD19 CAR-T cells demonstrated strong cytotoxicity towards Nalm 6 cells in vitro except in patients 3 and D. In the latter patients, the absolute numbers of all subsets, especially the CD8 + T-cell absolute numbers in peripheral blood were very low. Patients 3 and D showed relatively short durations from transplant to recurrence and received chemotherapy after relapse. In the patients receiving CD19 CAR-T cell therapy, the most commonly observed AE was grade 1 to 2 cytokine release syndrome. None of the cases showed acute graft-versus-host disease during treatment. Patients 1 and 2 achieved complete response with complete restoration of donor chimerism. Patient 3, who received the same CD19 CAR-T cell therapy, did not respond to this therapy.ConclusionCD19 CAR-T cells derived from patients relapsed after allo-HSCT with a low level of donor chimerism were effective for salvage therapy and could restore to complete donor chimerism after 12 days’ culture in vitro.Trial RegistrationHumanized CD19 CAR-T cell therapy for relapse or refractory B-cell lymphoma or acute B lymphocytic leukemia, ChiCTR1800019622, Registered 24 November 2018, http://www.chictr.org.cn/index.aspx.

The Natural History of Pre-Existing Donor Specific Antibody and Amnestic Responses after Heart Transplantation

Sensitized patients have prolonged wait times for heart transplant and often low-level donor specific antibodies (DSA) will be crossed when accepting a donor heart. The purpose of this study was to determine if positive low-level DSA at the time of transplant increase in strength post transplant and increase the risk of antibody mediated rejection (AMR). We reviewed 72 heart transplant patients between 2010 and 2018 in whom low-level DSA were crossed. DSA strength, as measured by mean fluorescence intensity (MFI) and AMR on biopsy was assessed at 1, 3, 6, 12 months post transplant. Other outcomes assessed: freedom from first-year rejection (acute cellular rejection (ACR), any treated rejection (ATR)), freedom from cardiac allograft vasculopathy (CAV, as defined by stenosis ≥30% by angiography), and freedom from graft dysfunction (LVEF ≤40%). Patients were compared to a contemporaneous control group conditional to 1-year survival without DSA at transplant. Approximately one-third of patients with pre-existing DSA at transplant demonstrated an increase in DSA strength. Patients with pre-existing DSA had decreased freedom from ATR and AMR but no differences in freedom from CAV, graft dysfunction, or ACR compared to the control group. Crossing low-level DSA may increase the risk of AMR, but there is no difference in clinically important sequelae such as CAV or graft dysfunction. Thus, accepting low-level DSA at the time of transplant appears feasible and could broaden the donor pool.

Matching Mitochondrial DNA Haplotypes for Circumventing Tissue-Specific Segregation Bias

SummaryMitochondrial DNA (mtDNA) segregation associated with donor-recipient mtDNA mismatch in mitochondria replacement therapy leads to unknown risks. Here, to explore whether matching mtDNA haplotypes contributes to ameliorating segregation, we reproduced various degrees of heteroplasmic mice with three single nucleotide polymorphisms to monitor segregation severity. “Segregation” presented in tissues of heteroplasmic mice containing low-level donor mtDNA heteroplasmy, and disappeared as donor mtDNA heteroplasmy levels ascended. Meanwhile, we found that distribution of donor mtDNA among the blastomeres of preimplantation embryos from the heteroplasmic mice shared the same tendency as that in adult tissues. Statistical analysis showed that no selective replication of donor mtDNA occurred during lifespan. Tracking donor mtDNA distribution showed that uneven distribution of donor mtDNA among embryonic blastomeres gradually became even as donor mtDNA heteroplasmy increased, indicating that the “segregation” in tissues was inherited from the uneven distribution. Our finding suggested that donor-recipient mtDNA matching could circumvent segregation in mitochondria replacement therapy.

CD45-Targeted Antibody Drug Conjugate Plus Post Transplant Cytoxan Is Sufficient to Enable Allogeneic Bone Marrow Transplant in a Minor Mismatch Mouse Model

Bone Marrow Transplant (BMT) is a potentially curative treatment for malignant and non-malignant blood disorders. Current regimens for patient preparation, or conditioning, prior to BMT limit the use of this curative procedure due to regimen-related mortality and morbidities, including risks of organ toxicity, infertility and secondary malignancies. Targeted preparation using antibody drug conjugates (ADCs) to mouse CD45 has previously been shown to be sufficient to enable bone marrow transplant (BMT) in syngeneic immune competent mice (Palchaudhuri et al. Nature Biotech 2016 34:738–745), and this approach to preparation has the potential to expand the utility of BMT if it can be successfully translated to patients. To further investigate the utility of this tool ADC in murine transplant models, we explored anti-CD45-saporin (CD45-SAP) in an allogeneic minor mismatch transplant model (Balb/c donor into DBA/2 recipients). The goal of the work was to identify the level of immune suppression, if any, that needs to be used in combination with CD45-SAP to enable high donor chimerism in the allogeneic setting. CD45-SAP (1.9 mg/kg, iv) was evaluated alone or in combination with additional immune modulating agents: clone 30F11 (25 mg/kg, IP), a naked anti-CD45 antibody that mimics ATG by relying on effector function to enable potent peripheral B- and T -cell depletion; pre-transplant Cytoxan (PreTCy, 200 mg/kg, IP), 2 Gy total body irradiation (TBI), and post-transplant Cytoxan (PTCy, 200 mg/kg, IP) to prevent graft versus host disease as well as block host versus graft rejection. 9 Gy TBI was used as the conventional conditioning positive control. Conditioned mice were transplanted with 2 × 107 whole bone marrow cells, and chimerism assessed over 12 weeks. CD45-SAP in combination with PTCy achieved comparable peripheral myeloid chimerism, a readout of stem cell engraftment, to 9 Gy TBI (>90%) at 8 weeks post-transplantation (Fig 1). The addition of 30F11 to the CD45-SAP/PTCy protocol had no effect on peripheral donor chimerism (59% vs 61%), suggesting additional lymphodepletion is not required. In contrast, the single agents alone, 2 Gy TBI in combination with 30F11 and PTCy resulted in These results indicate CD45-SAP in combination with PTCy is sufficient to enable high levels of donor chimerism in the minor mismatched setting without the need for additional immune suppression. CD45-SAP was more effective at conditioning than 2Gy TBI or PreTCy. Future experiments will investigate the CD45-SAP/PTCy protocol in haploidentical and fully mismatched allogeneic mouse models.

The Best of Both? Megadose Stem Cells with CD34 Selection and T-Cell Addback Enables Engraftment from Mismatched Unrelated Donors Using Reduced Toxicity Conditioning in Primary Immunodeficiency

▪Background: The optimal approach for transplanting patients with primary immunodeficiency from a mismatched unrelated donor (MMUD) remains unclear. With reduced intensity conditioning, when bone marrow (BM) was used as the stem cell source in such patients we have previously observed a high rate of very low level donor chimerism (4 of 12 patients) requiring a second transplant procedure. The use of peripheral blood stem cells (PBSC) can overcome this but results in a high incidence of acute (≥ Gde II 10/21, Gde III-IV 5/21) and chronic (10/21, extensive 7/21) graft-versus-host disease (GVHD) (Rao et al in press). We hypothesized that the use of megadose CD34 selected stem cells from PB with addback of a T-cell dose akin to BM might facilitate engraftment through improved competition for the stem cell niche without severe GVHD.Methods: We prospectively analysed outcomes on 20 consecutive patients with primary immunodeficiency (SCID n=5, HLH n=3, Other PID n=12) transplanted from 8/10 (n=4) or 9/10 (n=16) HLA MMUD at our institution between 2011-2015. The mean age at transplant was 5.1 years. All patients received reduced toxicity conditioning regimens (12/20 Fludarabine/Melphalan/Alemtuzumab; 8/20 Fludarabine/Treosulphan/Alemtuzumab) with Cyclosporin (CsA) and mycophenolate mofetil (MMF) GvHD prophylaxis. CD34 selection of donor PBSC was performed using CliniMACs and the mean CD34+ dose was 20.1x106/kg. At day 0 either 108 CD3/kg (cohort 1, n=6) or 3x108 CD3/kg (cohort 2, n=14) T-cells from the CD34- fraction were infused with the graft. Mean follow up was 31.9 months.Results: In cohort 1, all patients engrafted and 2 developed high level donor mixed chimerism (both curative) in the myeloid and lymphoid lineages at last follow up. Two patients had Grade II acute GvHD and 1 had moderate chronic GvHD. In view of the slow immune reconstitution in this cohort, the T-cell addback dose was increased to 3x108/Kg in subsequent patients. In cohort 2, all patients achieved full donor haemopoiesis initially, 6/14 developed high levels of donor chimerism in both myeloid and lymphoid lineages later and 1/14 progressed with 10% donor T-cell engraftment but remains disease-free. The incidence of significant aGVHD was low (grade II n=4, no grade III or IV) and no patient developed cGvHD. Overall across both cohorts, 10 patients had viral reactivations and there were 5 deaths (3 viral complications, 1 pulmonary vasculopathy, 1 cGVHD lungs). The disease free survival at 2 years 7 months follow up was 70% which compares favorably with a previous cohort transplanted with unmanipulated BM as the stem cell source (Fig 1). Immune reconstitution was delayed (mean CD3 and CD19 counts at day 100 post-transplant was 245 x 109/L and 315 x 109/L) with similar kinetics in both cohorts (Fig 2), comparable to RIC transplant using unmanipulated BM. By 1 year post transplant 11/14 evaluable patients achieved normal CD3+T-cell numbers and 8/14 normal CD19+ B-cell counts.Conclusion: The use of megadose peripheral blood stem cells with T-cell addback in the mismatched unrelated donor transplant setting results in high rates of curative engraftment and a low incidence of acute and chronic GvHD following reduced toxicity conditioning. However, T-cell reconstitution remains delayed (presumably reflecting the effect of Alemtuzumab on the infused T-cells) with a high incidence of viral complications. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

(784) - Low Level Donor Specific Antibodies at Transplant Does Not Appear to Be Associated With the development of Cardiac Allograft Vasculopathy After Heart Transplantation

Donor specific antibodies (DSA) following heart transplant has been reported to be associated with increased development of cardiac allograft vasculopathy (CAV) and risk of mortality following transplantation. In the era of virtual crossmatch, we may now elect to place donor hearts in patients with low level DSAs that are not placed as avoids. The effect of these low level DSAs on the development of CAV has not yet been established.

Heart Transplantation across Low Level Donor Specific Antibodies: Is It Bad?

Heart Transplantation across Low Level Donor Specific Antibodies: Is It Bad?

6-P

Flow cytometric crossmatches (FCXM) are used to detect low-level donor-specific antibody and determine transplantation eligibility. We measured HLA class I and II surface expression using different lymphocyte sources (peripheral blood (PBL), spleen, or nodes) to investigate the impact of HLA expression on detecting low-level donor specific antibody. HLA expression was measured on a BD FACSCanto II using fluorescently conjugated antibodies specific for monomorphic determinants on HLA class I and II; measurements were converted to Molecules of Soluble Fluorescence (MESF) units. FCXM reactivity was determined using the ratio of test serum to negative control serum and MESF units. Compared to Live Donor (LD) PBLs (n=249), mean HLA class I expression on lymphocytes isolated from Deceased Donor (DD) (n=25) spleen and lymph nodes was lower (5.8% and 10.4%, respectively). The mean HLA class II expression was significantly lower on DD splenic (90.4%, p < 0.05) and lymph node (125.6%, p < 0.05) lymphocytes. We then compared lymphocytes isolated from the same DD (n=4) to better assess the variability of HLA expression on cells derived from different tissues. Compared to DD PBL, HLA class I expression was higher on splenic (54%) and lymph node lymphocytes (34%). HLA class II expression on DD PBLs was lower than that from spleen (63%), but higher than the lymph node-derived lymphocytes (99%). Variation in HLA class II expression on cells from different tissues also impacted crossmatch reactivity. HLA class II donor specific sera, expected to yield a positive FCXM, consistently showed the highest reactivity with splenic lymphocytes; intermediate reactivity with PBL; and the lowest reactivity with lymph node cells. HLA expression may not be routinely analyzed; however, it can substantially impact crossmatch reactivity. Laboratories using a single positive threshold for all FCXMs should consider that splenic lymphocytes may yield reactivity most comparable to that of PBLs from LDs.

Differential immunoregulation in successful oocyte donation pregnancies compared with naturally conceived pregnancies

FOXP3 is critical for the development and function of CD4+CD25bright natural regulatory T cells (nTreg). Individuals harboring mutations in FOXP3 develop immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). We describe a child diagnosed with IPEX who underwent a reduced intensity, T and B cell depleted, matched unrelated donor bone marrow transplant followed by clinical resolution. Using lineage-specific donor chimerism studies, we demonstrate that non-myeloablative HSCT resolves disease in the context of low level donor hematopoietic stem cell (HSC) engraftment. Despite low-levels of donor HSC, thymically-derived nTreg and to a lesser extent CD4+ and CD8+ T cells, exhibit a selective in vivo growth advantage for populations containing a functional FOXP3 gene. Moreover, nTreg from this patient show regulatory function directly ex vivo. These results have implications for improving clinical therapy for patients with IPEX and provide mechanistic insight into the in vivo development of human nTreg and unexpectedly, non-regulatory T cells.

Therapeutic in vivo selection of thymic-derived natural T regulatory cells following non-myeloablative hematopoietic stem cell transplant for IPEX

FOXP3 is critical for the development and function of CD4(+)CD25(bright) natural regulatory T cells (nTreg). Individuals harboring mutations in FOXP3 develop immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). We describe a child diagnosed with IPEX who underwent a reduced intensity, T and B cell depleted, matched unrelated donor bone marrow transplant followed by clinical resolution. Using lineage-specific donor chimerism studies, we demonstrate that non-myeloablative HSCT resolves disease in the context of low level donor hematopoietic stem cell (HSC) engraftment. Despite low-levels of donor HSC, thymically-derived nTreg and to a lesser extent CD4(+) and CD8(+) T cells, exhibit a selective in vivo growth advantage for populations containing a functional FOXP3 gene. Moreover, nTreg from this patient show regulatory function directly ex vivo. These results have implications for improving clinical therapy for patients with IPEX and provide mechanistic insight into the in vivo development of human nTreg and unexpectedly, non-regulatory T cells.

Reduced Intensity Transplantation for Primary Immunodeficiency Disorders

Studies so far indicate that reduced intensity transplantation (RIT) may have an important role in treating patients with primary immunodeficiency disease (PID). Unlike more standard approaches, such regimens can be used without severe toxicity in patients with severe pulmonary or hepatic disease. RIT also offers the advantage that long-term sequelae such as infertility or growth retardation may be avoided or reduced. RIT appears to be most appropriate for those patients with significant co-morbidities (eg T cell deficiencies) and those undergoing unrelated donor haematopoietic cell transplantation. More studies are required using pharmacokinetic monitoring (eg busulphan, treosulfan and alemtuzumab) and varying stem cell sources to optimise graft vs marrow reactions and minimise graft vs host disease. In certain PID patients RIT will be the “first step” towards establishing donor cell engraftment; second infusions of donor stem cells, donor lymphocyte infusions, or a second myeloablative HCT, which appears to be well tolerated, may be required in some patients with low level donor chimerism or graft rejection.

11-P: Impact of Low Level Donor Specific Antibodies in Renal Transplant Outcomes

11-P: Impact of Low Level Donor Specific Antibodies in Renal Transplant Outcomes

Haploidentical In Utero Hematopoietic Cell Transplantation Improves Phenotype and Can Induce Tolerance for Postnatal Same-Donor Transplants in the Canine Leukocyte Adhesion Deficiency Model

In the murine model, in utero hematopoietic cell transplantation (IUHCT) has been shown to achieve low levels of allogeneic chimerism and associated donor-specific tolerance permitting minimal conditioning postnatal hematopoietic stem cell transplantation (HSCT). In this pilot study, we investigated IUHCT in the canine leukocyte adhesion deficiency (CLAD) model. Haploidentical IUHCT resulted in stable low-level donor cell chimerism in all dogs that could be analyzed by sensitive detection methodology (4 of 10) through 18 months of follow-up. In the 2 CLAD recipients, low-level chimerism resulted in amelioration and complete reversal of the CLAD phenotype, respectively. Six recipients of IUHCT (5 carriers and 1 CLAD) subsequently received postnatal HSCT from the same haploidentical prenatal donor after minimal conditioning with busulfan 10 mg/kg. Chimerism in 2 of 5 CLAD carriers that underwent HSCT increased from < 1% pre-HSCT to sustained levels of 35% to 45%. Control animals undergoing postnatal haploidentical HSCT without IUHCT had no detectable donor chimerism. These results demonstrate that haploidentical IUHCT in the CLAD model can result in low-level donor chimerism that can prevent the lethal phenotype in CLAD dogs, and can result in donor-specific tolerance that can facilitate postnatal minimal conditioning HSCT.

51-W: Transplant outcome in patients with low level donor specific antibody

51-W: Transplant outcome in patients with low level donor specific antibody

Chimerism and tolerance post in utero transplantation with ontogenically different sources of stem cells

Prenatal transplantation (IUT) is a promising strategy for the early treatment of hemoglobinopathies and genetic disorders. We have previously demonstrated that fetal liver cells injected prenatally induced tolerance, while cytokine stimulated peripheral blood stem cells induce allosensitization. In order to this hypothesis, we have transplanted Balb/c fetuses (H-2d) at day 13–14 of gestation with 5x103 129 embryonic, fetal liver and bone marrow stem cells from C57BL/6J mice (H-2b) by intra-peritoneal injection. Transplanted mice received boosts (transplants without conditioning) nine month after birth, by intravenous injection of 40x106 of the corresponding cells. The pre-boost donor chimerism detected by highly sensitive (0.01%) semi-quantitative PCR assay demonstrated 0.1–3% of donor cells at 6 weeks and ≤0.2% at 9 months. Our hypothesis is that prenatally induced allosensitization was responsible for this decrease in donor chimerism. This was supported by the observation that cytotoxicity assay showed shift towards immunity and the fact that postnatal injections of high dose donor cells resulted in low level donor chimerism. We conclude that the low level of donor chimerism obtained after prenatal transplantation may lead to allosensitization. The cell dose required for prenatal tolerance induction may be much higher than that used in this experiment and relevant experiments to test this hypothesis are in progress. Ongoing studies are pending to delineate differences between embryonic, fetal and adult sources of stem cells in chimerism and tolerance induction and will be presented.