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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.

Nineteen subjects have more than 18 months' follow-up in a phase IIb tolerance protocol in HLA-mismatched recipients of living donor kidney plus facilitating cell enriched hematopoietic stem cell allografts (FCRx). Reduced intensity conditioning preceded a kidney allograft, followed the next day by FCRx. Twelve have achieved stable donor chimerism and have been successfully taken off immunosuppression (IS). We prospectively evaluated immune reconstitution and immunocompetence. Return of CD4 and CD8 T central and effector memory cell populations was rapid. T-cell receptor (TCR) Excision Circle analysis showed a significant proportion of chimeric cells produced were being produced de novo. The TCR repertoires posttransplant in chimeric subjects were nearly as diverse as pretransplant donors and recipients, and were comparable to subjects with transient chimerism who underwent autologous reconstitution. Subjects with persistent chimerism developed few serious infections when off IS. The majority of infectious complications occurred while subjects were still on conventional IS. BK viruria and viremia resolved after cessation of IS and no tissue-invasive cytomegalovirus infections occurred. Notably, although 2 of 4 transiently or nonchimeric subjects experienced recurrence of their underlying autoimmune disorders, none of the chimeric subjects have, suggesting that self-tolerance is induced in addition to tolerance to alloantigen. No persistently chimeric subject has developed donor-specific antibody, and renal function has remained within normal limits. Patients were successfully vaccinated per The American Society for Blood and Marrow Transplantation guidelines without loss of chimerism or rejection. Memory for hepatitis vaccination persisted after transplantation. Chimeric subjects generated immune responses to pneumococcal vaccine. These data suggest that immune reconstitution and immunocompetence are maintained in persistently chimeric subjects.

OBJECTIVE—Plasmacytoid precursor dendritic cell facilitating cells (p-preDC FCs) play a critical role in facilitation of syngeneic and allogeneic hematopoietic stem cell (HSC) engraftment. Here, we evaluated the phenotype and function of CD8+/TCR− FCs from NOD mice.RESEARCH DESIGN AND METHODS—The phenotype of CD8+/TCR− FCs was analyzed by flow cytometry using sorted FCs from NOD, NOR, or B6 mice. The function of NOD FCs was evaluated by colony-forming cell (CFC) assay in vitro and syngeneic or allogeneic HSC transplantation in vivo.RESULTS—We report for the first time that NOD FCs are functionally impaired. They fail to facilitate engraftment of syngeneic and allogeneic HSCs in vivo and do not enhance HSC clonogenicity in vitro. NOD FCs contain subpopulations similar to those previously described in B6 FCs, including p-preDC, CD19+, NK1.1+DX5+, and myeloid cells. However, the CD19+ and NK1.1+DX5+ subpopulations are significantly decreased in number in NOD FCs compared with disease-resistant controls. Removal of the CD19+ or NK1.1+DX5+ subpopulations from FCs did not significantly affect facilitation. Notably, Flt3 ligand (FL) treatment of NOD donors expanded FC total in peripheral blood and restored facilitating function in vivo.CONCLUSIONS—These data demonstrate that NOD FCs exhibit significantly impaired function that is reversible, since FL restored production of functional FCs in NOD mice and suggest that FL plays an important role in the regulation and development of FC function. FCs may therefore be linked to diabetes pathogenesis and prevention.

DNA damage results in an increase in P53 levels, which is required to initiate a P53-mediated cell cycle arrest and/or apoptosis. P53 and MDM-2 form a feedback control loop: while P53 can transactivate the MDM-2 gene, high levels of MDM-2 inhibit P53 transactivation as well as promote rapid degradation of P53. In the present study, we investigated the interaction between endogenous MDM-2 and P53 following UV-induced DNA damage in an MDM-2 overexpression cell line. A human osteosarcoma cell line (OsACL, which contains wild-type P53 and overexpresses MDM-2 protein) was used in this study. Here we show that following UV treatment, P53 levels increased in the OsACL cells despite the presence of high-level endogenous MDM-2; however, CAT assays using a P53 reporter system revealed that this P53 was transcriptionally inactive. Although P53 transactivation was inhibited, MDM-2 levels rose markedly following UV irradiation. Northern blot analysis revealed that the increase in MDM-2 protein levels was a result of increased levels of MDM-2 mRNA, possibly due to increased transcription. Cell cycle analysis revealed that OsACL cells were markedly resistant to UV-induced apoptosis. Transfection of OsACL cells with an anti-sense MDM-2 plasmid dowregulated MDM-2 expression and increased UV-induced apoptosis. In conclusion, MDM-2 overexpression can block UV-induced cell cycle arrest and apoptosis by inhibiting P53 transcriptional activity. Furthermore, increased expression of MDM-2 in OsACL cells following UV irradiation appears to be related to P53-independent mechanisms.

Adenoviral-mediated gene transfer of the apoptotic gene E2F-1 has been shown to induce apoptosis in a variety of tumor cells and acts in an additive or cooperative fashion with several specific chemotherapeutic agents to induce tumor cell death. The apoptotic function of E2F-1 is dependent on its ability to bind DNA; cyclin A kinase activity has been shown to negatively regulate the DNA-binding capacity of E2F-1. In the present study, we sought to determine whether cyclin A kinase activity is involved in mediating the interaction between E2F-1 and chemotherapeutic agents in colon cancer cells. Therefore, human colon adenocarcinoma (SW620) cells were treated with an adenovirus expressing E2F-1 (Ad-E2F-1, multiplicity of infection 20). Immediately following infection, a panel of conventional chemotherapeutic agents with varying modes of cytotoxic action were administered at LD(25 )doses. Three days following treatment, viability and growth inhibition were determined by trypan blue exclusion assay. Apoptosis was confirmed using cellular morphology, poly (ADP-ribose) polymerase cleavage, and flow-cytometric analysis. E2F-1 overexpression and cyclin A protein expression were monitored by immunoblot, and cyclin A kinase activity was determined by kinase assay. Vincristine (VIN), camptothecin (CPT), and actinomycin D were found to have a cooperative (38% over the additive single therapy values) effect on E2F-1-mediated apoptosis. Etoposide, cisplatin (CIS), and 5-fluorouracil (5-FU) showed the least cooperation (or=11.5% over the additive single therapy values) with E2F-1. Ad-E2F-1 treatment alone results in 3.4-fold increase of cyclin A kinase activity compared to Ad-LacZ control (p0.05); when combined with chemotherapeutic agents, cyclin A kinase activity was inhibited significantly by VIN, actinomycin D, and etoposide (p0.005), but not with CPT, CIS, and 5-FU (p0.1) compared to Ad-E2F-1 treatment alone. Combination of Ad-LacZ/5-FU and Ad-LacZ/actinomycin D significantly inhibited cyclin A kinase activity compared to Ad-LacZ treatment alone (p0.005). No other Ad-LacZ/drug combinations significantly affected cyclin A kinase activity (p0.05). In conclusion, combinations of E2F-1 adenovirus and VIN, CPT, or actinomycin D at LD(25 )had significant cooperative effects on colon cancer apoptotic cell death in vitro. Although inhibition of cyclin A kinase activity was observed in most Ad-E2F-1/drug combination treatments compared to Ad-E2F-1 treatment alone, there was no consistent correlation between degree of inhibition of cyclin A kinase activity and the cooperative effect. Nonetheless, inhibition of cyclin A kinase activity may be an important mechanism by which the chemogene therapy effects involving E2F-1 are modulated.

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.

CD8+TCR− graft facilitating cells (FCs) enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic and syngeneic recipients. The mechanisms by which FCs promote HSC engraftment and tolerance induction have not been fully elucidated. Here, we provide data to support a critical role for dedicator of cytokinesis 2 (DOCK2) in multiple aspects of FCs function. DOCK2−/− FCs exhibit compromised facilitative function in vivo as evidenced by the loss of engraftment-enhancing capability for c-Kit+Sca-1+lineage− (KSL) cells, and compromised ability to promote KSL cell homing and lodgment in hematopoietic niche. Deletion of DOCK2 abrogates the ability of FCs to induce differentiation of naïve CD4+CD25− T cells into FoxP3+ regulatory T cells and interleukin-10-producing type 1 regulatory T cells in vitro. Moreover, DOCK2−/− FCs are unable to promote survival of KSL cells when cocultured with KSL cells. DOCK2−/− FCs also exhibit compromised migration to stroma-derived factor-1 in vitro and impaired homing to the bone marrow in vivo. In conclusion, our results demonstrate that DOCK2 is critical for FCs to maintain its immunomodulatory function and exert its trophic effects on KSL cells. These findings may have direct clinical relevance to promote HSC engraftment for treatment of autoimmunity, hemoglobinopathies, and to induce transplantation tolerance. Stem Cells 2014;32:2732–2743

BACKGROUND E2F-1 is a transcription factor that stimulates cellular proliferation and cell cycle progression from G1 to S-phase. Somewhat paradoxically, E2F-1 also has the properties of a tumor suppressor. Overexpression of E2F-1 has been shown to induce apoptosis in some cancer cells. In the current study, the effect of adenovirus-mediated E2F-1 gene transfer on human melanoma cell growth was investigated. METHODS Two human melanoma cell lines, SK-MEL-28 (wild-type p53) and SK-MEL-2 (mutant p53), were treated by mock infection, infection with a control vector expressing the β-galactosidase gene (Ad5CMV-LacZ), or infection with a vector expressing E2F-1 (Ad5CMV-E2F-1) at a multiplicity of infection of 100. Cell proliferation and viability were determined by WST-1 assay and trypan blue exclusion, respectively. Apoptosis was assessed by cell flow cytometry and confirmed by cell morphology, in situ terminal deoxynucleotidyl nick end labeling assay, and poly(ADP-ribose) polymerase cleavage assay. RESULTS Marked overexpression of E2F-1 was evident in both cell lines 24 hours after infection with Ad5CMVE2F-1 by Western blot analysis. E2F-1 overexpression resulted in growth inhibition and rapid loss of cell viability. Overexpression of E2F-1 also resulted in premature S-phase entry and G2 arrest at 24 hours followed by apoptotic cell death at 48 hours. After Ad5CMVE2F-1 infection, expression of Bax and Bak was unchanged, whereas Mcl-1 levels decreased markedly. In SK-MEL-28 cells, Bcl-XL levels also declined after E2F-1 expression. Bcl-2 was undetectable in SK-MEL-28 cells but was increased in SK-MEL-2 cells in response to E2F-1 overexpression. CONCLUSIONS Adenovirus-mediated E2F-1 gene transfer efficiently induces widespread apoptosis in human melanoma cells. E2F-1 overexpression induced apoptosis in cell lines containing wild-type and mutant p53, suggesting that this effect does not require wild-type p53 function. Anti-apoptotic proteins of the Bcl-2 family, notably Mcl-1 and Bcl-XL, may be involved in mediating the response to E2F-1. These data suggest that adenovirus-mediated E2F-1 gene therapy may be effective in the treatment of melanoma. Cancer 1999;86:2021–33. © 1999 American Cancer Society.

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.

Hematopoietic stem/progenitor cell (HSPC) transplantation is increasingly applied for treatment of hematologic malignancies and autoimmune diseases. Despite significant clinical advances of HSPC transplantation, the morbidity and mortality of ablative conditioning and graft-versus-host disease (GVHD) remain a challenge. These risks can be overcome through less toxic nonmyeloablative conditioning and approaches that selectively remove GVHD-causing cells while retaining engraftment-enhancing, tolerogenic cells. We were the first to discover CD8+ T cell receptor (TCR)- graft facilitating cells (FCs) in mouse bone marrow (BM). The addition of as few as 30,000 FCs to 10,000 HSPCs significantly enhances engraftment of hematopoietic stem cells (HSCs) in allogeneic recipients without causing GVHD. FCs also potently enhance engraftment of limiting numbers of syngeneic HSPCs. FCs are distinct from HSPCs since they do not generate multilineage engraftment when infused alone, nor do they function as a stem cell in vitro. The phenotype and mechanism of action of human FCs remains to be defined. We report here for the first time that two distinct FC subpopulations are critical to overall human FC function: CD8+TCR-CD56bright (CD56bright FCs) and CD8+TCR-CD56dim (CD56dim FCs). FACS analysis showed that the majority of CD56bright FCs are CD11c+CD11b+ and C-X-C motif chemokine receptor 4 (CXCR4)bright and keep a dendritic morphology after stimulation with CpG oligodeoxynucleotides (CpG ODN) (TLR9). The majority of CD56dim FCs express CD3ε and exhibit a lymphoid morphology. The CD56dim FC subpopulation significantly promotes homing of HSPCs to BM in non-obese diabetic/severe combined immunodeficiency/interleukin-2 receptor gnull (NSG) recipients and enhances hematopoietic colony formation. The CD56dim FC subpopulation produces rapid reconstitution of donor hematopoietic cells without causing GVHD. In contrast, recipients of CD56bright FCs plus HSPCs show low donor chimerism early after transplantation. However, the level of donor chimerism significantly increases with time. Both recipients of HSPCs plus CD56dim or CD56bright FC subpopulations showed durable donor chimerism at significantly higher levels in BM compared to recipients of HSPCs alone. Co-culture of CD56bright FCs with HSPCs up-regulates small innate immunity-related peptides such as cathelicidin or beta defensin-2, factors that prime responsiveness of HSCs to a low stromal cell-derived factor-1 (SDF-1) gradient. Both CD56bright and CD56dim FC subpopulations highly up-regulate mRNA expression of the HSC growth factors, stem cell factors and fms-related tyrosine kinase 3 (FLT3) Ligand (3,000 fold and 14 fold respectively). Taken together, our data indicate that: 1) human CD8+TCR- FCs are two distinct, complementary and synergistic subpopulations with multifactorial effects on HSCs; 2) CD56bright FCs significantly enhance durable chimerism in the BM of NSG mice and produce significantly increased levels of factors that prime HSC homing; 3) CD56dim FCs significantly enhance homing of human HSC to the BM in NSG recipients and promote HSC clonogenicity; and 4) recipients of HSCs plus CD56dim FCs or CD56bright FCs show durable donor human chimerism in peripheral blood, BM and spleen. Disclosures Bozulic: Regenerex, LLC, a start-up biotech company: Employment. Ildstad:Regenerex, LLC, a start-up biotech company: CEO Other.

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.

DNA damage results in an increase in P53 levels, which is required to initiate a P53-mediated cell cycle arrest and/or apoptosis. P53 and MDM-2 form a feedback control loop: while P53 can transactivate the MDM-2 gene, high levels of MDM-2 inhibit P53 transactivation as well as promote rapid degradation of P53. In the present study, we investigated the interaction between endogenous MDM-2 and P53 following UV-induced DNA damage in an MDM-2 overexpression cell line. A human osteosarcoma cell line (OsACL, which contains wild-type P53 and overexpresses MDM-2 protein) was used in this study. Here we show that following UV treatment, P53 levels increased in the OsACL cells despite the presence of high-level endogenous MDM-2; however, CAT assays using a P53 reporter system revealed that this P53 was transcriptionally inactive. Although P53 transactivation was inhibited, MDM-2 levels rose markedly following UV irradiation. Northern blot analysis revealed that the increase in MDM-2 protein levels was a result of increased levels of MDM-2 mRNA, possibly due to increased transcription. Cell cycle analysis revealed that OsACL cells were markedly resistant to UV-induced apoptosis. Transfection of OsACL cells with an anti-sense MDM-2 plasmid dowregulated MDM-2 expression and increased UV-induced apoptosis. In conclusion, MDM-2 overexpression can block UV-induced cell cycle arrest and apoptosis by inhibiting P53 transcriptional activity. Furthermore, increased expression of MDM-2 in OsACL cells following UV irradiation appears to be related to P53-independent mechanisms.

Melanoma has proven to be resistant to conventional chemotherapy; however,the mechanism of chemoresistance is still unclear. Recent reports show that the transcription factor, E2F-1, may play a role in mediating cytotoxicity of certain chemotherapeutic agents. We have shown in a previous study that adenovirus-mediated overexpression of E2F-1 can efficiently induce apoptosis in melanoma cells. In the present study, the effect of E2F-1 expression on drug sensitivity of melanoma cells was evaluated. Two human melanoma cell lines, SK-MEL-28 and SK-MEL-2, were treated with drugs (etoposide, Adriamycin, roscovitine, cisplatin, 5-fluorouracil, or cycloheximide), alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ) or E2F-1 (Ad-E2F-1) at a multiplicity of infection of 1 in vitro. E2F-1 expression was confirmed by Western blot analysis. Sublethal concentrations of each drug alone or infection with Ad-E2F-1 alone produced5% apoptosis by 3 days posttreatment. Conversely, cotreatment with Ad-E2F-1 and low concentrations of etoposide or Adriamycin markedly sensitized melanoma cells to apoptotic cell death. A slight enhancement of the cytotoxicity of roscovitine was demonstrated in combination with E2F-1 overexpression, but not to cisplatin, 5-fluorouracil, or cycloheximide. Ad-LacZ infection showed no obvious effects on drug sensitivity. Overexpression of p21 can block apoptosis induced by the combination chemogene therapy of Ad-E2F-1 and topoisomerase II poisons and does not require its proliferating cell nuclear antigen-binding ability. The protein synthesis inhibitor cycloheximide also has a cytotoxicity-protective effect against topoisomerase II inhibitor/E2F-1-induced apoptosis and suggests that new protein synthesis is required for this process. Topoisomerase II inhibitors also cooperated with Ad-E2F-1 to enhance antitumor activity in an in vivo model using xenografts in nude mice. When combined with Adriamycin or etoposide, E2F-1 adenovirus therapy resulted in an 87% or 91% decrease in tumor size, respectively, compared with controls (P0.002). Our results show that adenovirus-mediated E2F-1 gene transfer can sensitize melanoma cells to some chemotherapeutic agents, particularly topoisomerase II poisons, in vitro and in vivo. These results suggest a new chemosensitization strategy for melanoma gene therapy.

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

E2F-1 and p53 are sequence specific transcription factors that are intimately involved in the regulation of the cell cycle. In addition to their role in cell cycle control, both E2F-1 and p53 have been identified as tumor suppressors and mediators of apoptosis. We have shown previously that adenoviral-mediated E2F-1 overexpression induces efficient apoptosis in colon adenocarcinoma cells. Previous reports have suggested that E2F-1 and p53 cooperate to mediate apoptosis and therefore, in this study, we examined the efficacy of combination gene therapy using adenovirus vectors expressing E2F-1 and p53 in human colon adenocarcinoma cell lines, HT-29 and SW620 (both mutant p53). Cells were treated by mock infection or infection with adenoviral vectors expressing b-galactosidase (LacZ), E2F-1, p53 or a combination of E2F-1 and p53. IC25 concentrations of each virus were estimated and used for each treatment in order to detect any synergistic or cooperative effects on tumor cell death in the combination therapy. By 5 days post infection, E2F-1-overexpressing cells exhibited growth inhibition and approximately 40-50% cell death in both cell lines. Co-expression of p53 with E2F-1 abrogated E2F-1-mediated growth inhibition and cell death. Cell cycle analysis revealed that overexpression of E2F-1 resulted in an accumulation of cells in G2/M phase, while overexpression of p53 resulted in a G1 phase accumulation. However, co-expression of E2F-1 and p53 counteracted each other as fewer cells accumulated in G1 and G2/M when compared to either p53 or E2F-1 alone. Furthermore, co-expression of p53 with E2F-1 resulted in decreased levels of E2F-1 protein expression. Mechanistically, upregulation of the CDK inhibitory protein, p21(WAF1/CIP1), was demonstrated in HT-29 cells following overexpression of either E2F-1, p53 or the combination E2F-1/p53 therapy. However, in SW620 cells, only the cells infected with Ad-p53 alone or in combination resulted in upregulation of p21(WAF1/CIP1). These results suggest that p53 and p21(WAF1/CIP1) may cooperate to inhibit the expression and activity of E2F-1. In conclusion, combination adenoviral vector-mediated E2F-1 and p53 gene transfer was not therapeutically advantageous in this in vitro model of human colon adenocarcinoma.

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.

E2F -1 is a transcription factor that regulates cell cycle progression into S-phase. Deregulation of E2F-1 activity has been associated with cellular commitment to apoptosis. Also critical in the regulation of S-phase are the actions of the cyclin dependent kinases, Cdk2 and cdc2. Inhibition of these cyclin dependent kinases has been similarly associated with disrupting orderly S-phase progression and causing subsequent apoptosis in certain cancer cells. In this study, we examine the ability of adenovirus-mediated E2F-1 overexpression to induce apoptosis in human gastric carcinoma cells. Furthermore, we investigate the effect of the cyclin dependent kinase inhibitors, olomoucine and roscovitine, on E2F-1-mediated apoptosis in human gastric carcinoma cells. AGS and SNU-1 gastric adenocarcinoma cells were infected with adenoviral vectors expressing E2F-1 (Ad5CMVE2F-1) or control viruses expressing beta-galactosidase (Ad5CMVLacZ) or lacking a transgene (Ad5). Gastric adenocarcinoma cells were then independently treated with roscovitine or olomoucine. Finally, gastric adenocarcinoma cells were infected with the various adenoviral vectors in combination with roscovitine or olomoucine. E2F-1 overexpression resulted in an 85% reduction in cell viability at 72 h compared to controls. Combining E2F-1 overexpression with roscovitine resulted in >99% reduction in cell viability by 72 h. Overexpression of E2F-1 resulted in premature S-phase entry and G2/M arrest at 24 h, followed by apoptosis by 72 h. Combining E2F-1 overexpression with roscovitine resulted in an earlier G2/M arrest, followed by a more complete, widespread apoptotic response by 24 h. Caspase 3/CPP32 activation and PARP cleavage in response to E2F-1 overexpression, alone and in combination with roscovitine, implicate the caspase cascade in E2F-1-mediated apoptosis of gastric cancer cells. Bax levels also increased in response to E2F-1 gene transfer, alone and in combination with roscovitine. E2F-1 overexpression induces widespread apoptosis in human gastric carcinoma cells. Combining E2F-1 overexpression with cyclin-dependent kinase inhibitors results in an enhanced apoptotic response, causing nearly complete gastric tumor cell death within 72 h. E2F-1 gene therapy in combination with cyclin dependent kinase inhibitors is a potentially active chemogene therapy strategy for the treatment of human gastric cancer.

Defective apoptotic mechanisms are considered to play a role in both the development of malignancy and resistance to chemotherapeutic drugs. The Bcl-2 family of proteins regulate the cellular commitment to survive or die when challenged with various apoptotic stimuli. PURPOSE: The purpose of this study was to identify the point at which Bcl-2 interrupts the apoptotic cascade initiated following exposure of human tumor cells to etoposide. METHODS: A stable Bcl-2-expressing HeLa-transfected clonal cell line, along with its control-vector-transfected counterpart, were utilized in this study. Following etoposide exposure, cells were examined for cell cycle arrest, formation of hyperdiploid cells, apoptotic DNA degradation, loss of plasma membrane integrity, levels of expression of members of the Bcl-2 protein family, caspase activation, degradation of poly(ADP-ribose) polymerase and movement of Bax from cytosol to cellular membrane fractions. RESULTS: Caspase activation, poly(ADP-ribose) polymerase degradation and Bax membrane insertion were initiated rapidly following etoposide removal, concomitantly with cell cycle arrest. Whereas Bcl-2 had no effect on etoposide-induced cell arrest, it interrupted all aspects of apoptosis, including activation of caspases, poly(ADP-ribose) polymerase degradation, DNA fragmentation and loss of plasma membrane integrity. Surprisingly, Bcl-2 also blocked Bax membrane insertion. In addition, Bcl-2 also prevented the increase in cellular levels of Bak, Bax and Bcl-xL, along with degradation of actin and Bax. However, inhibition of etoposide-induced apoptosis by Bcl-2 resulted in the accumulation of giant, multinucleated cells that eventually lost the ability to exclude trypan blue without apoptotic morphology or DNA degradation. CONCLUSIONS: These results indicate that biochemical apoptotic processes are initiated concomitant with etoposide-induced cell cycle arrest and are interrupted by Bcl-2 overexpression. However, the aberrant mitotic events induced by etoposide are sufficient to kill these cells even in the absence of apoptosis.

A reduced capacity for apoptosis induction is considered to play a significant role both in the development of malignancy and in tumor cell resistance to chemotherapeutic drugs. The Bcl-2 oncoprotein inhibits apoptosis induced by antitumor agents at a point downstream of drug-target interactions. Stable expression of Bcl-2 in the human epithelial tumor (HeLa) cell line results in inhibition of apoptosis following exposure to the topoisomerase II poison, etoposide. However, Bcl-2 is unable to enhance clonogenic survival as a result of alternate pathways to reproductive death induced by the drug. Purpose: The purpose of this study was to further investigate the role of Bcl-2 in human epithelial tumor cell drug resistance using 5-fluoro-2′-deoxyuridine, staurosporine, and doxorubicin, in addition to etoposide. Methods: The ability of Bcl-2 to enhance clonogenic cell survival was studied by colony-forming assays, while delay of cell death induction was assessed by trypan blue viability measurements. The proportion of apoptotic cells was measured by morphological criteria, as well as detection of apoptotic DNA fragmentation using the terminal deoxynucleotidyl transferase assay. Results: Despite profound inhibition to loss of plasma membrane integrity for all agents tested, Bcl-2 was only able to significantly increase clonogenic survival following exposure to 5-fluoro-2′-deoxyuridine and staurosporine, but not following exposure to etoposide or doxorubicin. Furthermore, the time-course of apoptosis induction following exposure of HeLa cells to equitoxic concentrations of staurosporine and etoposide was profoundly different. Conclusions: These results indicate that Bcl-2 enhances clonogenic survival of human epithelial tumor cells in an agent-specific fashion, which may be determined by the initial cytotoxic lesion induced by a particular drug.

A phase 2 protocol was developed to attempt to induce donor-specific tolerance to renal allografts in related and unrelated donor/recipient combinations (IND 13947 phase 2). Subjects were conditioned with fludarabine (days -5, -4, -3), cyclophosphamide (50 mg/ky days -3 and +3), and 200 cGy total body irradiation. The kidney transplant was performed day 0. G-CSF mobilized peripheral blood stem cells processed to remove GVHD-producing cells and retain graft facilitating cells (FC) was administered on day +1. The conditioning was well tolerated and the subjects were managed as outpatients after post-operative day 2. This has resulted in high levels of durable chimerism and immunosuppression-free graft survival without GVHD or engraftment syndrome in mismatched related and unrelated recipients of living donor FC/hematopoietic stem cell/kidney allografts. Nine subjects are completely off immunosuppression from 2 months to 3 years. A number of others are in the process of tapering. In the present study, we have prospectively analyzed recovery of immune function, persistence of vaccination memory, and response to vaccination in subjects who exhibit high levels of chimerism. Chimerism testing was performed using molecular short tandem report (sensitivity ±5%). Three of four subjects who had been vaccinated to hepatitis B prior to transplantation and whose donors had not been vaccinated retained their immunity following transplantation. All subjects tested exhibited memory for varicella and the majority did as well for measles (9/11), mumps (8/11), and rubella (5/10). A blood group disparity was present in 9 chimeric donor/recipient pairs. One chimeric subject converted to donor blood type, 3 exhibited mixed donor/host RBC chimerism, and 5 retained their own blood type. Six chimeric subjects have been immunized with pneumococcal vaccine after transplantation. All generated an immune response to vaccination, confirming immunocompetence to generate an antibody response to antigen. Notably, recovery of CD8+ and CD4+ central memory, naïve, and effector memory T cells occurred within one year post-transplantation to levels that were not significantly different from pre-transplantation. In addition, CD31+/CD45RA+ CD8+ and CD4+ T cells representative of recent thymic emigrants were present by 3 months, demonstrating de novo thymic production of T cells after transplantation. Four patients were randomly selected for study of Tcell repertoire (TCR) generation after transplantation. Two had achieved durable full donor chimerism and the other two did not have durable chimerism. Peripheral blood samples freshly obtained from donors and recipients and T cell subsets were isolated using MACs microbead system. DNA was extracted and sequenced by ImmunoSeq (Adaptive Biotech, Seattle, WA) to evaluate TCR clonal repertoires in recipients. Although clonal diversity in TCR repertoire was reduced in post-Tx recipients (0.9 ± 0.05 pre-Tx vs. 0.79 ± 0.09 post-Tx), the repertoire was diverse enough to suggest recovery of immune competence. Interestingly, at least 97% of the unique sequences observed in post-Tx recipient were not present in either donor or recipient pre-Tx. Within the pool of shared sequences, full chimerism correlated with a shift towards homology with the donor, while loss of chimerism correlated with recipient pre-Tx. In addition, the chimeric patients also exhibited reduced diversity of TCR sequences and increased clonality. Top 20 “high frequency” clones are most stably expressed. CD8+ cells had the highest number of “high frequency” clones. Notably, the pattern of “low frequency” clones was highest in the CD127-CD4+CD25+ regulatory T cell subset, indicating an extensive and rapidly changing TCR repertoire. Taken together, these data suggest that immunologic recovery is robust in these nonmyeloablatively conditioned tolerant chimeric subjects. Disclosures: Bozulic: Regenerex, LLC: Employment. Badder:Regenerex, LLC: Employment. Ildstad:Regenerex, LLC: Equity Ownership.

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 1885 CD8+/TCR− graft facilitating cells (FC) significantly enhance engraftment of hematopoietic stem cells (HSC) in both allogeneic and syngeneic recipients. We have shown that DOCK2−/− FC are compromised in cell migration in vitro and facilitating HSC engraftment in vivo. DOCK2 activates the small GTPase Rac and is indispensable for migration of neutrophils, lymphocytes and plasmacytoid dendritic cells (DC). A recent study demonstrated that DOCK2−/− DC were defective in antigen uptake and presentation. FC are a heterogeneous cell population, with a predominant subpopulation resembling plasmacytoid precursor DC. We previously found that FC induced antigen-specific regulatory T cells (Treg) in vitro and in vivo. In the present study, we evaluated whether DOCK2−/− FC could induce Treg. 5×105 wild-type (WT) sorted CD4+CD25− splenocyte T cells were incubated with 1×105 WT FC or DOCK2−/− FC at a 5:1 ratio in U bottom 96-well plates with long-term culture medium (IMEM with 20% horse serum, 10−6M hydrocortisone and 50μM β-mercaptoethanol) for up to 6 days. The co-cultures were harvested and CD4+CD25+ T cells were analyzed for FoxP3 by flow cytometry intracellular staining. WT CD4+CD25− splenocytes were cultured as negative control. Phagocytosis of APC-labeled polystyrene beads by WT and DOCK2−/− FC was assessed after incubating FC with the beads for 3 hours. We found that FC induced the generation of CD4+CD25+FoxP3+ Tregin vitro as expected, while DOCK2 deficiency completely abrogated the ability of FC to induce Treg generation. Moreover, the uptake of beads by DOCK2−/− FC was significantly impaired compared to their uptake by WT FC. In summary, DOCK2−/− FC were unable to induce the generation of CD4+CD25+FoxP3+ Tregin vitro and were compromised in phagocytosis. Disclosures: Ildstad: Regenerex, a biotech start-up company: 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 142 CD8+/TCR− graft facilitating cells (FC) have a potent capability to facilitate relatively small numbers of highly purified hematopoietic stem cells (HSC) in both allogeneic and syngeneic recipients. The mechanisms by which FC promote HSC engraftment have not been fully elucidated. We previously found that FC from non-obese diabetic mice (NOD) were compromised in enhancing engraftment of syngeneic and allogeneic HSC compared with FC from diabetes free congenic NOR mice. We therefore compared gene expression profiling between NOD FC and control NOR FC by microarray analysis. Among 18 most significant differentially expressed genes revealed from 45101 genes by false discovery rate control analysis with a cut-off value at level 0.05, dedicator of cytokinesis 2 (DOCK2) was identified as the gene with the most significant difference (P = 1.07 × 10−7). DOCK2 is a hematopoietic cell-specific member of the Caenorhabditis elegans Ced-5, mammalian DOCK180 and Drosophila melanogaster myoblast city (CDM) family of guanine nucleotide exchange factors. DOCK2 activates the small GTPase Rac and is indispensable for migration of plasmacytoid dendritic cells (pDC). FC is a heterogeneous cell population, with a predominant subpopulation resembling plasmacytoid precursor DC (p-preDC FC). In vitro studies demonstrated that FC increased clonogenicity of HSC and improved the ability of the impaired stroma to support late cobblestone area formation by HSC, which suggests that FC homing to hematopoietic niche as a potential component might be a perquisite for FC to enhance HSC engraftment. Therefore, we hypothesized that signaling pathways controlling cell migration via DOCK2 are critical for FC to enhance HSC engraftment. To test our hypothesis, DOCK2 expression data from microarray analysis was further confirmed using relative quantitative real-time PCR and high content image analysis. The expression level of DOCK2 in FC was significantly lower in NOD mice compared with NOR mice (p < 0.01 vs. NOR FC). Functional phenotypes of DOCK2-deficient FC were determined by Transwell migration assay and colony-forming cell assay in vitro, and by co-transplantation of HSC with FC in vivo as well as enumeration of CellTrack Green labeled FC by flow cytometry in spleen, thymus, and bone marrow of femurs and tibias 18 hours post-transplantation. Deficiency of DOCK2 in FC did not affect the ability of FC in promoting HSC colony formation when the two were cultured together. However, DOCK2-deficient FC were compromised in migration to the α-cheemokine, stromal derived factor-1 (SDF-1) at dose 200 ng/ml (Fig. A, P < 0.01 vs. wild-type FC). Homing of FC to spleen and bone marrow of femurs and tibias was also significantly impaired in DOCK2-deficient FC. Moreover, deficiency of DOCK2 in FC abrogated enhancement of HSC engraftment by FC in the syngeneic and allogeneic in vivo assays (Fig. B, syngeneic model: 500 B6 HSC plus 30K B6 or DOCK2−/−FC into lethally irradiated B6 recipients; Fig. C, allogeneic model: 10K B6 HSC plus 30K B6 or DOCK2−/−FC into lethally irradiated B10.BR recipients, P < 0.05 vs. B6 HSC plus wild-type FC). Taken together, our results indicate that deficiency of DOCK2 in FC leads to the dysfunction in migration, and suggest that the signaling pathways involved in FC migration are crucial for FC to enhance HSC engraftment. 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.

CD8+/TCR− facilitating cells (FC) enhance engraftment of purified hematopoietic stem cells (HSC) in syngeneic and allogeneic recipients. FC also induce the production of regulatory T cells (Treg) in vivo and in vitro. The B220+/CD11c+/CD11b− precursor plasmacytoid dendritic cell (p-preDC) subpopulation in FC (p-preDC FC) is critical to FC function. However, p-preDC FC are significantly less efficient in function compared to FC total. In this study, we evaluated the phenotype and function of FC from diabetes-prone nonobese diabetes (NOD) mice. We found that NOD FC contain subpopulations similar to those previously described in B6 FC, including p-preDC, CD19+, NK1.1+DX5+ and myeloid cells. P-preDC represent the major FC subpopulation in NOD mice. The CD19+, DX5+ and B220−/CD11c+/CD11b+ subpopulations were significantly decreased in NOD FC compared to those from B6 or NOR mice (Figure 1a; * = P< 0.05; ** = P< 0.007). To test the function of NOD FC, 500 HSC (c-Kit+/Sca-1+/Lin−) were sorted and transplanted with or without 30,000 FC into conditioned 950 cGy recipients. MHC-matched diabetes-resistant NOR mice were served as a control strain for NOD mice. 5 (31%) of 16 recipients of NOR HSC were engrafted and survived up to 130 days. 7 (70%) of 10 recipients of NOR HSC plus FC have long-term engraftment and survival over 130 days, indicating that the NOR FC significantly enhanced engraftment of NOR HSC compared to HSC alone. In striking contrast, NOD FC were functionally impaired and did not enhance HSC engraftment in NOD recipients as evidenced by similar engraftment of HSC with FC (31%, n = 13) compared to the HSC alone (24%, n = 17; P = 0.579). Notably, when NOD mice were treated with Flt3 ligand (FL; 10 μg/ subcutaneous/daily, 10 days), FC were expanded in peripheral blood (PB). The DX5+ and B220−/CD11c+/CD11b+ subpopulations were significantly increased (Figure 1b). The FL-PB FC significantly facilitate engraftment of allogeneic HSC in vivo in the NOD → B10 model (Figure 1c; P = 0.02). These data demonstrate that NOD FC exhibit significantly impaired function and FL plays an important role in regulation and development of FC function. We propose that the defective function of NOD FC is mechanistically due either to the abnormal activation status of the p-preDC FC population and/or the absence of collaborative subpopulations in FC such as NK FC. This hypothesis offers an attractive explanation for the mechanism of FC to enhance HSC engraftment and to induce tolerance. Studies are underway to evaluate the mechanism by which FC contribute to diabetes-pathogenesis and/or prevention. Figure Figure

One hundred forty-four maritally distressed clinic couples and 138 nonclinic couples participated in a study investigating the relationship between individual sex role identity and marital adjustment. Subjects completed Baucom's (1976) masculinity (MSC) and femininity (FMN) scales, and either the Dyadic Adjustment Scale (DAS; Spanier, 1976) or the Marital Adjustment Scale (MAS; Locke & Wallace, 1959). As predicted, femininity was related to positive marital adjustment; masculinity was related as well, but perhaps to a lesser degree. Androgyny within a relationship was found to be highly desirable, whereas marriages involving undifferentiated individuals were more likely to be distressed.