Nonmyeloablative Bone Marrow Transplantation Research Articles

The Influence of Heterochronic Non-Myeloablative Bone Marrow Transplantation on the Immune System, Frailty, General Health, and Longevity of Aged Murine Recipients.

The stem cell theory of aging postulates that stem cells become inefficient at maintaining the original functions of the tissues. We, therefore, hypothesized that transplanting young bone marrow (BM) to old recipients would lead to rejuvenating effects on immunity, followed by improved general health, decreased frailty, and possibly life span extension. We developed a murine model of non-myeloablative heterochronic BM transplantation in which old female BALB/c mice at 14, 16, and 18(19) months of age received altogether 125.1 ± 15.6 million nucleated BM cells from young male donors aged 7–13 weeks. At 21 months, donor chimerism was determined, and the immune system’s innate and adaptive arms were analyzed. Mice were then observed for general health and frailty until spontaneous death, when their lifespan, post-mortem examinations, and histopathological changes were recorded. The results showed that the old mice developed on average 18.7 ± 9.6% donor chimerism in the BM and showed certain improvements in their innate and adaptive arms of the immune system, such as favorable counts of neutrophils in the spleen and BM, central memory Th cells, effector/effector memory Th and Tc cells in the spleen, and B1a and B1b cells in the peritoneal cavity. Borderline enhanced lymphocyte proliferation capacity was also seen. The frailty parameters, pathomorphological results, and life spans did not differ significantly in the transplanted vs. control group of mice. In conclusion, although several favorable effects are obtained in our heterochronic non-myeloablative transplantation model, additional optimization is needed for better rejuvenation effects.

Disease Burden and Pre-Transplant Health-Related Quality of Life in Pediatric Sickle Cell Disease Patients Receiving Nonmyeloablative HLA-Identical Sibling Donor Transplantation

Title: Disease Burden and Pre-transplant Health-related Quality of Life in Pediatric Sickle Cell Disease Patients Receiving Nonmyeloablative HLA-identical Sibling Donor TransplantationBackground: Sickle cell disease (SCD) is a hemoglobinopathy characterized by recurrent pain crises, severe organ damage, and reduced health-related quality of life (HRQL). Bone marrow transplantation (BMT) is a proven curative treatment for SCD that can improve both disease burden and HRQL. Despite the benefits, relatively few patients pursue a cure through BMT. The decision to receive a BMT is nuanced and likely to be influenced by a number of factors. One study by Roth et al. showed SCD patients interested in BMT had a higher HRQL than those uninterested in BMT, suggesting some variability between SCD patients pursuing transplant and the general SCD patient population. To date the factors that may affect pre-transplant HRQL of pediatric SCD patients remain unclear, particularly in the nonmyeloablative setting.Objective: To evaluate the impact of disease-burden, patient demographics, and hydroxyurea treatment on pre-transplant HRQL.Methods: Children and young adults with SCD set to receive a nonmyeloablative HLA-identical sibling donor BMT were administered HRQL surveys (PedsQL) in the six months leading up to the start of the conditioning regimen. Patients' disease history and demographic information were captured and analyzed for their relationship to HRQL. PedsQL parent surveys were used when patient surveys were unavailable. The mean difference in HRQL between groups were compared using an unpaired t test or linear regression.Results: 32 patients were enrolled onto this study. Six of the 32 patients HRQL were not included due to withdrawal, delay of transplant, or completion of the survey outside of window. The median age at the time of survey administration was 13.5 years (range 2-21). 57.7% of patients were male and 88% of patients had genotypes hemoglobin SS or Sβ 0thalassemia. 12% of patients had a history of overt stroke. 52% of patients were hospitalized while receiving HU treatment. 32% of patients received chronic blood transfusions leading up to transplant. The median number of hospitalizations in the two years leading up to transplant was 2 (range 0-10). 16.7% of patients met the AAPT diagnostic criteria for chronic pain. 75% of patients received HU treatment leading up to transplant. The median hemoglobin F percentage was 10.4. The median HRQL for all patients was 77.17 (range 36.9-98.91, SD +/- 15.99).Sickle cell genotype (P=0.88), history of overt stroke (P=0.79), hospitalization while receiving HU treatment (P=0.78), and the number of hospitalizations in the two years before transplant (P=0.39) were not associated with lower HRQL. History of chronic transfusion leading up to transplant approached statistical significance for lower HRQL (P=0.06). Older age (P=0.17) and female gender (P=0.89) were not associated with worse HRQL. Patients receiving HU treatment had significantly higher HRQL than patients not receiving HU (P=0.0034), with median PedsQL scores of 80.06 and 58.64, respectively. The date of survey administration was not associated with HRQL (P=0.40). The number of eligibility criteria met (P=0.63), chronic pain (P=0.17), total number of RBC transfusions (P=0.45), and pre-transplant hemoglobin (P=0.25) were not associated with HRQL.Discussion: Patient demographics and several markers of disease burden appear to have minimal impact on HRQL. Interestingly, the use of HU therapy was associated with significantly higher pre-transplant HRQL. The etiology of this finding is unclear given that disease burden was not associated with HRQL and warrants further investigation. The absence of association between disease burden and HRQL through current disease severity eligibility paradigms suggests that other factors may impact HRQL in SCD patients choosing a low toxicity nonmyeloablative BMT. DisclosuresNo relevant conflicts of interest to declare.

Grade II Acute Gvhd Improves Progression-Free Survival after Myeloablative HLA-Matched Bone Marrow Transplant with Posttransplantation Cyclophosphamide

Introduction: Posttransplant cyclophosphamide (PTCy) can be safely utilized as the sole graft-versus-host-disease (GVHD) prophylaxis after myeloablative conditioning (MAC) for HLA-matched bone marrow transplant (BMT). However, the effects of GVHD with this transplant platform are not well defined. Methods: We retrospectively analyzed 298 consecutive adult patients transplanted between 2004-2012 for hematologic malignancy who engrafted after MAC (busulfan with either fludarabine or Cy) HLA-matched related (MRD, n=187) or HLA-matched unrelated (MUD, n=111) T-cell-replete BMT with Cy 50 mg/kg on days 3 and 4. Results: Median recipient age was 48 and 47 years for MAC MRD and MUD BMT, respectively. Median follow up was 4.33 and 3.74 years in the two cohorts, respectively. After MAC MRD BMT, 35% of patients experienced grade II acute (a) GVHD, 12% grade III-IV aGVHD, and 9% chronic (c) GVHD. After MAC MUD BMT, 54% of patients experienced grade II aGVHD, 17% grade III-IV aGVHD, and 17% cGVHD. We choose landmark analyses that look at the time to an event after a pre-specified time point. By starting the failure-time clock later, one can regard events prior to the landmark as baseline factors in the analysis, allowing us to study the effects of a posttransplant event on subsequent outcomes. We selected landmark time points at 100 days and 1 year, which allowed us to capture 100% of the patients who developed maximal grade II aGVHD and 97% of the patients who developed cGVHD. Few patients died of acute or chronic GVHD before their respective landmark time points. In landmark analyses at 100 days after MAC MRD BMT, 4-year overall survival (OS) and progression-free survival (PFS) were 60% (95% CI: 0.50-0.72) and 41% (95% CI: 0.30-0.54) in patients without grade II-IV aGVHD, and 67% (95% CI: 0.55-0.82) and 56% (95% CI: 0.43-0.73) in patients with maximal grade II aGVHD (Figure 1). In landmark analyses at 100 days after MAC MUD BMT, 4-year OS and PFS were 51% (95% CI: 0.36-0.73) and 44% (95% CI: 0.28-0.69) in patients without grade II-IV aGVHD, and 65% (95% CI: 0.52-0.82) and 45% (95% CI: 0.31-0.64) in patients with grade II aGVHD. In landmark analysis at 1 year after MAC MRD BMT, 4-year OS and PFS were 77% (95% CI: 0.67-0.89) and 60% (95% CI: 0.46-0.77) in patients without GVHD, and 69% (95% CI: 0.45-1) and 75% (95% CI: 0.5-1) in patients with cGVHD. In landmark analysis at 1 year after MAC MUD BMT, 4-year OS and PFS were 57% (95% CI: 0.39-0.82) and 57% (95% CI: 0.36-0.90) in patients without GVHD, and 90% (95% CI: 0.73-1) and 75% (95% CI: 0.5-1) in patients with cGVHD. In time-dependent multivariable analyses adjusted for disease risk index, recipient age, and hematopoietic cell transplant-comorbidity index (HCT-CI), grade II aGVHD was associated with improved OS and PFS after both MAC MRD (HR 0.56 95% CI 0.3-1.05, p=.07 and HR 0.59 95% CI 0.35-0.99, p=.05, respectively) and MAC MUD BMT (HR 0.53 95% CI 0.26-1.06, p=.07 and HR 0.47 95% CI 0.25-0.91, p=.03, respectively). In contrast, after MAC MRD, grade III-IV aGVHD was associated with decreased OS (HR 2.25 95% CI 1.18-4.29, p=.01) with no significant difference in PFS (HR 1.5 95% CI 0.8-2.81, p=.20). In contrast to the univariable analysis, chronic GVHD was not significantly associated with OS or PFS in MAC MRD (HR 1.18 95% CI: 0.52-2.69, p=.69 and HR 0.77 95% CI: 0.34-1.74, p=.53) or MAC MUD (HR 0.73 95% CI: 0.27-1.97, p=.53 and HR 0.67 95% CI: 0.28-1.65, p=.39) BMT. Increasing nucleated cell graft dose as a continuous variable was associated with improved PFS after MAC MRD and MAC MUD BMT (HR 0.85 95% CI 0.71-1.01, p=.06 and HR 0.83 95% CI 0.69-1.0, p=.05, respectively). Conclusion: Development of maximal grade II aGVHD was associated with significantly improved PFS after HLA-matched BMT with PTCy. In this distinct cohort, we validated prior work from our group, which showed that grade II aGVHD improves PFS after nonmyeloablative HLA-haploidentical BMT with PTCy, mycophenolate mofetil, and tacrolimus (McCurdy et al. 2017 submitted for publication). This suggests that, in the setting of PTCy, grade II aGVHD improves PFS. Therefore intensifying GVHD prophylaxis to reduce grade II aGVHD may not improve transplant outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Establishment of A Mixed Chimeric Mouse Model of Allogeneic Bone Marrow Transplantation and Its Influencing Factors

To establish a mouse mixed chimerism (MC) model of nonmyeloablative allogeneic bone marrow transplantation(allo-BMT) and explore its affecting factors. The MC model was established by nonmyeloablative allo-BMT followed by high-dose post-transplant cyclophosphamide (PTCY). 123 mice in the experiments was retrospectively analyzed, and the factors related with the chimerism were explored with the univariate and multivariate logistic regression analysis. A multivariate linear regression was performed by R project to obtain a mathematical model for predicting the chimeric level with relevant affecting factors. The model presented mixed chimerism on day 14 after transplantation, and was characterized by a donor lymphocyte infusion (DLI) which significantly promoted donor engraftment on day 15, but transfplantation of PBS in control group was failed. Among 123 mice, 47 (38.21%) mice were MC, while 76 (61.79%) mice were non-MC in 123 mice, respectively; univariate analysis showed that the baseline body weight of mice (P=0.001, 17.84±1.19 g vs 18.50±0.94 g), total body irradiation(TBI，P=0.048) and the using of cyclophosphamide (P=0.16) were affected the chimeric state of mice, while the number of infusing cells and the time of detection showed no significant effects. Multivariate regression analysis showed that under certain conditions, the body weight of mice on day 0 was an independent factor affecting chimeric levels (OR=0.493, 95% CI 0.307-0.791, P=0.003). Through R project multiple linear regression, the math model was achieved, which was chimerism=6.09-12×weight(g)+80.03×TBI(Gy)-4.4×cell-counts (× 107) +0.38×CTX (mg/kg), R2=0.5841, P<0.001. The experiment presents a method for establishing a mixed chimeric mice model after non-myeloablative bone marrow transplantation and constructs a mathematical model with relevant factors affected chimerism status.

Effects of heterochronic, non-myeloablative bone marrow transplantation on age-related behavioural changes in mice

Experiments using heterochronic parabionts, i.e. young and old animals connected surgically and hence developing a shared circulation, have shown that blood-borne factors, transferred from young to old mice and vice versa, play a role in influencing a range of health outcomes associated with advanced age. Previous work has explored the contributory role of plasma-derived factors in mediating such parabiotic effects, including those on aging-associated neural and behavioural impairments. Here, we wanted to identify possible influences that blood-borne cellular factors may have on age-related behavioural phenotypes. Towards this end, we subjected old BALB/c H-2d mice to repetitive non-myeloablative bone marrow transplants (BMT) from young donor animals and assessed effects on behaviour and cognition. We detected expected age-related alterations in our behavioural assays but did not discern any obvious differences between old BMT mice and old control animals. Our study represents the first to look at possible behavioural and cognitive effects of heterochronic, non-myeloablative BMT. Future work should extend this study by including additional behavioural tests in the analysis, addressing whether beneficial effects of BMT may be detectable on other genetic backgrounds and reconciling our findings with those achieved by myeloablative BMT.

Haploidentical BMT for severe aplastic anemia with intensive GVHD prophylaxis including posttransplant cyclophosphamide

AbstractSevere aplastic anemia (SAA) is a stem cell disorder often treated with bone marrow transplantation (BMT) to reconstitute hematopoiesis. Outcomes of related HLA-haploidentical (haplo) donors after reduced-intensity conditioning with intensive graft-versus-host disease (GVHD) prophylaxis including posttransplantation cyclophosphamide are presented here from 37 SAA, 20 relapsed/refractory (R/R), and 17 treatment-naïve (TN) SAA patients. Median follow-up is 32 months (90% confidence interval [CI], 29-44). The median age was 25 (range, 4-69) years. The median time to neutrophil recovery was 17 days (range, 15-88). Four of 37 patients (11%) experienced graft failure (GF). There was 1 primary GF of 20 patients in the R/R group and 3 of 17 in the TN group at 200 cGy (1 primary, 2 secondary), but none in the 10 patients who received 400 cGy total body irradiation. Two patients with GF succumbed to infection and 2 were rescued with second haplo BMT. The overall survival for all patients is 94% (90% CI, 88-100) at 1 and 2 years. The cumulative incidence of grade II-IV acute GVHD at day 100 is 11%. The cumulative index of chronic GVHD at 2 years is 8%. Similar results were seen in 10 SAA patients who received the identical nonmyeloablative regimen with posttransplant cyclophosphamide but matched donor transplants. Haplo BMT with posttransplant cyclophosphamide represents a potential cure in SAA, with all 20 R/R currently alive, disease-free, and with no evidence of active GVHD. Extending this approach to TN patients was associated with higher GF rates, but an increase in total body irradiation dose to 400 cGy was associated with durable engraftment without greater early toxicity. Nonmyeloablative haplo BMT in TN SAA could lead to a paradigm shift, such that essentially all patients can proceed quickly to safe, curative BMT. These trials were registered at www.cincialtrials.gov as #NCT02224872) and #NCT02833805.

Non-Myeloablative Bone Marrow Transplant with Post-Transplant Cyclophosphamide Plus Thiotepa Improves Donor Engraftment in Patients with Transfusion Dependent Thalassemia: Results of an International Consortium (VGC2)

<h3>Background</h3> Post-transplant cyclophosphamide (PTCy) enables allogeneic hematopoietic cell transplant (Allo-HCT) across the human leukocyte antigen barrier. Allo-HCT for severe transfusion dependent thalassemia is restricted in its application due to limited donor availability, graft rejection, and regimen-related toxicities. We explored the use of a common non-myeloablative (NMA) conditioning regimens with PTCy based graft-versus-host disease (GvHD) prophylaxis for matched related, haploidentical, and unrelated donors in the setting of a collaborative consortium. <h3>Methods</h3> A multicenter learning collaboration involving 3 centers used an augmented NMA conditioning with PTCy developed by John Hopkins group (Bolaños-Meade, Blood 2012). All patients received common conditioning regimen consisting of ATG 4.5 mg/kg, fludarabine 150 mg/m², cyclophosphamide 29 mg/kg, thiotepa (10 mg/kg on day -7) and TBI 200 cGy. Graft versus host disease (GvHD) prophylaxis was PTCy 50 mg/kg on days +3 and +4, MMF, and sirolimus. To improve engraftment rates, all patients received preconditioning with hydroxyurea 30mg/kg x 60 days (Figure 1). Most patients received G-primed bone marrow as stem cell source. Two patients received matched related donor (MRD), 4 patients haploidentical, and 3 patients matched unrelated donor (I-MUD, 2-mismatched MUD) bone marrow transplants (Figure 2). <h3>Results</h3> A total of 9 patients with transfusion dependent thalassemia were included with a median follow-up time of 371 days. One case of primary graft failure was seen in the haploidentical cohort at D+32; 89% (8/9) patients successfully engrafted. Two cases each of mild gut and skin acute GvHD and one case each of limited gut and skin chronic GvHD were seen in transplanted patients. No cases of grades III-IV acute or severe, chronic GvHD were seen. All patients are alive, thalassemia free survival (TFS) was 100% in the MRD and M/MUD recipients, but 75% (3/4) in the haploidentical cohort. There was no statistical difference between groups in regards to neutrophil or platelet engraftment, age, TNC or CD34+ cell dose. No engrafted patient required long-term immunosuppression therapy, and all remain transfusion independent. <h3>Conclusion</h3> We have shown the feasibility of NMA conditioning with PTCy plus thiotepa for severe transfusion dependent thalassaemia. A phase II study will help determine the optimal regimen with least toxicity and improved thalassaemia free survival.

The Path to Cure: Using Haploidentical (haplo) Donors and High-Dose Post-Transplant Cyclophosphamide (PTCy) for Treatment-Naïve and Refractory Severe Aplastic Anemia (SAA)

SAA is a life-threatening hematopoietic stem cell disorder that is often treated with bone marrow transplantation (BMT) to reconstitute hematopoiesis and eliminate clonality. Our group has shown that PTCy after related HLA-haplo BMT can facilitate engraftment and yield low rates of relapsed and refractory (R/R) SAA. Given the risk of relapse or secondary clonal disease following immunosuppressive therapy (IST) is &amp;gt; 50% at five years, we decided to implement this treatment paradigm in untreated SAA. Here, we review the haplo outcomes in SAA, with an update in R/R SAA patients and new results in treatment-naïve (TN) SAA patients using haplo donors and PTCy. Patients were eligible with SAA. Fanconi anemia and short telomere patients were excluded. R/R patients had ≥ one course of IST. The conditioning regimen included standard published haplo backbone of antithymocyte globulin, low dose CY, and total body irradiation (TBI). All R/R patients received 200cGy. The TBI dose for the TN cohort was modified after 3 graft failures (GF) in initial 7 patients treated at 200cGy; thus, the last 8 TN patients received 400cGy. After the marrow graft was infused on day 0, cyclophosphamide 50mg/kg/day IV on days +3,+4 post-transplant was administered. All received mycophenolate mofetil beginning day +5 through 35 and tacrolimus day +5 to 365. Engraftment definitions include: absolute neutrophil count &amp;gt;1.0 x10E9/L for three consecutive days; days from last red blood cell transfusion for hemoglobin and platelet count &amp;gt;50,000 for 7 days without transfusion. Thirty-five SAA patients received haplo BMT: 20 R/R and 15 TN. The median follow-up is 31 months (90% CI: 26.0, 45.4). The median age was 25 (range, 4-69) years, with 23 patients &amp;gt; age 20 and 57% were males. The median related haplo donor age was 34 years (range, 19-57), including three non-first degree relatives. Table 1 shows data by cohort. Median time to neutrophil recovery was 17 days (range 15-88), to red cell recovery 23 days (range 6-58) and to platelet recovery 28 days (range 15-108). At the time of BMT, 25 of 35 patients had evidence of clonality (mostly PNH clones). Four of 35 patients (11.4%) (95% CI: 3.2, 26.7%) experienced GF. There was one GF out of 20 patients in the R/R group (5%) (95% CI: 0.1, 24.9%) and 3 of 15 in the TN group 20% (95% CI: 4.3,48.1%, p= 0.20). OS for all patients (Figure 1) is 94% (90% CI: 88,100%) at one and two years, 100% in the R/R group and 86% (90% CI: 72, 100%) in the TN group. The cumulative incidence of grade II-IV aGVHD at day 100 is 9% (90% CI: 1, 17%). The cumulative incidence of chronic GVHD at 2 years is 9% (90% CI: 1,17%). All R/R patients are alive and well, and fully engrafted with 100% donor chimerism; patient with primary GF is 100% chimeric after 2nd transplant from different haplo donor. One patient has extensive cGVHD. Of the first 7 TN patients who received 200cGy TBI, 4 are fully engrafted and well; of the 3 with GF, 2 died from infection and one is now 100% chimeric using second haplo donor. After increasing the TBI dose to 400cGy, the subsequent 8 patients are alive with full donor chimerism. Nonmyeloablative haplo BMT with PTCy represents a potential cure in SAA, with all 20 R/R currently alive, disease-free (including eradication of clonal diseases), and no evidence of active GVHD. Extending this approach to TN patients was associated with higher GF rates. Perhaps this is not surprising given that the potential beneficial effect of front-line IST on engraftment is absent. The increase in TBI dose to 400cGy in TN patients appears to achieve more durable engraftment without early greater toxicity (infection, delayed engraftment, GVHD); more patients and longer follow up are required to understand rates of fertility and other survivorship issues. Alternative donor BMT (including haplo donors) should be offered to all SAA patients with R/R SAA who are fit enough to tolerate non-myeloablative conditioning. A potential advantage of haplo donors over unrelated donors is that the transplant center has full control and oversight of allograft quality, which can be especially important with the use of the preferred allograft source for SAA, bone marrow. Non-myeloablative haplo BMT in TN SAA could lead to a paradigm-shift in the field, such that essentially all children and fit adults can proceed quickly to a relatively safe, curative BMT. Disclosures DeZern: Astex Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy. Cooke:Jazz pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Posttransplant cyclophosphamide in allogeneic bone marrow transplantation for the treatment of nonmalignant hematological diseases.

We present a single-center retrospective series of allogeneic bone marrow transplantation (BMT) with the use of posttransplant cyclophosphamide (PTCy) in the setting of nonmalignant hematological conditions. Nine patients were treated between 2013 and 2019. Nonmyeloablative conditioning consisted of antithymocyte globulin, fludarabine, low-dose cyclophosphamide, and total body irradiation (200cGy) followed by allogeneic bone marrow infusion. Post-BMT GVHD prophylaxis was with PTCy, tacrolimus, and mycophenolate mofetil. At a median follow-up of 24 months (range 4, 63), all patients are alive, with donor-derived hematopoiesis and free of significant acute or chronic GVHD. Donors were haploidentical (n = 6), fully matched unrelated (n = 2), and fully matched sibling (n = 1). Neutrophil and platelet engraftment occurred at a median of 21 days and 33 days, respectively, after transplantation. Three patients (3/9, 33%) experienced stage 1-2 acute skin GVHD. The only cases of chronic GVHD are in three patients (3/9, 33%) with ocular disease (two mild, one moderate). No patient has required systemic immunosuppression beyond 12 months after BMT. PTCy-based nonmyeloablative allogeneic BMT is safe and effective for nonmalignant hematologic conditions and should be prospectively compared with historical regimens.

Transplantation tolerance in nonhuman primates and humans.

This review focuses on our recent studies involving nonmyeloablative bone marrow transplantation as an approach to inducing organ allograft tolerance across MHC barriers in nonhuman primates and in patients. The clinical studies are focused on mechanisms of tolerance involved in a protocol carried out at Massachusetts General Hospital in HLA-mismatched haploidentical combinations for the induction of renal allograft tolerance. These studies, in which chimerism was only transient and GVHD did not occur, suggest an early role for donor-specific regulatory T cells in tolerance induction, followed by partial and gradual deletion of donor-reactive T cells. We utilized high-throughput sequencing methodologies in a novel way to identify and track large numbers of alloreactive T cell receptors (TCRs). This method has been shown to identify biologically significant alloreactive TCRs in transplant patients and pointed to clonal deletion as a major mechanism of long-term tolerance in these patients. More recently, we adapted this sequencing method to optimally identify the donor-specific regulatory T cell (Treg) repertoire. Interrogation of the early posttransplant repertoire demonstrated expansion of donor-specific Tregs in association with tolerance. Our studies suggest a role for the kidney graft in tolerance by these mechanisms in patients who had only transient chimerism. Nonhuman primate studies indicate that other organs, including the heart, the lungs and the liver, are less readily tolerated following a period of transient mixed chimerism. Our efforts to extend the reach of mixed chimerism for tolerance induction beyond the kidney are therefore focused on the addition of recipient Tregs to the protocol. This approach has the potential to enhance chimerism while further reducing the risk of GVHD.

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.

Endothelial cell fitness dictates the source of regenerating liver vasculature.

Neoangiogenesis plays a key role in diverse pathophysiological conditions, including liver regeneration. Yet, the source of new endothelial cells (ECs) remains elusive. By analyzing the regeneration of the liver vasculature in irradiation-based myeloablative and nonmyeloablative bone marrow transplantation mouse models, we discovered that neoangiogenesis in livers with intact endothelium was solely mediated by proliferation of resident ECs. However, following irradiation-induced EC damage, bone marrow-derived mononuclear cells were recruited and incorporated into the vasculature. Further experiments with direct bone marrow infusion or granulocyte colony-stimulating factor (G-CSF)-mediated progenitor cell mobilization, which resembles clinically relevant stem cell therapy, demonstrated that bone marrow-derived cells did not contribute to the regeneration of liver vasculature after two-thirds partial hepatectomy (PHx). Taken together, the data reconcile many of the discrepancies in the literature and highlight that the cellular source of regenerating endothelium depends on the fitness of the residual vasculature.

Abstract 4624: Natural killer cell licensing contributes to clearance of lung cancer

Abstract a) Natural Killer (NK) cell Inhibitory receptors function in a dichotomous role. Their engagement by MHC Class I upon encountering a target results in the inhibition of cytotoxicity. During NK development, however, their interaction with MHC Class I mediates the process of licensing or education, leading to an increase in responsiveness and cytotoxicity. For this reason the role of inhibitory receptors in controlling the clearance of MHC Class I+ solid tumors such as lung cancer is poorly defined. Since NK cells comprise the primary cytotoxic lymphocyte responsible for clearance of lung cancer, we set out to determine whether the licensing status of NK cells contributes to lung cancer susceptibility. b) Growth of MHC Class I+ murine lung cancer was evaluated in MHC Class I- mice, with unlicensed NK cells in direct comparison to MHC Class I+ mice with licensed NK cells. IFN-γ production was evaluated after plate bound antibody stimulation through the NKp46 and NKG2D pathways both flow cytometrically and by quantitative RT-PCR. Bone marrow transplantation was performed by injection of 10x106 unfractionated bone marrow cells after 500cGy irradiation and NK and CD4+ T cell depletion of recipient host. c) Injection of Lewis Lung Carcinoma (LLC) revealed a significant decrease in pulmonary tumor burden in mice with licensed NK cells (230.4±21.6 vs. 442.7±53.8 mg) that disappeared after NK depletion (421.4±36.8 vs. 421.9±66.3 mg). Freshly isolated licensed NK cells lysed LLC more robustly than unlicensed NK cells (28.7±.16% vs. 4.9±.2% specific lysis at 100:1 ratio respectively). Lung cancer expresses stress ligands recognized by NKG2D and NKp46 activating receptors. Licensed NK cells produced significantly more IFN-γ than their unlicensed counterparts after plate-bound stimulation with anti-NKp46 and anti-NKG2D (17.9±2.8% vs. 5.4±.8% respectively for dual NKp46 and NKG2D stimulation). No differences were evident between licensed and unlicensed NK cells in ribosomal mass, making it unlikely that translational defects were responsible for altered cytokine production. Licensing status also did not affect stability of IFN-γ but did affect mRNA levels. This suggests transcriptional control of licensing-mediated NK activity. IFN-γ production could be significantly increased in unlicensed NK cells by reintroduction of MHC Class I through non-myeloablative bone marrow transplantation from MHC Class I-sufficient congenic donors. Blockade of H2Kb-Ly49c (MHC Class I-inhibitory receptor) interaction during NK development in C57BL/6 mice using a non-depleting anti-Ly49c antibody (clone 4LO) decreased IFN-γ production in NK cells. d) The role of NK cell licensing and tumor-specific cytotoxicity is highly controversial. We now demonstrate that licensed NK cells mediate a superior lung cancer-specific immune response in a murine model. Our data suggests that altering the licensing status of NK cells may be an avenue of lung cancer-specific immunotherapy. Citation Format: Shi Lei, Kang Li, Saeed Arefanian, Alexander S. Krupnick. Natural killer cell licensing contributes to clearance of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4624. doi:10.1158/1538-7445.AM2017-4624

Bidirectional immune tolerance in nonmyeloablative MHC-mismatched BMT for murine β-thalassemia

AbstractNonmyeloablative conditioning using total lymphoid irradiation (TLI) and rabbit antithymocyte serum (ATS) (the murine preclinical equivalent of antithymocyte globulin [ATG]) facilitates immune tolerance after bone marrow transplantation (BMT) across major histocompatibility complex (MHC) disparities and may be a useful strategy for nonmalignant disorders. We previously reported that donor effector T-cell function and graft-versus-host disease (GVHD) are regulated via recipient invariant natural killer T-cell (iNKT) interleukin-4–driven expansion of donor Foxp3+ naturally occurring regulatory T cells (Tregs). This occurs via recipient iNKT- and STAT6-dependent expansion of recipient myeloid dendritic cells (MDCs) that induce contact-dependent expansion of donor Treg through PD-1/PD ligand signaling. After TLI/ATS + BMT, Gr-1lowCD11c+ MDCs and Gr-1highCD11cneg myeloid-derived suppressor cells (MDSCs) were enriched in GVHD target organs. We now report that the recovery of both recipient MDSCs (P < .01) and MDCs (P < .01) is significantly increased when the alkylator cyclophosphamide (CTX) is added to TLI/ATS conditioning. In a BALB/c → B6 lethal GVHD model, adoptive transfer of MDSCs from TLI/ATS/CTX-conditioned recipients is associated with significantly improved GVHD colitis and survival (P < .001), conversion of MDSCs to PD ligand–expressing MDCs, and increased donor naturally occurring Treg recovery (P < .01) compared with control treatment. Using BALB/c donors and β-thalassemic HW-80 recipients, we found significantly improved rates of engraftment and GVHD following TLI/ATS/CTX compared with TLI/ATS, lethal or sublethal total body irradiation/ATS/CTX, or CTX/ATS conditioning. These data provide preclinical support for trials of TLI/ATG/alkylator regimens for MHC-mismatched BMT for hemoglobinopathies. The data also delineate innate immune mechanisms by which TLI/ATS/CTX conditioning may augment transplantation tolerance.

Characterisation of central versus peripheral tumour associated macrophages in glioblastoma multiforme

BackgroundGlioblastoma multiforme is the most biologically aggressive brain tumour, with only 12 month survival, despite maximum treatment. Tumour-associated macrophages and microglia (TAMM) are myeloid cells populating 30–50% of glioblastoma multiforme tumours and are identified as potential immunotherapy targets. However, characterising functional roles of TAMM subpopulations remains elusive, because of flawed mouse models and inadequate cell-surface markers. In normal rodent brain, antibody markers CD11bhi and CD45hi are proposed to highlight infiltrating monocytes, whereas CD11bhi and CD45lo cells represent microglia and differentiated peripheral macrophages. This marker-set provides conflicting results in the context of the tumour microenvironment. We redressed this issue and aimed to determine whether peripheral or central TAMMs have independent roles in glioblastoma multiforme. MethodsWith our non-myeloablative bone-marrow transplant model, we can replace a mouse immune system using congenic donor cells marked with green fluorescence protein (GFP) without damaging the blood-brain barrier or inadvertently activating microglia; when applying this technique we subsequently distinguish between central microglial and peripheral macrophage populations. By stereotaxic intracranial implantation of murine glioblastoma multiforme (GL261), we recapitulated the normal development of a glioblastoma multiforme tumour and performed sham intracranial injections as a comparative control. Fluorescence-activated cell sorting was done to separate TAMM subpopulations for RNA sequencing. FindingsOur chimeric mouse model consistently produced 70–80% peripheral blood chimerism and overcame the issue surrounding cell-surface expression markers, distinguishing peripherally infiltrating and central tissue-resident brain cells by GFP marker status. Cerebral digestion and analysis of tumour-implanted mice revealed a novel third population (FSChi/SSChi cells) on flow cytometry that stained greater than 90% CD11b/CD45+ suggestive of TAMMs; but this cell population was absent in hemispheres of both non-tumour bearing and sham-injected mice. InterpretationOur preliminary data showed that in chimeric mice implanted with glioblastoma multiforme, peripheral cells (GFP high) were the dominant infiltrating population. With RNA extraction for downstream transcriptional analysis we aim to reliably track trafficking myeloid populations and subsequently address the ambiguity surrounding TAMMs. Thereafter, we aim to develop an immunotherapy against glioblastoma multiforme using the findings from our study. FundingNone.

Minor Antigen Disparities Impede Induction of Long Lasting Chimerism and Tolerance through Bone Marrow Transplantation with Costimulation Blockade.

Mixed chimerism and tolerance can be successfully induced in rodents through allogeneic bone marrow transplantation (BMT) with costimulation blockade (CB), but varying success rates have been reported with distinct models and protocols. We therefore investigated the impact of minor antigen disparities on the induction of mixed chimerism and tolerance. C57BL/6 (H2b) mice received nonmyeloablative total body irradiation (3 Gy), costimulation blockade (anti-CD40L mAb and CTLA4Ig), and 2 × 107 bone marrow cells (BMC) from either of three donor strains: Balb/c (H2d) (MHC plus multiple minor histocompatibility antigen (mHAg) mismatched), B10.D2 (H2d) or B10.A (H2a) (both MHC mismatched, but mHAg matched). Macrochimerism was followed over time by flow cytometry and tolerance was tested by skin grafting. 20 of 21 recipients of B10.D2 BMC but only 13 of 18 of Balb/c BMC and 13 of 20 of B10.A BMC developed stable long-term multilineage chimerism (p < 0.05 for each donor strain versus B10.D2). Significantly superior donor skin graft survival was observed in successfully established long-term chimeras after mHAg matched BMT compared to mHAg mismatched BMT (p < 0.05). Both minor and major antigen disparities pose a substantial barrier for the induction of chimerism while the maintenance of tolerance after nonmyeloablative BMT and costimulation blockade is negatively influenced by minor antigen disparities.

Quality of Life Survey in Nonmyeloablative Bone Marrow Transplant Patients at a Large Academic Center

Introduction:Patients who undergo hematopoietic stem cell transplant (HCT) experience various qualities of life post-transplant as a consequence of the disease, comorbities and/or transplant-related complications. Reports of post-allotransplant quality of life (QOL) describe those who received myeloablative conditioning. The consequences of the illness and its treatment are often more challenging for a person of advanced age. While less intense nonmyeloablative (NMAT) or reduced-toxicity conditioning permits allotransplantation for older or infirm transplant candidates, such patients also come to transplant with decreased life-expectancy and comordities that may differ from younger recipients. There is scarcity of reports concerning post-NMAT QOL; knowledge in this area would help guide patients in making decisions about their treatment. We sought to study the QOL of survivors of NMAT at the Indiana University (IU) School of Medicine and the IU Simon and Bren Cancer Center.Goal:Document QOL of patients who underwent NMAT for acute myeloid leukemia (AML) or myelodysplasia (MDS) at Indiana University (IU) School of Medicine and the IU Simon and Bren Cancer Center from 2000-2013.Methods:We utilized the Functional Assessment of Cancer Therapy-Bone Marrow Transplant Scale (FACT-BMT) which measures the effect of transplant on domains in physical well-being (PWB), social well-being (SWB), emotional well-being (EWB), functional well-being (FWB) along with BMT-specific concerns. Each response is scored between 0 and 4, with 0 being the "not at all true" and 4 being "very much true". There are 37 total questions and the highest possible score is 148, with higher scores corresponding to better QOL. The study was approved by the IRB and the surveys were conducted over the phone following obtainment of consent.Results:Thirty-three consecutive survivors of NMAT were asked to participate in a telephone survey and 27 (82%) agreed to participate. Twelve males and 15 females completed the survey; 2 declined and 4 could not be contacted. Sixteen had undergone transplant for the treatment of MDS and 11 for the treatment of AML. The median age at transplant was 59 years (range, 44-67) and median age at time of interview was 65 years (56-75). The time from transplant ranged from 2 to 15 years. All participants had received identical conditioning (cyclophosphamide, 60mg/m2 x 2 days; fludarabine, 25mg/kg x 5 days) and a variety of graft-versus-host prophylaxis regimens. ECOG performance status at the time of the transplant was 0 (n=22), 1 (n=4) or 2 (n=1). One patient experienced relapse shortly after transplant; all others were in remission.The average total FACT-BMT score for QOL was 120. Mean category-specific scores were as follows: PWB, 22/28; SWB, 25/28; EWB, 20/24; FWB 22/28; BMT-specific concerns, 31/40. Overall, patients scored highest in the SWB category, despite a number of patients lacking a spouse or main support (n=7). Lack of energy was rated the most important problem in the PWB category, with an average score of 2.03. Nausea was rated the least problematic, with an average score of 0.25. In the EWB category, the majority of patients were satisfied with how they were coping with their illness (3.51), and there was surprisingly little concern about dying (0.33). In the FWB category, all but four patients were "quite a bit" to "very much" content with the quality of their life. Twenty-six were able to work at a job or at home at least "somewhat"; 1 was able to work "not at all". In the BMT-specific category, every patient responded "very much" to the query "I have confidence in my nurse."Conclusion:Patients who participated in this single institution study appear to have generally favorable perceptions of their QOL post-NMAT. We found the FACT-BMT tool easy to use in a telephone survey, the level of participation was surprisingly high and the study was accomplished rather quickly with minimal resources. Future efforts will be directed at attempts to correlate QOL with additional transplant outcome variables, such as conditioning intensity, ECOG performance status at transplant and the presence or absence of chronic GVHD. DisclosuresNo relevant conflicts of interest to declare.

Comparative Cost Analysis of Therapy in Sickle Cell Anemia: Supportive Care Vs. Bone Marrow Transplant

Objective: A comparative cost analysis of sickle cell admissions vs. stem cell transplants in sickle cell patients.Hypothesis: We believe the overall cost of a bone marrow transplant for a sickle cell patient will be less than that of a patient with multiple sickle cell admissions.Background: Sickle cell disease remains an increasing burden to the cost of health care and health care providers. The disease results in a variety of serious organ system complications that can lead to life-long disabilities and/or early death. Despite the advent of hydroxyurea, sickle cell admissions and cost have been increasing over the course of several years. Various contributing factors may include socioeconomic status, complications of sickle cell anemia itself, narcotic dependence and noncompliance with medications.Bone marrow transplants were introduced in 1982 as an option for the treatment of sickle cell anemia, and are currently the only curative option in this disease. A study conducted at the NIH from 2004-2013, found that bone marrow transplant reversed the disease in 26 of 30 patients (87%) (Hseih et al, N Engl J Med 2009; 361:2309-2317). The patients ultimately had a normal hemoglobin, fewer hospitalizations, and lower use of narcotics to treat pain from the disease. However, the underutilization of bone marrow transplant continues to exist and may in part be secondary to a lack of fully matched donors. To overcome this challenge, the Johns Hopkins group developed a nonmyeloablative bone marrow transplantation platform using HLA haploidentical donors for patients using posttransplant cyclophosphamide. As a result, 17 patients were successfully transplanted, 14 from HLA-haploidentical and 3 from HLA-matched related donors (Meade et al, Blood. 2012; 120(22):4285-4291). Due to this, most patients with sickle cell disease have the potential to undergo a successful bone marrow transplant. However, an analysis comparing the cost of admissions vs. transplant has yet to be determined. In order to create an effective cost comparison, we utilized a nationwide database.Methods: US hospital admissions were identified from discharge data from the Nationwide Inpatient Sample (NIS), Healthcare Cost and Utilization Project (HCUP), Agency for Healthcare Research and Quality from 1998 to 2011 using ICD9 codes. Admissions were included if they had an ICD9 code for Sickle cell anemia (282.5).Results:Table 1Year1998199920002001200220032004Length of Stay (Days)6.216.136.376.196.466.316.05Cost ($)15,724.0016,465.0018,654.0018,750.0022,587.0024,501.0023,743.00Table 2Year2005200620072008200920102011AverageLength of stay (Days)6.166.136.005.915.625.715.616.04Cost ($)25,129.0027,471.0028,425.0027,314.0029,767.0029,929.0031,683.0024,687.00The cost per hospitalization has almost doubled since 1998, despite a slight decrease in length of stay from an average of 6.5 days to 5.6 days from 1998-2011. According to the data base from our study, the average cost per patient per hospitalization was $24,687. The average number of admissions per year for a single patient with sickle cell anemia is 6 (Ballas et al, Am J Hem 2009) for an estimated overall cost per year of $148,000. However, this value underestimates the true cost since this does not include emergency room visits, medicine costs, and readmission rates.Conclusion: In the age of cost effective medicine, clinicians struggle to find a balance between low cost and optimal patient care. An underutilized modality of care and even cure for sickle cell disease is bone marrow transplant. The sickle cell information center website estimates the cost of the transplant process for most patients to be $150,000 to $250,000 which includes pre-transplant evaluation, transplant stay, and post-transplant follow-up (https://scinfo.org). We estimate that a bone marrow transplant is approximately equivalent to the cost of ten sickle cell hospital admissions. According to this analysis, undergoing a bone marrow transplant would ultimately prove to be more cost efficient while decreasing the rate of complications associated with this debilitating disease. DisclosuresNo relevant conflicts of interest to declare.

Alloreactive Regulatory T Cells Allow the Generation of Mixed Chimerism and Transplant Tolerance.

The induction of donor-specific transplant tolerance is one of the main goals of modern immunology. Establishment of a mixed chimerism state in the transplant recipient has proven to be a suitable strategy for the induction of long-term allograft tolerance; however, current experimental recipient preconditioning protocols have many side effects, and are not feasible for use in future therapies. In order to improve the current mixed chimerism induction protocols, we developed a non-myeloablative bone-marrow transplant (NM-BMT) protocol using retinoic acid (RA)-induced alloantigen-specific Tregs, clinically available immunosuppressive drugs, and lower doses of irradiation. We demonstrate that RA-induced alloantigen-specific Tregs in addition to a NM-BMT protocol generates stable mixed chimerism and induces tolerance to allogeneic secondary skin allografts in mice. Therefore, the establishment of mixed chimerism through the use of donor-specific Tregs rather than non-specific immunosuppression could have a potential use in organ transplantation.

Allogeneic inducible regulatory T cells generated with retinoic acid allow the generation of mixed chimerism via nonmyeloablative conditioning in mice models

Allogeneic inducible regulatory T cells generated with retinoic acid allow the generation of mixed chimerism via nonmyeloablative conditioning in mice models