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Background: Among people with ASCVD, those with T2D have excess CVD risk and derive greater absolute benefit from CVD risk reduction therapies. We describe the use of evidence-based therapies for secondary prevention in participants (pts) with T2D. Methods: We report a registry to longitudinally describe LDL-C treatment patterns among U.S. pts with ASCVD and LDL-C ≥70mg/dL taking lipid-lowering therapy. Pts (n=5006) were enrolled in 1 of 3 cohorts: taking PCSK9i (11%); LDL-C 70-99mg/dL (53%); and LDL-C ≥100mg/dL (36%). Results: Overall, 1655 (33%) had diabetes (96% with T2D): 146 in the PCSK9i cohort, 874 in the LDL-C 70-99mg/dL cohort, and 635 in the LDL-C ≥100mg/dL cohort. Mean age among pts with diabetes was 68 y, 60% were male, 81% had CAD (including 33% with prior MI), 17% with PAD, and 17% had HF. Mean LDL-C was 100mg/dL. Although overall statin use was at 87%, high-intensity statins were used in only 45% and ezetimibe in only 9.5% of pts. Aspirin and ACE-I/ARB were each used in 72% and beta blockers in 71% of pts. The most commonly used glucose-lowering medications among patients with T2D were metformin (56%), insulin (34%), and sulfonylurea (22%). Only 10% and 8% of pts with T2D were treated with SGLT2i or GLP-1RA, respectively. The proportion of pts with T2D that were receiving all guideline-recommended therapies for optimal risk reduction (high-intensity statins, aspirin, ACE-I/ARB, and either SGLT2i or GLP-1RA) was just 3%. Conclusion: In GOULD, among pts with T2D, ASCVD, and LDL-C ≥70mg/dL, most are not receiving high intensity statin therapy at baseline and few receive ezetimibe. Furthermore, less than 20% are treated with glucose-lowering therapies that have a proven CVD benefit, and a strikingly low proportion receive all guideline-recommended risk reduction strategies. These results highlight an opportunity to improve care and outcomes. Disclosure M.N. Kosiborod: Consultant; Self; Amgen Inc., AstraZeneca, Bayer AG, Boehringer Ingelheim International GmbH, Eisai Co., Ltd., GlaxoSmithKline plc., Glytec, LLC, Intarcia Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc., Novartis AG, Novo Nordisk A/S, Sanofi. Research Support; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc. J. De Lemos: Other Relationship; Self; Amgen Inc., Novo Nordisk A/S, Regeneron Pharmaceuticals. R.S. Rosenson: Consultant; Self; CVS/Caremark, Regeneron Pharmaceuticals. Research Support; Self; Akcea Therapeutics, Amgen Inc., Medicines Company, Regeneron Pharmaceuticals. Other Relationship; Self; Amgen Inc., Kowa Pharmaceutical Europe Co. Ltd. C.M. Ballantyne: Consultant; Self; Abbott, Akcea Therapeutics, Amarin Corporation, Amgen Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Esperion, Gilead Sciences, Inc., Matinas BioPharma, Merck & Co., Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Regeneron Pharmaceuticals, Roche Diagnostic USA, Sanofi. Research Support; Self; Abbott, Akcea Therapeutics, Amarin Corporation, Amgen Inc., Esperion, Novartis Pharmaceuticals Corporation, Regeneron Pharmaceuticals, Roche Diagnostic USA, Sanofi. Other Relationship; Self; Roche Diagnostic USA. K. Philip: Employee; Self; Amgen Inc. Stock/Shareholder; Self; Amgen Inc. Y. Liu: None. K.E. Mues: Employee; Self; Amgen Inc. Stock/Shareholder; Self; Amgen Inc. S. Alam: Employee; Self; Amgen Inc. Stock/Shareholder; Self; Amgen Inc. M. Elliott: Employee; Self; Amgen Inc. Stock/Shareholder; Self; Amgen Inc. D.L. Bhatt: Research Support; Self; Abbott, Amarin Corporation, Amgen Inc., AstraZeneca, Bayer Vital GmbH, Boehringer Ingelheim International GmbH, Bristol-Myers Squibb Company, Chiesi USA, Inc., Eisai Inc., Eli Lilly and Company, Ethicon, Inc., FlowCo, Forest Laboratories, Inc., Idorsia, Ironwood Pharmaceuticals, Inc., Ischemix, Medicines Company, Medtronic, Merck & Co., Inc., Novo Nordisk Inc., Pfizer Inc., PhaseBio Pharmaceuticals, Inc., PLx Pharma, Regeneron Pharmaceuticals, Roche Pharma, Sanofi, Synaptic, Takeda Pharmaceutical Company Limited. C.P. Cannon: Consultant; Self; Alnylam, Amarin Corporation, Amgen Inc., Corvia Medical, Inc., Eisai Inc., Kowa Pharmaceutical Europe Co. Ltd., Pfizer Inc., Regeneron Pharmaceuticals, Sanofi. Research Support; Self; Daiichi Sankyo Company, Limited. Other Relationship; Self; Amgen Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Bristol-Myers Squibb Company, Janssen Pharmaceuticals, Inc., Merck & Co., Inc. Funding Amgen Inc.

CD8(+)/TCR(-) facilitating cells (FCs) in mouse bone marrow (BM) significantly enhance engraftment of hematopoietic stem/progenitor cells (HSPCs). Human FC phenotype and mechanism of action remain to be defined. We report, for the first time, the phenotypic characterization of human FCs and correlation of phenotype with function. Approximately half of human FCs are CD8(+)/TCR(-)/CD56 negative (CD56(neg)); the remainder are CD8(+)/TCR(-)/CD56 bright (CD56(bright)). The CD56(neg) FC subpopulation significantly promotes homing of HSPCs to BM in nonobese diabetic/severe combined immunodeficiency/IL-2 receptor γ-chain knockout mouse recipients and enhances hematopoietic colony formation in vitro. The CD56(neg) FC subpopulation promotes rapid reconstitution of donor HSPCs without graft-versus-host disease (GVHD); recipients of CD56(bright) FCs plus HSPCs exhibit low donor chimerism early after transplantation, but the level of chimerism significantly increases with time. Recipients of HSPCs plus CD56(neg) or CD56(bright) FCs showed durable donor chimerism at significantly higher levels in BM. The majority of both FC subpopulations express CXCR4. Coculture of CD56(bright) FCs with HSPCs upregulates cathelicidin and β-defensin 2, factors that prime responsiveness of HSPCs to stromal cell-derived factor 1. Both FC subpopulations significantly upregulated mRNA expression of the HSPC growth factors and Flt3 ligand. These results indicate that human FCs exert a direct effect on HSPCs to enhance engraftment. Human FCs offer a potential regulatory cell-based therapy for enhancement of engraftment and prevention of GVHD.

Aim: To examine the efficacy of 12 weeks of monthly evolocumab (EvoMab) 420 mg vs. placebo (Pbo) in lowering LDL-C in those with type 2 diabetes mellitus (T2DM) and hypercholesterolemia or mixed dyslipidemia and on statin therapy. Methods: Patients ≥18 years with T2DM, HbA1c Results: Mean participant age (SD) was 62 (8) years; 44% women; 77% Caucasian. In modified, intent to treat analyses (all randomized and dosed patients) EvoMab significantly reduced LDL-C and non-HDL-C vs. Pbo, and did not impact glycemic control. The incidence of AEs was comparable between EvoMab and Pbo, with no clinically meaningful differences in serious AEs. See Table.Table.Placebo(N=141)Evolocumab(N=280)Baseline LDL-C (mg/dL), mean (SD)110.4 (33.0)108.6 (31.0)Baseline non-HDL-C (mg/dL), mean (SD)145.5 (33.9)144.6 (34.9)Any adverse event (AE), n (%)52 (36.9)110 (39.3)Serious AE, n (%)2 (1.4)14 (5.0) 14 (5.0)14 (5.0)aMean of Weeks 10 and 12LipidsPercent change from baseline in LDL-C, mean (SE)–0.8 (1.8)–65.0 (1.3)bPercent change from baseline in non-HDL-C, mean (SE)–0.(1.6)–56.6 (1.2)bAchievement of LDL-C < 70 mg/dL, n, %20 (14.8)253 (92.7)bAchievement of ≥50% reduction in LDL-C, n, %1 (0.7)230 (84.2)bWeek 12Glycemic ControlChange from baseline in FSG in mg/dL, median (Q1, Q3)4.0(–15.0, 29.0)5.0(–13.5, 29.5)Change from baseline in HbA1c in percent, median (Q1, Q3)0.1(–0.2, 0.5)0.1(–0.2, 0.5)aNo pattern in differences in serious AEs was observed. Chronic obstructive pulmonary disease was the only serious AE reported by ≥ 1% of patients in the evolocumab treatment group.bP < 0.0001 for evolocumab versus placebo comparisonFSG, fasting serum glucose; HbA1c, glycated hemoglobin; LDL-C, low-density lipoprotein cholesterol; non-HDL-C, non-high-density lipoprotein cholesterol; SE, standard errorFunding: Amgen Inc. Conclusion: In statin-treated patients with T2DM and hypercholesterolemia or mixed dyslipidemia, evolocumab safely and effectively lowered LDL-C. Disclosure R.S. Rosenson: Other Relationship; Self; Akcea Therapeutics, Amgen Inc., AstraZeneca, Eli Lilly and Company, The Medicines Company, Regeneron Pharmaceuticals, Inc., Sanofi US, Kowa Pharmaceuticals America, Inc., UpToDate. M.L. Daviglus: Advisory Panel; Self; Amgen Inc. P. Reaven: Research Support; Self; AstraZeneca, Amgen Inc., Bristol-Myers Squibb Company. P. Pozzilli: Research Support; Self; Sanofi. Speaker's Bureau; Self; Eli Lilly and Company. Research Support; Self; Merck Sharp & Dohme Corp. H. Bays: Research Support; Self; Amgen Inc. M. Monsalvo: Employee; Self; Amgen Inc.. Stock/Shareholder; Self; Amgen Inc. M. Elliott: Employee; Self; Amgen Inc.. Stock/Shareholder; Self; Amgen Inc. R. Somaratne: Employee; Self; NGM Biopharmaceuticals. Stock/Shareholder; Self; NGM Biopharmaceuticals. Y. Handelsman: Consultant; Self; Amarin Corporation. Speaker's Bureau; Self; Amarin Corporation. Board Member; Self; American Association of Clinical Endocrinologists. Consultant; Self; Amgen Inc.. Research Support; Self; Amgen Inc.. Speaker's Bureau; Self; Amgen Inc.. Consultant; Self; AstraZeneca. Research Support; Self; AstraZeneca. Speaker's Bureau; Self; AstraZeneca. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Bristol-Myers Squibb Company, Gan & Lee Pharmaceuticals. Consultant; Self; Intarcia Therapeutics, Inc., Janssen Pharmaceuticals, Inc.. Speaker's Bureau; Self; Janssen Pharmaceuticals, Inc.. Research Support; Self; Lexicon Pharmaceuticals, Inc.. Consultant; Self; Merck & Co., Inc.. Research Support; Self; Merck & Co., Inc.. Consultant; Self; Novo Nordisk Inc.. Research Support; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Novo Nordisk Inc., Regeneron Pharmaceuticals, Inc.. Consultant; Self; Sanofi. Research Support; Self; Sanofi. Speaker's Bureau; Self; Sanofi.

Pancreatic cancer is often resistant to conventional chemotherapy. In this study, we examined the role of adenovirus-mediated overexpression of E2F-1 in inducing apoptosis and increasing the sensitivity of pancreatic cancer cells to chemotherapeutic agents. MIA PaCa-2 pancreatic head exocrine adenocarcinoma cells (mutant p53) were treated by mock infection or adenoviruses expressing beta-galactosidase or E2F-1 (Ad-E2F-1) alone or in combination with sublethal concentrations of each chemotherapeutic drug. Cell growth and viability were assessed at selected time points. Apoptosis was evaluated by flow cytometry, characteristic changes in cell morphology and poly (ADP-ribose) polymerase (PARP) cleavage. Western blot analysis was used to examine the expression of E2F-1 and Bcl-2 family member proteins and PARP cleavage. Western blot analysis revealed marked overexpression of E2F-1 at a multiplicity of infection (MOI) of 20 and 70. By 3 days after infection, Ad-E2F-1 treatment at an MOI of 70 resulted in approximately a 20-fold reduction in cell growth and 60% reduction in cell viability as compared to mock-infected cells. Cell cycle analysis, PARP cleavage and changes in cell morphology supported apoptosis as the mechanism of cell death in response to E2F-1. In order to test the efficacy of treatment with a combination of gene therapy and chemotherapy, we utilized concentrations of Ad-E2F-1 which reduced viability to 50% in combination with each chemotherapeutic agent. Cotreatment of the cells with E2F-1 virus and roscovitine (ROS) or etoposide resulted in an additive effect on cell growth inhibition and induction of apoptosis. Interestingly, 5-fluorouracil did not cooperate with Ad-E2F-1 in the mediation of tumor death or inhibition of cell growth. Immunoblotting for Bcl-2 family members revealed no significant changes in the expression levels of Bcl-2, Bcl X(L), Bax or Bak following gene or 'chemogene' therapy with E2F-1. However, a Bax cleavage product was noted which was substantially increased by cotreatment with ROS or etoposide. E2F-1 overexpression initiates apoptosis and suppresses growth in pancreatic MIA PaCa-2 cells in vitro. E2F-1-mediated apoptosis was not associated with significant changes in the expression of Bcl-2 family member proteins in these pancreatic cancer cells. ROS and etoposide, when combined with E2F-1 overexpression, induce apoptosis in an additive manner. This chemogene combination may provide a potentially useful therapeutic strategy for advanced pancreatic cancer.

The toxicity of chronic immunosuppressive agents required for organ transplant maintenance has prompted investigators to pursue approaches to induce immune tolerance. We developed an approach using a bioengineered mobilized cellular product enriched for hematopoietic stem cells (HSCs) and tolerogenic graft facilitating cells (FCs) combined with nonmyeloablative conditioning; this approach resulted in engraftment, durable chimerism, and tolerance induction in recipients with highly mismatched related and unrelated donors. Eight recipients of human leukocyte antigen (HLA)-mismatched kidney and FC/HSC transplants underwent conditioning with fludarabine, 200-centigray total body irradiation, and cyclophosphamide followed by posttransplant immunosuppression with tacrolimus and mycophenolate mofetil. Subjects ranged in age from 29 to 56 years. HLA match ranged from five of six loci with related donors to one of six loci with unrelated donors. The absolute neutrophil counts reached a nadir about 1 week after transplant, with recovery by 2 weeks. Multilineage chimerism at 1 month ranged from 6 to 100%. The conditioning was well tolerated, with outpatient management after postoperative day 2. Two subjects exhibited transient chimerism and were maintained on low-dose tacrolimus monotherapy. One subject developed viral sepsis 2 months after transplant and experienced renal artery thrombosis. Five subjects experienced durable chimerism, demonstrated immunocompetence and donor-specific tolerance by in vitro proliferative assays, and were successfully weaned off all immunosuppression 1 year after transplant. None of the recipients produced anti-donor antibody or exhibited engraftment syndrome or graft-versus-host disease. These results suggest that manipulation of a mobilized stem cell graft and nonmyeloablative conditioning represents a safe, practical, and reproducible means of inducing durable chimerism and donor-specific tolerance in solid organ transplant recipients.

Solid organ transplant recipients generally require life-long immunosuppressive agents to maintain graft acceptance. This is associated with a number of toxicities, including renal dysfunction, metabolic abnormalities, opportunistic infection and malignancies. Calcineurin inhibitors, the most frequently used agents for immunosuppression, induce nephrotoxicity, with gradual renal decline in many transplant recipients. As a result, approaches to induce tolerance remain a major focus of research. We report here the long-term results from a reduced-intensity nonmyeloablative conditioning approach to establish high levels of donor chimerism without graft-versus-host disease (GVHD) or engraftment syndrome in mismatched related and unrelated recipients of combined living donor kidney/hematopoietic stem cell transplant (HSCT). Recipients were conditioned with 200 cGy total body irradiation plus fludarabine (30 mg/kg day -5, -4, -3) and cyclophosphamide (50 mg/kg days -3 and +3) and administered donor granulocyte colony-stimulating factor mobilized stem cells, enriched for hematopoietic stem cells (HSC) and graft facilitating cells (FCs) and depleted of GVHD-producing cells (FCRx). Subjects ranged in age from 18 to 65 years. All subjects were HLA-disparate from their living kidney/HSCT donors, ranging from 5 of 6 related to 0 of 6 unrelated. Eight were unrelated and 11 related to their donors. Subjects were maintained on tacrolimus and mycophenolate mofetil (MMF) for 6 months. At 6 months, if protocol biopsy is normal and chimerism present, the MMF is discontinued. Similarly, the tacrolimus is discontinued at 12 months if stable renal function, normal protocol biopsy and chimerism are present. 30 subjects have been enrolled and 25 transplanted. Nineteen subjects have > 1 year follow up. Twelve have achieved stable donor chimerism and have been successfully taken off immunosuppression. Time off IS ranges from 8 months to 48 months. None have developed GVHD. We have herein prospectively evaluated immune reconstitution and immunocompetence. Return of CD4+ and CD8+ T central and effector memory cell populations was rapid. Examination for recent thymic emigrants (T cell staining for CD31+ CDRA+, TCR excision circle analysis) indicated that the majority (>97%) of these chimeric cells are being produced de novo. The TCR repertoires post-transplant in chimeric subjects were as diverse as pre-transplant donors and recipients but were distinct from donor or recipient pre-transplant. An increase in the CD4+ regulatory T cell/CD4+ effector T cell ratio was seen early in durably and transiently chimeric patients, suggesting active immunoregulation. Patients have been successfully vaccinated per American Society of Bone and Marrow Transplantation guidelines without loss of chimerism or development of allograft rejection. Of four subjects immunized for hepatitis B and whose donors were not, three retained immunologic memory after transplantation. Most chimeric subjects retained memory for measles, mumps, rubella and varicella. Chimeric subjects generated immune responses to pneumococcal vaccine. Opportunistic viral infections such as BK viremia and cytomegalovirus activation were absent after cessation of immunosuppression. These findings suggest that immunologic recovery is robust in chimeric subjects who receive kidney/FCRx and that durable chimerism is associated with donor-specific tolerance to renal allografts. Disclosures Bozulic: Regenerex, LLC: Employment. Tollerud:Regenerex, LLC: Chief Operating Officer Other. Ildstad:Regenerex, LLC: Chief Executive Officer Other.

The present study was designed to investigate the efficacy of combination gene therapy using adenoviral vectors expressing gene products shown to possess apoptotic activity: E2F-1 (Ad-E2F-1) and a C-terminal deletion mutant of p21(WAF1/cIP1) (Ad-p21(-PCNA)), on growth inhibition and apoptosis of human colon cancer cells in vitro and in vivo. Marked E2F-1 and p21(-PCNA) overexpression in response to adenovirus infection was evident by Western blot analysis. IC(25) concentrations of each virus were used for each treatment in vitro to detect cooperative effects on cell death. Coexpression of E2F-1 and p21(-PCNA) resulted in an additive effect on cell death compared to infection with either virus alone. Cell cycle analysis, poly(ADP-ribose) polymerase (PARP) cleavage and analysis of cell morphology also revealed that coinfection with both Ad-E2F-1 and Ad-p21(-PCNA) enhanced cellular apoptosis compared to either virus alone. Interestingly, E2F-1 protein expression was markedly enhanced in the E2F-1/p21(-PCNA) adenovirus combination compared to Ad-E2F-1 infection alone. However, these same effects were not evident in cells coinfected with Ad-E2F-1 and an adenovirus expressing wild-type human p21(WAF1/CIP1) (Ad-p21(WT)). The increase in E2F-1 expression with coexpression of E2F-1 and p21(-PCNA) was not a result of increased E2F-1 protein stability, but was related to increased transcriptional activity from the CMV promoter. Cell cycle analysis revealed G1 arrest 72 hours following single-gene therapy with either the wild-type or mutant p21, whereas increased accumulation of cells in G2/M phase was demonstrated in the E2F-1-overexpressing cells. In the combined therapies, E2F-1/p21(-PCNA) treatment still resulted in G1 arrest, but E2F-1 was able to counteract the G1 arrest when coinfected with p21(WT). These results provide further evidence of the importance of the p21:PCNA-binding domain in mediating the complex cell cycle interaction between E2F-1 and p21. Simultaneous intratumoral injection of Ad-E2F-1 and Ad-p21(-PCNA) dramatically reduced tumor burden of SW620 xenografts compared to either treatment alone in our in vivo model but not in HT-29 colon cancer xenografts. When combined with Ad-p21(-PCNA), E2F-1 adenovirus therapy resulted in approximately 95% decrease in tumor volume of SW620 tumor xenografts compared with controls (P

Recently, it has been demonstrated that Etoposide, a topoisomerase II inhibitor, can induce apoptosis in MDM2-overexpressing tumor cells by inhibition of MDM2 synthesis. We have previously shown that E2F-1 overexpression induces apoptosis of MDM2-overexpressing sarcoma cells, which is related to the inhibition of MDM2 expression. Therefore, the present study was designed to investigate the in vitro and in vivo effect of combined treatment of adenovirus-mediated E2F-1 and topoisomerase II inhibitors on the growth inhibition and apoptosis in human sarcoma cells. Two human sarcoma cell lines, OsACL and U2OS, were treated with topoisomerase II inhibitors (Etoposide and Adriamycin), alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ) or E2F-1 (Ad-E2F-1). E2F-1 expression was confirmed by Western blot analysis. Ad-E2F-1 gene transfer at a low dose (multiplicity of infection, 2) markedly increased the sensitivity of human sarcoma cells to topoisomerase II inhibitor treatment. This cooperative effect of E2F-1 and topoisomerase II inhibitors was less marked in SAOS-2 cells (p53 and pRb null). Topoisomerase II inhibitors also cooperated with E2F-1 overexpression to enhance tumor cell killing in an in vivo model using xenografts in nude mice. When combined with Adriamycin or Etoposide, E2F-1 adenovirus therapy resulted in approximately 95% and 85% decrease in tumor size, respectively, compared to controls (P.05). These results suggest a new chemosensitization strategy that is effective in MDM2-overexpressing tumors and may have clinical utility.

Abstract 4093 Hematopoietic stem cell (HSC) transplantation has become a common procedure for treatment of hematopoietic malignancies and autoimmune disease. Despite significant advances in HSC transplantation, the morbidity and mortality of ablative conditioning and graft-versus-host disease (GVHD) remain limitations to application in the clinic. However, these risks can be overcome through less toxic nonmyeloablative conditioning and cell depletion strategies to remove GVHD causing-cells while retaining engraftment enhancing-tolerogeneic cells. We were the first to discover CD8+/TCR− graft facilitating cells (FC) in mouse bone marrow. The addition of as few as 30,000 FC to 10,000 HSC significantly enhances engraftment of HSC in allogeneic recipients without causing GVHD. FC also potently enhance engraftment of limiting numbers of syngeneic HSC. Human CD8+/TCR- FC comprised 1.1% ± 0.27% of total G-CSF-mobilized peripheral blood mononuclear cells (mPBMC). In the CD8+/TCR- FC, 48% of cells expressed CD3ε+, 43% were FoxP3+, 43% were CD11c+, 19% were CD19+, and 30% were HLA-DR+. Approximately 55% of FC are also CD56dim/-, and the remaining population is CD56bright. The morphology of human CD8+/TCR− FC with Wright-Giemsa staining under light microscopy suggested that the human FC population is heterogeneous. Here we evaluated if human FC enhance human HSC or progenitor homing to bone marrow of NOD/SCID/IL-2rγnull (NSG) mouse recipients. CD45+CD34+ HSC and CD8+/TCR−/CD56dim/-FC were sorted from mPBMC. NSG recipients were conditioned with 1100 cGy of total body irradiation (TBI). 24 hours after TBI, 100,000 HSC with or without 300,000 CD8+/TCR−/CD56dim/- FC were transplanted into conditioned NSG recipients. Recipients were euthanized 16 hours after transplantation. Bone marrow was harvested from femurs and tibias of recipients and plated in Colony Forming Culture (CFC) Assays. Recipients of HSC plus FC generated significantly more colony formation (colonies = 110) compared with HSC alone (colonies = 65) (P = 0.011), suggesting that CD8+/TCR−/CD56dim/- FC enhanced homing of HSC or progenitors to bone marrow. To test if human CD8+/TCR−/CD56dim/- FC facilitate engraftment of human HSC in NSG mice, 300,000 CD8+/TCR−/CD56dim/- FC were mixed with 100,000 HSC and transplanted into NSG recipient mice conditioned with 325 cGy TBI. Mice that received HSC alone served as controls. At 30 days after transplantation, PBL typing showed that 34% (10 of 29) recipients of HSC alone engrafted. In contrast, 78% of recipients (n = 23) of HSC plus CD8+/TCR−/CD56dim/- FC engrafted, and donor chimerism in PB was 1.1% ± 0.8% and 4.1% ± 1.3% (P Disclosures: Bozulic: Regenerex, LLC: Employment. Ildstad:Regenerex, LLC: Equity Ownership.

Abstract 1969 Background: It has been known for over 50 years that hematopoietic stem cell (HSC) chimerism induces tolerance to transplanted tissues and cells. However, the widespread application of this approach has been constrained by graft-versus-host disease (GVHD), the need for close genetic matching between donor and recipient, and the toxicity of conditioning the recipient to establish chimerism. We have demonstrated that full donor chimerism can be established with minimal toxicity in highly-mismatched unrelated and related kidney allograft recipients through nonmyeloablative conditioning followed by infusion of a bioengineered CD8+/TCR− facilitating cell stem cell graft (FCRx), to avoid the risk of GVHD while achieving chimerism. Methods: Twelve HLA-mismatched living donor renal transplant recipients have been entered into a phase 2 trial (IDE 13947) involving low-intensity conditioning (fludarabine, cyclophosphamide, 200 cGy TBI days −4 to −1). Patients received a living donor kidney transplant on day 0, followed by infusion of G-CSF cryopreserved FCRx on day +1. All subjects were discharged by post-operative day 3 and managed as outpatients. We herein present data regarding the immunologic recovery observed in our first 8 evaluable patients with > 6 months follow up. Results: All patients experienced an expected nadir period affecting leukocytes (ANC < 500, range 2–14 days) and platelets (< 50K, range 0–20 days). All patients demonstrated peripheral blood macrochimerism at 1 month post-transplant, ranging from 6% to 100%. Chimerism was gradually lost in two patients at 3 and 6 months post-transplant. Patients demonstrated in vitro evidence of donor-specific hyporesponsiveness (DSH) by MLR +/− CML as early as 3 months post-transplant; of interest, DSH also was observed and persisted in the two patients who lost peripheral blood chimerism. Patients at > 1 yr post-transplant are immunocompetent to respond to mitogen (PHA), MHC-disparate third-party alloantigen, and tetanus in in vitro proliferative assays. Immunologic reconstitution in kidney + FCRx recipients was characterized by a blunted return in CD4+ T cells, with inversion of the CD4/CD8 ratio. A preferential recovery of memory (CD4+/CD45RO+/CD62L+/−) vs. naïve (CD4+/CD45RA+/CD62L+) T cells was observed. Although total number of CD4+/CD25+/CD127lo/FoxP3+ Treg was reduced initially, an increase in the CD4+ Treg /CD4+Teff (CD4+/CD45RA+/CD62L−) ratio was seen in patients exhibiting durable chimerism. In addition, an expansion of central memory CD8+ T cells was observed in durably chimeric recipients. No patient developed donor-specific antibodies as assessed by flow cytometric analysis. The absence of GVHD correlated with in vitro hyporesponsiveness of the fully chimeric recipients against archived pre-treatment recipient APC. Conclusions: Combined kidney + FCRx recipients demonstrate characteristic immunophenotypic and functional changes associated with reconstitution following transplantation; additional studies are required to determine whether these changes are mechanistically related to the persistence of chimerism and/or prevention of GVHD. Disclosures: Ildstad: Regenerex LLC: Equity Ownership.

Abstract 3709 CD8+/TCR− graft facilitating cells (FC) enhance engraftment of hematopoietic stem cells (HSC). They are distinct from HSC since they do not generate multilineage engraftment when infused alone, nor do they form colonies in vitro. The function of human FC remains to be defined. Here, we phenotypically characterized the human CD8+/TCR− FC subpopulations and evaluated their function in vivo and in vitro. Human CD8+/TCR− FC comprised 1.1% ± 0.27% of total G-CSF-mobilized peripheral blood mononuclear cells (PBMC). Approximately 55% of FC express CD56dim/−, and the remaining population is CD56bright. In the CD8+/TCR−/CD56bright cell population, approximately 65% of cells express CD11c+ and 67% of cells express CD11b+. In CD8+/TCR−/CD56dim/− cell population, the majority of cells express CD3ε+ (80%), 17% were CD11c+, 16% were CD19+, 14% were CD11b+, 11% were CD123+, and 30% were HLA-DR+. To evaluate whether human CD8+/TCR−/CD56dim/− FC enhance engraftment of human HSC in vivo, we transplanted 100,000 HSC alone or plus 300,000 CD8+/TCR−/CD56dim/− FC into NOD/SCID/IL2rγnull (NSG) recipient mice conditioned with 325 cGy of total body irradiation. At 30 days after transplantation, 8 of 21 (38%) recipients of HSC alone engrafted. In contrast, 81% of recipients (n = 16) receiving HSC plus CD8+/TCR−/CD56dim/− FC engrafted, and donor lymphocyte and monocyte chimerism in PB was 0.53% ± 0.16% and 3.93% ± 1.28%, respectively. At 6 months after transplantation, NSG recipients of HSC alone lost donor chimerism in PB and no donor cells were detected in spleen and BM. In contrast, NSG recipients of HSC + CD8+/TCR−CD56dim/− FC exhibited durable donor chimerism in PB and showed significantly higher levels of donor chimerism in spleen (13%) and BM (9.43%) compared to recipients of HSC alone. To evaluate the function of CD8+/TCR−/CD56dim/− FC in vitro, HSC were incubated with CD8+/TCR−CD56dim/− FC for 18 hrs and then cultured in methylcellulose for 14 days in colony-forming cell assay. HSC plus CD8+/TCR−CD56dim/− FC generated significantly more colonies compared with HSC alone (P = 0.0038), suggesting that human CD8+/TCR−/CD56dim/− FC have a direct effect on the clonogenicity of HSC (Figure). In summary, our data indicate that (1) human CD8+/TCR− FC are a heterogeneous cell population; (2) CD8+/TCR−/CD56dim/− FC enhance engraftment of human HSC in vivo and promote HSC clonogenicity in vitro; and (3) recipients of CD8+/TCR−/CD56dim/− FC maintain a durable donor chimerism in BM and spleen. Disclosures: Bozulic: Regenerex, LLC: Employment. King:Regenerex, LLC: Employment, Equity Ownership. Ildstad:Regenerex, LLC: Equity Ownership.