Durable Chimerism Research Articles

Cellular trafficking and fate mapping of cells within the nervous system after in utero hematopoietic cell transplantation

In utero hematopoietic cell transplantation (IUHCT) utilizes fetal immune tolerance to achieve durable chimerism without conditioning or immunosuppression during a unique window in fetal development. Though donor cells have been observed within the nervous system following in utero injection, the timeline and distribution of cellular trafficking across the blood-brain barrier following IUHCT is not well understood. We injected 20 × 106 adult bone marrow mononuclear cells intravenously at gestational age (GA) 12–17 days and found that donor cells were maximally concentrated in the brain with treatment between GA 13–14. Donor cell engraftment persisted within the brain at every timepoint analyzed and concentrated within the hindbrain with significantly more grafted cells than in the forebrain. Additionally, transplanted cells terminally differentiated into various nervous system cellular morphologies and also populated the enteric nervous system. This study is the first to document the timeline and distribution of donor cell trafficking into the immune-protected nervous system and serves as a foundation for the application of IUHCT to treat neurogenetic diseases.

Durable mixed chimerism may permit subsequent immunosuppression-free intestinal transplantation—A proof-of-principle study

Intestinal transplantation (ITx) is the definitive treatment for intestinal failure but has the highest rejection rate among solid organ transplants, requiring high doses of immunosuppressive medication, which is associated with high rates of infection, graft-versus-host disease, and malignancy. Transplant tolerance would overcome the need for long-term immunosuppression (ISP). Using nonmyeloablative conditioning, our laboratory has developed a novel swine model of hematopoietic stem cell transplantation (HSCT) that produces durable mixed chimerism (MC) and immune tolerance without toxicity. We investigated whether durable MC would promote tolerance of subsequently transplanted donor-matched intestinal allografts without ISP. Using miniature swine with a defined major histocompatibility complex (MHC), we performed HSCT across an MHC-class-I haplotype mismatch. Immunosuppressive therapy was stopped by day 45. MC was evaluated using flow cytometry, and mixed lymphocyte reaction assays were used to evaluate cellular responses. Subsequently, orthotopic ITx was performed without ISP using a donor that was MHC-matched to the HSCT donor. The recipients were observed for 4 weeks and euthanized for tissue collection and mechanistic assays. After HSCT, the recipients developed durable multilineage MC and apparent deletional tolerance. After ITx, recipients showed no clinical or histologic signs of rejection, and chimerism was unchanged. These results demonstrate the potential value of generating durable MC to achieve transplant tolerance.

Correction of T-Cell Repertoire and Autoimmune Diabetes in NOD Mice by Non-myeloablative T-Cell Depleted Allogeneic HSCT.

The induction of partial tolerance toward pancreatic autoantigens in the treatment of type 1 diabetes mellitus (T1DM) can be attained by autologous hematopoietic stem cell transplantation (HSCT). However, most patients treated by autologous HSCT eventually relapse. Furthermore, allogeneic HSCT which could potentially provide a durable non-autoimmune T-cell receptor (TCR) repertoire is associated with a substantial risk for transplant-related mortality. We have previously demonstrated an effective approach for attaining engraftment without graft versus host disease (GVHD) of allogeneic T-cell depleted HSCT, following non-myeloablative conditioning, using donor-derived anti-3rd party central memory CD8 veto T cells (Tcm). In the present study, we investigated the ability of this relatively safe transplant modality to eliminate autoimmune T-cell clones in the NOD mouse model which spontaneously develop T1DM. Our results demonstrate that using this approach, marked durable chimerism is attained, without any transplant-related mortality, and with a very high rate of diabetes prevention. TCR sequencing of transplanted mice showed profound changes in the T-cell repertoire and decrease in the prevalence of specific autoimmune T-cell clones directed against pancreatic antigens. This approach could be considered as strategy to treat people destined to develop T1DM but with residual beta cell function, or as a platform for prevention of beta cell destruction after transplantation of allogenic beta cells.

Leveraging the lymphohematopoietic graft-versus-host reaction (LGVHR) to achieve allograft tolerance and restore self tolerance with minimal toxicity.

Mixed allogeneic chimerism has considerable potential to advance the achievement of immune tolerance to alloantigens for transplantation and the restoration of self-tolerance in patients with autoimmune disease. In this article, I review evidence that graft-versus-host (GVH) alloreactivity without graft-vs-host disease (GVHD), termed a lymphohematopoietic graft-vs-host reaction (LGVHR), can promote the induction of mixed chimerism with minimal toxicity. LGVHR was originally shown to occur in an animal model when non-tolerant donor lymphocytes were administered to mixed chimeras in the absence of inflammatory stimuli and was found to mediate powerful graft-vs-leukemia/lymphoma effects without GVHD. Recent large animal studies suggest a role for LGVHR in promoting durable mixed chimerism and the demonstration that LGVHR promotes chimerism in human intestinal allograft recipients has led to a pilot study aiming to achieve durable mixed chimerism.

Bone marrow chimerism breaks the barrier to pancreatic islet transplantation.

In their recent Cell Reports paper, Chang and colleagues report on a successful strategy to achieve durable mixed hematopoietic chimerism that promotes the engraftment and long-term function of pancreatic islet allotransplants in fully immunocompetent mice without immunosuppression.

Curative islet and hematopoietic cell transplantation in diabetic mice without toxic bone marrow conditioning.

Mixed hematopoietic chimerism can promote immune tolerance of donor-matched transplanted tissues, like pancreatic islets. However, adoption of this strategy is limited by the toxicity of standard treatments that enable donor hematopoietic cell engraftment. Here, we address these concerns with a non-myeloablative conditioning regimen that enables hematopoietic chimerism and allograft tolerance across fully mismatched major histocompatibility complex (MHC) barriers. Treatment with an αCD117 antibody, targeting c-Kit, administered with Tcell-depleting antibodies and low-dose radiation permits durable multi-lineage chimerism in immunocompetent mice following hematopoietic cell transplant. In diabetic mice, co-transplantation of donor-matched islets and hematopoietic cells durably corrects diabetes without chronic immunosuppression and no appreciable evidence of graft-versus-host disease (GVHD). Donor-derived thymic antigen-presenting cells and host-derived peripheral regulatory Tcells are likely mediators of allotolerance. These findings provide the foundation for safer bone marrow conditioning and cell transplantation regimens to establish hematopoietic chimerism and isletallograft tolerance.

Favorable outcomes with durable chimerism after hematopoietic cell transplantation using busulfan and fludarabine-based reduced-intensity conditioning for pediatric patients with hemophagocytic lymphohistiocytosis

BackgroundThe incorporation of a reduced-intensity conditioning (RIC) regimen in hematopoietic cell transplantation (HCT) for patients with hemophagocytic lymphohistiocytosis (HLH) has decreased early mortality but is associated with a high rate of mixed chimerism and graft failure. Here, we present a successful single-center experience using busulfan and a fludarabine-based RIC regimen for the treatment of HLH.MethodsThe medical records of pediatric patients with HLH who underwent HCT using a busulfan/fludarabine-based RIC regimen between January 2008 and December 2017 were reviewed retrospectively.ResultsNine patients received HCT with a busulfan/fludarabine-based RIC regimen. Three patients had primary HLH, and the other six patients had secondary HLH with multiple reactivations. All three patients with primary HLH had UNC13D mutations. All patients achieved neutrophil and platelet engraftment at a median of 11 days (range, 10‒21) and 19 days (range, 13‒32), and all eight evaluable patients had sustained complete donor chimerism at the last follow-up. Two patients (22%) experienced grade 2 acute graft-versus-host disease (GVHD). Two patients (22%) developed chronic GVHD, and one died from chronic GVHD. One patient (11%) experienced reactivation 4 months after HCT from a syngeneic donor and died of the disease. The 8-year overall survival and event-free survival rates were 78%. No early treatment-related mortality within 100 days after HCT was observed.ConclusionOur experience suggests that a busulfan/fludarabine-based RIC regimen is a viable option for pediatric patients with HLH who require HCT.

Using mTOR Inhibitor Nanoimmunotherapy to Induce Cardiac Allograft Tolerance in Non-Human Primates

<h3>Purpose</h3> Though tolerance to kidney allografts has been consistently achieved using a mixed chimerism model, cardiac allografts remain resistant to tolerance induction. Lipoprotein-based nanobiologics loaded with a mammalian target of rapamycin inhibiting prodrug (mTOR inhibiting nanobiologics, mTORi-NBs) inhibit trained immunity and promote graft infiltrating M2-macrophages that provide a favorable local immunologic milieu for tolerance induction. In this study, we evaluate the ability of mTORi-NBs to induce tolerance in a cardiac allograft model in NHPs. <h3>Methods</h3> Six NHPs underwent simultaneous heterotopic heart and bone marrow transplantation for mixed chimerism induction. Group A (n=3) was conditioned with 3 Gy total body irradiation (TBI), 7 Gy thymic irradiation (TI), ATGAM, anti-CD40L monoclonal antibody on days 0, 2, 5,7, 9, and 12 post-bone marrow transplant (pBMTx), and cyclosporine until day 28 pBMTx. Group B recipients (n=3) underwent the same conditioning regimen with the addition of mTORi-NBs at a dose of 0.15 mg/kg on days 2, 5, 12, 19, and 26 pBMTx. Group B donors were also treated with a single dose of mTORi-NBs two days before transplant. <h3>Results</h3> In group A, all allografts had end stage rejection by day 175 pBMTx with a mean duration of lymphocyte chimerism of 74 days. The first recipient in group B developed durable full chimerism and mild graft versus host disease (GVHD) which resolved after a course of steroids and cyclosporine. After resolution of his GVHD, his graft continued beating strongly with no evidence of rejection through day 239 pBMTx. The second and third recipients received half dose (1.5 Gy) TBI and both allografts are contracting strongly at days 271 and 26 pBMTx, though the second recipient had some evidence of rejection at day 212 pBMTx. Currently, the mean duration of lymphocyte chimerism in Group B is 149 days. <h3>Conclusion</h3> Preliminary studies suggest that adding mTORi-NB nanoimmunotherapy to a mixed chimerism protocol prolongs both donor lymphocyte chimerism and heart graft survival in NHPs even when reducing pre-operative TBI.

87 - Establishment of Durable Chimerism with Minimal GvHD in Highly Mismatched Recipients Receiving an Investigational Facilitated Allo-HSCT

87 - Establishment of Durable Chimerism with Minimal GvHD in Highly Mismatched Recipients Receiving an Investigational Facilitated Allo-HSCT

Chimerism-Based Tolerance to Kidney Allografts in Humans: Novel Insights and Future Perspectives

Chronic rejection and immunosuppression-related toxicity severely affect long-term outcomes of kidney transplantation. The induction of transplantation tolerance – the lack of destructive immune responses to a transplanted organ in the absence of immunosuppression – could potentially overcome these limitations. Immune tolerance to kidney allografts from living donors has been successfully achieved in humans through clinical protocols based on chimerism induction with hematopoietic cell transplantation after non-myeloablative conditioning. Notably, two of these protocols have led to immune tolerance in a significant fraction of HLA-mismatched donor-recipient combinations, which represent the large majority of cases in clinical practice. Studies in mice and large animals have been critical in dissecting tolerance mechanisms and in selecting the most promising approaches for human translation. However, there are several key differences in tolerance induction between these models and humans, including the rate of success and stability of donor chimerism, as well as the relative contribution of different mechanisms in inducing donor-specific unresponsiveness. Kidney allograft tolerance achieved through durable full-donor chimerism may be due to central deletion of graft-reactive donor T cells, even though mechanistic data from patient series are lacking. On the other hand, immune tolerance attained with transient mixed chimerism-based protocols initially relies on Treg-mediated suppression, followed by peripheral deletion of donor-reactive recipient T-cell clones under antigenic pressure from the graft. These conclusions were supported by data deriving from novel high-throughput T-cell receptor sequencing approaches that allowed tracking of alloreactive repertoires over time. In this review, we summarize the most important mechanistic studies on tolerance induction with combined kidney-bone marrow transplantation in humans, discussing open issues that still need to be addressed and focusing on techniques developed in recent years to efficiently monitor the alloresponse in tolerance trials. These cutting-edge methods will be instrumental for the development of immune tolerance protocols with improved efficacy and to identify patients amenable to safe immunosuppression withdrawal.

Engraftment Effects after Intra-Bone Marrow versus Intravenous Allogeneic Stem Cell Transplantation in a Reduced-Intensity Conditioning Dog Leukocyte Antigen-Identical Canine Model

Following conventional i.v. hematopoietic stem cell transplantation (IV-HSCT), most of the hematopoietic stem cells get trapped in peripheral organs and do not reach the bone marrow niche. A promising approach to overcome this cell loss during the homing process seems to be the infusion of hematopoietic stem cells directly into the bone marrow cavity (intra-bone marrow [IBM]-HSCT). This study aimed to investigate the engraftment efficiency of IBM-HSCT compared with IV-HSCT following reduced-intensity conditioning in a canine HSCT model. Furthermore, the impact of 2 different graft infusion rates during IBM-HSCT on the engraftment was evaluated. Dogs received 4.5 Gy total body irradiation for conditioning at day -1 and 15mg/kg cyclosporin A twice daily at days -1 to +35 as immunosuppression. The IV-HSCT group (n=7) received unmodified bone marrow. The IBM-HSCT cohorts received buffy coat-enriched bone marrow that was applied into the humerus and femur simultaneously with an infusion time of either 10 minutes (IBM10; n=8) or 60 minutes (IBM60; n=7). Statistical analyses were performed using the Kruskal-Wallis test followed by the Mann-Whitney U test with Bonferroni correction for multiple comparisons. Statistical significance was declared at Bonferroni-adjusted P < .017. All dogs initially engrafted. One dog of the IBM10 cohort died at day +15 from infection. All 21 evaluable dogs developed a durable mixed donor chimerism over the course of 112 days. Engraftment kinetics did not differ significantly across the 3 groups. Leukocyte and platelet nadirs, as well as the durations of leukopenia and thrombocytopenia, were comparable in the 3 groups. Signs of toxicity for ingestion, body temperature, activity, and defecation did not show statistically significant differences among the 3 groups; only weight loss was greater in the IBM60 group compared with the IV group. IBM-HSCT following reduced-intensity conditioning resulted in an engraftment efficiency and hematopoietic recovery comparable to that seen with conventional IV-HSCT. In addition, modification of the graft infusion rate had no impact on engraftment and hematopoietic recovery in the canine IBM-HSCT model.

Establishment of Durable Chimerism with Minimal GvHD in Highly Mismatched Recipients Receiving an Investigational Facilitated Allo-HSCT

IntroductionWe previously reported the induction of kidney transplant tolerance by the establishment of durable whole blood and T-cell chimerism and withdrawal of immunosuppression (IS) in 26 of 37 highly mismatched recipients of combined stem cell and living donor kidney transplant recipients (KTx) with low risk of graft versus host disease (GvHD), using FCR001, an investigational cell therapy. The focus of the current analysis is to evaluate the impact of degree of bidirectional donor/recipient mismatching using high resolution allele typing at HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1, on the ability to establish durable chimerism, allowing full immunosuppression (IS) withdrawal and the induction of transplant tolerance.MethodsIn this phase 2 trial, recipients received nonmyeloablative conditioning with fludarabine 30mg/m2 (Days -5, -4, -3), cyclophosphamide 50mg/kg (Days-3 and +3), 200 cGy TBI (Day-1) followed by KTx (Day 0). G-CSF mobilized peripheral blood mononuclear cells were apheresed from the donor >2 weeks prior to KTx, processed to remove GvHD-producing cells yet retain CD34 +cells and tolerogenic CD8+/TCR facilitating cells (FC) and cryopreserved until infusion day+1 after KTx. IS consisted of mycophenolate and tacrolimus. IS was weaned and discontinued in the presence of sustained donor T-cell chimerism (>50%), stable renal function and no evidence of biopsy-proven rejection.ResultsSamples from 32 of the 37 subject pairs were available for analysis; of these, three subjects did not have sufficient DNA to test for locus DPB1.All 32 recipients, ranging from age 18 - 65 years, have reached at least 4.5 years of follow-up up to 12 years. Two recipients were re-transplants. Of the 29 D/R pairs with data from all 12 alleles, 21 were mismatched between 6 to 12 alleles (6 related, 15 unrelated), 8 were mismatched between 2 and 5 alleles (all related). Of the three D/R pairs missing DP data, one was mismatched 6 of 10, another 10 of 10, and a third 2 of 10 alleles. Despite the high degree of mismatch, durable chimerism allowed for full IS withdrawal in 25 of these 32 subjects (time off IS from 3.5 - 11 years). 12/25 off IS were from unrelated D/R pairs and ≥ 8 HLA mismatches; the majority showed >95% donor whole blood/T cell chimerism. Three have exhibited stable mixed chimerism ranging between 40% - 60%. Of the subjects not off IS, two failed to engraft their cells; four lost chimerism by 4 months, and one developed GVHD. Durably chimeric patients retained chimerism after removal of IS, remain rejection-free without donor-specific antibody (DSA) with up to 12 years following KTx and have not resumed IS. Transiently chimeric subjects resumed endogenous hematopoiesis and are maintained on low dose IS with stable renal function. There have been two cases of GvHD: one grade 2 lower GI acute GvHD that developed during conversion from tacrolimus to sirolimus and responded to steroids; this patient has developed moderate chronic GvHD of the skin. He is off IS with normal renal function. The second presented late following development of severe gastrointestinal symptoms and manifested treatment-resistant lower GI GvHD with associated tissue-invasive CMV colitis that proved fatal at 11 months post-transplant.ConclusionsIn summary, high levels of durable chimerism and tolerance with a low (5.5%) incidence of GvHD has been achieved in highly mismatched related and unrelated recipients of FCR001 + kidney transplant. [Display omitted] DisclosuresKrieger: Talaris Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Tambur: Viela Bio and Sanofi: Consultancy; ThermoFisher: Speakers Bureau. Schneider: Talaris Therapeutics, Inc.: Current Employment. Olsen: Talaris Therapeutics, Inc.: Current Employment. Ildstad: Talaris Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

Using Selective Bcl2 Inhibition to Induce Cardiac Allograft Tolerance

<h3>Purpose</h3> Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group using a mixed chimerism model with allogeneic bone marrow transplant after non-myeloablative conditioning but not without kidney co-transplantation. B cell lymphoma 2 (Bcl2) inhibition has allowed for durable mixed chimerism with reduced conditioning in the murine skin transplant model. We investigated whether the Bcl2 inhibitor ABT199 could enhance mixed chimerism and induce cardiac allograft tolerance in the absence of kidney cotransplantation. <h3>Methods</h3> Fifteen cynomolgus recipients underwent mixed chimerism conditioning (ATGAM, total body and thymic irradiation) then organ and bone marrow transplantation. Group 1 recipients received heart alone (n=5), Group 2 recipients received heart and kidney cotransplants (n=7), and in Group 3, heart recipients received ABT199 in lieu of kidney cotransplants (n=3).Group 1 and 2 received 3Gy total body irradiation (TBI) while Group 3 received 1.5Gy TBI. All recipients underwent a 28-day course of immunosuppression with a calcineurin inhibitor and anti-CD154 mAb after which all immunosuppression was stopped. <h3>Results</h3> All five Group 1 recipients demonstrated cellular rejection (ISHLT 3R) by 96 days post bone marrow transplant (pBMTx). In contrast, no Group 2 recipients showed early rejection and 6/7 survived >400 days pBMTx. The first two Group 3 recipients developed GvHD and succumbed by days 59 and 68 pBMTx without evidence of rejection. However, after reducing the size of the donor BMT, the third recipient is now 76 days pBMTx with excellent allograft contractility and no evidence of GVHD. Mean peak lymphoid chimerism was 10% on day 26 pBMTx for Group 1, 26% on day 33 pBMTx for Group 2, and 57% on day 54 pBMTx for Group 3. (Figure 1) <h3>Conclusion</h3> After further refinement of the conditioning regimen, ABT199 may allow for the induction of heart allograft tolerance without the need for kidney co-transplantation and with a reduction in TBI thereby improving safety.

Correction of murine sickle cell disease by allogeneic haematopoietic cell transplantation with anti-3rd party veto cells.

Despite advances in gene therapy allogeneic hematopoietic stem cell transplants (HSCT) remains the most effective way to cure sickle cell disease (SCD). However, there are substantial challenges including lack of suitable donors, therapy-related toxicity (TRM) and risk of graft-versus-host disease (GvHD). Perhaps the most critical question is when to do a transplant for SCD. Safer transplant protocols for HLA-disparate HSCT is needed before transplants are widely accepted for SCD. Although risk of GvHD and TRM are less with T-cell-deplete HSCT and reduced-intensity conditioning (RIC), transplant rejection is a challenge. We have reported graft rejection of T cell-depleted non-myeloablative HSCT can be overcome in wild type fully mis-matched recipient mice, using donor-derived anti-3rd party central memory CD8-positive veto cells combined with short-term low-dose rapamycin. Here, we report safety and efficacy of this approach in a murine model for SCD. Durable donor-derived chimerism was achieved using this strategy with reversal of pathological parameters of SCD, including complete conversion to normal donor-derived red cells, and correction of splenomegaly and the levels of circulating reticulocytes, hematocrit, and hemoglobin.

Long term disease burden post-transplantation: three decades of observations in 25 Hurler patients successfully treated with hematopoietic stem cell transplantation (HSCT)

BackgroundMucopolysaccharidosis type I-Hurler syndrome (MPSI-H) is a lysosomal storage disease characterized by severe physical symptoms and cognitive decline. Early treatment with hematopoietic cell transplant (HSCT) is critical to the survival of these patients. While survival rates and short-term outcomes are known to be improved by HSCT, the long-term cognitive, adaptive and psychosocial functional outcomes of children with (MPSI-H) post-HSCT are not well documented. This manuscript focuses on retrospective long-term follow-up (7–33 years) of 25 MPSI-H patients, transplanted between 1986 and 2011.ResultsThe median age at transplantation was 21 months (range 12–57 months). Except for one death, all successfully transplanted MPSI-H patients surviving at least 1 year after HSCT are alive to-date, with a median age of 21 years (range 8–36 years) at the last follow-up evaluation. A majority of HSCT grafts were bone marrow transplants (BMT), resulting in durable full chimerism in 18 (72%). Pre-HSCT, the onset of first symptoms occurred very early, at a median age of 3 months (range birth-16 months). The most prevalent symptoms before MPSI-H diagnosis involved progressive dysostosis multiplex; almost all patients suffered from hip dysplasia and thoracolumbar spine Kyphosis. Despite HSCT, considerable residual disease burden and ensuing corrective surgical interventions were observed in all, and at every decade of follow-up post HSCT. Late-onset psychiatric manifestations were significant (n = 17 patients; 68%), including depression in 13 patients at a median onset age of 18 years (range 13–31 years), hyperactivity and attention deficit disorder (n = 4), and multiple acute psychotic episodes (APE), independent of depression observed (n = 3) at a median onset age of 18 years (range 17–31 years). The adult Welscher Intelligence Scale results (n = 16) were heterogenous across the four scale dimensions; overall lower scores were observed on both working memory index (median WMI = 69.5) and processing speed index (median PSI = 65), whereas verbal comprehension index (median VCI = 79) and perceptual reasoning index (median PRI = 74) were higher.ConclusionWith advanced treatment options, MPSI-H are living into 3rd and 4th decades of life, however not disease free and with poor adaptation. Residual disease (loss of mobility, limited gross and fine motor skills; low cognitive ability; suboptimal cardiopulmonary function, vision and hearing) negatively impacts the quality of life and psychosocial functioning of affected individuals.

Distinct roles for major and minor antigen barriers in chimerism-based tolerance under irradiation-free conditions.

Eliminating cytoreductive conditioning from chimerism‐based tolerance protocols would facilitate clinical translation. Here we investigated the impact of major histocompatibility complex (MHC) and minor histocompatibility antigen (MiHA) barriers on mechanisms of tolerance and rejection in this setting. Transient depletion of natural killer (NK) cells at the time of bone marrow (BM) transplantation (BMT) (20 × 106 BALB/c BM cells → C57BL/6 recipients under costimulation blockade [CB] and rapamycin) prevented BM rejection. Despite persistent levels of mixed chimerism, BMT recipients gradually rejected skin grafts from the same donor strain. Extending NK cell depletion did not improve skin graft survival. However, F1 (C57BL/6×BALB/c) donors, which do not elicit NK cell‐mediated rejection, induced durable chimerism and tolerance. In contrast, if F1 donors with BALB/c background only were used (BALB/c×BALB.B), no tolerance was observed. In the absence of MiHA disparities (B10.D2 donors, MHC‐mismatch only), temporal NK cell depletion established stable chimerism and tolerance. Conversely, MHC identical BM (BALB.B donors, MiHA mismatch only) readily engrafted without NK cell depletion but no skin graft tolerance ensued. Therefore, we conclude that under CB and rapamycin, MHC disparities provoke NK cell‐mediated BM rejection in nonirradiated recipients whereas MiHA disparities do not prevent BM engraftment but impede skin graft tolerance in established mixed chimeras.

SAT-317 INFLEUNZA VACCINATION RECALL IMMUNE RESPONSES QUANTIFICATION IN CHIMERIC RENAL TRANSPLANT RECEIPITS AT A SINGLE CELL LEVEL & HIA

Drug-based immunosuppression significantly increases the risk of infection and cancer. Development of new approaches to minimize or eliminate graft rejection is of great interest to the transplant community. Establishment of durable hematopoietic chimerism through hematopoietic stem cell transplantation (HSCT) has been shown in preclinical models and patients to lead to donor specific tolerance. Those chimeric patients immune competence to virus infection never been investigated. Herein, we investigated the recall immune responses in a series of chimeric renal transplant recipients received CD8+/TCR- facilitating cell-based hematopoietic stem cell transplant (termed FCRx) to induce tolerance to mismatched renal allografts. This study took advantage of recent breakthroughs in the single-cell quantification of human peripheral blood B-cell responses to prospectively evaluate both B- and T-cell responses to the seasonal influenza vaccine (A/California[H1N1], A/SWIZERLAND [H3N2], B/BRISBANE, B/PHUKET] at day 0 before vaccination and day 30 post vaccination in 3 chimeric renal transplant recipients (proved by HLA PCR], and 3 healthy controls. Utilizing ELISA, flowcytometry analysis of T & B phenotypes, intracellular IFN-Y, B-cell culture supernatant and haemaggultination inhibition assay. there was a significant overall B-cell response to influenza vaccination, quantified by the frequency of influenza-specific antibody-secreting cells (ASC) in peripheral blood of chimeric patients but the magnitude was significantly reduced compared to healthy controls. The magnitude of the sero-response and the rate of sero-conversion were also blunted. The influenza-specific interferon-gamma (IFNγ) T-cell response was significantly reduced in transplant recipients; however, there was no correlation between the magnitude of the influenza specific IgG ASC, IFNγ responses and HIA. The induction of memory T- and B-cell responses to influenza vaccination supports the recommendation to vaccinate while the blunted responses in chimeric transplant patients.

Favorable Outcome with Durable Chimerism after Allogeneic Hematopoietic Cell Transplantation Using Busulfan and Fludarabine-Based Reduced-Intensity Conditioning for Children with Hemophagocytic Lymphohistiocytosis

Purpose Hematopoietic Stem Cell Transplantation (HSCT) is the only curative treatment for patients with familial, relapsing, or persistent hemophagocytic lymphohistiocytosis (HLH). Here, we present a single center experience of using busulfan (Bu) and fludarabine (Flu) based reduced intensity conditioning regimen (RIC) for treatment of HLH. Methods Medical records of pediatric patients with HLH, who received HSCT using Bu/Flu based RIC regimen.from January 2008 to December 2017, were reviewed retrospectively. HSCT outcomes including engraftment, survival, and GVHD were analyzed. Results Nine patients were received HSCT using Bu/Flu based RIC regimen. Three patients received HSCT after diagnosis of familial HLH and the other 6 received after reactivation. All 3 patients with familial HLH had UNC13D mutations. Median 8.35 × 106 /kg (range, 2.84-10.05 × 106) CD34 positive cells were infused. All patients achieved WBC and PLT engraftment at median 11 days (range, 10-21) and 19 days (range, 13-32) from HSCT with full donor chimerism which sustained until last visit. Cyclosporine and mycophenolate mofetil were applied as GVHD prophylaxis except one patient who received HSCT from syngeneic sibling. Three patients (33%) experienced acute graft-versus-host disease (GVHD) of grade 2 which was well controlled after conventional steroid treatment. Two patients (22%) underwent chronic GVHD, and one of them died. One patient (11%) had reactivation 4 months after HSCT from syngeneic sibling donor, and died. The 5-year overall survival was 78%. Two patients died; one died of disease and the other died of treatment related mortality (TRM). No TRM other than chronic GVHD was existed. Conclusion Our experience suggests that Bu/Flu based RIC regimen can be effective option for pediatric patients with HLH who need HSCT showing durable chimerism and long-term survival with decreased toxicity.

Outcomes in Children with Primary Immunodeficiencies Undergoing HSCT Complicated By BCG-Osis

<h3>Introduction</h3> BCG vaccination is an important global health measure in countries with endemic tuberculosis but poses significant risks when administered to patients with Severe Combined Immune Deficiency (SCID) or genetic defects associated with Mendelian Susceptibility to Mycobacterial Diseases (MSMD). A study by Marciano et al. reported that BCG-osis occurred in over 50% of BCG-vaccinated patients with SCID. The impact of this infection on allogeneic hematopoietic stem cell transplant (HSCT) outcomes is unclear. Importantly, BCG-directed antimicrobial regimens are variable and little is known about whether these drugs impact engraftment when administered at the time of transplant. <h3>Methods</h3> Our study retrospectively reviewed transplant outcomes for children with primary immunodeficiencies (PIDs) complicated by BCG-osis between 2013 and 2019. Data analyzed include diagnosis, conditioning regimen, antimicrobial therapies, severity of BCG disease, survival and donor engraftment. <h3>Results</h3> 5 patients underwent allogeneic HSCT complicated by BCG-osis. Patient demographics and BCG treatment details are shown in Table 1. Patients received reduced intensity conditioning (RIC) with alemtuzumab/fludarabine/melphalan (n=3) or a reduced toxicity myeloablative conditioning (MAC) regimen with alemtuzumab/fludarabine/busulfan (n=2). All patients engrafted in less than 15 days with at least 99% whole blood donor chimerism. Two patients subsequently developed mixed chimerism. Both had SCID and received RIC. Whole blood donor chimerism for all patients is shown in Figure 1 and sorted T-cell donor chimerism for patients with mixed chimerism is shown in Figure 2. Four out of five patients (80%) remain alive at a median follow up of 894 days (range 92-2152 days). <h3>Conclusion</h3> Our experience suggests that children with PIDs undergoing HSCT complicated by BCG-osis have favorable outcomes with BCG-directed antimicrobial therapy. In addition, BCG-directed antimicrobial therapy was well tolerated and did not immediately appear to affect engraftment in our study. Patients who received MAC had durable donor chimerism. Expected mixed chimerism was observed in patients with SCID who received RIC, but donor T-cell chimerism was durable. As post-transplant BCG-osis occurred in several patients, our experience suggests that antimicrobial therapy is likely needed for several months until immune reconstitution is achieved.

HLA-Haploidentical Transplantation in Children with Primary Immunodeficiency Using Post Cyclophosphamide

Introduction Hematopoietic Stem Cell Transplantation (HCT) is considered the curative treatment for patients with Primary Immune Deficiencies (PIDs). Finding a full matched Human leukocyte donor remains an obstacle for performing the bone marrow transplant in a timely fashion. Haplo-identical HCT is an alternative treatment option for patients, who do not have access to full-matched donor. We are reporting preliminary data on our experience of HCT in PID patients using haplo-identical donor with post-transplant Cyclophosphamide. Methods Eleven transplant naive PID patients were infused from June 2017 to August 2018 using a haplo-identical HLA donor at our institution. Eight (72.7%) of the recipients were male. Median age at infusion was 0.9 years (Min: 5.5 months Max: 2.3 years). Primary indications for transplantation included Severe Combined Immune Deficiency (SCID) in seven (63.6%), Omenn Syndrome in two (18.2%), and Chediak-Higashi syndrome and Combined Immunedeficiency in one (9.1%) each. All received GVHD prophylaxis comprising of Cyclophosphamide, Mycophenolate mofetil (MMF), and Cyclosporine regimen. Conditioning regimen was BU/FLU/Thiotepa (±ATG) in six (54.5%) recipients followed by BU/FLU/ATG in four (36.4%). Source of stem cells was bone marrow in ten patients (90.9%) while for the remaining one child PBSC was utilized. Results Eight patients (72.7%) had ANC engraftment with a median time to ANC recovery 15.5 days (Minimum: 13, maximum: 22) whereas, seven (63.6%) of our patients achieved platelets recovery (Median: 33 days, minimum: 16, maximum: 150). In terms of chimeric studies, all but one patient, engrafted at Day +100. Cumulative incidence of aGVHD was 27.3% (n=3); no chronic GVHD was recorded. At a median follow-up of 18.8 months (95% CI: 14.6-23.1), cumulative probability of overall survival at one year from infusion, for our patients is 72.7% (±13.4%). Hemorrhagic cystitis was seen in only one (9.1%) patient while two (18.2%) experienced mucositis (Grade1 or 2). Three (27.3%) contracted CMV infection and were successfully treated.At one-year follow-up lymphoid chimerism remains >95% donor type and CD3:1717 mm3 and CD19 521 mm3 Conclusion Preliminary outcome data on our small cohort of immunodeficiency patients who underwent HCT using Haplo-identical donor with Post-Cy is quite encouraging with durable and stable donor chimerism and CD3 and CD19 engraftment.