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From www.bloodjournal.org by guest on June 11, 2017. For personal use only. Blood First Edition Paper, prepublished online November 17, 2009; DOI 10.1182/blood-2009-08-239509 Induction of immune tolerance to FIX by intramuscular AAV gene transfer is independent of the activation status of dendritic cells Running Head: DC activation and FIX tolerance Arpita S. Bharadwaj,1 Meagan Kelly,1 Dongsoo Kim1 & Hengjun Chao1 1 Division of Hematology/Oncology, Department of Medicine, Cancer Institute, Immunology Institute Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029 Correspondence: Dr. Hengjun Chao Division of Hematology/Oncology, Box 1079 Mount Sinai School of Medicine One Gustave L. Levy Place, New York, NY 10029 Phone: 212-241-9567, Fax: 212-426-4390, Email: [email protected] Abbreviations: AAV, adeno-associated virus. DC, dendritic cells. FIX, coagulation factor IX. hFIX, human FIX. 1 of 39 Copyright © 2009 American Society of Hematology From www.bloodjournal.org by guest on June 11, 2017. For personal use only. Abstract The nature of viral vectors is suggested to be a significant contributor to undesirable immune responses subsequent to gene transfer. Such viral vectors recognized as “danger signals” by the host immune system, activate dendritic cells (DCs), causing unwanted anti-vector and/or transgene product immunity. We recently reported efficient induction of immune tolerance to FIX by direct intramuscular injection of AAV1-FIX. AAV vectors are non-pathogenic and elicit minimal inflammatory response. We hypothesized that the non-pathogenic nature of AAV plays a critical role in induction of tolerance following AAV gene transfer. We observed inefficient recruitment and activation of DCs subsequent to intramuscular injection of AAV. To further validate our hypothesis, we examined immune responses to FIX following intramuscular injection of AAV with simultaneous activation of DCs. We were able to achieve phenotypic and functional activation of DCs following administration of LPS and anti-CD40 antibody. However, we observed efficient induction of FIX tolerance irrespective of DC activation in mice with different genetic and MHC backgrounds. Furthermore, activation of DCs did not exaggerate the immune response induced following intramuscular injection of AAV2. Our results demonstrate that induction of FIX tolerance following AAV gene transfer is independent of DC activation status. 2 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. Introduction Gene therapy is emerging as a valuable alternative treatment for human diseases. However, adverse immune responses subsequent to gene transfer, such as severe cytotoxic T lymphocyte response (CTL) and formation of inhibitory antibodies against transgene products,1-4 need to be addressed for successful application of gene therapy in human patients. The primary step would be to determine critical factors that may decide or control the ultimate immunological outcomes in gene transfer. This will provide a fundamental insight into the mechanism accounting for the immune responses subsequent to gene transfer, leading to a better understanding and then final resolution of the adverse immune responses. A variety of inherent factors, in conjunction with gene transfer, can be encountered and recognized as a class of “danger signals” by the host immune system. 2,5 Such danger signals elicit the innate immunity of the host, thus stimulating and causing the maturation and activation of quiescent antigen presenting cells (APC). 2,5,6 The activated APCs in turn present the processed antigen with appropriate MHC molecules to antigen-specific T cells, to initiate the relevant immune response. 2,5 Dendritic cells (DC) are a major type of professional APCs, which act as the central decision-maker of the immune system.6-8 Activation of dendritic cells by the danger signal is a critical step in deciding the ultimate immunological outcome. 5,6,8,9 Quiescent (immature or mature) DCs are considered tolerogenic and capable of inducing T cell deletion, anergy or regulatory T cells. Whether a T cell is tolerized or activated to become an effector cell, depends on the activation status of the antigen presenting cells. The mature and activated DCs initiate priming of the antigen-specific CD4+ helper T cells, leading to immune responses to 3 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. relevant targets such as the delivery vector.6,9,10 Not all transgene products are immunogenic, therefore not recognized as “danger” by the host immune system. However, the anti-viral vector immunity can instigate collateral adverse immune responses against the transgene product.2 If the transgene product itself is immunogenic, the anti-viral vector immunity can worsen the intensity of the undesirable immune responses against the transgene product. A critical factor in relation to gene transfer that can be identified as a “danger signal” by the host organisms is the nature of the viral gene delivery vector. Many gene transfer vectors are recombinant derivatives of viruses, such as adenovirus and retrovirus, the majority of which are pathogenic and immunotoxic.1,2 Although the final recombinant viral vectors that are used in gene delivery are devoid of potential pathogenic viral component, vector-related immunotoxic incidents have been observed in many gene transfer studies.1 Among all viral vectors tested in gene therapy studies, adeno-associated virus (AAV) is the only virus that is not associated with any known human disease. The non-pathogenic nature of AAV does not present itself as a “ danger signal” to the host. It therefore causes only a minimum level of vector-related toxicity and immune responses in AAV-based gene transfers.2,11,12 This makes AAV an attractive gene transfer vector compared with other gene transfer vectors derived from pathogenic viruses. FIX gene transfer for hemophilia B treatment is a good model for gene therapy studies. Multiple strategies have been used for FIX gene transfer including the use of viral vectors. AAV has been extensively tested for hemophilia B gene therapy. Direct intramuscular injection of AAV has been shown to be a convenient, safe and potentially effective approach for FIX gene transfer.13-15 Intramuscular injection of AAV does not 4 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. cause severe cellular immune response such as CTL in rodents, canine, or human patients. Such severe immune responses were however substantial following gene transfers using recombinant adenoviral vector.16 Formation of inhibitory anti-FIX antibodies, which is the major complication in FIX replacement to treat hemophilia B, has also been observed in pre-clinical studies of intramuscular AAV gene transfer.13,17-19 Efficient induction of immune tolerance to FIX is critical for the success of hemophilia treatment. Many factors have been proposed to contribute to induction of immune tolerance or immunity to FIX following muscular AAV gene transfer.13,14,17-19 It is however inconclusive and still an ongoing debate as to the immunological consequence and the significance of these factors. It needs further investigation. We recently reported, efficient induction of immune tolerance to FIX by direct intramuscular injection of AAV serotype one vector (AAV1) in mice with diverse genetic and immunological backgrounds.13-15 AAV has proven to be much less immunogenic in contrast to other pathogenic viral vectors such as recombinant adenoviral vectors.11,12 It is conceivable that lower incidence of immunity against transgene product in AAV gene transfer may be ascribed to the non-pathogenicity of AAV and the consequent inefficiency of activated DC to stimulate an immune response. It was also reported that excess local immunity at the injection site could increase anti-FIX immunity in the context of intramuscular AAV gene transfer.20 We therefore hypothesized that the nonpathogenic property of the AAV vector is one of the major factors that facilitates induction of immune tolerance to FIX following intramuscular AAV1-FIX gene transfer. In the current study, we first examined the status of DCs in the context of intramuscular AAV gene transfer. We found that the DCs remained in a steady state 5 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. following intramuscular injection of either AAV1 or AAV2. We then investigated immune responses to FIX following intramuscular injection of AAV1 concomitant with full activation of DCs by LPS and anti-CD40 antibody. Our data demonstrated that despite activation of DCs, no change in the ultimate outcome of immune tolerance induced by intramuscular injection of AAV1 vectors was observed. We accordingly concluded that, induction of FIX tolerance by intramuscular AAV gene transfer is independent of the activation status of dendritic cells. Material and Methods AAV vector production, animal care and procedures The AAV-hFIX vectors were made using a three plasmid transfection scheme as previously described.13,15 C57BL/6, Balb/c and C3H mice were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). All the mice were maintained in pathogen-free animal facilities at Mount Sinai School of Medicine, and treated in accordance with the guidelines of the Institutional Animal Care and Use Committee of Mount Sinai School of Medicine, which approved this study. AAV injections, animal procedures and plasma sample collection were conducted as previously described.13,15 For in vivo dendritic cell activation, eight to ten week old mice were injected with 100 μg/mouse of anti-CD40 antibody (Rat anti-mouse CD40, IgG2a, Clone FGK45, Axxora, LLC. San Diego, CA) in the footpad and 100 μg/mouse LPS (Lipopolysaccharide, Sigma Aldrich, St. louis, MO) via intraperitoneal injection or anti-CD40 antibody alone on day 0. The draining lymph nodes and spleens were collected 6, 24, 72 and 120 hours after AAV and anti-CD40 antibody±LPS injection for characterization of dendritic cells. Two units of recombinant 6 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. hFIX (rhFIX; Genetics Institute, Cambridge, MA) emulsified in 100 μl of complete Freund’s adjuvant (CFA; Pierce Biotechnology, Rockford, IL) was injected subcutaneously for boosting the immune response to hFIX or for verification of hFIX immune tolerance. Detection of hFIX antigen and anti-hFIX antibodies Human FIX antigen was measured by an enzyme-linked immunosorbent assay (ELISA) as previously described.13 Anti-hFIX antibody-specific ELISA was performed to detect anti-hFIX antibodies as previously described.13,15 Phenotypic characterization of DCs Single cell suspensions were prepared by mechanical disintegration of the isolated draining lymph nodes and spleen, followed by red blood cell lysis (BD Pharmingen, CA). The cells were suspended in 100 μl of FACS staining buffer (5 ml PBS, 100 μl each of normal mouse, rabbit and human serum, 333 μl 30% BSA, 5 ml HBSS complete with BSA and 100mM EDTA), and then stained with the following florochrome conjugated antibodies CD11c-PE (1:200), CD80-APC (1:200), CD86-APC (1:200) (BD Pharmingen, CA) and MHCII-Biotin (eBiosciences, CA). The cells were incubated with the antibody mixture for 30 minutes on ice and washed twice by the addition of 1ml FACS buffer. Cells that received biotin conjugated antibodies were incubated an additional 30 minutes with avidin-APC-Cy7 (BD Pharmingen, CA). The cells were then washed twice and stored in 1x formalin buffer prior to analysis. The cells were read on BD LSRII flow cytometer equipped with the BD FACSDiva software (Becton Dickinson, San Jose, CA). The data was analyzed using FlowJo analysis software (TreeStar, OR). 7 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. Cytokine Profile Splenocytes were suspended in complete RPMI-1640 and cultured for 4 hours at 37°C in a CO2 incubator. GolgiSTOP (4µl/6ml culture,BD Pharmingen, CA) was added and incubated for an additional 4 hours at 37°C. The cells were collected and stained for surface antigen CD11c-PE (1:200) (BD Pharmingen, CA). The cells were then fixed and permeabilized as per manufacturers recommendation (BD Cytofix/Cytoperm Plus Fixation/Permeabilization kit, BD Pharmingen, CA). The cells were subsequently stained for intracellular cytokines IL-12-APC, IL-6-PE and IL-10-FITC (BD Pharmingen, CA). The cells were evaluated on BD LSRII flow cytometer equipped with the BD FACSDiva software (Becton Dickinson, San Jose, CA). The data was analyzed using FlowJo analysis software (TreeStar, OR). Statistical analysis The data was analyzed using GraphPad Prism (GraphPad Software, CA). Statistical differences between the various experimental groups were evaluated by twotailed, unpaired T test. p<0.05 was considered statistically significant. Results In the current study we proposed to examine the role of DCs in AAV1 induced immune tolerance to FIX. For a sensitive as well as dependable evaluation of DC motivation, recruitment and activation following intramuscular injection of AAV, we performed preliminary experiments to determine the appropriate duration, route and mode of DC activation. We compared different agents that are known and defined for DC activation, such as LPS (Lipopolysaccharide) and anti-CD40 antibody administered either 8 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. subcutaneously (footpad) or intraperitoneally. 21-23 We examined number of CD11c+ cells as well as expression of MHCII and co-stimulatory molecules (CD80 and CD86) on CD11c+ cells in draining lymph nodes (Figure 1A) and spleen (Figure 1B) of the mice at 6, 24, 72 and 120 hours after administration of anti-CD40 antibody alone or LPS plus anti-CD40 antibody or PBS. We observed that the combination of LPS and anti-CD40 antibody was optimal for the activation of DCs (Figure 1A-B). We also observed that DC activation peaked at 24 hours upon administration of the DC stimulator via the subcutaneous (footpad) route (Figure 1A-B). We thus decided to evaluate the status of DCs at 24 hours following intramuscular injection of AAV and/or administration of antiCD40 antibody together with LPS. Direct intramuscular injection of AAV vectors does not motivate or activate DCs We first examined the numbers and activation status of DCs subsequent to intramuscular injection of AAV vectors. Cohorts of eight to ten week old C57BL/6 mice (n=4 per cohort) received intramuscular injection of 1 x 1011 vg (vector genomes) of AAV1-hFIX or 6x1010 vg of AAV2-hFIX vectors as described. Twenty-four hours after AAV injection, cells from the draining lymph nodes and spleen of the mice were harvested for detection of cell surface markers by flow cytometry. The size of draining lymph nodes and spleen in the AAV1-injected and AAV2-injected mice were similar to those in the naïve, untreated mice. We also observed no change in the number of CD11c+ cells in the draining lymph nodes and spleen of AAV1 or AAV2-injected mice when compared to naïve, untreated mice (Figure 2A). Baseline levels of cells expressing CD11c remained around 2% in draining lymph nodes and around 5-6% in the spleen in 9 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. both experimental and untreated groups. We observed no statistically significant difference in the expression of co-stimulatory molecules on the CD11c+ cells in both the draining lymph nodes and spleen of AAV1 and AAV2-injected mice when compared with naïve untreated mice (Figure 2B). In summary, we did not observe significant upregulation in the numbers or activation of DCs after intramuscular injection of AAV. LPS and Anti-CD40 antibody can efficiently recruit DCs and induce complete activation of DCs To further validate our preliminary observation and hypothesis, we proposed to investigate the effect of DC activation on immune responses to FIX following intramuscular injection of AAV1. Intramuscular injection of AAV alone does not affect the number and activation status of DCs. Therefore, we injected mice with LPS and antiCD40 antibody to activate DCs as described. Anti-CD40 antibody has been proven to be a potent activator of DCs, B cells, epithelial cells, etc, causing strong inflammation.22,23 Cohorts of eight to ten week old C57BL/6 mice received intramuscular injection of 1 x 1011 vg of AAV1-hFIX and 6 x 1010 vg of AAV2-hFIX with or without administration of LPS and anti-CD40 antibody (n=4 for each cohort). Twenty-four hours after intramuscular injection of AAV1/AAV2 and/or administration of LPS and anti-CD40 antibody, cells from the draining lymph nodes and spleen of the mice were harvested for detection of cell surface markers by flow cytometry. The size of draining lymph nodes and spleen in the mice that received LPS and anti-CD40 antibody were considerably larger when compared to naïve, untreated mice, or the mice that received only intramuscular injection of AAV. We observed a significant increase in the number of 10 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. CD11c+ cells in both the draining lymph nodes as well as spleen of mice that received LPS and anti-CD40 antibody when compared to naïve and untreated mice (Figure 3A). Significant expression of MHCII and co-stimulatory molecules (CD80 and CD86) was also detected in the CD11c+ cells in both the draining lymph nodes and spleen of the mice that received LPS and anti-CD40 antibody, compared to naïve and untreated mice (Figure 3B). These results demonstrated significantly increased numbers and complete phenotypic activation of DCs in experimental mice following a single administration of LPS and anti-CD40 antibody. Upon activation, DCs express pro-inflammationary cytokines. We thereby examined cytokine expression of the activated DCs to further validate the complete activation of DCs following administration of LPS and anti-CD40 antibody .We analyzed the expression of cytokines in these cells by flow cytometry. We observed that there was a significant increase in the number of CD11c+ cells expressing proinflammatory cytokines IL-6 and IL-12 after administration of either LPS and anti-CD40 antibody (p=0.0111 for IL-12, p= 0.0139 for IL-6) or anti-CD40 antibody alone (p= 0.0030 for IL12) in comparison to CD11c+ cells from naïve and untreated mice (Figure 3C). However, there was no difference in the expression of IL-10 amongst the experimental and naïve groups. This further validated complete activation of these DCs by LPS and anti-CD40 antibody. Activation of DCs does not abrogate the FIX tolerance induced by intramuscular injection of AAV1 11 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. In steady state without inflammatory mediators, DCs are known to capture, process and present antigen to T cells leading to immune tolerance. Upon inflammatory stimulation, DCs are activated to initiate and promote immunity. We thereby deduced that induction of inflammation and DC activation at the same time as AAV injection would have the potential to change the immunological outcome following intramuscular injection of AAV. We then evaluated the effect of DC activation on FIX tolerance induced by intramuscular injection of AAV1 vector. Following intramuscular injection of AAV1 with or without co-administration of LPS and anti-CD40 antibody, we examined levels of human FIX antigen and anti-hFIX antibody in the circulation of the experimental mice . Low levels of anti-hFIX antibody, and equivalent levels of circulating hFIX antigen were detected in the mice regardless of the activation of DCs by co-administration of LPS and anti-CD40 antibody (Figure 4A-B, n=5 for each cohort). The levels of anti-hFIX antibody in mice that received LPS and anti-CD40 antibody are comparable to those that did not receive LPS and anti-CD40 antibody. We are aware of the detection of low levels of anti-hFIX antibody in mice after intramuscular injection of AAV1, which are slightly higher than background anti-hFIX antibody in naïve isogenic mice. Such low levels of anti-hFIX antibodies do not exhibit inhibitory activity.15 We also observed that activation of DCs does not exaggerate the immune response induced by intramuscular injection of AAV2. Eight to ten week old C57BL/6 mice received intramuscular injection of 6 x 1010 vg of AAV2-hFIX with or without anti-CD40 antibody (n=5 for each cohort). We observed barely detectable levels of hFIX antigen levels in both experimental groups. Very high levels of anti-hFIX antibodies were observed which was further increased on immunogenic challenge (Figure 4C-D). 12 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. Furthermore, no change of hFIX antigen levels and anti-FIX antibodies titers in the AAV1 and DC activator-injected mice after immunogenic challenge of rhFIX emulsified in CFA, demonstrated definite immune tolerance to FIX in these mice (Figure 4A-B). On the other hand, injection of rhFIX/CFA in AAV2-injected mice, irrespective of whether they received anti-CD40 antibody or not, increased the titers of anti-FIX antibodies (Figure 4C-D). These results clearly demonstrate that activation of DCs cannot affect the immunological outcome of FIX tolerance induced by intramuscular injection of AAV1. We also examined expression of FIX antigen at early time points after intramuscular injection of AAV1-FIX. We detected human FIX antigen in AAV1injected mice as early as 24 hours after intramuscular injection of AAV1-FIX, which became significant by day 5 (Mean=165.3+/-SEM=55.27 ng/ml, n=5). Detection of immediate expression of FIX antigen at early time points post AAV injection demonstrates exposure of the host immune system to the FIX antigen when DCs are activated by the inflammatory stimulation of LPS and anti-CD40 antibody. DC activation-independent FIX tolerance induced by intramuscular injection of AAV1 is irrespective of genetic and MHC backgrounds of mice It was reported that mice with C57BL/6 are more tolerogenic than other strains of mice such as Balb/c and C3H.24,25 We then moved on to test whether activation of DCs could reverse the immune outcome in mice with different genetic and MHC backgrounds by using Balb/c (H2d) and C3H (H2k) mice. Eight to ten week old Balb/c and C3H mice received intramuscular injection of 3 x 1011 vg of AAV1-hFIX (a dose required to induce 13 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. FIX tolerance)13 alone or with LPS and anti-CD40 antibody (n=5 for each cohort). We examined levels of human FIX antigen and anti-hFIX antibodies in the circulation of the experimental mice. A peak in circulating FIX antigen levels were observed at 8 weeks followed by a stable expression of FIX until week 16 in both Balb/c (Figure 5A) and C3H mice (data not shown). A slight peak in anti-FIX antibodies was observed initially at week 4 followed by very low levels until week 16 in both Balb/c (Figure 5B) and C3H mice (data not shown). The levels of both circulating hFIX antigen and anti-hFIX antibodies were comparable irrespective of whether the DCs were activated with LPS and anti-CD40 antibody or not. DC activation failed to reverse the tolerance observed after intramuscular injection of AAV1 (Figure 5 depict Balb/c results, C3H mice data not shown). This indicates that activation of DCs does not change the immunological outcome in mice with diverse genetic and immunological backgrounds. Discussion Adverse immune responses following gene transfer are considered one of the major causes of the minimal success in gene therapy clinical trials to date.2-4 It is of great importance to elucidate the effect and the extent of gene transfer vector on adverse immunity for successful gene therapy. The nature of the gene transfer vectors is suggested as a foundation accounting for undesirable immune responses subsequent to gene transfer.1,2 Vectors, the majority of which are derived from viruses, are encountered and recognized as “danger signals” by the host immune system. Such viral vectors cause a certain extent of inflammation by stimulating and initiating activation of APCs, leading to unwanted anti-vector and/or transgene product immunity.1,2 Very little is known about 14 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. the exact nature of APCs that are implicated in the initiation of anti-hFIX immune responses subsequent to intramuscular injection of AAV. DCs are the most potent and the major type of professional APCs with a unique T cell stimulatory aptitude.6,7 DCs are the essential link between the innate and the adaptive immune responses.7 The activation status of DCs has been proven to play a major role in determining the immunological outcome. Immature or mature DCs in a steady state have been shown to lead to T cell tolerance.6,9,10 The activation of the DCs and their function in immune regulation, in turn, is dictated by the innate immune responses to different pathogens and the consequent “danger signals”.6,7 Such innate immune responses and the relevant “danger signal” triggers migration, maturation, differentiation and activation of the DCs by significantly up-regulating expression of MHC and co-stimulatory molecules, which are critical in initiating/promoting T cell priming and proliferation for effective immunity.6 Activation of DCs not only precedes adaptive immune responses, it also determines and controls the final outcomes of the immune responses.6 The non-pathogenic AAV vectors elicit negligible inflammation and thus have minimal effect on DC activation.11,12 It is conceivable that inefficiency in activation of DCs would favor lower immunity against vector as well as the transgene product. It was reported that there was insignificant immunity subsequent to AAV gene transfer.26 We hypothesized that inefficiency of DC activation subsequent to AAV gene transfer is a factor that promotes FIX tolerance. The objective of this study was to investigate the effect of activation status of the major APC on humoral immune responses to FIX (formation of inhibitory anti-FIX antibodies or FIX tolerance) following intramuscular AAV gene transfer for hemophilia 15 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. B treatment. The results of our studies first revealed absence of, or inefficient recruitment and activation of DC subsequent to intramuscular AAV gene transfer. This is consistent with the non-pathogenic nature of the AAV vector, thus in support of our hypothesis. If the absence of DC activation were essential for induction of FIX tolerance in the context of intramuscular injection of AAV, activation of the DCs would abrogate the FIX tolerance and elicit anti-FIX immunity. We however observed efficient induction of immune tolerance to FIX subsequent to intramuscular injection of AAV1 regardless of full phenotypic and functional activation of DCs. Such an observation is contrary to our hypothesis and is also inconsistent with the danger signal theory. Accordingly, complete activation of dendritic cells locally and systemically, failed to initiate an anti-FIX immunity or obliterate the immune tolerance to FIX induced by intramuscular AAV1 gene transfer. This indicates the likelihood of the limited role the effect of inflammation, innate immunity and the subsequent activation of DCs plays in determining the immune responses to transgene product in the context of intramuscular gene transfer. It also suggests that the non-pathogenic nature of AAV vectors and the consequent minimal inflammation of AAV gene transfer may not contribute as much to induction of immune tolerance to the transgene product, as postulated. In support of our results, immune tolerance to FIX was also observed in FIX gene transfer using adenoviral and lentiviral vectors targeting the liver.16,27 However, significant inflammation and DC activation was observed subsequent to gene transfer of adenoviral and lentiviral vectors.2,16,27 This also seemed to be inconsistent with the “danger signal” theory of immunology.2,5,6 A body of recent experimental evidence strongly suggests that 16 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. maturation of DCs can also render them to be tolerogenic.6,9,10 We would like to point out that the LPS and anti-CD40 antibody-stimulated DCs in this study, are not only fully matured, but also completely activated. Administration of anti-CD40 antibody also causes a substantial level of general inflammation in addition to complete activation of many types of APCs such as DCs, B cells, even endothelial cells.22,23 To our knowledge, there is no report or proposal suggesting tolerance induction under the condition of complete activation of DCs. Unique characteristics of gene transfer may make immune responses to trangene product in the context of gene transfer distinctive from the immunology of classical protein replacement. As for the immune responses to FIX following intramuscular injection of AAV vector, multiple factors including property of the gene transfer vectors, vector dose, targeting organ/tissue, efficiency and amount of the transgene product expressed, persistent expression of the transgene product, etc. may play a certain part in determining the ultimate immunological outcome. High levels of FIX antigen were reported as critical in deciding the immune responses to FIX following muscular AAV gene transfer.13-15,18 It is however elusive as to whether there is a single decisive factor, or a synergy of several factors that command the eventual immunological outcome in the context of AAV gene transfer. Our results in the current study clearly demonstrated that activation of DCs alone could not reverse induction of FIX tolerance in the context of intramuscular injection of AAV vectors. Ongoing efforts in our laboratory are investigating the interaction among multiple factors such AAV dose, antigen (FIX) level, DC differentiation status, etc in deciding the ultimate immunological outcome to FIX in the context of intramuscular AAV gene transfer. 17 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. It was also reported that the inefficiency of local inflammatory activation by AAV at injection sites could be overcome by an increase of “adjuvant”-like component(s) in the AAV vector preparation upon elevated dose of AAV vectors.20 It was proposed that the intensified local inflammation at the AAV injection site consequently led to higher risk of formation of anti-FIX antibodies.20 Inefficient expression of adequate levels of FIX antigen rather than the local inflammation caused by high dose AAV seemed to play a major role in the higher risk of anti-FIX antibodies formation in that report.13,15,18 In summary, we demonstrated that activation of dendritic cells could not abrogate the FIX immune tolerance induced by intramuscular injection of AAV1 vectors. Our results provided critical insight into not only the immune responses to the transgene product and the relevant mechanisms following gene transfer, but also the universal principles and mechanisms governing general immune responses. Better understanding of the mechanisms involved in the immune responses to FIX following gene transfer will facilitate development of a successful gene therapy approach for hemophilia treatment and FIX tolerance induction. Acknowledgements: The authors claim no conflict of interest. This work is supported by grant NIH R01-HL076699 to HJC. HJC is an NHLBI/NHF researcher. Author contributions: HJC designed the study, analyzed the data and wrote the paper. AB, MK and DK performed the experiments. AB summarized the data and participated in data analysis and paper writing. 18 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. References 1. Hackett NR, Kaminsky SM, Sondhi D, Crystal RG. Antivector and antitransgene host responses in gene therapy. Curr Opin Mol Ther. 2000;2(4):376-382. 2. Brown BD, Lillicrap D. Dangerous liaisons: the role of "danger" signals in the immune response to gene therapy. Blood. 2002;100(4):1133-1140. 3. Manno CS, Pierce GF, Arruda VR, et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med. 2006;12(3):342-347. 4. Raper SE, Chirmule N, Lee FS, et al. Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab. 2003;80(1-2):148-158. 5. Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol. 1994;12991-1045. 6. Diebold SS. Determination of T-cell fate by dendritic cells. Immunol Cell Biol. 2008;86(5):389-397. 7. Mellman I, Steinman RM. Dendritic cells: specialized and regulated antigen processing machines. Cell. 2001;106(3):255-258. 8. Liu YJ, Kanzler H, Soumelis V, Gilliet M. Dendritic cell lineage, plasticity and cross-regulation. Nat Immunol. 2001;2(7):585-589. 9. Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol. 2003;21685-711. 10. Rutella S, Danese S, Leone G. Tolerogenic dendritic cells: cytokine modulation comes of age. Blood. 2006;108(5):1435-1440. 19 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 11. McCaffrey AP, Fawcett P, Nakai H, et al. The host response to adenovirus, helper-dependent adenovirus, and adeno-associated virus in mouse liver. Mol Ther. 2008;16(5):931-941. 12. Stilwell JL, Samulski RJ. Role of viral vectors and virion shells in cellular gene expression. Mol Ther. 2004;9(3):337-346. 13. Cohn EF, Zhuo J, Kelly ME, Chao HJ. Efficient induction of immune tolerance to coagulation factor IX following direct intramuscular gene transfer. J Thromb Haemost. 2007;5(6):1227-1236. 14. Chao H, Monahan PE, Liu Y, Samulski RJ, Walsh CE. Sustained and complete phenotype correction of hemophilia B mice following intramuscular injection of AAV1 serotype vectors. Mol Ther. 2001;4(3):217-222. 15. Kelly ME, Zhuo J, Bharadwaj AS, Chao H. Induction of immune tolerance to FIX following muscular AAV gene transfer is AAV-dose/FIX-level dependent. Mol Ther. 2009;17(5):857-863. 16. Dai Y, Schwarz EM, Gu D, Zhang WW, Sarvetnick N, Verma IM. Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc Natl Acad Sci U S A. 1995;92(5):1401-1405. 17. Ge Y, Powell S, Van Roey M, McArthur JG. Factors influencing the development of an anti-factor IX (FIX) immune response following administration of adeno-associated virus-FIX. Blood. 2001;97(12):3733-3737. 20 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 18. Zhang TP, Jin DY, Wardrop RM, 3rd, et al. Transgene expression levels and kinetics determine risk of humoral immune response modeled in factor IX knockout and missense mutant mice. Gene Ther. 2007;14(5):429-440. 19. Fields PA, Arruda VR, Armstrong E, et al. Risk and prevention of anti-factor IX formation in AAV-mediated gene transfer in the context of a large deletion of F9. Mol Ther. 2001;4(3):201-210. 20. Herzog RW, Fields PA, Arruda VR, et al. Influence of vector dose on factor IX- specific T and B cell responses in muscle-directed gene therapy. Hum Gene Ther. 2002;13(11):1281-1291. 21. Pulendran B, Kumar P, Cutler CW, Mohamadzadeh M, Van Dyke T, Banchereau J. Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo. J Immunol. 2001;167(9):5067-5076. 22. Quezada SA, Jarvinen LZ, Lind EF, Noelle RJ. CD40/CD154 interactions at the interface of tolerance and immunity. Annu Rev Immunol. 2004;22307-328. 23. Maxwell JR, Campbell JD, Kim CH, Vella AT. CD40 activation boosts T cell immunity in vivo by enhancing T cell clonal expansion and delaying peripheral T cell deletion. J Immunol. 1999;162(4):2024-2034. 24. Fields PA, Armstrong E, Hagstrom JN, et al. Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice. Gene Ther. 2001;8(5):354-361. 25. Tulone C, Tsang J, Prokopowicz Z, Grosvenor N, Chain B. Natural cathepsin E deficiency in the immune system of C57BL/6J mice. Immunogenetics. 2007;59(12):927935. 21 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 26. Tenenbaum L, Lehtonen E, Monahan PE. Evaluation of risks related to the use of adeno-associated virus-based vectors. Curr Gene Ther. 2003;3(6):545-565. 27. Follenzi A, Battaglia M, Lombardo A, Annoni A, Roncarolo MG, Naldini L. Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic factor IX in mice. Blood. 2004;103(10):3700-3709. 22 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. Figure Legends Figure 1. Kinetics of dendritic cell activation upon administration of LPS and antiCD40 antibody. Eight to ten week old C57BL/6 mice (n=4 per cohorts) received LPS (100μg/mouse) plus anti-CD40 antibody (100μg/mouse) (red line) or anti-CD40 antibody alone (blue line) or phosphate buffered saline (PBS) (green line). Cells from draining lymph nodes (A) and splenocytes (B) were collected at 6, 24, 72, and 120 hours after injection and stained for cell surface antigens of CD11c (top panel), CD80 (middle panel) and CD86 (bottom panel). The cells were first gated on the CD11c+ population of live cells. Expression of CD80 and CD86 were determined from the gated CD11c population. A representative of 2 independent experiments for each time point is shown. Figure 2. Direct intramuscular injection of AAV vectors does not activate DCs. Eight to ten week old C57BL/6 mice (n=4 per cohorts) received intramuscular injection of 1x1011 vg of AAV1-hFIX or 6x1010 vg of AAV2-hFIX. Control mice of similar age were administered phosphate buffered saline (PBS). After 24 hours, cells from the draining lymph nodes and spleen were collected and stained for the appropriate antibodies and flow cytometry analyses were performed as described. (A) No change in DC numbers in draining lymph nodes and spleen following intramuscular injection of AAV. The dot plots show the expression of CD11c on cells. Numbers indicate the percentage of CD11c+ cells amongst the total live cells in either the draining lymph nodes or spleen. (B) No activation of DCs in draining lymph nodes and spleen following intramuscular injection of AAV. The dot plots show the expression of either 23 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. MHCII and CD80 or MHCII and CD86 on cells gated with CD11c. Numbers indicate the percentage of MHCII+CD80+ or MHCII+CD86+ amongst the CD11c+ population. Data shown are mean ± SEM. A representative of 2 independent experiments is shown. Figure 3. Anti-CD40 antibody and LPS recruits and activates DCs in the draining lymph nodes and spleen. Eight to ten week old C57BL/6 mice (n=4 per cohorts) received intramuscular injection of 1x1011 vg of AAV1-hFIX + 100μg of LPS + 100μg of anti-CD40 antibody (in the footpad) or 6x1010 vg of AAV2-hFIX + 100μg of LPS + 100μg of anti-CD40 antibody (in the footpad) on the same day. Control mice received phosphate buffered saline (PBS). Cells from the draining lymph nodes and spleen were collected after 24 hours, stained with the appropriate antibodies and flow cytometry analysis was performed as described. (A) Increase in DC numbers in draining lymph nodes and spleen following administration of LPS and anti-CD40 antibody in comparison to naïve mice. The dot plots show the expression of CD11c on cells. Numbers indicate the percentage of CD11c+ cells amongst the total live cells in either the draining lymph nodes or the spleen. (B) Activation of DCs in draining lymph nodes and spleen following administration of LPS and anti-CD40 antibody. The dot plots show the expression of either MHCII and CD80 or MHCII and CD86 on cells gated with CD11c. Numbers indicate the percentage of MHCII+CD80+ or 24 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. MHCII+CD86+ amongst the CD11c+ population. Data shown are mean ± SEM. A representative of 2 independent experiments is shown. (C) Cytokine profile of splenocytes after 24 hours following administration of LPS and anti-CD40 antibody. The histograms show the expression of IL-12 (top panel), IL-6 (middle panel) and IL-10 (bottom panel) on cells gated with CD11c. Numbers indicate the percentage of CD11c+ cells that are expressing the particular cytokine. Data shown are mean ± SEM. n= 4 for each cohort. A representative of two independent experiments is shown. Figure 4. Activation of Dendritic cells does not abrogate FIX tolerance induced by intramuscular injection of AAV1 in C57BL/6 mice. Plasma was collected every 4 weeks after AAV and LPS + anti-CD40 antibody injection, and measured for hFIX antigen and anti-hFIX IgG antibodies by ELISA. Data shown are mean ± SEM. n=5 for each cohort. (A) Human FIX antigen. Eight to ten week old C57BL/6 mice received intramuscular injection of 1x1011 vg of AAV1-hFIX alone (Closed square) or with LPS and anti-CD40 antibody (Open square). (B) Anti-hFIX IgG antibodies. Eight to ten week old C57BL/6 mice received intramuscular injection of 1x1011 vg of AAV1-hFIX alone (Closed square) or with LPS and anti-CD40 antibody (Open square). (C) Human FIX antigen. Eight to ten week old C57BL/6 mice received intramuscular injection of 6x1010 vg of AAV2-hFIX alone (Closed square) or with anti-CD40 antibody (Open square). 25 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. (D) Anti-hFIX IgG antibodies. Eight to ten week old C57BL/6 mice received intramuscular injection of 6x1010 vg of AAV2-hFIX alone (Closed square) or with anti-CD40 antibody (Open square). Figure 5. Activation of DCs does not abrogate FIX tolerance induced by intramuscular injection of AAV1 in Balb/c mice. Eight to ten week old Balb/c mice received intramuscular injection of 3x1011 vg of AAV1-hFIX alone (Closed square) or with LPS and anti-CD40 antibody (Open square) (n=5 for each cohort). Plasma was collected every 4 weeks after AAV and LPS + anti-CD40 antibody injection, and measured for hFIX antigen (A) and anti-hFIX IgG antibodies (B) by ELISA. Data shown are mean ± SEM. 26 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 27 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 28 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 29 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 30 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 31 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 32 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 33 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 34 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 35 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 36 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 37 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 38 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 39 of 39 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. Prepublished online November 17, 2009; doi:10.1182/blood-2009-08-239509 Induction of immune tolerance to FIX by intramuscular AAV gene transfer is independent of the activation status of dendritic cells Arpita S. Bharadwaj, Meagan Kelly, Dongsoo Kim and Hengjun Chao Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in the paper journal (edited, typeset versions may be posted when available prior to final publication). Advance online articles are citable and establish publication priority; they are indexed by PubMed from initial publication. Citations to Advance online articles must include digital object identifier (DOIs) and date of initial publication. 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