Mixed Hematopoietic Chimerism Research Articles

IL-2 receptor engineering enhances regulatory T cell function suppressed by calcineurin inhibitor

Clinical trials utilizing regulatory T cell (Treg) therapy in organ transplantation have shown promising results, however, the choice of a standard immunosuppressive regimen is still controversial. Calcineurin inhibitors (CNIs) are one of the most common immunosuppressants for organ transplantation, although they may negatively affect Tregs by inhibiting IL-2 production by conventional T cells. As a strategy to replace IL-2 signaling selectively in Tregs, we have introduced an engineered orthogonal IL-2 (ortho IL-2) cytokine/cytokine receptor (R) pair that specifically binds with each other but does not bind with their wild-type counterparts. Murine Tregs were isolated from recipients and retrovirally transduced with ortho IL-2Rβ during ex vivo expansion. Transduced Tregs (ortho Tregs) were transferred into recipient mice in a mixed hematopoietic chimerism model with tacrolimus administration. Ortho IL-2 treatment significantly increased the ortho IL-2Rβ(+) Treg population in the presence of tacrolimus without stimulating other T cell subsets. All the mice treated with tacrolimus plus ortho IL-2 achieved heart allograft tolerance, even after tacrolimus cessation, whereas those receiving tacrolimus treatment alone did not. These data demonstrate that Treg therapy can be adopted into a CNI-based regimen by utilizing cytokine receptor engineering.

Naïve T Cell-Depleted Hematopoietic Graft Combined with Solid Organ Transplantation As Strategy to Induce Tolerance By Transient Mixed Chimerism

BACKGROUND The major challenge of solid organ transplantation (SOT) is to prevent graft rejection. The chronic use of pharmacological immunosuppression has been associated with increased rates of infection, cardiovascular, metabolic, endocrine disorders, and cancer. The induction of chimerism by donor hematopoietic stem cell transplantation (HSCT) together with SOT is an active tolerance induction approach. Here, we present a new model for inducing transient mixed chimerism in 2 patients undergoing SOT employing a non-myeloablative conditioning (NMA) regimen and a partially T cell-depleted (TCD) graft consisting of CD34+ hematopoietic progenitor cells and CD45RA− memory lymphocytes. METHODS SOT and HSCT were performed at University Hospital La Paz, Madrid. Bone marrow harvesting was performed on the iliac crest and lumbar and dorsal vertebrae of the deceased donor for patient#1 and mobilized leukoapheresis from sibling donor for patient#2. First we obtained the enriched by CD34+ selection using the CliniMACS system (Miltenyi Biotec) and afterwards the CD45RA− cells were purified. Lineage-specific chimerism analysis was analyzed using quantitative real-time polymerase chain reaction. Immune cell recovery was monitored by multiparametric flow cytometry. We obtained the permission of the Organización Nacional de Trasplantes and the informed consent was obtained from the patient and donor and/or caregivers. RESULTSPatient 1 A 15-year-old boy was diagnosed with intestinal epithelial dysplasia in 2008. After two previous intestinal and multivisceral acute cellular rejections forced a third MVT. With the imptemption to induce donor tolerance, a T cell-depleted bone marrow graft from the same deceased donor was employed. Bone marrow harvesting was performed obtaining a total volume of 141 ml with 4x109 total nucleated cells. Peripheral blood was obtained after unclasping the thoracic aorta. The stem cell fraction from bone marrow was enriched by CD34+ selection, obtaining 8.75x105/kg CD34+cells/kg. Memory lymphocytes were purified and aliquots containing 1x106/kg CD3+CD45RO+ and 2.1x104/kg CD3+CD45RA+ T cells. Both products were cryopreserved. The conditioning regimen before HSCT included fludarabine and cyclophosphamide. On day 88 after MVT, we infused all the CD34+ cells and 1x106/Kg CD3+CD45RO+ T cells. The CD3+CD45RO+ T cells were also infused periodically on a monthly basis for 1 year. Donor chimerism in CD3+ T cells ranged from 0.05 to 5.1 during the first year after HSCT. We observed a peak of the percentage of regulatory T cells (Tregs). No degree of graft-versus-host disease (GvHD) was observed, and the patient became independent of total parenteral and enteral nutrition. Serial follow-up intestinal biopsies did not show any sign of immune rejection. The patient continued to receive sirolimus to maintain drug concentrations at 6 ng/ml as the only immunosuppression treatment. No signs of rejection were detected after 27 months of follow-up. Patient 2 A 49-year-old woman was diagnosed with chronic kidney failure secondary to rapidly progressive glomerulonephritis. The patient had received 3 previous kidney transplants from deceased donors. A kidney transplant from her sibling was performed in March 2021. Hematopoietic progenitor cells were obtained from mobilized peripheral blood leukapheresis. A NMA conditioning regimen employing total lymphoid irradiation (8 Gy) with antithymocyte globulin (7.5 mg/kg). Two weeks after the kidney transplantation, we infused a graft consisting of 1.27x107/kg CD34+, 4.43x106/kg CD3+CD45RO+, and 5x104/kg CD3+CD45RA+ cells. Controls for hematopoietic chimerism demonstrated the presence of transient mixed hematopoietic chimerism during the first 2 months after SOT of between 0.1% and 40% CD3+ cells. The patient continued on monotherapy with tacrolimus. Immune reconstitution analysis showed a Treg peak. The patient did not have acute rejection, GvHD, viral reactivations or episodes of bacterial infection during the 17 months of follow-up. CONCLUSIONS We describe for the first time the use of non-myeloablative conditioning and a CD45RA− T cell-depleted graft to induce transitory mixed chimerism using both hematopoietic progenitors and a solid organ coming from the same donor, for tolerance induction purposes. The patients continue to progress favorably 17 months or more after SOT, receiving immunosuppressive monotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transplantation of Hematopoietic Stem and Progenitor Cells from Cadaveric Organ Donors Leads to Long-Term Multilineage Engraftment in NSG Mice

Background Infusion of Hematopoietic Stem and Progenitor Cells (HSPC) from cadaveric organ donors (CD) has been used anecdotally in the setting of allogeneic Hematopoietic Stem Cell transplants (HSCT) most commonly with the intention for induction of mixed hematopoietic chimerism and subsequent immunological tolerance. No systematic study has addressed the functional capacity of these cells for inducing long-term multi-lineage engraftment. Since the introduction of post-transplant Cyclophosphamide (PTCy) it has been possible to reliably overcome HLA barriers and achieve stable engraftment across extensively HLA-mismatched donor and recipient pairs. Banked HSPC from cadaveric donors may provide a new approach to expand the number of donors for HSCT. Usage of HSPC from bone marrow (BM) of banked cadaveric donors is viewed as no different than live donor aspirated marrow by the FDA but is not yet available on the registry. This emerging source could be of high importance to allow rapid on-demand access to transplants for patients who lack HSPC donors or induce stable mixed chimerism and immune tolerance in patients undergoing combined organ and BM transplantation. The objective of our studies was to investigate whether hCD34⁺HSC selected from cadaveric BM retain their capacity for long-term multi-lineage engraftment. Methods and Results We compared CD34⁺-selected HSPC obtained from CD and living donors (LD) regarding their in vitro and in vivo function and with particular focus on their ability to achieve long-term multi-lineage engraftment following transplantation into NSG mice. Bone marrow was recovered from vertebral bodies of CD and CD34⁺ selected. 5x10⁵ hCD34⁺ cells from 24 different CD were transplanted i.v. in to NSG or SGM3 mice (n=5 per donor) following 1Gy total body irradiation (TBI). Blood was drawn every two weeks, starting week 4 and human CD45⁺ chimerism was analyzed via flow cytometry. All donors engrafted successfully and robust long-term multi-lineage engraftment >16 weeks was seen in 19 out of 22 donors. To further investigate the long-term repopulating capacity, we performed secondary transplants. BM of 15 primary engrafted donors was harvested, hCD45⁺ were selected with immunobeads and transplanted into 1Gy irradiated NSG mice. Successful multi-lineage engraftment after secondary transfer was observed in 12 out of 15 donors in the peripheral blood (PB). Results of tissue engraftment are still outstanding for most experiments. So far they showed mean engraftment of human CD45⁺ cells of 6.16%+/-2.4% for donor 1 and 8.48%+/-0.74% for donor 2 in the BM. To compare CD and LD we set up two side-by-side transplantation experiments and intra-mouse competitive engraftment studies using HLA-mismatched donors. Using side-by-side experiments no significant difference in human CD45⁺ chimerism was detected in blood measured at weeks 6, 12 and 16 pos-transplant. The tissues showed a mean engraftment of human CD45⁺ cells of 87% +/- 9.6% vs 71% +/- 9.5% in spleen and 76% +/- 5.3% vs 78% +/- 4% (NS) in BM upon takedown 20 weeks post-transplant. For our competitive assays, we transplanted 2.5x10⁵ hCD34⁺ i.v. from LD and CD simultaneously into NSG mice (n=5 per donor pair) following 1Gy TBI. In total 8 CD and 3 LD were used, out of whom 4 CD reached higher engraftment than the LD. Conclusion Our data suggest that CD34+HSPC from CD retain their long-term multi-lineage engraftment capacity, reaching at least equipotent levels as HSPC from living donors. Based on our findings cadaveric bone marrow should be considered as HSPC source for HSCT.

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.

CT-368 Discordance of Chimerism in the Blood and Bone Marrow as a Possible Sign of Adverse Outcome After Allo-HSCT

DNA isolated from bone marrow (BM) or peripheral blood (PB) of patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) is used to assess hematopoietic chimerism. Both sources are presumably equivalent for this purpose. Here, we report several allo-HSTC cases with BM chimerism not matching that of PB and vice versa. To investigate the possible association between the disparity of chimerism in paired PB and BM samples and disease features in post-transplant acute myeloid leukemia patients. This study analyzed the STR profiles in the DNA of paired archival BM and PB samples of 43 patients who received allo-HSCT in the National Medical Research Center for Hematology (Moscow, Russia) from 2016 to 2022. Paired BM and PB samples were collected with a time difference of one day or less. Chimerism was assessed by STR-PCR. STR profiles were evaluated by PCR with a COrDIS Plus multiplex kit (Gordiz Ltd, Russia). Patients with mixed hematopoietic chimerism at certain time points during monitoring were included. In 8 patients out of 43 (18.6%), a disparity of chimerism between PB and BM samples was found. In 3 patients (7%) in the early stages after allo-HSCT, chimerism measured in the PB sample was ≥2 times higher than in BM. In 5 (11.6%) patients, the proportion of recipient DNA in BM was twice as high as in PB. In 2 patients, this phenomenon might be due to donor lymphocytes transfusion before measuring chimerism. For 3 patients, no simple explanation for this phenomenon is evident so far. However, all 5 relapsed. Four patients died from the progression of the disease, and 1 underwent a second transplant and maintains remission. The predominance of recipient DNA in PB samples compared to BM, noted in the early stages after allo-HSCT, may not correspond to the real picture due to delayed hematopoietic reconstitution. The study of chimerism in sorted cell populations might provide an explanation for host predominance in BM over PB. Probably, such patients relapsing in the early stages after allo-HSCT could have chimerism split between certain cell lineages. Further studies of chimerism in paired PB and BM samples on extended cohorts of post-transplant patients are required.

Islet cell replacement and transplantation immunology in a mouse strain with inducible diabetes

Improved models of experimental diabetes are needed to develop cell therapies for diabetes. Here, we introduce the B6 RIP-DTR mouse, a model of experimental diabetes in fully immunocompetent animals. These inbred mice harbor the H2b major histocompatibility complex (MHC), selectively express high affinity human diphtheria toxin receptor (DTR) in islet β-cells, and are homozygous for the Ptprca (CD45.1) allele rather than wild-type Ptprcb (CD45.2). 100% of B6 RIP-DTR mice rapidly became diabetic after a single dose of diphtheria toxin, and this was reversed indefinitely after transplantation with islets from congenic C57BL/6 mice. By contrast, MHC-mismatched islets were rapidly rejected, and this allotransplant response was readily monitored via blood glucose and graft histology. In peripheral blood of B6 RIP-DTR with mixed hematopoietic chimerism, CD45.2 BALB/c donor blood immune cells were readily distinguished from host CD45.1 cells by flow cytometry. Reliable diabetes induction and other properties in B6 RIP-DTR mice provide an important new tool to advance transplant-based studies of islet replacement and immunomodulation to treat diabetes.

In Utero Hematopoietic Cell Transplantation of Lineage- c-Kit- Sca-1- Cells Is Responsible for Tolerance Induction

IntroductionIn utero hematopoietic cell transplantation (IUHCT) of allogenic bone marrow mononuclear cells (BM MNC) results in mixed hematopoietic chimerism in the murine model. In contrast, highly enriched HSCs (LSKs: Lin- c-Kit+ Sca-1+ cells) do not engraft after allogeneic IUHCT. Tracking studies in the first 72 hours after IUHCT demonstrate relatively large numbers of donor cells in the fetal thymus after IUHCT of BM MNCs, but not after IUHCT of LSK cells. This suggests that a sub-population of BM MNCs are capable of homing to the thymus within a window of opportunity for tolerance induction and that this population is lost, with enrichment of HSC.The aim of this study is to identify the specific cell population within BM MNCs that is responsible for tolerance induction. To achieve this goal, we developed a practical model for determination of prenatal tolerance induction and tested subpopulations of BM MNC for their ability to induce tolerance.MethodsDonor BM MNCs and subpopulations were isolated from 6-week old C57BL/6TgN(act-EGFP)OsbY01) (B6GFP) mice. Lineage (CD3, B220, CD11b, Gr-1) or CD3 depletion of donor cells was performed using magnetic sorting (Auto-MACS Pro Separator) and anti-Biotin microbeads.To isolate the Lin- subpopulations, anti-biotin antibody was used to exclude Lin+ cells, and then the remaining cells were stained for c-Kit and Sca-1 markers.Donor subpopulations were generated by sorting using the FACSAria.IUHCT was performed at E14 in Balb/c fetuses via vitelline vein injection. Post-natal injections were performed at P0 through the facial vein. Donor cell engraftment was assessed in peripheral blood at 4 and 12 weeks of age by flow cytometry (% GFP+ cells within CD45+ population).Thymi and livers of E16 fetuses were harvested and GFP cells were detected using a fluorescence stereomicroscope.Statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Results are expressed as mean±SEM. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children's Hospital of Philadelphia.ResultsTo develop the model for tolerance assessment we determined that injection of 10^7 CD3 depleted BM MNCs into Balb/c pups at P0 resulted in no engraftment. In contrast, IUHCT at E14 of 10^7 BM MNCs resulted in chimerism (13.2±2.01% chimerism) at 4 weeks with associated donor specific tolerance. Finally, P0 injection of 10^7 CD3 depleted BM MNCs into in mice that had received E14 injections of 10^7 BM MNCs resulted in engraftment with enhancement of chimerism at 4 weeks (38.02±3.67% in E14 plus P0 group vs 0.008±0.005 in P0 only group, p=0.0001).After establishing the model to test tolerance, we performed IUHCT of subpopulations of cells derived from BM MNCs, using the dose of each subpopulation found in 10^7 BM MNCs, to test their potential for tolerance induction. Mice that underwent IUHCT of 10^5 Lin- cells prior to the P0 injection of 10^7 CD3depleted BM MNCs had higher chimerism at 4 weeks than mice that received E14 injections of 10^5 Lin+ cells (9.843±0.79% vs 0.088±0.056%, p=0.0001).Thymuses were harvested from E16 fetuses that were injected at E14 with different subsets of the Lin- population (c-Kit+ Sca-1+, c-Kit- Sca-1+, c-Kit+ Sca-1-, c-Kit- Sca-1int, and c-Kit- Sca-1- cells). The only thymuses to show GFP cells at E16 were those that had received E14 Lin- c-Kit- Sca-1- cells. Mice that received Lin- c-Kit- Sca-1- cells at E14 prior to P0 injections showed chimerism at 4 weeks when compared to those that had received Lin- c-Kit+ Sca-1-, Lin- c-Kit+ Sca-1+, Lin- c-Kit- Sca-1int, or Lin- c-Kit- Sca-1hi cells at E14, in which chimerism was absent (6.755±0.95% vs 0.0%; p=0.02.)ConclusionWe have demonstrated that it is the Lin- c-Kit- Sca-1- subpopulation of BM MNCs that induces tolerance and prevents rejection of subsequent injections from the same donor. We have also shown that only the Lin- c-Kit- Sca-1- population migrates to the thymus in time for the induction of central tolerance. Furthermore, mouse fetuses that were given Lin- c-Kit- Sca-1- intravascularly at E14 were able to become chimeric with a postnatal CD3 depleted MNCs transplantation without immunosuppression or myeloablation. These findings suggest that a c-Kit- Sca-1- progenitor is responsible for tolerance induction, likely via common dendritic progenitors that home to the thymus. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mixed xenogeneic porcine chimerism tolerizes human anti-pig natural antibody-producing cells in a humanized mouse model.

A major obstacle to the success of organ transplantation from pigs to humans, necessitated by the shortage of human organs, is robust humoral immune rejection by pig-reactive human antibodies. Mixed xenogeneic hematopoietic chimerism induces xenoreactive B cell tolerance in rodents, but whether mixed pig/human chimerism could induce tolerance of human B cells to pig xenoantigens is unknown. We investigated this question using a humanized mouse model in which durable mixed (pig-human) xenogeneic chimerism can be established. Human natural anti-pig cytotoxic antibodies, predominantly IgM, are detectable in non-chimeric humanized mouse serum, and pig-reactive antibodies were reduced in mixed chimeric versus non-chimeric humanized mice. This difference required persistent mixed chimerism and was not due to the adsorption of antibodies on pig cells in vivo. Furthermore, human B cells from spleens of mixed chimeric mice produced lower levels of anti-pig antibodies when stimulated in vitro compared with those from non-chimeric mice. Our findings demonstrate that mixed chimerism reduces human natural antibodies to pig xenoantigens, providing the first in vivo evidence of human B cell tolerance induction by mixed xenogeneic chimerism and supporting further evaluation of this approach for inducing human B cell tolerance to xenografts.

Selective expansion of regulatory T cells using an orthogonal IL-2/IL-2 receptor system facilitates transplantation tolerance.

Adoptive transfer of Tregs has been shown to improve alloengraftment in animal models. However, it is technically challenging to expand Tregs ex vivo for the purpose of infusing large numbers of cells in the clinic. We demonstrate an innovative approach to engineering an orthogonal IL-2/IL-2 receptor (IL-2R) pair, the parts of which selectively interact with each other, transmitting native IL-2 signals, but do not interact with the natural IL-2 or IL-2R counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this orthogonal IL-2R into Tregs. Upon adoptive transfer in a murine mixed hematopoietic chimerism model, orthogonal IL-2 injection significantly promoted orthogonal IL-2R+Foxp3GFP+CD4+ cell proliferation without increasing other T cell subsets and facilitated donor hematopoietic cell engraftment followed by acceptance of heart allografts. Our data indicate that selective target cell stimulation enabled by the engineered orthogonal cytokine receptor improves Treg potential for the induction of organ transplantation tolerance.

Cardiac Allograft Tolerance Induction with Ox40L Costimulatory Blockade

<h3>Purpose</h3> Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group via hematopoietic mixed chimerism with allogeneic bone marrow and kidney co-transplantation. This model has failed without the regulatory mechanisms associated with the donor kidney. We hypothesized that rapamycin and anti-OX40L mAb (KY1005, Kymab Ltd) could substitute for kidney co-transplantation and induce cardiac allograft tolerance by providing a favorable immunologic milieu for regulatory T cells (Tregs). <h3>Methods</h3> Fourteen cynomolgus NHPs underwent mixed chimerism conditioning (ATGAM, total body and thymic irradiation) then organ and bone marrow transplant. Heart Alone recipients received a heart transplant (n=5), Heart/Kidney recipients received a heart and kidney co-transplant (n=7). Heart/OX40L recipients received a heart transplant plus 40 days of anti-OX40L mAb (n=2). All groups underwent a 12 day course of aCD154 mAb. The Heart Alone and Heart Kidney groups received 28 days of cyclosporine while the Heart aOX40L group received 100 days of rapamycin. <h3>Results</h3> All Heart Alone recipients had cellular rejection (ISHLT 3R) by 200 days post bone marrow transplant (pBMT). No Heart/Kidney recipients showed early rejection. Similarly, no Heart/OX40L recipients showed early rejection and one is currently 258 days pBMT without clinical or pathologic evidence of rejection (Figure 1). The other recipient died of viral complications. Mean peak lymphoid chimerism was 10% for the Heart Alone group, 26% for the Heart/Kidney group, and 29% for the Heart/OX40L group. The anti-OX40L protocol also showed an expansion of the Treg population with a peak of 35% Tregs in the CD4+ population. <h3>Conclusion</h3> The OX40L protocol achieved robust lymphoid chimerism with concomitant expansion of Tregs. The surviving Heart/OX40L recipient achieved longer freedom from allograft rejection when compared to the Heart Alone group. OX40L costimulatory blockade may allow for heart allograft tolerance without kidney co-transplantation.

Tregs and Mixed Chimerism as Approaches for Tolerance Induction in Islet Transplantation.

Pancreatic islet transplantation is a promising method for the treatment of type 1 and type 3 diabetes whereby replacement of islets may be curative. However, long-term treatment with immunosuppressive drugs (ISDs) remains essential for islet graft survival. Current ISD regimens carry significant side-effects for transplant recipients, and are also toxic to the transplanted islets. Pre-clinical efforts to induce immune tolerance to islet allografts identify ways in which the recipient immune system may be reeducated to induce a sustained transplant tolerance and even overcome autoimmune islet destruction. The goal of these efforts is to induce tolerance to transplanted islets with minimal to no long-term immunosuppression. Two most promising cell-based therapeutic strategies for inducing immune tolerance include T regulatory cells (Tregs) and donor and recipient hematopoietic mixed chimerism. Here, we review preclinical studies which utilize Tregs for tolerance induction in islet transplantation. We also review myeloablative and non-myeloablative hematopoietic stem cell transplantation (HSCT) strategies in preclinical and clinical studies to induce sustained mixed chimerism and allograft tolerance, in particular in islet transplantation. Since Tregs play a critical role in the establishment of mixed chimerism, it follows that the combination of Treg and HSCT may be synergistic. Since the success of the Edmonton protocol, the feasibility of clinical islet transplantation has been established and nascent clinical trials testing immune tolerance strategies using Tregs and/or hematopoietic mixed chimerism are underway or being formulated.

Mixed Chimerism after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with β Thalassemia: Impact on Outcome and Risk Factors

Background: Hematopoietic stem cell transplantation (HSCT) is the most effective curative option for patients with thalassemia major(TM). Early post-transplant mixed chimerism (MC) has known to be a predictor of secondary graft rejection. However, the impact of the persisting mixed chimerism on transplant outcome remains controversial. Recently Thiotepa(TT) has been decreased to reduce toxicity.There was no data available on reduced intensity conditioning on mixed chimersim in a large group of patients with thalassemia to date. Aims: To assess risk factors of mixed chimerism and to evaluate possible correlations between mixed chimerism and the development of complications after HSCT, in terms of graft versus host disease (GVHD) development, OS, TFS and GR. Patients and methods: From December2008 until December 2019, 618 patients with TM underwent HSCT at our center with median age of 6 (1-23) and median follow up time of 70 months(1-135), from HLA-identical sibling donors (SD) (n=212; 34.3%), unrelated donors (UD, n=313; 50.6%),sibling Cord blood(CB, n=49,7.9%)and parent donors (PD, n=44,7.1%). Source of graft were 538 (87.1%) from peripheral stem cells, 32(5.2%) from bone marrow and 48(7.8%)from sibling cord blood. Cy+Bu+Flu+TT+ATG conditioning regimen was used in 416 pts while a reduced conditioning regimen Cy+Bu+Flu+ATG was used for 167pts and Cy+Bu+Flu for the other 35 pts. Results Overall Survival (OS), Thalassemia-Free survival (TFS), Transplantation-related mortality (TRM) and graft rejection (GR) for the entire group were 94.8%, 92.7% ,5.2% and 2.4% respectively. MC was presented in 70 pts with 52 male and 18 female, defined as the presence of &amp;gt;5% residual recipient cells. Cumulative incidence of MC was 12.4% (Figure1). 5 pts had secondary graft failure and another 5 patients persisting MC post-transplant refractory to DLI following a second HSCT. There were two deaths ineffective to DLI and one die of idiopathic pneumonia and one of hemolytic uremic syndrome. The median MC present time was 4 months (1-59 months).No significant difference was observed in OS and Thalassemia-Free survival (TFS) between MC and PC pts. The cumulative probability of OS, TFS between MC pts and FC pts was 97.1% vs. 94.4% (P=0.343) and 89.6% vs. 93.2% (P=0.272). However, the incidence of MC was significantly associated with the development of graft failure/rejection. Graft failure occurred in 5 pts in the MC group vs.7 pts in the full donor chimerism (PC) group, the corresponding incidence were 7.6% vs. 1.6 % respectively. (P =0.001, Figure 2). Acute GVHD incidence was much lower in MC pts than in PC pts (2.9% vs. 12.7%, P=0.022, Figure 3 A), whereas MC pts had significantly higher chronic GVHD(16.8% vs. 4.2%, P=0.000 Figure 3 B). 2 of 70 MC pts developed I-II aGVHD. No MC pts develop II-IV aGVHD. MC patients were unlikely to have severe acute GVHD. MC was associated with an increased risk of chronic GVHD, mostly were DLI derived Patients receiving stem cells from cord blood of matched sibling donors had a high risk of MC.MC incidence of stem cell source from peripheral blood(PB), bone marrow(BM), cord blood (CB)were 10.6%, 10%, 34.2% respectively.(P=0.000, Figure 4). Unrelated donors had a lower risk of MC than sibling donors. Incidence of MC from unrelated donors was 9.5% vs. 16.1% from sibling donors (P=0.007, Figure 5) Intravenous Thiotepa (TT) in combination with Busulfan (Bu) , cyclophosphamide (Cy) anti-thymocyte globulin(ATG) on HSCT could reduce the risk of MC. The corresponding incidence of MC in Cy+Bu+Flu+TT+ATG, Cy+Bu+Flu+ATG, Cy+Bu+Flu conditioning regimen were 9.2%,15.9%,40.9% resepectively(P=0.000, Figure 6). There was also a significant difference in chimerism based on donor-recipient gender pairing (P = 0.041): male grafts into male patients having the greatest risk of MC and female grafts into female patients having the least. Patients who under 9 years old had a higher risk of MC than that of pts&amp;gt;9 years old (P=0.018, Figure 7), Summary: Our results show that patients who presented mixed chimerism showed a similar post-transplant outcome in OS and TFS but was associated with higher risk of graft failue.Patients receiving stem cells from CB had a high risk of MC than from PBSC. Reduced nonmyeloablative approach capable of achieving allogeneic mixed hematopoietic chimerism. Our data suggests that Cy+Bu+Flu+TT+ATG is a preferred conditioning regimenfor TM patients could reduce the risk of mixed chimerism. Disclosures No relevant conflicts of interest to declare.

Characterization of a Novel CD8+ B Cell Subset in Monkeys Tolerant of Heart Allografts

We have previously reported that transient mixed hematopoietic chimerism induced via donor bone marrow transplantation can achieve tolerance to cardiac allografts in non-human primates (NHPs). The purpose of this study was to measure the frequency and properties of a unique subset of CD8+ B cells, recently identified in our laboratory, in tolerant monkeys. Cynomolgus Macaques underwent organ transplantation followed by injection of donor bone marrow cells with mixed chimerism conditioning consisting of 3Gy TBI, 7Gy thymic irradiation, anti-thymocyte globulin, and a short course of anti-CD154 mAb and CyA therapy. The frequencies of different B cell subsets were measured in the peripheral blood of ten monkeys pre- and post-transplantation. In addition, PBMCs were isolated from peripheral blood of two tolerant animals (recipients of heart plus kidney or heart plus thymus cotransplants) 847 and 274 days post-bone marrow and FACS-sorted into five groups: T cells (CD3+), whole B cells (CD20+), mature B cells (CD20+CD8-CD27+), naïve B cells (CD20+CD8-CD27-), and newly identified CD8+ B cells (CD20+CD8+). mRNA was extracted from lysates of each cell population and sequenced using an Illumina NextSeq 500. During the reconstitution phase of the conditioning protocol, CD8+ B cells exhibited a phenotype reminiscent of transitional B cells (CD20+CD27-IgMhiCD38hi) and the percentage of these cells among peripheral B cells was much higher than that observed in naïve animals (23.4% vs 2.5% respectively; p<0.001). In the two tolerant animals, the CD8+ B cells gene expression suggested an immature profile which included the expression of CD21, CD38, and CD24. Interestingly, however, CD8+ B cells showed an increased expression of the gene encoding for the regulatory protein CD72. In addition, CD8+ B cells had greater expression of CD69 which is involved with TGFbeta production. The prevalence of CD8+ B cells expressing genes involved in regulatory immunity after bone marrow transplantation suggests that these cells may play a role in tolerance induction to heart allografts in NHPs. Work is in progress to further characterize these novel cells.

Inducing Donor MHC Chimerism with Bone Marrow Derived Exosomes in Non-Human Primates

Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group via hematopoietic mixed chimerism consisting of an allogeneic bone marrow (BM) and kidney co-transplantation from the same donor after non-myeloablative conditioning. In this setting, T cell tolerance is believed to rely on expression of donor MHC molecules in the host's primary and secondary lymphoid organs. Here, we investigated whether donor MHC chimerism could be achieved using donor bone marrow derived extracellular vesicles (exosomes) instead of cells. Exosomes were isolated through serial ultracentrifugation from supernatants of donor BM cells cultured for 48 hours (expressing H38 MHC class I epitope). Two H38- cynomolgus macaque recipients received conditioning consisting of ATGAM and thymic irradiation, with or without total body irradiation (TBI) before a heart and kidney co-transplantation. Both recipients underwent a short course of immunosuppression with a calcineurin inhibitor and co-stimulation blockade with anti-CD154 mAb. A single dose of H38+ donor bone marrow derived exosomes was infused into H38- recipients on post-operative day 2. Image flow cytometry was used to detect the presence of recipient cells displaying donor MHC class I on their surface (cross-dressed) in the blood, thymus, spleen and lymph nodes. Infusion of donor bone marrow derived exosomes resulted in the presence of recipient peripheral blood leukocytes displaying donor MHC class I molecules in both animals. Flow cytometric analysis showed that MHC class I chimerism was restricted to the myeloid cell compartment. Tissues collected from the total body irradiated NHP showed that H38+ cross-dressed cells were also present in the lymph nodes, thymus, and spleen. In a separate study, a NHP recipient treated with an exosome protocol without TBI is currently 54 days post-transplantation with a well-functioning cardiac allograft. Donor MHC chimerism in the form of MHC cross-dressing can be achieved in a NHP model using donor bone marrow derived exosomes. These findings suggest that bone marrow derived exosomes could be used in place of whole bone marrow to induce allograft tolerance. The use of exosomes would allow for the reduction of recipient conditioning and would eliminate the risk of GVHD making this approach clinically safer than traditional BMT.

Translational impact of NIH-funded nonhuman primate research in transplantation.

The National Institutes of Health (NIH) has long supported using nonhuman primate (NHP) models for research on kidney, pancreatic islet, heart, and lung transplantation. The primary purpose of this research has been to develop new treatments for down-modulating or preventing deleterious immune responses after transplantation in human patients. Here, we discuss NIH-funded NHP studies of immune cell depletion, costimulation blockade, regulatory cell therapy, desensitization, and mixed hematopoietic chimerism that either preceded clinical trials or prevented the human application of therapies that were toxic or ineffective.

P1.10: Donor’s graft ex vivo T cell depletion with fludarabine reduces GvHD signs and improves survival after intestine transplantation

Introduction: Intestine passenger T leukocytes are responsible of graft versus host disease (GvHD) in intestine transplantation (ITx). These lymphocytes are known to have inferior tolerogenic qualities compared with leukocytes in the liver, other solid organs and bone marrow. Fludarabine is a routinely used anti neoplasic agent with high cytotoxicity against T cells. We hypothesized that ex vivo fludarabine treatment of the bowel graft could diminish the risk of GvHD and improve post Tx overall survival. Methods: We performed isolated heterotopic ITx from Lewis (LEW) to Brown Norway (BN) rat strains. The grafts of one of the experimental groups were imbided and sealed in Celsior preserving solution with 1000 µM fludarabine during surgery (1 h), before its implantation into recipient animals. We compared a group of untreated (n = 7) vs a group of fludarabine-treated bowel recipients (n = 5). The mixed hematopoietic chimerism was determined by flow cytometry using strain-specific anti HLA antibodies. Clinical signs of GvHD or as well as post-Tx overall survival were also monitored. Results: One hour fludarabine treatment of the bowel grafts induced specific apoptosis of its passenger T cells at concentrations from 100 μM while no histological signs of intestinal tissue alterations were observed after 1000 μM fludarabine treatment. After heterotopic LEW -> BN ITx, untreated intestine recipients showed GvHD signs from the fourth day post-Tx (n = 7). These symptoms include: rash (n = 4), weight loss (n = 3), piloerection (n = 2) and diarrhea (n = 1). The chimerism or percentage of donor’s lymphocytes in the recipient rat, determined in peripheral blood, reached 6.61% (range 1.7–10.8%) at day 3 post-Tx and 2.56% (range 0–9.8%) at day 7 post-Tx (Fig. 1). Rats transplanted with a fludarabine-conditioned intestine showed statistically signifcant later and milder clinical signs of GvHD. Additionally, fludarabine treatment reduced total donor cells chimerism at day 7 and the percentage of chimeric T cells at days 3 and 7 post-Tx (Fig. 2). Both experimental groups died showing clinical signs of graft rejection. Untreated bowel grafts recipients died within 9.2 days ± 0.3 days, while fludarabine-treated graft recipients showed prolonged survival (13.5 days ± 0.3 days). Conclusions: Graft immunosupresion with fludarabine during surgical procedure protects bowel recipients of GvHD risk and improves post-Tx overall survival.

P3.60: Native spleen preservation attenuate graft versus host disease in an experimental model of modified multivisceral transplantation.

Introduction: Native spleen used to be removed in patients undergoing modified multivisceral transplantation (MMVT) increasing the risk of sepsis and graft-versus-host disease (GVHD). The mechanistic basis of these effects including mixed chimerism level is poorly understood. Based on a novel experimental procedure of MMVT that triggers GVHD, we aimed to evaluate the effects of native spleen preservation in this model in order to gain insight into the mechanisms that may mediate this phenomenon. Material and Methods: Heterotopic MMVT from Lewis to Brown Norway rats was performed. MMVT graft consisted of stomach, duodenum, pancreas, spleen and the small bowel of the donor. The native spleen was removed (MMVT-S) in half of the MMVT recipients (N=5), while the remaining preserved their own spleen (MMVT+NSP) (N=5). After MMVT mixed hematopoietic chimerism was determined in native spleen and peripheral blood samples by flow cytometry using strain-specific HLA antibodies. Also, histopathological (using Pintar score) and clinical signs of GVHD such as skin rash were evaluated. Results: all animals in the group MMVT-S presented clinical signs of GVHD such as skin rash, weight loss, and diarrhea, among others, between 7–10 days after transplantation. Skin rash was particularly remarkable in the ears and periocular area. Clinical signs of GVHD were less frequent in MMVT+NSP recipients (40%; p<0.05). Also, significant differences between groups were observed in the histopathological study and mixed chimerism in peripheral blood 3 days after MMVT (36.6 and 23.62 % in MMVT-S and MMVT+NSP respectively) (Figure 1). Also, native spleen showed an 18±5 % of chimerism (CD3+ donor cells) 1 week after transplantation. After day 10 post-MMVT, when the signs of transient GVHD disappeared, both groups presented graft rejection. Conclusions: Native spleen preservation in MMVT recipients attenuated the occurrence of GVHD and reduced the level of chimerism compared to recipients that underwent spleen removal during MMVT. Despite more studies are necessary, our preliminary results suggest that native spleen preservation increase the efficacy in removal of anti-recipient reactive clones that would explain the protective effect of native spleen preservation against GVHD.

Hematopoietic stem cell transplantation gene therapy for hemophilia a using non-genotoxic ANTI-CD117 immunotoxin conditioning

Background & Aim Hematopoietic stem cell transplantation (HSCT) for coagulation factor VIII (fVIII) gene therapy is a potentially curative approach for the treatment of the most common severe bleeding disorder, hemophilia A (HA). Our laboratory has previously established the ability to produce stable therapeutic levels of fVIII in a mouse model of HA following irradiation or chemotherapy and transplantation of Sca-1+ cells, a population containing both HSC and progenitors, genetically modified ex vivo using recombinant lentiviral vector encoding a bioengineered high-expression fVIII variant, termed ET3. Conventional myeloablative regimens for HSCT such as total body irradiation and myeloablative alkylating chemotherapy are associated with adverse side effects, including sterility, hormonal dysregulation and genotoxicity. These risks represent a primary barrier to clinical translation of HSCT gene therapy for HA and many other monogenic diseases. Therefore, the development of targeted, non-genotoxic conditioning agents is critical. Methods, Results & Conclusion We have successfully implemented the use of an immunotoxin bioconjugate comprised of a monoclonal antibody against the stem cell factor receptor c-Kit (anti-CD117) and the ribosome-inactivating protein toxin saporin (sap) for non-genotoxic HSCT conditioning. We achieved consistent depletion of bone marrow LSK (lineage-negative Sca-1+ c-Kit+) and long-term HSC (LSK+ CD150+ CD48−) compartments five days after retro-orbital venous sinus injection of 0.5 mg/kg CD117-sap compared to control (59.2% LSK reduction; 38.9% LT-HSC reduction) in both C57BL/6J wild-type and HA mice. Following CD117-sap conditioning, HA mice were transplanted with 1 × 106 ET3-transduced Sca-1+ cells from GFP+ congenic mice. We observed robust mixed hematopoietic chimerism and myeloid engraftment at two and four weeks post-transplantation in recipients conditioned with CD117-sap compared to control mice (2 wk: 86.4 ± 9.2% vs. 1.2 ± 0.6% GFP+ granulocytes, p = 0.00009; 4 wk: 93.9 ± 5.6% vs. 0.2 ± 0.3% GFP+ granulocytes, p = 0.0018). Moreover, these mice exhibit therapeutic levels of circulating fVIII activity as early as 2 weeks post-transplantation (0.11 IU/ml, p = 0.005). Taken together, these preliminary data are a promising proof-of-concept demonstrating the feasibility of using non-genotoxic immunotoxin conditioning for HA gene therapy.

Importance of Hematopoietic Mixed Chimerism for Induction of Renal Allograft Tolerance in Nonhuman Primates.

Although induction of durable mixed chimerism is required for murine skin allograft tolerance (TOL), renal allograft TOL has been achieved after induction of only transient mixed chimerism in nonhuman primates (NHPs) and humans. To better define the level/duration of chimerism required for stable renal allograft TOL, we retrospectively analyzed these parameters and compared them with transplant outcomes in NHP combined kidney and bone marrow transplant recipients. Peripheral blood levels and duration of myeloid or lymphoid chimerism were retrospectively analyzed in 34 NHP combined kidney and bone marrow transplantation recipients which were divided into 3 groups: TOL, n = 10; chronic antibody-mediated rejection (CAMR), n = 12; and T cell-mediated rejection (TCMR), n = 12. All 4 of the recipients that failed to develop any chimerism lost their allografts due to TCMR after discontinuation of immunosuppression (56 ± 3 d). Among 30 recipients who successfully developed multilineage chimerism, 10 achieved long-term immunosuppression-free survival without rejection (1258 ± 388 d), 12 eventually developed CAMR (932 ± 155 d), and 8 developed TCMR (82 ± 10 d). The maximum level but not duration of lymphoid chimerism was significantly higher in TOL recipients compared with both CAMR (P = 0.0159) and TCMR (P = 0.0074). On the other hand, the maximum myeloid chimerism was significantly higher in TOL than in TCMR (P = 0.0469), but not in CAMR. Receiver operating characteristic analyses revealed that lymphoid chimerism levels of 3.1% or greater could reliably predict long-term immunosuppression-free renal allograft survival (P < 0.0001). This retrospective study confirmed that induction of chimerism is essential for long-term immunosuppression-free survival, which best correlates with lymphoid chimerism levels higher than 3.1%.

Combination therapy of an iNKT cell ligand and CD40-CD154 blockade establishes islet allograft acceptance in nonmyeloablative bone marrow transplant recipients.

Islet transplantation is an effective therapeutic option for type 1 diabetes. Although maintenance immunosuppression therapy is required to prevent allogeneic rejection and recurrence of autoimmunity, long-term allograft survival has not yet been achieved partly because of its adverse effects. The induction of donor-specific immunotolerance is a promising approach for long-term allograft survival without maintenance immunosuppression therapy. We previously reported that combination therapy using a liposomal ligand for invariant natural killer T cells, RGI-2001, and anti-CD154 antibody established mixed hematopoietic chimerism for the induction of donor-specific immunotolerance. This study investigated whether the protocol could promote islet allograft acceptance in experimental diabetes. Streptozotocin-induced diabetic BALB/c mice were transplanted with bone marrow cells from C57BL/6 donors and received combination therapy of RGI-2001 and anti-CD154 antibody after 3-Gy total body irradiation. 3Weeks after bone marrow transplantation, islets isolated from C57BL/6 donors were transplanted under the kidney capsule. Mixed chimerism was established in diabetic mice receiving the tolerance induction protocol. After islet transplantation, blood glucose levels improved and normoglycemia persisted for over 100days. Hyperglycemia recurred after islet grafts were removed. Histopathological examinations showed insulin-positive staining and absence of cellular infiltration in the islet grafts. T cells of recipients showed donor-specific hyporesponsiveness, and anti-donor antibodies were not detected. The tolerance induction protocol with combination therapy of RGI-2001 and anti-CD154 antibody promoted islet allograft acceptance in a mouse diabetic model. This protocol may be clinically applied to islet transplantation for type 1 diabetes mellitus.