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Phase I (Safety) Study of Autologous Tolerogenic Dendritic Cells in Type 1 Diabetic Patients

Phase I (Safety) Study of Autologous Tolerogenic Dendritic Cells in Type 1 Diabetic Patients Emerging Treatments and T echnologies OR IGIN AL AR TIC L E Phase I (Safety) Study of Autologous Tolerogenic Dendritic Cells in Type 1 Diabetic Patients 1,2 1 NICK GIANNOUKAKIS, PHD JO HARNAHA, MS undergo apoptosis. Immature dendritic 1 1 BRETT PHILLIPS, PHD MASSIMO TRUCCO, MD cells also modulate networks of suppres- DAVID FINEGOLD, MD sive immune cells, such as T cells express- ing the Foxp3 transcription factor. Our preclinical data in the NOD mouse OBJECTIVE—The safety of dendritic cells to selectively suppress autoimmunity, especially strain demonstrating prevention and rever- in type 1 diabetes, has never been ascertained. We investigated the safety of autologous sal of type 1 diabetes with costimulation- dendritic cells, stabilized into an immunosuppressive state, in established adult type 1 di- impaired, immunosuppressive dendritic abetic patients. cells (bone marrow–derived dendritic cells RESEARCH DESIGN AND METHODS—A randomized, double-blind, phase I study treated ex vivo with a mixture of antisense was conducted. A total of 10, otherwise generally healthy, insulin-requiring type 1 diabetic oligonucleotides targeting the primary patients between 18 and 60 years of age, without any other known or suspected health con- transcripts of CD40, CD80, and CD86) ditions, received autologous dendritic cells, unmanipulated or engineered ex vivo toward an (6) compelled us to determine the safety immunosuppressive state. Ten million cells were administered intradermally in the abdomen of, and possible immune reactions against, once every 2 weeks for a total of four administrations. The primary end point determined the such dendritic cells in humans. We there- proportion of patients with adverse events on the basis of the physician’sglobal assessment, fore generated human dendritic cells anal- hematology, biochemistry, and immune monitoring for a period of 12 months. ogous to the ones successfully used in those NOD studies (6), concurrently tar- RESULTS—The dendritic cells were safely tolerated. There were no discernible adverse events in any patient throughout the study. Other than a significant increase in the frequency of pe- geting the expression of the same costim- ripheral B220+ CD11c2 B cells, mainly seen in the recipients of engineered dendritic cells during ulatory molecules ex vivo, envisaging the dendritic cell administration period, there were no statistically relevant differences in other type 1 diabetes cell therapy. We hypoth- immune populations or biochemical, hematological, and immune biomarkers compared with esized that immunosuppressive den- baseline. dritic cells would primarily be safe and well tolerated and, secondarily, could al- CONCLUSIONS—Treatment with autologous dendritic cells, in a native state or directed ex ter the frequency of immune cell popu- vivo toward a tolerogenic immunosuppressive state, is safe and well tolerated. Dendritic cells lations potentially beneficial in type 1 upregulated the frequency of a potentially beneficial B220+ CD11c2 B-cell population, at least in type 1 diabetes autoimmunity. diabetes. Diabetes Care 34:2026–2032, 2011 RESEARCH DESIGN AND METHODS—This phase I study ype 1 diabetes autoimmunity selec- self (4). Many animal studies confirm that (ClinicalTrials.gov identifier NCT00445913) tively impairs and destroys pancre- exogenous dendritic cell administration was conducted at the University of Pitts- Tatic b-cells. Thymic and peripheral prevents autoimmunity and facilitates burgh Medical Center Clinical Transla- tolerance failure (1,2) involves dendritic allograft survival (5). Such dendritic cells tional Research Center after review and cells, which are as important in diabetes often are phenotypically and functionally approval by the Food and Drug Admin- onset and progression as pathogenic T immature and are largely defined by istration, the University of Pittsburgh cells (3). In general, dendritic cells coor- impaired T-cell costimulation ability. Institutional Review Board, and the Data dinate immune responses to microenvi- Without costimulation, T cells, including Safety Monitoring Board and after written ronmental anomalies (i.e., infection and autoreactive cells, either enter into a state informed consent was obtained from each tissue damage) and orchestrate tolerance to of functional impairment (anergy) or patient. The data herein were reviewed by the Data Safety Monitoring Board and the Food and Drug Administration. ccccc cccccccc cccccccc cccccccc cccccccc cccccccc ccc c Patients (Table 1) were eligible for en- From the Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh School of Medicine, rollment if they were between 18 and 60 Pittsburgh, Pennsylvania; the Department of Pathology, University of Pittsburgh School of Medicine, years of age, had insulin-requiring diabe- Pittsburgh, Pennsylvania; and the Department of Human Genetics, University of Pittsburgh School of tes for at least 5 years between the time Medicine, Pittsburgh, Pennsylvania. of clinical diagnosis and the first dendritic Corresponding author: Massimo Trucco, [email protected]. cell injection, and met all the inclusion Received 10 March 2011 and accepted 12 May 2011. DOI: 10.2337/dc11-0472. Clinical trial reg. no. NCT00445913, clinicaltrials.gov. and exclusion criteria (Supplementary This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/suppl/doi:10. Methods Table T1). The patient-selection 2337/dc11-0472/-/DC1. criteria were recommended by the Food © 2011 by the American Diabetes Association. Readers may use this article as long as the work is properly and Drug Administration with institu- cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/ licenses/by-nc-nd/3.0/ for details. tional review board concurrence. 2026 DIABETES CARE, VOLUME 34, SEPTEMBER 2011 care.diabetesjournals.org Giannoukakis and Associates Table 1—Study group characteristics injection sites; of any grade $2toxicity or hypersensitivity, including chills, ma- laise, fever, shortness of breath, palor, or Control Immunosuppressive light headedness; or any subjective report dendritic cells dendritic cells of discomfort. Patients maintained their n 37 insulin-administration regimen through- Age (years) out the study, with the objective of main- Mean 30.3 6 4.5 31.6 6 13.3 taining glycated hemoglobin levels within Median 30 27 the age-specified range according to the Range 26–35 19–57 American Diabetes Association guidelines Male sex [n (%)] 2 (67) 4 (57) (8,9). In the intervening weeks between Race or ethnicity (self-reported) [n (%)] administrations, patients continued to be White 3 (100) 7 (100) evaluated (physical, biochemical, hemato- Non-Hispanic 3 (100) 7 (100) logic, and immune monitoring) to identify Type 1 diabetes autoantibodies [n (%)] possible adverse events. This evaluation 0 0 (0) 2 (29) was then conducted twice monthly for 1 2 (67) 3 (43) the first 6 months after the first dendritic 2 1 (33) 1 (14) cell administration and then once monthly Years since diagnosis 18.0 6 3.0 15.0 6 7.5 for the remainder of the study period (6 Years from diagnosis to first injection months). of dendritic cells Median 18 14 Safety evaluations Range 14–18 5–26 Patients were observed for treatment- Weight (kg) 84.6 6 12.5 87.0 6 25.4 related toxicity or adverse reactions dur- BMI 28.2 6 3.4 27.3 6 6.4 ing and after each course of dendritic cell 3 6 Total white blood cells (per mm )(310 /mL) 8.3 6 1.9 5.9 6 1.6 administration. The Common Toxicity Glycated HbA at baseline (%) 8.73 6 1.76 8.26 6 2.22 1c Criteria (ctep.info.nih.gov/reporting/ctc. C-peptide at baseline Undetectable Undetectable html) defined toxicity type and grade. Total insulin dose (units/kg) 0.49 6 0.20 0.32 6 0.05 The definition of adverse events con- Received all four rounds of dendritic cell formed to 21 CFR 312.32 (a) (serious) injections [n (%)] 3 (100) 7 (100) and 21 CFR 312.32 (a) (unexpected). Data are means 6 SD, unless otherwise indicated. BMI is weight in kilograms divided by the square of height Safety evaluations also probed for auto- in meters. immunity other than type 1 diabetes in serum, indices of systemic immunosup- pression measured as an in vitro cellular A power analysis was conducted were generated ex vivo from leukaphere- response to alloantigens and vaccination using simulations of continuously moni- sis products. The methods for generating antigens, white blood cell counts, and tored, trial-stopping boundaries to de- control and immunosuppressive den- flow cytometry–based measurement of termine the accrual buffer needed to dritic cells are available in the Supple- frequencies of specificimmune cellpop- ulations (Supplementary Methods Tables suspend a trial after an adverse event mentary Methods. Once generated, the T2 and T3). (7). This analysis concluded that in a total cells were frozen in aliquots of 1 3 10 sample size of 10 patients, the occurrence cells until the day of administration. Par- Laboratory measurements of an adverse event in 2 patients would ticipants and research staff were blinded Standard hematology and biochemistry give a 75% probability, and the occur- to the type of dendritic cell product ad- rence of an adverse event in 3 patients ministered. were used to screen blood, serum, and would give a 90% probability of hitting Each patient received 1 3 10 den- urine samples and also to detect specific the boundary where the boundary is dritic cells once every 2 weeks for a total pathogens, antinuclear, antithyroglobulin, defined as trial suspension (7). Thus, 10 of four administrations. For each admin- antithyroperoxidase, and type 1 diabetes– patients who met all inclusion and ex- istration, four aliquots of cells were intra- relevant autoantibodies. Immune monitor- clusion criteria (Supplementary Methods dermally injected; each aliquot into one of ing included serum cytokine detection, Table T1) were enrolled. Peripheral blood the vertices of a rectangular quadrant of multiparameter flow cytometry for im- was obtained to measure baseline levels 3–4 square inches overlying the anterior mune cell subsets, and cellular prolifera- of immune cell populations, immune- abdominal wall perpendicularly above tion assays to vaccination and alloantigens reactivity indices, serum immune bio- the physical location of the stomach and in vitro (see also Supplementary Methods markers, and autoantibodies, as well as pancreas. After thawing, the cells were Tables T3 and T4). for biochemistry and hematology evalua- slowly delivered by a tuberculin syringe tion. Urine was collected to determine attached to a 27-g 1/2 needle underneath Statistical analysis kidney function. Enrolled patients were a raised “bleb” of skin at each of the four The trial aimed at assuring that the tox- randomly assigned in a 2-to-1 ratio of im- individual injection sites. Patients were icity rate was acceptably low to warrant munosuppressive dendritic cells to con- then monitored by the physician for at additional study of the dendritic cell prod- trol dendritic cells. Control dendritic cells least 2 h for evidence of local or systemic ucts. The following stopping rule was im- and immunosuppressive dendritic cells allergic reaction; inflammation at the posed: If at any time during the trial the care.diabetesjournals.org DIABETES CARE, VOLUME 34, SEPTEMBER 2011 2027 Phase I tolerogenic dendritic cell safety trial observed proportion of the sum of all grade $2 toxicities or adverse events and grade 2 autoimmune toxicities equaled or exceeded 33% of treated patients, addi- tional treatment would be held pending review by theDataSafetyMonitoring Board. An upper bound on the rate of se- rious toxicity was chosen to claim, with at least 90% probability, that the true serious toxicity rate was no greater than 20% (7,10). To determine whether the differ- ences in specific cell-population frequen- cies between baseline and in-trial time points were statistically relevant, the Wilcoxon signed-rank test for paired observations was used. Elsewhere, and as indicated, standard two-tailed and repeated- measures ANOVA and two-tailed t tests were used to ascertain statistical signifi- cance to apparent differences in trends and values. RESULTS—There was no apparent ef- fect of sex or ethnicity on any of the out- comes or results of the trial. Dendritic cell administration is well tolerated without any adverse events Ten insulin-requiring type 1 diabetic pa- tients were enrolled in the study (Table 1). There were no detectable adverse events in any patient throughout the study dura- tion. There were no difficulties experi- enced during the series of intradermal injections in any patient. During follow- up, no patient reported experiences out- side routine activities of daily living. None of the patients exhibited any acute changes in diabetes control. No patient demon- strated acute illnesses that met the stan- dard of an adverse event. The physical examination outcomes of all patients dur- ing the course of the study were un- changed from pretreatment/baseline and were considered normal and unremark- able. The average insulin dose remained unchanged for each patient throughout the duration of the study when compared with pretreatment/baseline even though the specific insulin formulation varied (Lantus long-acting insulin with multiple Figure 1—Effects of control and immunosuppressive dendritic cells on nonfasting blood glucose daily injections of Humalog, Humulin in- levels (A), nonfasting glycated HbA levels (B), and nonfasting, nonstimulated C-peptide levels in 1c jectable or pump, or Novolog injectable). serum (C). Week 0 represents pretreatment/baseline levels. The symbols and lines in blue rep- The only notable physical observation resent the immunosuppressive dendritic cell recipients and those in red represent the control was a predictable “wheal and flare” reac- dendritic cell recipients. The legend to the right of each graph shows the symbols that correspond tion at the abdominal rectangular quad- to each individual patient (P). The values were measured in freshly obtained blood and serum at rant defined by the injection-site vertices, each of the weeks of the trial, shown on the x-axis in the graph. For C-peptide levels, only values which resolved within the first 60 min, on .0.5 ng/mL are shown based on standard clinical reference. average. We did not observe or detect any subjective or objective evidence of fever, systemic hypersensitivity, malaise, chills, 2028 DIABETES CARE, VOLUME 34, SEPTEMBER 2011 care.diabetesjournals.org Giannoukakis and Associates Table 2—Immune cell populations pains, or cardiac or ventilation abnormalities. None of the enrolled patients reported any adverse subjective sensation by 72 h Week 12 after each of the dendritic cell administra- Cell population Baseline (DC4) 6 Months 12 Months tions suggestive of a treatment-related CD3+ CD4+ anomaly. Control dendritic cells 37.7 6 0.5 33.9 6 9.9 47.1 6 1.9 48.2 6 2.7 Immunosuppressive dendritic cells 46.3 6 2.7 45.5 6 3.7 40.8 6 4.6 42.9 6 3.7 Laboratory outcomes CD3+ CD8+ Nonfasting blood glucose levels fluctu- Control dendritic cells 20.9 6 3.4 21.8 6 3.2 28.6 6 3.0 29.7 6 2.7 ated compared with baseline in all the Immunosuppressive dendritic cells 23.6 6 1.9 18.6 6 2.9 20.4 6 2.2 22.4 6 1.8 patients treated with control or immuno- CD4+ CD69+ suppressive dendritic cells (Fig. 1A). Control dendritic cells 0.6 6 0.4 2.4 6 1.2 0.6 6 0.3 0.8 6 0.5 Compared with pretreatment/baseline, Immunosuppressive dendritic cells 1.7 6 0.8 2.0 61.0 0.7 60.2 1.4 6 1.0 glycated hemoglobin levels did not change CD8+ CD69+ in any significant manner in control or Control dendritic cells 1.7 6 1.2 4.2 6 2.4 0.8 6 0.2 1.1 6 0.5 immunosuppressive dendritic cell recipi- Immunosuppressive dendritic cells 3.4 6 0.7 3.8 6 1.6 0.6 6 0.1 1.7 6 0.4 ents (Fig. 1B) with the exception of one CD4+ CD45RA+ immunosuppressive dendritic cell recipi- Control dendritic cells 25.2 6 2.2 23.9 6 6.7 24.4 6 1.6 23.4 612.3 ent (patient 13, Fig. 1B), who had histor- Immunosuppressive dendritic cells 29.5 6 3.9 27.2 6 6.0 29.7 6 4.6 29.5 6 2.1 ically exhibited poor glycemic control. CD8+ CD45RA+ However, more intriguing was the obser- Control dendritic cells 12.3 6 2.0 15.8 6 0.5 16.7 62.6 16.9 61.5 vation that C-peptide levels in some of the Immunosuppressive dendritic cells 13.9 6 1.7 15.0 62.8 13.8 6 2.4 15.7 61.8 subjects became detectable, whereas pre- HIGH CD4+ CD25 FOXP3+ viously and at the time of enrollment they Control dendritic cells 2.1 6 1.2 2.9 6 1.9 1.3 6 0.5 1.8 6 0.7 were undetectable (Fig. 1C). Immunosuppressive dendritic cells 1.1 6 0.3 1.7 60.5 1.8 6 0.7 1.9 6 0.7 Hematology assessment at all times B220+ CD11c2 during and after the dendritic cell admin- Control dendritic cells 3.5 6 2.9 9.6 6 5.1 3.2 6 2.6 5.0 6 2.1 istrations did not reveal any differences in Immunosuppressive dendritic cells 5.0 6 2.9 13.9 6 8.8 5.3 6 2.7 1.6 6 0.96 total leukocyte frequency or specific gen- CD11c+ CD83+ HLA-DR+ eral leukocyte population compared with Control dendritic cells 73.9 6 6.7 62.6 6 13.1 65.6 6 10.1 63.6 6 4.7 the expected standard reference range Immunosuppressive dendritic cells 71.9 6 6.9 71.0 6 5.4 70.2 6 4.9 57.8 6 8.0 (Supplementary Fig. 1). The frequency of relevant periph- Data are means 6 SEM and indicate the percentage of gated cells by fluorescence-activated cell sorter. “Immunosuppressive dendritic cells” refers to antisense oligonucleotide-treated dendritic cell recipients. eral blood immune cell populations DC4 at week 12 indicates the measurement of the cell populations at 1 week after the last dendritic cell (total and activated CD4+ and CD8+ administration. T cells, total B cells, and peripheral blood CD11c+ dendritic cells) was measured by fluorescence-activated cell sorter at key time points of the trial (Table 2). There Two of three control dendritic cell were interleukin (IL)-4 and IL-10, even were no statistically distinguishable differ- recipients and five of seven immunosup- if inconsistently (Fig. 2A). Thesourceof ences in the frequencies of any of these pressive dendritic cell recipients exhibited these cytokines currently is unknown, populations between control and immuno- GAD autoantibodies at baseline. There but their established role in immune toler- suppressive dendritic cell recipients as well were no apparent differences in GAD ance suggests that control or immunosup- as within each group compared with base- autoantibody concentrations at all times pressive dendritic cell administration may line at any time during the trial, with one the serum was collected before and after providesomebenefit by upregulating exception. Compared with baseline, con- dendritic cell administration. One of three their production in one or more immune trol and immunosuppressive dendritic control dendritic cell recipients and one cell populations. We note that control and cell recipients exhibited a statistically rel- of seven immunosuppressive dendritic cell immunosuppressive dendritic cells do not evant increase in B220+ CD11c2 cells recipients exhibited insulinoma-associated themselves produce IL-4 or IL-10 in vitro, during the dendritic cell administration protein-2 antibodies, whose concentra- although whether they do so in vivo is period (Supplementary Fig. 2). However, tions did not demonstrate any apparent unknown. despite sustained increases compared change before and after dendritic cell ad- To determine their potential to in- with baseline, statistical significance was ministration (Supplementary Fig. 4). Also, duce systemic immunosuppression, we lost by the end of the treatment period the levels of antinuclear and antithyroglo- conducted a standard allogeneic mixed- (Table 2). The frequencies returned to bulin antibodies were below the threshold leukocyte reaction (MLR) in vitro, mea- baseline through month 12, although im- considered to be clinically relevant (Sup- suring the proliferation of dendritic munosuppressive dendritic cell recipi- plementary Fig. 5). cell–recipient T cells in culture with allo- ents exhibited lower-than-baseline levels For what concerns serum cytokine geneic peripheral blood mononuclear at this time (Table 2). Additional charac- profiles and concentration, the only two cells (PBMCs). The stimulation indices re- terization of this population uncovered cytokines whose levels increased in con- mained at the baseline level and even in- immunosuppressive activity in vitro (Sup- trol and immunosuppressive dendritic creased in some dendritic cell recipients plementary Fig. 3A and B). cell recipients compared with baseline over the study period (Fig. 2B). We also care.diabetesjournals.org DIABETES CARE, VOLUME 34, SEPTEMBER 2011 2029 Phase I tolerogenic dendritic cell safety trial Figure 2—A: Serum levels of IL-4 and IL-10 in dendritic cell recipients. The presence and the concentration of serum cytokines was measured by Luminex multianalyte assay systems (LincoPLEX and Beadlyte). Serum IL-4 and IL-10 were reproducibly detectable. The points and lines in red indicate cytokine concentration in the serum of control dendritic cell recipients and the blue points and lines show the concentration in immuno- suppressive dendritic cell recipients. The graph at the top shows the levels of IL-4 and that on the bottom shows IL-10. The legend to the right shows the symbols that correspond to each individual patient (P). B: Dendritic cell administration does not confer systemic immunosuppression. Pro- liferation of dendritic cell–recipient T cells in allogeneic MLR in vitro. 23 10 PBMCs from the freshly obtained blood of the dendritic cell recipients were cultured alone or mixed with an equal number of allogeneic irradiated PBMCs in standard one-way MLR. The cells were cultured for 4 days before BrdU addition. BrdU incorporation into proliferating cells was measured on day 5 by flow cytometry. The graph shows the ratio of BrdU+ cells in the presence of allogeneic stimulators to BrdU+ cells in the absence of stimulators at each of the indicated weeks of the trial in seven individual patients compared with baseline (visit 2). Patients 6, 8, 10, 12, and 13 received immunosuppressive dendritic cells; patients 9 and 14 received control dendritic cells. The volumes of blood (and hence cell number) obtained from the remainder of the patients were not permissive to properly conduct biologically meaningful MLR. The legend to the right shows the symbols that correspond to each individual patient (P). C: The ELISPOT response of dendritic cell–recipient T cells to vaccination antigens in vitro. PBMCs from freshly isolated blood were frozen and, for this assay, thawed at a later 5 5 time. The thawed cells were plated on interferon-g ELISPOT strips (Human Interferon ELISPOT Pro; Mabtech) at densities of 1 3 10 or 3 3 10 cells. PBMCs were plated alone, with a viral peptide pool (CEF Peptide Pool; Mabtech) at 2 mg/mL, or with a kit-supplied CD3 agonistic antibody. An ELISPOT assay was conducted as recommended by the manufacturer, and the spots were counted electronically (KS ELISPOT; Zeiss) in an Axioplan 2 microscope with ELISPOT adapter/module. The data in the graph are shown for each patient where frozen PBMCs from each of the indicated weeks of the trial were available. The data are represented as the ratio of interferon-g spots in viral peptide–stimulated PBMCs to the spot number in unstimulated cells. Viability of frozen PBMCs after thawing from the remainder of the patients precluded the conduct of a biologically meaningful MLR assay. The legend to the right shows the symbols that correspond to each individual patient (P). measured the reactivity of dendritic cell– prevention or reversal of autoimmunity and Dendritic cells are strong T-cell modula- treated patient PBMCs to a panel of viral in facilitating allograft survival (5,11,12), tors, and control and/or immunosuppressive peptide antigens by interferon g enzyme- no human trials have ever been con- dendritic cell administration could po- linked immunosorbent spot (ELISPOT). ducted to determine immunosuppressive tentially cause general T-cell activation In all screened individuals, there was no dendritic cell safety and potential benefits. (with or without proliferation). By mea- loss of response compared with baseline Herein, we report, for the first time, that suring the frequency of CD69+ T cells at (Fig. 2C). Altogether, these data indicate multiple intradermal injections of autolo- key trial time points, we concluded that that control and immunosuppressive den- gous dendritic cells, untreated or stabilized neither control nor immunosuppressive dritic cells do not induce nonspecific sys- ex vivo toward an immunoregulatory tol- dendritic cells induced any changes in temic immunosuppression. erogenic state into an abdominal region CD69+ T-cell frequency compared with overlying the anatomical location of the baseline (Table 2). This observation is in CONCLUSIONS—To date, all known pancreas, are well tolerated and safe in adult line with the tolerogenic nature of imma- dendritic cell clinical trials aimed at boost- type 1 diabetic patients with established ture dendritic cells, which, on their own, ing immunity. Despite the convincing evi- disease longer than 5 years. There were no do not activate T cells. Nevertheless, we dence supporting dendritic cell–based observable adverse events or toxicities. cannot rule out the possibility that T cells 2030 DIABETES CARE, VOLUME 34, SEPTEMBER 2011 care.diabetesjournals.org Giannoukakis and Associates underwent unmeasurable differentiation B cells referred to as B-regulatory cells patients with cytokines such as IL-2 activity not involving activation, per se. (Bregs) (14–16). In murine models of in- and granulocyte macrophage–colony- Next, we probed the possibility that flammation and autoimmunity, includ- stimulating factor (20–22). control or immunosuppressive dendritic ing collagen-induced arthritis, colitis, Our findings demonstrate, for the cells could affect the frequency of naive lupus, and type 1 diabetes, exogenously first time, that nonmanipulated autologous antigen-inexperienced T cells (CD45RA+ administered Bregs suppress disease dendritic cells, or autologous dendritic cells T cells), which, if activated to proliferate, (14–16). Possible roles of Bregs in human directed ex vivo toward a costimulation- could induce diabetes-unrelated autoimmu- autoimmunity recently have been identi- impaired immunologically suppressive nities. The frequency of CD4+ CD45RA+ fied (17,18). Our putative Bregs do not state, are well tolerated and safe. Our ob- and CD8+ CD45RA+ T cells was statisti- seem to be like the recently described B10 servations herein also portend beneficial HIGH HIGH cally indistinguishable at all trial time and CD24 CD38 Bregs (Supple- outcomes when considering tolerogenic points compared with baseline, between mentary Fig. 7), even though CD24 and dendritic cell intervention in autoimmune and within control and immunosuppres- CD27, characteristic of the recently de- diseases such as type 1 diabetes at a time sive dendritic cell recipients (Table 2), in- scribed human B10 Bregs, were expressed closer to the clinical onset of the disease. dicating that this possibility was unlikely. strongly on the surface of the parental Administration of immature dendritic B220+ CD11c2 cells (17,18). This does Acknowledgments—This study was sup- cells in vivo increases the prevalence of not exclude the possibility that our Bregs ported by National Institutes of Health Award Foxp3+ T-regulatory cells (Tregs), and these couldinfactbea heterogeneouspopula- R33-DK-63499-03 (to M.T.). cells partially mediate the beneficial effects tion consisting of memory and transitional No potential conflicts of interest relevant to of immunoregulatory dendritic cells (13). B cells in different states of activation. That this article were reported. N.G. wrote and edited all draft versions and We observed a slight increase in CD4+ even untreated dendritic cells upregulate HIGH the final version of the manuscript, performed CD25 Foxp3+ T cells in immunosup- potentially beneficial B cells in humans the immunological and mechanistic assays, pressive dendritic cell recipients compared is not surprising in light of analogous ob- analyzed the immunological and mechanistic with control dendritic cell recipients, servations of B-cell upregulation in pio- data, and had full access to all data in the study mainly during the dendritic cell adminis- neering diabetes prevention studies with and had the final responsibility for the decision tration period (Supplementary Fig. 6). The pancreatic immature dendritic cells in to submit the manuscript for publication. B.P. differences, however, between immuno- NOD mice (19). It would be of significant performed the immunological and mechanis- suppressive and control dendritic cell re- interest to determine whether type 1 dia- tic assays and analyzed the immunological cipients as well as the difference between betic patients and high-risk relatives ex- and mechanistic data. D.F. analyzed the safety before and after dendritic cell injections, hibit perturbations in one or more Breg and physical patient data. J.H. performed the immunological and mechanistic assays. M.T. with the exception of three patients (Sup- populations. We currently are exploring wrote and edited all draft versions and the final plementary Fig. 6), were not statistically this possibility. version of the manuscript, analyzed the im- relevant (Table 2). Autoantibodies often reflect an under- munological and mechanistic data, analyzed We noted an increased frequency of lying autoimmunity and are an appropri- the safety and physical patient data, and had PBMC B220+ CD11c2 lymphocytes of ate biomarker of de novo autoimmunity. full access to all data in the study and had the all dendritic cell recipients as early as One concern of our trial was the potential final responsibility for the decision to submit 1 week after dendritic cell administration. to induce de novo autoimmunity, espe- the manuscript for publication. On average, this cell population compri- cially thyroid-specific autoimmunity, The authors thank Carl Engman, Robert ses between 1.0 and 3.3% of the total whichhas been showninsomepopula- Lakomy, and Alexis Styche for excellent tech- PBMCs in normal individuals (data not tions to cosegregate with type 1 diabetes. nical support and assistance. Carl Engman, J.H., shown). In four of seven immunosup- We did not observe any antinuclear anti- B.P., Robert Lakomy, and Alexis Styche are fully compensated employees of the Children’s pressive dendritic cell–treated patients, bodies or any de novo thyroid-specific Hospital of Pittsburgh. N.G., M.T., and D.F. are the increases were substantial during the autoantibodies arguing against the pos- fully compensated employees of the University dendritic cell administration period com- sibility that control or immunosuppres- of Pittsburgh School of Medicine. pared with baseline. These differences sive dendritic cell administration could were statistically relevant only during provoke a generalized autoimmunity or the first 6 weeks after the first round of a potential type 1 diabetes cosegregating References immunosuppressive dendritic cell admin- secondary autoimmunity. 1. Eisenbarth GS. Insulin autoimmunity: istration (P , 0.05; one-tailed Wilcoxon All dendritic cell recipients exhibited immunogenetics/immunopathogenesis of signed-rank test) (Supplementary Fig. 2). normal immune responses to vaccination type 1A diabetes. Ann N Y Acad Sci 2003; Although there were no statistically antigens and alloantigen stimulation in 1005:109–118 distinguishable differences in B220+ vitro, indicating that control and immu- 2. Fan Y, Rudert WA, Grupillo M, He J, Sisino G, Trucco M. Thymus-specificde- CD11c2 cell frequency at any time during nosuppressive dendritic cells do not in- letion of insulin induces autoimmune di- the trial between immunosuppressive duce any measurable level of systemic abetes. EMBO J 2009;28:2812–2824 dendritic cell and control dendritic cell immunosuppression. In light of the out- 3. Lo J, Clare-Salzler MJ. Dendritic cell sub- recipients (P . 0.05, two-tailed Mann- come of this study, we believe, like others, sets and type I diabetes: focus upon DC- Whitney U test), further characterization that the widely reported adverse events, based therapy. Autoimmun Rev 2006;5: and in vitro functional assays suggest that including elevated proinflammatory cyto- 419–423 this population contains at least one novel kines, fever, chills, and general malaise, 4. Steinman RM, Nussenzweig MC. Avoid- suppressive subpopulation (Supplementary associated with immunostimulatory den- ing horror autotoxicus: the importance of Fig. 3A and B). A number of studies con- dritic cell immunotherapy are attributed dendritic cells in peripheral T cell tolerance. firm the existence of immunosuppressive to the normally adopted priming of the Proc Natl Acad Sci USA 2002;99:351–358 care.diabetesjournals.org DIABETES CARE, VOLUME 34, SEPTEMBER 2011 2031 Phase I tolerogenic dendritic cell safety trial 5. McCurry KR, Colvin BL, Zahorchak AF, transplantation. Ann Rheum Dis 2008;67 18. Iwata Y, Matsushita T, Horikawa M, et al. Thomson AW. Regulatory dendritic cell (Suppl. 3):iii90–iii96 Characterization of a rare IL-10-competent therapy in organ transplantation. Transpl 12. Phillips BE, Giannoukakis N, Trucco M. B-cell subset in humans that parallels mouse Int 2006;19:525–538 Dendritic cell mediated therapy for immu- regulatory B10 cells. Blood 2011;117:530– 6. Machen J, Harnaha J, Lakomy R, Styche A, noregulation of type 1 diabetes mellitus. 541 Trucco M, Giannoukakis N. Antisense Pediatr Endocrinol Rev 2008;5:873–879 19. Clare-Salzler MJ, Brooks J, Chai A, Van oligonucleotides down-regulating costimu- 13. Yamazaki S, Dudziak D, Heidkamp GF, HerleK,AndersonC.Preventionofdiabetes lation confer diabetes-preventive properties et al. CD8+ CD205+ splenic dendritic cells in nonobese diabetic mice by dendritic cell to nonobese diabetic mouse dendritic are specialized to induce Foxp3+ regulatory transfer. J Clin Invest 1992;90:741–748 cells. J Immunol 2004;173:4331–4341 T cells. J Immunol 2008;181:6923–6933 20. Dhodapkar MV, Steinman RM, Krasovsky 7. Lazaridis E, Gonin R. A new program to 14. Bouaziz JD, Yanaba K, Tedder TF. Regu- J, Munz C, Bhardwaj N. Antigen-specific compute and evaluate continuously moni- latory B cells as inhibitors of immune re- inhibition of effector T cell function in tored stopping boundaries for clinical trials. sponses and inflammation. Immunol Rev humans after injection of immature den- Comput Methods Programs Biomed 2000; 2008;224:201–214 dritic cells. J Exp Med 2001;193:233–238 61:187–194 15. Mauri C, Ehrenstein MR. The ‘short’ his- 21. Redman BG, Chang AE, Whitfield J, et al. 8. American Diabetes Association. Standards tory of regulatory B cells. Trends Immunol Phase Ib trial assessing autologous, tumor- of medical care in diabetes: 2010. Diabetes 2008;29:34–40 pulsed dendritic cells as a vaccine adminis- Care 2010;33(Suppl. 1):S11–S61 16. Fillatreau S, Gray D, Anderton SM. Not tered with or without IL-2 in patients with 9. American Diabetes Association. Diagnosis always the bad guys: B cells as regulators metastatic melanoma. J Immunother 2008; and classification of diabetes mellitus. Dia- of autoimmune pathology. Nat Rev Im- 31:591–598 betes Care 2010;33(Suppl. 1):S62–S69 munol 2008;8:391–397 22. Correale P, Campoccia G, Tsang KY, et al. 10. Simon R, Thall PF, Ellenberg SS. New 17. Yanaba K, Bouaziz JD, Haas KM, Poe JC, Recruitment of dendritic cells and en- designs for the selection of treatments to Fujimoto M, Tedder TF. A regulatory B hanced antigen-specific immune reactivity be tested in randomized clinical trials. Stat cell subset with a unique CD1dhiCD5+ in cancer patients treated with hr-GM-CSF Med 1994;13:417–429 phenotype controls T cell-dependent in- (Molgramostim) and hr-IL-2. Results from 11. Thomson AW, Robbins PD. Tolerogenic flammatory responses. Immunity 2008;28: a phase Ib clinical trial. Eur J Cancer 2001; dendritic cells for autoimmune disease and 639–650 37:892–902 2032 DIABETES CARE, VOLUME 34, SEPTEMBER 2011 care.diabetesjournals.org http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Diabetes Care Pubmed Central

Phase I (Safety) Study of Autologous Tolerogenic Dendritic Cells in Type 1 Diabetic Patients

Giannoukakis, Nick; Phillips, Brett; Finegold, David; Harnaha, Jo; Trucco, Massimo
Diabetes Care , Volume 34 (9) – Aug 19, 2011
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© 2011 by the American Diabetes Association.
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0149-5992
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1935-5548
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10.2337/dc11-0472
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Abstract

Emerging Treatments and T echnologies OR IGIN AL AR TIC L E Phase I (Safety) Study of Autologous Tolerogenic Dendritic Cells in Type 1 Diabetic Patients 1,2 1 NICK GIANNOUKAKIS, PHD JO HARNAHA, MS undergo apoptosis. Immature dendritic 1 1 BRETT PHILLIPS, PHD MASSIMO TRUCCO, MD cells also modulate networks of suppres- DAVID FINEGOLD, MD sive immune cells, such as T cells express- ing the Foxp3 transcription factor. Our preclinical data in the NOD mouse OBJECTIVE—The safety of dendritic cells to selectively suppress autoimmunity, especially strain demonstrating prevention and rever- in type 1 diabetes, has never been ascertained. We investigated the safety of autologous sal of type 1 diabetes with costimulation- dendritic cells, stabilized into an immunosuppressive state, in established adult type 1 di- impaired, immunosuppressive dendritic abetic patients. cells (bone marrow–derived dendritic cells RESEARCH DESIGN AND METHODS—A randomized, double-blind, phase I study treated ex vivo with a mixture of antisense was conducted. A total of 10, otherwise generally healthy, insulin-requiring type 1 diabetic oligonucleotides targeting the primary patients between 18 and 60 years of age, without any other known or suspected health con- transcripts of CD40, CD80, and CD86) ditions, received autologous dendritic cells, unmanipulated or engineered ex vivo toward an (6) compelled us to determine the safety immunosuppressive state. Ten million cells were administered intradermally in the abdomen of, and possible immune reactions against, once every 2 weeks for a total of four administrations. The primary end point determined the such dendritic cells in humans. We there- proportion of patients with adverse events on the basis of the physician’sglobal assessment, fore generated human dendritic cells anal- hematology, biochemistry, and immune monitoring for a period of 12 months. ogous to the ones successfully used in those NOD studies (6), concurrently tar- RESULTS—The dendritic cells were safely tolerated. There were no discernible adverse events in any patient throughout the study. Other than a significant increase in the frequency of pe- geting the expression of the same costim- ripheral B220+ CD11c2 B cells, mainly seen in the recipients of engineered dendritic cells during ulatory molecules ex vivo, envisaging the dendritic cell administration period, there were no statistically relevant differences in other type 1 diabetes cell therapy. We hypoth- immune populations or biochemical, hematological, and immune biomarkers compared with esized that immunosuppressive den- baseline. dritic cells would primarily be safe and well tolerated and, secondarily, could al- CONCLUSIONS—Treatment with autologous dendritic cells, in a native state or directed ex ter the frequency of immune cell popu- vivo toward a tolerogenic immunosuppressive state, is safe and well tolerated. Dendritic cells lations potentially beneficial in type 1 upregulated the frequency of a potentially beneficial B220+ CD11c2 B-cell population, at least in type 1 diabetes autoimmunity. diabetes. Diabetes Care 34:2026–2032, 2011 RESEARCH DESIGN AND METHODS—This phase I study ype 1 diabetes autoimmunity selec- self (4). Many animal studies confirm that (ClinicalTrials.gov identifier NCT00445913) tively impairs and destroys pancre- exogenous dendritic cell administration was conducted at the University of Pitts- Tatic b-cells. Thymic and peripheral prevents autoimmunity and facilitates burgh Medical Center Clinical Transla- tolerance failure (1,2) involves dendritic allograft survival (5). Such dendritic cells tional Research Center after review and cells, which are as important in diabetes often are phenotypically and functionally approval by the Food and Drug Admin- onset and progression as pathogenic T immature and are largely defined by istration, the University of Pittsburgh cells (3). In general, dendritic cells coor- impaired T-cell costimulation ability. Institutional Review Board, and the Data dinate immune responses to microenvi- Without costimulation, T cells, including Safety Monitoring Board and after written ronmental anomalies (i.e., infection and autoreactive cells, either enter into a state informed consent was obtained from each tissue damage) and orchestrate tolerance to of functional impairment (anergy) or patient. The data herein were reviewed by the Data Safety Monitoring Board and the Food and Drug Administration. ccccc cccccccc cccccccc cccccccc cccccccc cccccccc ccc c Patients (Table 1) were eligible for en- From the Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh School of Medicine, rollment if they were between 18 and 60 Pittsburgh, Pennsylvania; the Department of Pathology, University of Pittsburgh School of Medicine, years of age, had insulin-requiring diabe- Pittsburgh, Pennsylvania; and the Department of Human Genetics, University of Pittsburgh School of tes for at least 5 years between the time Medicine, Pittsburgh, Pennsylvania. of clinical diagnosis and the first dendritic Corresponding author: Massimo Trucco, [email protected]. cell injection, and met all the inclusion Received 10 March 2011 and accepted 12 May 2011. DOI: 10.2337/dc11-0472. Clinical trial reg. no. NCT00445913, clinicaltrials.gov. and exclusion criteria (Supplementary This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/suppl/doi:10. Methods Table T1). The patient-selection 2337/dc11-0472/-/DC1. criteria were recommended by the Food © 2011 by the American Diabetes Association. Readers may use this article as long as the work is properly and Drug Administration with institu- cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/ licenses/by-nc-nd/3.0/ for details. tional review board concurrence. 2026 DIABETES CARE, VOLUME 34, SEPTEMBER 2011 care.diabetesjournals.org Giannoukakis and Associates Table 1—Study group characteristics injection sites; of any grade $2toxicity or hypersensitivity, including chills, ma- laise, fever, shortness of breath, palor, or Control Immunosuppressive light headedness; or any subjective report dendritic cells dendritic cells of discomfort. Patients maintained their n 37 insulin-administration regimen through- Age (years) out the study, with the objective of main- Mean 30.3 6 4.5 31.6 6 13.3 taining glycated hemoglobin levels within Median 30 27 the age-specified range according to the Range 26–35 19–57 American Diabetes Association guidelines Male sex [n (%)] 2 (67) 4 (57) (8,9). In the intervening weeks between Race or ethnicity (self-reported) [n (%)] administrations, patients continued to be White 3 (100) 7 (100) evaluated (physical, biochemical, hemato- Non-Hispanic 3 (100) 7 (100) logic, and immune monitoring) to identify Type 1 diabetes autoantibodies [n (%)] possible adverse events. This evaluation 0 0 (0) 2 (29) was then conducted twice monthly for 1 2 (67) 3 (43) the first 6 months after the first dendritic 2 1 (33) 1 (14) cell administration and then once monthly Years since diagnosis 18.0 6 3.0 15.0 6 7.5 for the remainder of the study period (6 Years from diagnosis to first injection months). of dendritic cells Median 18 14 Safety evaluations Range 14–18 5–26 Patients were observed for treatment- Weight (kg) 84.6 6 12.5 87.0 6 25.4 related toxicity or adverse reactions dur- BMI 28.2 6 3.4 27.3 6 6.4 ing and after each course of dendritic cell 3 6 Total white blood cells (per mm )(310 /mL) 8.3 6 1.9 5.9 6 1.6 administration. The Common Toxicity Glycated HbA at baseline (%) 8.73 6 1.76 8.26 6 2.22 1c Criteria (ctep.info.nih.gov/reporting/ctc. C-peptide at baseline Undetectable Undetectable html) defined toxicity type and grade. Total insulin dose (units/kg) 0.49 6 0.20 0.32 6 0.05 The definition of adverse events con- Received all four rounds of dendritic cell formed to 21 CFR 312.32 (a) (serious) injections [n (%)] 3 (100) 7 (100) and 21 CFR 312.32 (a) (unexpected). Data are means 6 SD, unless otherwise indicated. BMI is weight in kilograms divided by the square of height Safety evaluations also probed for auto- in meters. immunity other than type 1 diabetes in serum, indices of systemic immunosup- pression measured as an in vitro cellular A power analysis was conducted were generated ex vivo from leukaphere- response to alloantigens and vaccination using simulations of continuously moni- sis products. The methods for generating antigens, white blood cell counts, and tored, trial-stopping boundaries to de- control and immunosuppressive den- flow cytometry–based measurement of termine the accrual buffer needed to dritic cells are available in the Supple- frequencies of specificimmune cellpop- ulations (Supplementary Methods Tables suspend a trial after an adverse event mentary Methods. Once generated, the T2 and T3). (7). This analysis concluded that in a total cells were frozen in aliquots of 1 3 10 sample size of 10 patients, the occurrence cells until the day of administration. Par- Laboratory measurements of an adverse event in 2 patients would ticipants and research staff were blinded Standard hematology and biochemistry give a 75% probability, and the occur- to the type of dendritic cell product ad- rence of an adverse event in 3 patients ministered. were used to screen blood, serum, and would give a 90% probability of hitting Each patient received 1 3 10 den- urine samples and also to detect specific the boundary where the boundary is dritic cells once every 2 weeks for a total pathogens, antinuclear, antithyroglobulin, defined as trial suspension (7). Thus, 10 of four administrations. For each admin- antithyroperoxidase, and type 1 diabetes– patients who met all inclusion and ex- istration, four aliquots of cells were intra- relevant autoantibodies. Immune monitor- clusion criteria (Supplementary Methods dermally injected; each aliquot into one of ing included serum cytokine detection, Table T1) were enrolled. Peripheral blood the vertices of a rectangular quadrant of multiparameter flow cytometry for im- was obtained to measure baseline levels 3–4 square inches overlying the anterior mune cell subsets, and cellular prolifera- of immune cell populations, immune- abdominal wall perpendicularly above tion assays to vaccination and alloantigens reactivity indices, serum immune bio- the physical location of the stomach and in vitro (see also Supplementary Methods markers, and autoantibodies, as well as pancreas. After thawing, the cells were Tables T3 and T4). for biochemistry and hematology evalua- slowly delivered by a tuberculin syringe tion. Urine was collected to determine attached to a 27-g 1/2 needle underneath Statistical analysis kidney function. Enrolled patients were a raised “bleb” of skin at each of the four The trial aimed at assuring that the tox- randomly assigned in a 2-to-1 ratio of im- individual injection sites. Patients were icity rate was acceptably low to warrant munosuppressive dendritic cells to con- then monitored by the physician for at additional study of the dendritic cell prod- trol dendritic cells. Control dendritic cells least 2 h for evidence of local or systemic ucts. The following stopping rule was im- and immunosuppressive dendritic cells allergic reaction; inflammation at the posed: If at any time during the trial the care.diabetesjournals.org DIABETES CARE, VOLUME 34, SEPTEMBER 2011 2027 Phase I tolerogenic dendritic cell safety trial observed proportion of the sum of all grade $2 toxicities or adverse events and grade 2 autoimmune toxicities equaled or exceeded 33% of treated patients, addi- tional treatment would be held pending review by theDataSafetyMonitoring Board. An upper bound on the rate of se- rious toxicity was chosen to claim, with at least 90% probability, that the true serious toxicity rate was no greater than 20% (7,10). To determine whether the differ- ences in specific cell-population frequen- cies between baseline and in-trial time points were statistically relevant, the Wilcoxon signed-rank test for paired observations was used. Elsewhere, and as indicated, standard two-tailed and repeated- measures ANOVA and two-tailed t tests were used to ascertain statistical signifi- cance to apparent differences in trends and values. RESULTS—There was no apparent ef- fect of sex or ethnicity on any of the out- comes or results of the trial. Dendritic cell administration is well tolerated without any adverse events Ten insulin-requiring type 1 diabetic pa- tients were enrolled in the study (Table 1). There were no detectable adverse events in any patient throughout the study dura- tion. There were no difficulties experi- enced during the series of intradermal injections in any patient. During follow- up, no patient reported experiences out- side routine activities of daily living. None of the patients exhibited any acute changes in diabetes control. No patient demon- strated acute illnesses that met the stan- dard of an adverse event. The physical examination outcomes of all patients dur- ing the course of the study were un- changed from pretreatment/baseline and were considered normal and unremark- able. The average insulin dose remained unchanged for each patient throughout the duration of the study when compared with pretreatment/baseline even though the specific insulin formulation varied (Lantus long-acting insulin with multiple Figure 1—Effects of control and immunosuppressive dendritic cells on nonfasting blood glucose daily injections of Humalog, Humulin in- levels (A), nonfasting glycated HbA levels (B), and nonfasting, nonstimulated C-peptide levels in 1c jectable or pump, or Novolog injectable). serum (C). Week 0 represents pretreatment/baseline levels. The symbols and lines in blue rep- The only notable physical observation resent the immunosuppressive dendritic cell recipients and those in red represent the control was a predictable “wheal and flare” reac- dendritic cell recipients. The legend to the right of each graph shows the symbols that correspond tion at the abdominal rectangular quad- to each individual patient (P). The values were measured in freshly obtained blood and serum at rant defined by the injection-site vertices, each of the weeks of the trial, shown on the x-axis in the graph. For C-peptide levels, only values which resolved within the first 60 min, on .0.5 ng/mL are shown based on standard clinical reference. average. We did not observe or detect any subjective or objective evidence of fever, systemic hypersensitivity, malaise, chills, 2028 DIABETES CARE, VOLUME 34, SEPTEMBER 2011 care.diabetesjournals.org Giannoukakis and Associates Table 2—Immune cell populations pains, or cardiac or ventilation abnormalities. None of the enrolled patients reported any adverse subjective sensation by 72 h Week 12 after each of the dendritic cell administra- Cell population Baseline (DC4) 6 Months 12 Months tions suggestive of a treatment-related CD3+ CD4+ anomaly. Control dendritic cells 37.7 6 0.5 33.9 6 9.9 47.1 6 1.9 48.2 6 2.7 Immunosuppressive dendritic cells 46.3 6 2.7 45.5 6 3.7 40.8 6 4.6 42.9 6 3.7 Laboratory outcomes CD3+ CD8+ Nonfasting blood glucose levels fluctu- Control dendritic cells 20.9 6 3.4 21.8 6 3.2 28.6 6 3.0 29.7 6 2.7 ated compared with baseline in all the Immunosuppressive dendritic cells 23.6 6 1.9 18.6 6 2.9 20.4 6 2.2 22.4 6 1.8 patients treated with control or immuno- CD4+ CD69+ suppressive dendritic cells (Fig. 1A). Control dendritic cells 0.6 6 0.4 2.4 6 1.2 0.6 6 0.3 0.8 6 0.5 Compared with pretreatment/baseline, Immunosuppressive dendritic cells 1.7 6 0.8 2.0 61.0 0.7 60.2 1.4 6 1.0 glycated hemoglobin levels did not change CD8+ CD69+ in any significant manner in control or Control dendritic cells 1.7 6 1.2 4.2 6 2.4 0.8 6 0.2 1.1 6 0.5 immunosuppressive dendritic cell recipi- Immunosuppressive dendritic cells 3.4 6 0.7 3.8 6 1.6 0.6 6 0.1 1.7 6 0.4 ents (Fig. 1B) with the exception of one CD4+ CD45RA+ immunosuppressive dendritic cell recipi- Control dendritic cells 25.2 6 2.2 23.9 6 6.7 24.4 6 1.6 23.4 612.3 ent (patient 13, Fig. 1B), who had histor- Immunosuppressive dendritic cells 29.5 6 3.9 27.2 6 6.0 29.7 6 4.6 29.5 6 2.1 ically exhibited poor glycemic control. CD8+ CD45RA+ However, more intriguing was the obser- Control dendritic cells 12.3 6 2.0 15.8 6 0.5 16.7 62.6 16.9 61.5 vation that C-peptide levels in some of the Immunosuppressive dendritic cells 13.9 6 1.7 15.0 62.8 13.8 6 2.4 15.7 61.8 subjects became detectable, whereas pre- HIGH CD4+ CD25 FOXP3+ viously and at the time of enrollment they Control dendritic cells 2.1 6 1.2 2.9 6 1.9 1.3 6 0.5 1.8 6 0.7 were undetectable (Fig. 1C). Immunosuppressive dendritic cells 1.1 6 0.3 1.7 60.5 1.8 6 0.7 1.9 6 0.7 Hematology assessment at all times B220+ CD11c2 during and after the dendritic cell admin- Control dendritic cells 3.5 6 2.9 9.6 6 5.1 3.2 6 2.6 5.0 6 2.1 istrations did not reveal any differences in Immunosuppressive dendritic cells 5.0 6 2.9 13.9 6 8.8 5.3 6 2.7 1.6 6 0.96 total leukocyte frequency or specific gen- CD11c+ CD83+ HLA-DR+ eral leukocyte population compared with Control dendritic cells 73.9 6 6.7 62.6 6 13.1 65.6 6 10.1 63.6 6 4.7 the expected standard reference range Immunosuppressive dendritic cells 71.9 6 6.9 71.0 6 5.4 70.2 6 4.9 57.8 6 8.0 (Supplementary Fig. 1). The frequency of relevant periph- Data are means 6 SEM and indicate the percentage of gated cells by fluorescence-activated cell sorter. “Immunosuppressive dendritic cells” refers to antisense oligonucleotide-treated dendritic cell recipients. eral blood immune cell populations DC4 at week 12 indicates the measurement of the cell populations at 1 week after the last dendritic cell (total and activated CD4+ and CD8+ administration. T cells, total B cells, and peripheral blood CD11c+ dendritic cells) was measured by fluorescence-activated cell sorter at key time points of the trial (Table 2). There Two of three control dendritic cell were interleukin (IL)-4 and IL-10, even were no statistically distinguishable differ- recipients and five of seven immunosup- if inconsistently (Fig. 2A). Thesourceof ences in the frequencies of any of these pressive dendritic cell recipients exhibited these cytokines currently is unknown, populations between control and immuno- GAD autoantibodies at baseline. There but their established role in immune toler- suppressive dendritic cell recipients as well were no apparent differences in GAD ance suggests that control or immunosup- as within each group compared with base- autoantibody concentrations at all times pressive dendritic cell administration may line at any time during the trial, with one the serum was collected before and after providesomebenefit by upregulating exception. Compared with baseline, con- dendritic cell administration. One of three their production in one or more immune trol and immunosuppressive dendritic control dendritic cell recipients and one cell populations. We note that control and cell recipients exhibited a statistically rel- of seven immunosuppressive dendritic cell immunosuppressive dendritic cells do not evant increase in B220+ CD11c2 cells recipients exhibited insulinoma-associated themselves produce IL-4 or IL-10 in vitro, during the dendritic cell administration protein-2 antibodies, whose concentra- although whether they do so in vivo is period (Supplementary Fig. 2). However, tions did not demonstrate any apparent unknown. despite sustained increases compared change before and after dendritic cell ad- To determine their potential to in- with baseline, statistical significance was ministration (Supplementary Fig. 4). Also, duce systemic immunosuppression, we lost by the end of the treatment period the levels of antinuclear and antithyroglo- conducted a standard allogeneic mixed- (Table 2). The frequencies returned to bulin antibodies were below the threshold leukocyte reaction (MLR) in vitro, mea- baseline through month 12, although im- considered to be clinically relevant (Sup- suring the proliferation of dendritic munosuppressive dendritic cell recipi- plementary Fig. 5). cell–recipient T cells in culture with allo- ents exhibited lower-than-baseline levels For what concerns serum cytokine geneic peripheral blood mononuclear at this time (Table 2). Additional charac- profiles and concentration, the only two cells (PBMCs). The stimulation indices re- terization of this population uncovered cytokines whose levels increased in con- mained at the baseline level and even in- immunosuppressive activity in vitro (Sup- trol and immunosuppressive dendritic creased in some dendritic cell recipients plementary Fig. 3A and B). cell recipients compared with baseline over the study period (Fig. 2B). We also care.diabetesjournals.org DIABETES CARE, VOLUME 34, SEPTEMBER 2011 2029 Phase I tolerogenic dendritic cell safety trial Figure 2—A: Serum levels of IL-4 and IL-10 in dendritic cell recipients. The presence and the concentration of serum cytokines was measured by Luminex multianalyte assay systems (LincoPLEX and Beadlyte). Serum IL-4 and IL-10 were reproducibly detectable. The points and lines in red indicate cytokine concentration in the serum of control dendritic cell recipients and the blue points and lines show the concentration in immuno- suppressive dendritic cell recipients. The graph at the top shows the levels of IL-4 and that on the bottom shows IL-10. The legend to the right shows the symbols that correspond to each individual patient (P). B: Dendritic cell administration does not confer systemic immunosuppression. Pro- liferation of dendritic cell–recipient T cells in allogeneic MLR in vitro. 23 10 PBMCs from the freshly obtained blood of the dendritic cell recipients were cultured alone or mixed with an equal number of allogeneic irradiated PBMCs in standard one-way MLR. The cells were cultured for 4 days before BrdU addition. BrdU incorporation into proliferating cells was measured on day 5 by flow cytometry. The graph shows the ratio of BrdU+ cells in the presence of allogeneic stimulators to BrdU+ cells in the absence of stimulators at each of the indicated weeks of the trial in seven individual patients compared with baseline (visit 2). Patients 6, 8, 10, 12, and 13 received immunosuppressive dendritic cells; patients 9 and 14 received control dendritic cells. The volumes of blood (and hence cell number) obtained from the remainder of the patients were not permissive to properly conduct biologically meaningful MLR. The legend to the right shows the symbols that correspond to each individual patient (P). C: The ELISPOT response of dendritic cell–recipient T cells to vaccination antigens in vitro. PBMCs from freshly isolated blood were frozen and, for this assay, thawed at a later 5 5 time. The thawed cells were plated on interferon-g ELISPOT strips (Human Interferon ELISPOT Pro; Mabtech) at densities of 1 3 10 or 3 3 10 cells. PBMCs were plated alone, with a viral peptide pool (CEF Peptide Pool; Mabtech) at 2 mg/mL, or with a kit-supplied CD3 agonistic antibody. An ELISPOT assay was conducted as recommended by the manufacturer, and the spots were counted electronically (KS ELISPOT; Zeiss) in an Axioplan 2 microscope with ELISPOT adapter/module. The data in the graph are shown for each patient where frozen PBMCs from each of the indicated weeks of the trial were available. The data are represented as the ratio of interferon-g spots in viral peptide–stimulated PBMCs to the spot number in unstimulated cells. Viability of frozen PBMCs after thawing from the remainder of the patients precluded the conduct of a biologically meaningful MLR assay. The legend to the right shows the symbols that correspond to each individual patient (P). measured the reactivity of dendritic cell– prevention or reversal of autoimmunity and Dendritic cells are strong T-cell modula- treated patient PBMCs to a panel of viral in facilitating allograft survival (5,11,12), tors, and control and/or immunosuppressive peptide antigens by interferon g enzyme- no human trials have ever been con- dendritic cell administration could po- linked immunosorbent spot (ELISPOT). ducted to determine immunosuppressive tentially cause general T-cell activation In all screened individuals, there was no dendritic cell safety and potential benefits. (with or without proliferation). By mea- loss of response compared with baseline Herein, we report, for the first time, that suring the frequency of CD69+ T cells at (Fig. 2C). Altogether, these data indicate multiple intradermal injections of autolo- key trial time points, we concluded that that control and immunosuppressive den- gous dendritic cells, untreated or stabilized neither control nor immunosuppressive dritic cells do not induce nonspecific sys- ex vivo toward an immunoregulatory tol- dendritic cells induced any changes in temic immunosuppression. erogenic state into an abdominal region CD69+ T-cell frequency compared with overlying the anatomical location of the baseline (Table 2). This observation is in CONCLUSIONS—To date, all known pancreas, are well tolerated and safe in adult line with the tolerogenic nature of imma- dendritic cell clinical trials aimed at boost- type 1 diabetic patients with established ture dendritic cells, which, on their own, ing immunity. Despite the convincing evi- disease longer than 5 years. There were no do not activate T cells. Nevertheless, we dence supporting dendritic cell–based observable adverse events or toxicities. cannot rule out the possibility that T cells 2030 DIABETES CARE, VOLUME 34, SEPTEMBER 2011 care.diabetesjournals.org Giannoukakis and Associates underwent unmeasurable differentiation B cells referred to as B-regulatory cells patients with cytokines such as IL-2 activity not involving activation, per se. (Bregs) (14–16). In murine models of in- and granulocyte macrophage–colony- Next, we probed the possibility that flammation and autoimmunity, includ- stimulating factor (20–22). control or immunosuppressive dendritic ing collagen-induced arthritis, colitis, Our findings demonstrate, for the cells could affect the frequency of naive lupus, and type 1 diabetes, exogenously first time, that nonmanipulated autologous antigen-inexperienced T cells (CD45RA+ administered Bregs suppress disease dendritic cells, or autologous dendritic cells T cells), which, if activated to proliferate, (14–16). Possible roles of Bregs in human directed ex vivo toward a costimulation- could induce diabetes-unrelated autoimmu- autoimmunity recently have been identi- impaired immunologically suppressive nities. The frequency of CD4+ CD45RA+ fied (17,18). Our putative Bregs do not state, are well tolerated and safe. Our ob- and CD8+ CD45RA+ T cells was statisti- seem to be like the recently described B10 servations herein also portend beneficial HIGH HIGH cally indistinguishable at all trial time and CD24 CD38 Bregs (Supple- outcomes when considering tolerogenic points compared with baseline, between mentary Fig. 7), even though CD24 and dendritic cell intervention in autoimmune and within control and immunosuppres- CD27, characteristic of the recently de- diseases such as type 1 diabetes at a time sive dendritic cell recipients (Table 2), in- scribed human B10 Bregs, were expressed closer to the clinical onset of the disease. dicating that this possibility was unlikely. strongly on the surface of the parental Administration of immature dendritic B220+ CD11c2 cells (17,18). This does Acknowledgments—This study was sup- cells in vivo increases the prevalence of not exclude the possibility that our Bregs ported by National Institutes of Health Award Foxp3+ T-regulatory cells (Tregs), and these couldinfactbea heterogeneouspopula- R33-DK-63499-03 (to M.T.). cells partially mediate the beneficial effects tion consisting of memory and transitional No potential conflicts of interest relevant to of immunoregulatory dendritic cells (13). B cells in different states of activation. That this article were reported. N.G. wrote and edited all draft versions and We observed a slight increase in CD4+ even untreated dendritic cells upregulate HIGH the final version of the manuscript, performed CD25 Foxp3+ T cells in immunosup- potentially beneficial B cells in humans the immunological and mechanistic assays, pressive dendritic cell recipients compared is not surprising in light of analogous ob- analyzed the immunological and mechanistic with control dendritic cell recipients, servations of B-cell upregulation in pio- data, and had full access to all data in the study mainly during the dendritic cell adminis- neering diabetes prevention studies with and had the final responsibility for the decision tration period (Supplementary Fig. 6). The pancreatic immature dendritic cells in to submit the manuscript for publication. B.P. differences, however, between immuno- NOD mice (19). It would be of significant performed the immunological and mechanis- suppressive and control dendritic cell re- interest to determine whether type 1 dia- tic assays and analyzed the immunological cipients as well as the difference between betic patients and high-risk relatives ex- and mechanistic data. D.F. analyzed the safety before and after dendritic cell injections, hibit perturbations in one or more Breg and physical patient data. J.H. performed the immunological and mechanistic assays. M.T. with the exception of three patients (Sup- populations. We currently are exploring wrote and edited all draft versions and the final plementary Fig. 6), were not statistically this possibility. version of the manuscript, analyzed the im- relevant (Table 2). Autoantibodies often reflect an under- munological and mechanistic data, analyzed We noted an increased frequency of lying autoimmunity and are an appropri- the safety and physical patient data, and had PBMC B220+ CD11c2 lymphocytes of ate biomarker of de novo autoimmunity. full access to all data in the study and had the all dendritic cell recipients as early as One concern of our trial was the potential final responsibility for the decision to submit 1 week after dendritic cell administration. to induce de novo autoimmunity, espe- the manuscript for publication. On average, this cell population compri- cially thyroid-specific autoimmunity, The authors thank Carl Engman, Robert ses between 1.0 and 3.3% of the total whichhas been showninsomepopula- Lakomy, and Alexis Styche for excellent tech- PBMCs in normal individuals (data not tions to cosegregate with type 1 diabetes. nical support and assistance. Carl Engman, J.H., shown). In four of seven immunosup- We did not observe any antinuclear anti- B.P., Robert Lakomy, and Alexis Styche are fully compensated employees of the Children’s pressive dendritic cell–treated patients, bodies or any de novo thyroid-specific Hospital of Pittsburgh. N.G., M.T., and D.F. are the increases were substantial during the autoantibodies arguing against the pos- fully compensated employees of the University dendritic cell administration period com- sibility that control or immunosuppres- of Pittsburgh School of Medicine. pared with baseline. These differences sive dendritic cell administration could were statistically relevant only during provoke a generalized autoimmunity or the first 6 weeks after the first round of a potential type 1 diabetes cosegregating References immunosuppressive dendritic cell admin- secondary autoimmunity. 1. Eisenbarth GS. Insulin autoimmunity: istration (P , 0.05; one-tailed Wilcoxon All dendritic cell recipients exhibited immunogenetics/immunopathogenesis of signed-rank test) (Supplementary Fig. 2). normal immune responses to vaccination type 1A diabetes. Ann N Y Acad Sci 2003; Although there were no statistically antigens and alloantigen stimulation in 1005:109–118 distinguishable differences in B220+ vitro, indicating that control and immu- 2. Fan Y, Rudert WA, Grupillo M, He J, Sisino G, Trucco M. Thymus-specificde- CD11c2 cell frequency at any time during nosuppressive dendritic cells do not in- letion of insulin induces autoimmune di- the trial between immunosuppressive duce any measurable level of systemic abetes. EMBO J 2009;28:2812–2824 dendritic cell and control dendritic cell immunosuppression. In light of the out- 3. Lo J, Clare-Salzler MJ. Dendritic cell sub- recipients (P . 0.05, two-tailed Mann- come of this study, we believe, like others, sets and type I diabetes: focus upon DC- Whitney U test), further characterization that the widely reported adverse events, based therapy. Autoimmun Rev 2006;5: and in vitro functional assays suggest that including elevated proinflammatory cyto- 419–423 this population contains at least one novel kines, fever, chills, and general malaise, 4. Steinman RM, Nussenzweig MC. Avoid- suppressive subpopulation (Supplementary associated with immunostimulatory den- ing horror autotoxicus: the importance of Fig. 3A and B). A number of studies con- dritic cell immunotherapy are attributed dendritic cells in peripheral T cell tolerance. firm the existence of immunosuppressive to the normally adopted priming of the Proc Natl Acad Sci USA 2002;99:351–358 care.diabetesjournals.org DIABETES CARE, VOLUME 34, SEPTEMBER 2011 2031 Phase I tolerogenic dendritic cell safety trial 5. McCurry KR, Colvin BL, Zahorchak AF, transplantation. Ann Rheum Dis 2008;67 18. Iwata Y, Matsushita T, Horikawa M, et al. Thomson AW. Regulatory dendritic cell (Suppl. 3):iii90–iii96 Characterization of a rare IL-10-competent therapy in organ transplantation. Transpl 12. Phillips BE, Giannoukakis N, Trucco M. B-cell subset in humans that parallels mouse Int 2006;19:525–538 Dendritic cell mediated therapy for immu- regulatory B10 cells. Blood 2011;117:530– 6. Machen J, Harnaha J, Lakomy R, Styche A, noregulation of type 1 diabetes mellitus. 541 Trucco M, Giannoukakis N. Antisense Pediatr Endocrinol Rev 2008;5:873–879 19. Clare-Salzler MJ, Brooks J, Chai A, Van oligonucleotides down-regulating costimu- 13. Yamazaki S, Dudziak D, Heidkamp GF, HerleK,AndersonC.Preventionofdiabetes lation confer diabetes-preventive properties et al. CD8+ CD205+ splenic dendritic cells in nonobese diabetic mice by dendritic cell to nonobese diabetic mouse dendritic are specialized to induce Foxp3+ regulatory transfer. J Clin Invest 1992;90:741–748 cells. J Immunol 2004;173:4331–4341 T cells. J Immunol 2008;181:6923–6933 20. Dhodapkar MV, Steinman RM, Krasovsky 7. Lazaridis E, Gonin R. A new program to 14. Bouaziz JD, Yanaba K, Tedder TF. Regu- J, Munz C, Bhardwaj N. Antigen-specific compute and evaluate continuously moni- latory B cells as inhibitors of immune re- inhibition of effector T cell function in tored stopping boundaries for clinical trials. sponses and inflammation. Immunol Rev humans after injection of immature den- Comput Methods Programs Biomed 2000; 2008;224:201–214 dritic cells. J Exp Med 2001;193:233–238 61:187–194 15. Mauri C, Ehrenstein MR. The ‘short’ his- 21. Redman BG, Chang AE, Whitfield J, et al. 8. American Diabetes Association. Standards tory of regulatory B cells. Trends Immunol Phase Ib trial assessing autologous, tumor- of medical care in diabetes: 2010. Diabetes 2008;29:34–40 pulsed dendritic cells as a vaccine adminis- Care 2010;33(Suppl. 1):S11–S61 16. Fillatreau S, Gray D, Anderton SM. Not tered with or without IL-2 in patients with 9. American Diabetes Association. Diagnosis always the bad guys: B cells as regulators metastatic melanoma. J Immunother 2008; and classification of diabetes mellitus. Dia- of autoimmune pathology. Nat Rev Im- 31:591–598 betes Care 2010;33(Suppl. 1):S62–S69 munol 2008;8:391–397 22. Correale P, Campoccia G, Tsang KY, et al. 10. Simon R, Thall PF, Ellenberg SS. New 17. Yanaba K, Bouaziz JD, Haas KM, Poe JC, Recruitment of dendritic cells and en- designs for the selection of treatments to Fujimoto M, Tedder TF. A regulatory B hanced antigen-specific immune reactivity be tested in randomized clinical trials. Stat cell subset with a unique CD1dhiCD5+ in cancer patients treated with hr-GM-CSF Med 1994;13:417–429 phenotype controls T cell-dependent in- (Molgramostim) and hr-IL-2. Results from 11. Thomson AW, Robbins PD. Tolerogenic flammatory responses. Immunity 2008;28: a phase Ib clinical trial. Eur J Cancer 2001; dendritic cells for autoimmune disease and 639–650 37:892–902 2032 DIABETES CARE, VOLUME 34, SEPTEMBER 2011 care.diabetesjournals.org

Journal

Diabetes Care – Pubmed Central

Published: Aug 19, 2011

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