Alpha Beta T-cell Research Articles

A Pilot Trial of Pre-Transplant Risk Stratification and Prophylactic Defibrotide to Prevent Serious Thrombotic Microangiopathy in High-Risk Pediatric Hematopoietic Stem Cell Transplant Patients

Background: Transplant associated thrombotic microangiopathy (TA-TMA) is a known complication of hematopoietic stem cell transplant (HSCT) associated with endothelial injury that leads to end organ damage and high morbidity and mortality. No proven method for prevention of TA-TMA exists. One strategy is to decrease or even prevent the initial endothelial injury during conditioning. Defibrotide is an anti-inflammatory and anti-thrombotic agent that has been shown to protect the endothelium from damage and treat TA-TMA. We hypothesize that prophylactic use of defibrotide during HSCT conditioning and acute recovery may potentially prevent TA-TMA. Methods: We initiated a pilot single-arm phase II trial (NCT#03384693) to evaluate the safety and feasibility of administering prophylactic defibrotide to pediatric patients at high risk for the development of TA-TMA following HSCT. Additionally, we sought to determine if administration of prophylactic defibrotide would prevent the development of TA-TMA in clinically high-risk patients compared to historic controls. High-risk patients were defined as either patients with high-risk neuroblastoma whose treatment plan included autologous tandem transplants conditioned with cyclophosphamide/thiotepa (Cy/TT) and carboplatin/etoposide/melphalan (CEM), or allogeneic transplant patients undergoing myeloablative conditioning, with at least three of the following: age ≥10 years old, race/ethnicity other than Caucasian, ABO minor incompatibility, or haploidentical donor. Based on prior analysis at our center, we anticipated an incidence of TA-TMA of 28% in the high-risk patients undergoing allogeneic transplants and 40% in the neuroblastoma patients. Patients received defibrotide 6.25mg/kg IV q6h the day prior to the start of conditioning through day +21 and received supportive care per institution guidelines, including maintaining platelets above 30 x10^9/L. Patients were prospectively monitored for TA-TMA from admission through week 24. Results: Twenty-five patients were enrolled, 14 with neuroblastoma and 11 undergoing allogeneic HSCT. The median age was 3.45 (2.1-10.1) and 15.7 (0.8-27.4) years for autologous and allogeneic patients, respectively. Ten of the allogenic transplants were with an alpha-beta T-cell depleted haploidentical HSCT, while one was with a matched unrelated HSCT. Follow-up is complete on all patients. Defibrotide was stopped early due to clinically significant bleeding in 3 patients (day +3, +6 and +10, respectively). Bleeding episodes were excessive bleeding from oral hematoma, diffuse alveolar hemorrhage, and melanic stools and epistaxis and were classified as "possibly related" to the study drug. The other 22 patients missed a median of 0.7% of doses (0-5.2%), most often due to concomitant medication infusions. There were no allergic or hypersensitivity reactions. One patient with acute lymphoblastic leukemia (ALL) in CR4 died during the study period from respiratory failure in the setting of disseminated aspergillus and veno-occlusive disease (VOD), leading to a TRM of 0% and 9.1% in the autologous and allogeneic patients, respectively. Of the neuroblastoma patients, 4 did not go on to receive a second transplant; three due to development of moderate to severe VOD and one per parental preference. Five of the 14 neuroblastoma patients developed VOD, 4 after Cy/TT (1 mild, 2 moderate, 1 severe) and 1 after CEM (mild); all of which resolved. One patient developed non-severe TA-TMA, diagnosed 12 days post-HSCT (4% incidence). She received an alpha-beta T-cell depleted peripheral blood HSCT from her mother for ALL after conditioning with total body irradiation, cyclophosphamide, thiotepa, and ATG. Her course was complicated by diffuse alveolar hemorrhage on day +6 for which defibrotide was stopped. Her TA-TMA was treated with eculizumab and resolved without sequelae. Conclusions: 12% (3/25) patients required early discontinuation of defibrotide due to clinically significant bleeding; no other severe adverse events occurred due to the study intervention. The observed TA-TMA incidence of 4% was far below the anticipated rate of 28-40%. Our study provides preliminary evidence that defibrotide prophylaxis is safe and feasible in children and adolescents undergoing HSCT at high risk for TA-TMA and may reduce the risk of TA-TMA. Future larger randomized studies are needed to verify these findings. Disclosures Higham: Jazz Pharmaceuticals: Research Funding. Shimano:Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Research Funding. OffLabel Disclosure: Defibrotide- off label use as prophylaxis for transplant associated thrombotic microangiopathy (TA-TMA)

Abstract 900: Intermittent cyclophosphamide and vinorelbine reshape the immune cell environment, induce T cell clonal replacement and increase the efficacy of PD-1 inhibition in models of triple negative breast cancer

Abstract The clinical success of cyclophosphamide (Cy)-based haploidentical stem cell transplants in hematological malignancies indicates that this drug has the potential to reorchestrate the immune system against cancer cells. Along a similar way, Cy is administered before the infusion of CAR-T cells to improve their clinical efficacy. We have previously found that daily, low/medium-dose Cy (20mg/Kg), in association with vinorelbine (V) was able to improve the preclinical efficacy of checkpoint inhibitors (CIs) anti-PD-1 and anti-PD-L1 in preclinical models of breast cancer and lymphoma (Orecchioni et al, 2018). Randomized clinical trials have recently indicated that the addition of CIs to other chemotherapy drugs such as taxanes (T), doxorubicin (D), or platinum (P) might be beneficial in triple negative breast cancer (TNBC) patients. Thus, we designed the present study in two orthotopic, immunocompetent, local and metastatic models of TNBC (CI-resistant 4T1 and EMT6, moderately responsive to CIs) to investigate whether Cy and V, in different schedules and doses, were more effective than T, D, or P. In vivo studies in local and metastatic models using both 4T1 and EMT6 TNBC cells clearly indicated that intermittent (ie every 6 days), medium-dosage (140 mg/Kg) Cy, was more effective than any other combinatorial regimens including CIs plus daily low/medium-dose Cy, or T, D, or P at their regular dosages. The association of V further increased the preclinical efficacy of intermittent Cy plus CIs, and abrogated tumor growth in both 4T1 and EMT6 models. In vivo studies with neutralizing monoclonal antibodies targeting in separate experiments T, B, NK, and myeloid cells demonstrated that CD3+CD4+, CD3+CD8+ T cells as well as CD11b+ monocyte/macrophage dendritic cells were crucial to abrogate tumor growth in TNBC-bearing mice treated with intermittent Cy, V and CIs. Single-cell transcriptome analysis of more than 60,000 intratumoral immune cells and flow cytometry studies in circulating and intratumoral subsets of immune cells indicated that V promoted the generation and maturation of myeloid APC cells, and that intermittent Cy generated new clones of tumor-infiltrating CD3+CD4+ and CD3+CD8+ TCR alpha beta cells. After treatment with intermittent Cy, V and CIs, intratumoral immune cells showed a unique gene signature of 42 genes (CD3D, Ptprc, CD69, Lat, Lck, CD2, CD28, CD27, and CD3E among others) suggestive of a change in morphology and behavior resulting from exposure to a mitogen, cytokine, chemokine, cellular ligand, or an antigen for which it is specific, as well as APC-to-T cell adhesion. Taken together, our data support the hypothesis that APC priming and T cell clonal replacement can significantly improve CI efficacy in vivo in TNBC models. We are further investigating the Cy-related mechanisms inducing intratumoral T-cell clonal replacement and how these T cells cooperate with V-induced APCs. Citation Format: Paolo Falvo, Stefania Orecchioni, Roman Hillje, Alessandro Raveane, Patrizia Mancuso, Chiara Camisaschi, Lucilla Luzi, Francesco Bertolini. Intermittent cyclophosphamide and vinorelbine reshape the immune cell environment, induce T cell clonal replacement and increase the efficacy of PD-1 inhibition in models of triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 900.

Islet cell dysfunction in patients with chronic pancreatitis.

Chronic pancreatitis (CP) is characterized by progressive inflammation and fibrosis of the pancreas that eventually leads to pancreatic exocrine and endocrine insufficiency. Diabetes in the background of CP is very difficult to manage due to high glycemic variability and concomitant malabsorption. Progressive beta cell loss leading to insulin deficiency is the cardinal mechanism underlying diabetes development in CP. Alpha cell dysfunction leading to deranged glucagon secretion has been described in different studies using a variety of stimuli in CP. However, the emerging evidence is varied probably because of dependence on the study procedure, the study population as well as on the stage of the disease. The mechanism behind islet cell dysfunction in CP is multifactorial. The intra-islet alpha and beta cell regulation of each other is often lost. Moreover, secretion of the incretin hormones such as glucagon like peptide-1 and glucose-dependent insulinotropic polypeptide is dysregulated. This significantly contributes to islet cell disturbances. Persistent and progressive inflammation with changes in the function of other cells such as islet delta cells and pancreatic polypeptide cells are also implicated in CP. In addition, the different surgical procedures performed in patients with CP and antihyperglycemic drugs used to treat diabetes associated with CP also affect islet cell function. Hence, different factors such as chronic inflammation, dysregulated incretin axis, surgical interventions and anti-diabetic drugs all affect islet cell function in patients with CP. Newer therapies targeting alpha cell function and beta cell regeneration would be useful in the management of pancreatic diabetes in the near future.

254-LB: Characterization of Ghrelin Receptor Expression in Mouse Islets Reveals Pancreatic Polypeptide Cells as a Key Ghrelin Target

Objective: While expression of the ghrelin receptor, GHSR (growth hormone secretagogue receptor), by pancreatic islets has been investigated, data regarding the specific islet cell types that express GHSR are inconsistent and incomplete. For instance, transcriptomic studies indicate marked GHSR expression by somatostatin (SST)-secreting delta cells vs. little to no GHSR expression by alpha cells and beta cells, which contrasts studies suggesting direct actions of ghrelin on GHSRs expressed by alpha cells and beta cells. Also, to our knowledge, GHSR expression by pancreatic polypeptide (PP) cells has not previously been directly investigated. The aims of this study were to determine GHSR expression within the four principal islet endocrine cell types and the effects of ghrelin on circulating PP. Methods: Ghsr-IRES-Cre mice, which express Cre recombinase directed by the GHSR promoter, crossed to Cre-dependent ROSA26-YFP reporter mice were used to histologically identify GHSR expression in islets. Double and triple labeling for YFP-immunoreactivity (IR) with insulin-IR, glucagon-IR, SST-IR, and/or PP-IR was performed to determine GHSR co-expression with islet hormones. Pharmacological manipulations were used to examine in vivo effects of acyl-ghrelin on plasma PP. Results: Eighty-five % of YFP-IR cells expressed PP-IR, 50% expressed SST-IR, and none expressed insulin-IR or glucagon-IR. GHSR antagonist raised plasma PP in overnight-fasted C57BL/6N mice. However, acyl-ghrelin administration had no effect on plasma PP in C57BL/6N mice. Conclusions: These results suggest that within mouse pancreatic islets, GHSR expression occurs most abundantly in PP cells and that pharmacologic blockade of ghrelin action raises plasma PP. Disclosure D. Gupta: None. B.K. Mani: None. K. Shankar: None. S. Osborne-Lawrence: None. N. Metzger: None. J.M. Zigman: Stock/Shareholder; Self; Medtronic. Funding Diana and Richard C. Strauss Professorship in Biomedical Research; Mr. and Mrs. Bruce G. Brookshire Professorship in Medicine; Kent and Jodi Foster Distinguished Chair in Endocrinology; University of Texas Southwestern Medical Center (to J.M.Z.)

Stem Cell Transplant From Donors After Alpha Beta Cell Depletion in Children and Young Adults

Stem Cell Transplant From Donors After Alpha Beta Cell Depletion in Children and Young Adults

The epidemiology of subcutaneous panniculitis-like alpha-beta T-cell lymphoma in New Zealand.

Subcutaneous panniculitis-like alpha-beta T-cell lymphoma is a rare disease, which comprises less than 1% of all non-Hodgkin lymphoma. The epidemiology of this disorder has not been established in New Zealand, particularly the ethnic variation. A retrospective study was conducted from 2005 to 2017 in South Auckland, New Zealand. Patients with histopathologically confirmed subcutaneous panniculitis-like alpha-beta T-cell lymphoma, according to the WHO-EORTC classification, were included. Cases were identified from multiple data sources to ensure the study population was completely surveyed. Census data were used as the denominator. Ten cases were identified. Five were men and five women. The median age at diagnosis was of 38.5 years (range 17-73years). Nine were Māori/Pacific, and one was European. The ethnic relative risk of subcutaneous panniculitis-like alpha-beta T-cell lymphoma in Māori/Pacific versus European was 11.1 (95% Cl 1.83-246.1, P=0.005). Māori and Pacific have a higher relative risk of subcutaneous panniculitis-like alpha-beta T-cell lymphoma compared to non-Māori/Pacific.

Comparative Analysis of Alpha-Beta T-Cell and B-Cell Depleted (abTCD) HLA-Haploidentical Hematopoietic Stem Cell Transplantation (haplo-HSCT) Versus Abtcd Haplo-HSCT with T-Cell Add-Back of Rivogenlecleucel Cell [Donor T Cells Transduced with the Inducible Caspase 9 (iC9) Gene Safety Switch] in Children with High-Risk Acute Leukemia (AL) in Remission

Background: For children with AL candidate to receive an allograft and lacking a suitable HLA-matched donor, HLA-haplo-HSCT after abTCD may represent a valid alternative. Due to delayed recovery of adaptive T-cell immunity with abTCD-haplo-HSCT alone, we conducted a phase I/II trial testing the safety and efficacy of post-transplant infusion of a titrated number of donor-derived T cells transduced with the iC9 gene (rivogenlecleucel; ClinicalTrials.gov id: NCT02065869) in children with malignant and non-malignant diseases. Here, we report on the cohort of 70 patients with AL treated in Italy with abTCD-haplo-HSCT+rivogenlecleucel, comparing the results with those of 88 patients given abTCD-haplo-HSCT alone and previously published by our group (Blood 2018; 132:2594-2607). Patients and methods: Patients (age < 18 years) were transplanted between 2010 and 2018. Patient and disease characteristics are shown in Table 1. Median age at HSCT was 6 years (range 0.3-18), and median follow-up was 71 and 31 months for abTCD-haplo-HSCT and abTCD-haplo-HSCT+rivogenlecleucel, respectively. Compared to the control group, abTCD-haplo-HSCT+rivogenlecleucel recipients were transplanted more recently and from a younger donor, and received a higher number of CD34+ cells (Table 1). Diagnosis did not differ between the 2 groups, acute lymphoblastic leukemia (ALL) being the most frequent diagnosis. All patients were transplanted in morphological complete remission (CR1 and CR2) and received myeloablative preparation. Graft composition is reported in Table 1; notably all patients received >10x106 CD34+cells/Kg and <1x105 alpha-beta+ T-cells/Kg. Patients did not receive any pharmacological post-transplant graft-versus-host disease (GvHD) prophylaxis. Results: Graft failure occurred in 2% of patients in each group. Median time to neutrophil and platelet engraftment was 14 (6-23) and 11 (5-56) days, with no differences between groups (p=0.28). Rivogenlecleucel were infused at a median time of 21 days (range 11-59). Treatment was well tolerated; no infusion-related side effects were recorded. Cumulative incidence (CI) of 100-day grade II-IV acute GvHD was 18.9% vs 15.9% (p=0.77) and CI of 1-year chronic GvHD was 6.9% vs 5.7% (p=0.56) in abTCD-haplo-HSCT and abTCD-haplo-HSCT+rivogenlecleucel, respectively. The 4-year non-relapse-mortality (NRM) was significantly lower in abTCD-haplo-HSCT+rivogenlecleucel (1.4% vs 8%, p=0.05) (Figure 1). There was no statistically significant difference in the 4-year CI of relapse (RI) (17% vs. 25%, p=0.30), respectively. Disease recurrence was the most common causes of death in both groups, viral and fungal infections being the most frequent non-relapse fatalities. The 4-year overall survival (OS) and leukemia-free survival (LFS) was 70% vs 87%, p=0.01 (Figure 2) and 67% vs 82%, p=0.05, for abTCD-haplo-HSCT and abTCD-haplo-HSCT+rivogenlecleucel, respectively. There was no difference in 4-year CI of CMV reactivation between the 2 groups (p=0.68), median time to CMV reactivation being 29 and 30 days (p=0.29), respectively. Once infused, rivogenlecleucel expanded (mainly in the CD8+ subset), reaching a peak at 9 months after infusion. At 6-months, median CD3+, CD3/CD4, CD3/CD8, CD3-/CD56 and CD20/CD19 count/microL were 820, 265, 225, 141, 171, for abTCD-haplo-HSCT and 898, 294, 288, 214, and 161 for abTCD-haplo-HSCT+rivogenlecleucel, (p=ns, p=ns, p=0.02, p=0.03, p=ns), respectively. The advantage in the recovery of CD3/CD8 and CD3-/CD56 after abTCD-haplo-HSCT+rivogenlecleucel persisted at 1 year (p=0.01, p=0.03, respectively). In multivariable analysis, abTCD-haplo-HSCT+rivogenlecleucel was associated with better OS (HR 0.27, p=0.003) and LFS (HR 0.40, p=0.001); there was also a trend for lower relapse risk (HR 0.50, p=0.098). Age below the median value at HSCT (HR 2.62, p=0.01), CR1 at HSCT (HR 0.35, p=0.03) and use of irradiation in the conditioning regimen (HR 0.32, p=0.02) were other factors correlating with OS and LFS. Conclusions: These data confirm that the infusion of donor-derived rivogenlecleucel is safe and well tolerated. Rivogenlecleucel cells infusion contributed to enhance recovery of cytotoxic T and NK cells, improving patients NRM and OS/LFS. Rivogenlecleucel (with the possibility of inducing apoptosis of donor T cells) may facilitate the cellular therapy approaches aimed at optimizing immune recovery after HSCT. Disclosures Merli: Amgen: Honoraria; Bellicum: Consultancy; Novartis: Honoraria; Sobi: Consultancy. Algeri:Bluebird bio: Consultancy, Honoraria; Miltenyi: Honoraria; Atara Biotherapeutics: Consultancy, Honoraria. Woodard:Bellicum Pharmaceuticals, Inc: Employment, Other: Stock, Stock options. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy.

Clinical Outcomes after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Transfusion-Dependent β-Thalassemia Treated at the Bambino Gesù Children's Hospital, Rome, Italy

β-thalassemia is one of the most common monogenic blood disorders worldwide, and is highly prevalent in Mediterranean countries. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been the only curative treatment for transfusion-dependent β-thalassemia (TDT; the most severe disease form) for many years, but it is limited by donor availability and has a significant risk of morbidity and mortality. We conducted a chart review of patients with β-thalassemia who underwent allo-HSCT (N=80) at the Bambino Gesù Children's Hospital, Rome, Italy, between March 2011 and August 2018. Median (range) age at allo-HSCT was 5.5 (0.3-20.0) years [<12 years: n=70 (87.5%), ≥12-18 years: n=7 (8.8%), ≥18 years: n=3 (3.8%)] and all patients but one had TDT [n=79 (98.8%)]. Prior to allo-HSCT, patients received a median (range) of 17.4 (4-52) transfusions per year (n=64) and had a median (range) serum ferritin concentration of 1217 (135-9123) ng/mL and a median (interquartile range) hemoglobin level of 10.3 (9.3-11.4) g/dL. All patients had received regular iron chelation therapy prior to transplantation. In total, 18 (22.5%), 28 (35.0%), and 34 (42.5%) patients received allo-HSCT from human leukocyte antigen (HLA)-identical sibling donors, HLA-haploidentical donors, and unrelated donors (fully matched donor: n=28, donor with a single HLA disparity: n=6), respectively. Of these donors, 42 (52.5%) were carriers for thalassemia-associated mutations. In total, 53 (66.3%) donors and 35 (43.8%) recipients were cytomegalovirus-positive. Bone marrow was the stem cell source in 51 cases (63.8%), while 28 patients received an alphabeta T-cell depleted peripheral blood haploidentical HSCT (35.0%); the remaining child (1.3%) received both bone marrow and cord blood from the same related donor. All patients continued to receive transfusions immediately after allo-HSCT; however, only 7 (8.8%) received a transfusion in the 3 to 12-month post-transplantation period (2 due to underlying disease; 5 due to other reasons including GI bleeding). Median (range) time to reach transfusion-free status was 3.8 (1.1-47.8) weeks. Median (interquartile range) hemoglobin levels at 6 and 12 months after allo-HSCT were 10.9 (10.2-11.9) and 11.9 (10.6-13.0) g/dL, respectively. The cumulative incidences of primary and secondary graft failure were 10.0% and 12.5% at 24 months (HLA-identical donor: 0% and 11.1%, haploidentical donor: 17.9% and 3.6%, unrelated donor: 8.8% and 20.6%). Eleven out of 14 patients experiencing graft failure were successfully rescued with a second allograft, while 2 patients were not retransplanted due to parental decision and 1 patient died after the engraftment of the second allograft. Eight patients developed grade II-IV acute graft-versus-host disease (GVHD) and one patient developed moderate chronic GVHD. Cumulative incidence rates of grades II-IV and III-IV acute GVHD were 12.7% and 8.0% at 24 months (HLA-identical donor: 0% and 0%, haploidentical donor: 7.3% and 0%, unrelated donor: 23.8% and 18.8%). Three patients (3.8%) died of transplant-related causes (1 case each of hemophagocytic lymphohistiocytosis, sepsis, and multi-organ failure [the patient receiving the second allograft]) with a median (range) time from transplantation to death of 8.7 (3.7-11.0) months. Of these patients, all had been transplanted from an unrelated donor and 2 had reached sustained full-donor chimerism. The probability of overall and event-free (event defined as either death or primary/secondary graft failure) survival was 96.2% and 81.2% at 24 months (HLA-identical sibling donor: 100% and 88.9%, haploidentical donor: 100% and 78.6%, unrelated donor: 91.2% and 79.4%). The probability of thalassemia-free survival (event defined as either death or primary/secondary graft failure not rescued by a second allograft) was 93.7% at 24 months (HLA-identical sibling donor: 100%, haploidentical donor: 92.9%, unrelated donor: 91.2%). In this large single-center cohort of children with predominantly TDT, allo-HSCT led to beneficial outcomes for most patients, resulting in the discontinuation of transfusions with 93.7% of patients being thalassemia-free. Nevertheless, HSCT is still associated with GVHD, graft failure, and mortality, and only 22.5% of patients had an HLA-identical sibling donor, illustrating a key limitation of allo-HSCT. Emerging research is addressing such barriers to treatment. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Algeri:Miltenyi: Honoraria; Atara Biotherapeutics: Consultancy, Honoraria; Bluebird bio: Consultancy, Honoraria. Gruppioni:Bluebird bio: Employment, Equity Ownership. Kommera:Bluebird bio: Employment, Equity Ownership. Maa:Bluebird bio: Employment, Equity Ownership. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Successful Salvage Haploidentical Alpha-Beta T Cell–Depleted Stem Cell Transplantation After Busulfan-Based Myeloablation in a Patient With IPEX Syndrome: A Case Report

BackgroundX-linked immunodysregulation syndrome with polyendocrinopathy and enteropathy (IPEX) is caused by FOXP3 gene mutations that block the generation of regulatory T lymphocytes. We report an 18-month-old boy with classic IPEX who underwent 2 hematopoietic stem cell transplantations (HSCTs). MethodsThe first HSCT from an unrelated 8/10 HLA-matched umbilical cord blood donor (UCB) was performed after a conditioning regimen consisting of treosulfan, fludarabine, thiotepa, and thymoglobulin. Due to complete rejection of the UCB transplant, a second transplantation from a 6/10 HLA-matched mother was performed after alpha-beta T-cell depletion. The second conditioning regimen consisted of busulfan, fludarabine, a single dose of cyclophosphamide 1 g/m2, and Grafalon (Neovii Pharmaceuticals, Rapperswil, Switzerland). The T-cell depletion product contained 15.06 x 106 CD34+ cells per kilogram body weight (BW) and 4.19 x 105 alpha-beta T lymphocytes per kilogram BW. Due to acute graft rejection, the boy was treated with thymoglobulin, and full donor chimerism in both T lymphocytes and mononuclear cells was achieved. The immunosuppressive therapy was stopped 1 year after transplantation. To date, the patient remains free from graft-vs-host disease (GVHD) and immunosuppression. ConclusionsHSCT after busulfan-based reduced-toxicity conditioning in patients with IPEX syndrome is feasible and well tolerated and can result in full donor engraftment. Monitoring of chimerism and aggressive therapy in cases of graft rejection are warranted due to the high reactivity of residual autologous T lymphocytes. T-cell depletion reduces the risk of GVHD and the need for steroid therapy, which is especially challenging in patients with diabetes.

110-LB: The Comparison of Glycemic Remission and Nonremission Patients after Sitagliptin Combination with Short-Term Intensive Insulin Therapy

Short-term intensive insulin therapy (IIT) could induce long-term glycemic remission. Sitagliptin increased endogenous GLP-1 content to achieved glucose control. The combination of sitagliptin with IIT might enhance the effect of improving alpha cell and beta cell function. 72 newly diagnosed type 2 diabetes patients with hypoglycemic therapy naïve were enrolled, with 47.6±9.0 years in age, with BMI of 25.2±2.7 kg/m2, FPG of 11.1±3.4mmol/L, and HbA1c of 10.6±2.3%. Continuous subcutaneous insulin infusion (CSII) were used as IIT, which were maintained for 14 days after patients achieving euglycemia. The treatment course of sitagliptin was 90 days. After cessation of sitagliptin, 45.8% of patients (33/72) achieved 1-year glycemic remission without any hypoglycemic medications. Compared the baseline of remission and non-remission groups, the baseline fasting plasma glucose (FPG) of remission group was lower (10.0±3.0 vs. 12.0±3.5mmol/L, p=0.012), but the levels of HbA1c, acute insulin response (AIR), HOMA-B and HOMA IR were not different between groups. After CSII cessation, FPG of remission group was still lower than non-remission group (5.8±0.8 vs. 6.9±1.1mmol/L, p<0.001), beta cell function and insulin resistance were improved significantly(HOMA-B: 62.96(25.94) vs. 40.67(41.79), p=0.026; HOMA-IR: 1.56(0.54) vs. 1.74(0.99), p=0.003). Analyzed insulin dosage of the 14th day after achieving glycemic target, basal insulin dosage was lower in remission group (9.6±5.6IU/d, vs. 14.4±7.1IU/d, p=0.002). In conclusion, lower levels of FPG at baseline and after IIT, less basal insulin usage during IIT, and better improvement of beta-cell function and insulin resistance suggested the possibility of glycemic remission after combination of IIT and sitagliptin therapy. Disclosure W. Ke: None. L. Liu: None. X. He: None. Y. Li: None.

Design, synthesis and evaluation of DNA nano-cubes as a core material protected by the alginate coating for oral administration of anti-diabetic drug

Poor control towards glycemic levels among diabetic patients may lead to severe micro/macro-vascular and neuropathic complexities. Proper functioning of alpha-beta cells of pancreases is required to attain long term glycemic control among type 2 diabetics. The recent developments to manage diabetes are focused on controlling the insulin-glucagon secretions from the pancreases. DPP-4 inhibitors class of drugs after elevating GLP-1/GIP (incretins) levels in the blood, not only raise the insulin levels but also suppress the glucagon level. Vildagliptin (VI) is a potent DPP-4 inhibitor with least adverse events compared to other DPP-4 inhibitors. We encapsulated VI into 3D nanocube that gets bind to the DNA due to secondary amine in its chemical structure. DNA-nanocube being negatively charged was incubated with the PLL to attain positive surface. Ultimately VI loaded nanocubes were coated with the negatively charged Na-alginate via electrostatic attraction method to get stable spherical nanospheres for oral delivery of VI. Nanospheres were evaluated physically through native PAGE analysis, DSC, TGA, dissolution testing, XRD and FTIR. We attained uniformed and spherical nanospheres with stable topology, nanoscale size precision (40–150 nm in diameter), Entrapment efficiency (up to 90%), prolonged drug release (13 ± 4 h) at basic pH, and superior oral antidiabetic effects with improved GLP1 and glycemic levels. The formulated nanospheres attained size uniformity and better therapeutic outcomes in terms of reduced adverse events and better control of glycemic levels than previously reported methods with decreased dosage frequency tested in Db/Db mice.

Conformational Dynamics of T-Cell Receptor Chassis for Mechanosensing

AlphaBeta T-cell receptors (TCRαβ) are the major components of the adaptive immune system, sensing foreign antigens displayed on the surface of mammalian host cells altered by infection or other transformations with a remarkably high signal to noise ratio. Despite their high sensitivity, the affinity of each to the antigen peptide-bound major histocompatibility complex (pMHC) is low in thermal equilibrium, typically in the micromolar range. An emerging concept that reconciles the conundrum of high sensitivity versus low affinity is that the TCRαβ functions as a mechanosensor. Mechanical load on the TCRαβ-pMHC complex enhances the bond lifetime via activation of a catch bond pathway, a mechanism which is largely uncharacterized in structural terms. Past studies focused on behaviors of contacts directly involving TCRαβ and pMHC surfaces. However, such localized interactions alone cannot account for the catch bond since there is an allostery that modulates the catch bond behavior through TCR subdomains distal to the TCRαβ-pMHC interface. Thus, understanding of the conformational behavior of the entire TCR protein is required. Here we perform atomistic molecular dynamics simulations of TCRαβ as well as TCR gamma-delta (TCRγδ), receptors of cognate and innate T-cell immunity subsets, respectively. We also study a TCRγδ-αβ chimera that has the antigen-binding variable domains of TCRγδ with the membrane-proximal TCRαβ constant domains. By building ball-and-stick models of TCRs based on simulation trajectories, we examine relative motions of the four subdomains of each TCR type. We find that interdomain motion affects the organization of the pMHC-binding complementarity determining loops. These results suggest a TCR catch bond mechanism whereby an applied load alters the conformational dynamics of the subdomains and in turn, controls the TCR-pMHC interaction including resultant bond lifetime.

T- and B-Cell Neogenesis Recovers Efficiently in Children with Acute Leukemia Given an Alpha-Beta T-Cell Depleted Haplo-HSCT Followed By Infusion of Donor T-Cells Genetically Modified with Inducible Caspase 9 Suicide Gene (BPX-501 cells)

Backgroundαβ T- and B-cell depleted HLA-haploidentical HSCT (αβ haplo-HSCT) is an alternative promptly available and virtually applicable to any children with malignant and non-malignant disorders lacking an HLA-matched donor or in need of an urgent allograft (Locatelli et al. 2017). Through this approach, recipients can benefit immediately after transplantation from the anti-leukemia effect mediated by donor natural killer (NK) and γδ T cells, which confers protection also against infections. Nonetheless, even with this type of HSCT, the recovery of adaptive immunity is suboptimal. We have developed a further refinement of the approach based on the post-transplant infusion of a titrated number of donor T cells transduced with the inducible suicide gene iC9 (BPX-501 cells). This strategy has the potential to accelerate the recovery of adaptive immunity and to restore an efficient T-cell mediated graft-versus-leukemia (GvL) effect without the risk of inducing uncontrollable graft-versus-host disease (GvHD). Since reconstitution of a naïve T- and B-cell repertoire plays a key role in the long-term ability to respond to a broad range of pathogens, as well as to tumor antigens, we quantified T-cell receptor and kappa chain-deleting recombination excision circles (TREC and KREC), which represent reliable surrogate of T and B-cell neogenesis respectively.Study design and patientsWe analyzed samples of 48 children (M/F: 26/22) with acute leukemia (31 ALL, 17 AML) given an ab T-cell and B-cell depleted haplo-HSCT after a myeloablative regimen followed by the infusion of a titrated number of donor BPX-501 cells (1 million/Kg) after a median of 27 days (range: 11-87). Median age at transplantation was 8.3 years (range, 0.9-18). Thirty-two and 16 patients did or did not receive a preparative regimen containing total body irradiation (TBI). Anti-T lymphocyte globulin (ATLG Grafalon®, Neovii Biotech, 12 mg/Kg) was administered from day -4 to -2 for preventing graft rejection and GvHD. Moreover, to reduce the risk of EBV-related PTLD, on day -1, patients received rituximab (200 mg/m2). We analyzed T-cell reconstitution by measuring the TREC, small DNA excision circles of the TCR d locus deleted during recombination of the a-locus and present in the majority of functional ab-T-cells (sjTREC), and during the TCR b-chain recombination (bTREC). We also analyzed B-cell reconstitution by measuring coding-joint (Cj), signal-joint KREC (sjKREC) which reflect newly produced naïve B cells and the mean number of B-cell division (n) using the formula n=LOG(Cj/sjKREC)/LOG2. We performed real-time quantitative PCR, as recently described (Arruda et al. 2018), on genomic DNA extracted from PBMC collected at 6 different time points (before and 1, 3, 6, 12 and 18 months after the allograft).ResultsRecovery of thymic function started at 3 months after transplant: sjTREC and bTREC amounts reached and overcame pre-transplant values at 6 months, continuing to increase until 18 months after HSCT. Number of B-cell divisions obtained by the measurement of cj and sjKREC found in peripheral blood matched with the newly-generated B-cells bone marrow output and showed the same kinetics of sj and bTREC, rising from the 3rd month after HSCT. All these data correlate with immunophenotyping findings, which show a progressive increase over time of both total CD3+ and ab-T-cells. Furthermore, we observed a negative impact on both T- and B-cell neogenesis played by aGvHD (occurring in 14 patients), which became significant from 3 months after HSCT for sjTREC (p=0.013) and at 6 months for sjKREC (p= 0.03). We did not observe any impact of the use of TBI during the preparative regimen on T- and B-cells generation in this cohort.ConclusionsBPX-501 cell infusion aims to contribute to the acceleration of immune reconstitution after a/b haplo-HSCT. In this study, we show that naïve T- and B- cells are quickly and efficiently generated in the early post-transplant period. This finding represents an interesting result considering the fully myeloablative conditioning regimens with the large use of TBI and the Ta/b and B-cells depletion (also in vivo by the administration of Rituximab) received by the patients. We observed an expected detrimental effect of aGvHD on immune reconstitution, but we emphasize the low frequency of this complication in this transplant setting despite the absence of any post-transplant pharmacological prophylaxis. DisclosuresFoster:Bellicum: Employment, Equity Ownership. Locatelli:bluebird bio: Consultancy; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Chimeric Antigen Receptor T-Cells for the Treatment of Gamma-Delta T-Cell Malignancies

▪IntroductionCancers derived from the malignant transformation of gamma delta T-cells are rare but carry very poor prognosis. Hepatosplenic T-cell lymphoma is a highly aggressive condition characterised by hepatosplenic and bone marrow involvement. It has among the worst outcomes of all lymphoma subtypes, with a median survival of only 6-8 months. 95% of cases express the gamma delta T-cell receptor (GDTCR), which is also expressed on a proportion of cases of T-ALL. Treatment for these cancers is based on cytotoxic chemotherapy, with no tumour-specific therapies including immunotherapy available. We have developed a novel chimeric antigen receptor targeting GDTCR and here demonstrate specific in vitro and in vivo efficacy against gamma delta T-cell malignancies.ResultsWe cloned anti-GDTCR antibody as a single chain variable fragment (ScFv), and confirmed specific binding to GDTCR-positive T-cell cell lines and primary GD cells. Next, we cloned anti-GDTCR ScFv into a 2ndgeneration chimeric antigen receptor (CAR) format, including a spacer derived from CD8-stalk, CD28 transmembrane domain and 41BB-zeta endodomain. This construct was stably introduced to primary alpha-beta T-cells by retroviral transduction and surface expression was confirmed by flow cytometry.We established 48-hour co-cultures of anti-GDTCR CAR T-cells or control anti-CD19 CAR T-cells with T-cell lines positive (Loucy, BE13, MOLT-13) or negative for surface GDTCR (Jurkat, SupT1-CD19). While control anti-CD19 CAR killed only SupT1-CD19 cells, specific cytotoxicity was seen by anti-GDTCR CAR T-cells only against GDTCR-positive cell lines. In addition, anti-GDTCR CAR T-cells demonstrated specific secretion of cytokines including interferon gamma and IL-2, and robust antigen-specific proliferation only in co-culture with GDTCR-positive cells. Expression of exhaustion, activation and differentiation markers in long term co-cultures with target cells was similar to that seen with control anti-CD19 CAR.To assess the in vivo potency of anti-GDTCR CAR T-cells, we established a murine model of disseminated GDTCR-positive leukaemia. NSG mice were intravenously injected with 4x10^6 Loucy cells, engineered to stably express Firefly luciferase. Tumour engraftment in bone marrow was confirmed at D7 following injection, and mice were treated with 0.8x10^6 anti-GDTCR or control anti-CD19 CAR T-cells. Disease burden was monitored by bioluminescence imaging. Mice receiving anti-GDTCR CAR demonstrated substantial reduction of tumour burden, increased expansion of CAR T-cells and prolonged survival compared to control-CAR treated animals.ConclusionsWe have developed a novel chimeric antigen receptor T-cell treatment for gamma-delta TCR-positive malignancies, including hepatosplenic T-cell lymphoma and some cases of T-ALL. Our approach is, to our knowledge, the first immunotherapeutic strategy proposed for these conditions. Given the restricted expression of GD-TCR on a small subset (0.5-5%) of peripheral T-cells and the absence of a clear human phenotype associated with GD T-cell deficiency, we suggest that this therapy may be well tolerated. Given the very poor prognosis and lack of effective therapies for GD-TCR-positive malignancies, as well as the considerable efficacy of CAR T-cell therapy in analogous B-cell disorders, our approach could bring much needed benefit to patients suffering these conditions. DisclosuresMaciocia:Autolus: Equity Ownership, Patents & Royalties: UCLB. Pule:UCLB: Patents & Royalties; Autolus: Employment, Equity Ownership.

Subchronic exposure to acrylamide leads to pancreatic islet remodeling determined by alpha cell expansion and beta cell mass reduction in adult rats

Acrylamide (AA) is a toxic substance, used to synthesize polymers for industrial and laboratory processes. Also, AA is a food contaminant formed during the high temperature preparation of carbohydrate-rich food. The main subject of this study was to examine effects of subchronic AA treatment on the islets of Langerhans of adult rats. Adult male Wistar rats were orally treated with 25 or 50 mg/kg bw of AA for 3 weeks. Qualitative and quantitative immunohistochemical evaluation of glucagon and insulin expression and stereological analyses of pancreatic alpha and beta cells were performed. Serum insulin and glucose levels were measured. Analysis of glucagon-immunostained sections revealed a dose-dependent increase of intensity of glucagon immunopositive signal, alpha cell surface and numerical densities, volume density of alpha cell nuclei and nucleocytoplasmic ratio in AA-treated groups compared to the control. In insulin-immunolabeled pancreatic sections in AA-treated animals was observed decrease of intensity of insulin immunopositive signal, beta cell surface, numerical and volume densities and volume density of beta cell cytoplasm. Serum insulin and glucose concentrations remained unchanged after both AA treatments. The number of islets of Langerhans was not affected by AA treatment. Our results suggest that AA subchronic treatment of adult rats leads to remodeling of islet of Langerhans characterized by alpha cell expansion and beta cell mass reduction.

Two-Photon Dye Cocktail for Dual-Color 3D Imaging of Pancreatic Beta and Alpha Cells in Live Islets.

Insulin-secreting beta cells together with glucagon-producing alpha cells play an essential role in maintaining the optimal blood glucose level in the body, so the development of selective probes for imaging of these cell types in live islets is highly desired. Herein we report the development of a 2-glucosamine-based two-photon fluorescent probe, TP-β, that is suitable for imaging of beta cells in live pancreatic islets from mice. Flow cytometry studies confirmed that TP-β is suitable for isolation of primary beta cells. Moreover, two-photon imaging of TP-β-stained pancreatic islets showed brightly stained beta cells in live islets. Insulin enzyme-linked immunosorbent assays revealed that TP-β has no effect on glucose-stimulated insulin secretion from the stained islet. Finally, to develop a more convenient islet imaging application, we combined our recently published alpha-cell-selective probe TP-α with TP-β to make a "TP islet cocktail". This unique dye cocktail enabled single excitation (820 nm) and simultaneous dual-color imaging of alpha cells (green) and beta cells (red) in live pancreatic islets. This robust TP islet cocktail may serve as a valuable tool for basic diabetic studies.

T Cell Repertoire after Alpha/Beta-T Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Patients

Allogeneic transplantation of hematopoietic cells (HSCT) is an established method to treat different hematologic malignancies and disorders of hematopoietic and lymphoid system. Graft-versus-host-disease is one of the main risk factor for success of the procedure. Simultaneous depletion of alpha-beta T-cells and CD19+ cells in graft is the promising way to reduce the risk. The approach was recently introduced in clinical practice and many aspects of immune system reconstitution are still unknown. We applied improved technology for T cell receptor (TCR) repertoire sequencing to study origin and dynamics of T cell clones during 1 year follow-up period after allogeneic TCRαβ/CD19-depleted HSCT in children.We performed TCR repertoire sequencing for peripheral blood samples of patients before HSCT, at 2, 6 and 12 months after HSCT (n=21, 21, 17, 16 respectively), and for respective donor blood apheresis samples before abT/CD19 depletion. Twelve of the patients were diagnosed with acute leukemia and the others with non-malignant inherited and acquired blood disorders. For each patient data on recipient’s T cell chimerism and counts of CD3+, naïve CD3+, alpha-beta T-cells and recent thymic emigrants (RTE) have been collected during 1 year follow-up period.Barcoding of each original TCR mRNA molecule passed to massive parallel sequencing allowed us to: (1) reduce sample preparation biases and quantitatively reconstruct of TCR repertoires; (2) equalize repertoire data analysis depth which is absolutely necessary for correct comparison of samples; (3) prevent risk of cross-contamination between samples and increase confidence of T clone origin determination.Two months after TCRαβ/CD19-depleted HSCT T cell repertoire mostly consists of several hundreds highly abundant clones. For patients with low recipient T cell chimerism from 13 to 504 largest T-cell clones (median 255, IQR 219, n=9, T cell chimerism <=20%) represented 80% of all T cells in peripheral blood. For comparison in healthy age-matched donors we found from 32,000 to 47,000 largest T-cell clones in identical analysis (median 43191, IQR 6493, n=14, data from Britanova O.V. et.al., JI 2014). The overall diversity at d60 after HSCT was also much less compared with the healthy subjects. We also found that most expanded T cell clones at d60 do not represent just a replica of the most expanded clones in graft samples, originating from low-abundant graft T cell clones.The diversity of T repertoire early after HSCT positively correlated with recipient T cell chimerism (the diversity was higher for those patients with higher percentage of recipient’s T cells). Also patients with low chimerism had higher number of T clones originating from the graft than from d0 pre-transplant recipient repertoire in contrast to the patients with high T cell chimerism who had inverse ratio (median number of patient’s clonotypes shared between graft and d0 was 56 or 3 for patients with low or absent chimerism (IQR = 24 or 19.25, n = 5) and 21.5 or 321.5 for patients with T cell chimerism >2% (IQR = 46.5 or 724.75, n = 10)). In addition CD4+ RTE count was higher for patients with high T cell chimerism. This observation was additionally confirmed by analysis of flow cytometry data for the expanded cohort of 105 patients at d60 after αβT-cell depleted HSCT (Wilcoxon rank sum test p-value = 0.002).Our results demonstrate that early after αβT-cell depleted HSCT repertoire of T cells are extremely skewed and unlikely able protect recipient efficiently. Observed recovery of T cell count mostly results from expansion of a few clones that have to divide intensely for the whole 60 days period in order to achieve the observed counts. Early reconstitution of TCR diversity and RTE counts in patients with substantial recipient T cell chimerism is mostly explained by surviving recipient T cells and intrathymic T cell progenitors, respectively.This work was supported by the Russian Science Foundation project №14-35-00105. Zvyagin I. is supported by grant MK-4583.2015.4. DisclosuresNo relevant conflicts of interest to declare.

Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR

Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha-beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients' tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.

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Glucagon is essential for alpha cell transdifferentiation and beta cell neogenesis.

The interconversion of cell lineages via transdifferentiation is an adaptive mode of tissue regeneration and an appealing therapeutic target. However, its clinical exploitation is contingent upon the discovery of contextual regulators of cell fate acquisition and maintenance. In murine models of diabetes, glucagon-secreting alpha cells transdifferentiate into insulin-secreting beta cells following targeted beta cell depletion, regenerating the form and function of the pancreatic islet. However, the molecular triggers of this mode of regeneration are unknown. Here, using lineage-tracing assays in a transgenic zebrafish model of beta cell ablation, we demonstrate conserved plasticity of alpha cells during islet regeneration. In addition, we show that glucagon expression is upregulated after injury. Through gene knockdown and rescue approaches, we also find that peptides derived from the glucagon gene are necessary for alpha-to-beta cell fate switching. Importantly, whereas beta cell neogenesis was stimulated by glucose, alpha-to-beta cell conversion was not, suggesting that transdifferentiation is not mediated by glucagon/GLP-1 control of hepatic glucose production. Overall, this study supports the hypothesis that alpha cells are an endogenous reservoir of potential new beta cells. It further reveals that glucagon plays an important role in maintaining endocrine cell homeostasis through feedback mechanisms that govern cell fate stability.