Immune Tolerance Induction to Factor IX through B Cell Gene Transfer: TLR9 Signaling Delineates between Tolerogenic and Immunogenic B Cells

© The American Society of Gene & Cell Therapy

original article

Immune Tolerance Induction to Factor IX through B Cell Gene Transfer: TLR9 Signaling Delineates between Tolerogenic and Immunogenic B Cells Xiaomei Wang1, Babak Moghimi2, Irene Zolotukhin1, Laurence M Morel3, Ou Cao1 and Roland W Herzog1 Department of Pediatrics, University of Florida, Gainesville, Florida, USA; 2Department of Pediatrics, University of Miami, Miami, Florida, USA; Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, Florida, USA

1 3

A subset of patients with severe hemophilia B, the X-linked bleeding disorder resulting from absence of coagulation factor IX (FIX), develop pathogenic antibody responses during replacement therapy. These inhibitors block standard therapy and are often associated with anaphylactic reactions to FIX. Established clinical immune tolerance induction protocols often fail for FIX inhibitors. In a murine model of this immune complication, retrovirally transduced primary B cells expressing FIX antigen fused with immunoglobulin-G heavy chain prevented antibody formation to FIX and was also highly effective in desensitizing animals with preexisting response. In contrast, transplant of B cells that received the identical expression cassette via nucleofection of plasmid vector substantially heightened antibody formation against FIX, a response that could be blocked by toll-like receptor 9 (TLR9) inhibition. While innate responses to TLR4 activation or to retrovirus were minimal in B cells, plasmid DNA activated TLR9, resulting in CpG-dependent NF-κB activation/IL-6 expression and adaptor protein 3 dependent, CpG-independent induction of IFN-I. Neither response was seen in TLR9-deficient B cells. Therefore, TLR9 signaling in B cells, in particular in response to plasmid vector, is highly immunogenic and has to be avoided in design of tolerance protocols. Received 10 May 2013; accepted 3 March 2014; advance online publication 1 April 2014. doi:10.1038/mt.2014.43

INTRODUCTION Hemophilia B is the X-linked bleeding disorder caused by deficiency of coagulant factor IX (FIX), which in its severe form results in frequent bleeding, pain, reduced quality of life, and early death if left untreated. Current clinical treatment is based on intravenous administration of FIX concentrate. Currently, the most problematic complication is the development of neutralizing antibodies (inhibitors), which compromise therapy, create immunetoxicity, and increase treatment costs. Compared with hemophilia A, little attention is paid on prevention and management of FIX

inhibitors, largely because hemophilia B is less common and inhibitor formation is less frequent.1 Inhibitors to FIX occur in 1.5–3% of hemophilia B patients.1 One recent report showed that out of 282 hemophilia B patients in Italy, 8 patients were found to develop inhibitors, ~2.8%.2 However, several important factors regarding FIX inhibitors should not be overlooked: allergic/ anaphylactic reactions frequently and simultaneously accompany with the appearance of inhibitors in hemophilia B, which rarely occurs in hemophilia A, and complicate attempts to eradicate FIX inhibitors.1 Patients with gene deletions or other null mutations are at elevated risk for inhibitor development, and ~80% of the FIX inhibitors are of high responding type (with Bethesda titers >5 BU (Bethesda unit)), which cause a strong anamnestic response to FIX and precludes the ongoing replacement therapy.1 For those patients, morbidity is more severe and potentially life threatening. Bypass therapy, e.g., in the form of activated factor VII, is available but is of high cost and has to be more carefully monitored to minimize risk of thrombosis.1 When using certain bypass agents such as plasma-derived activated prothrombin complex concentrate, an anamnestic response and anaphylaxis might occur. Some patients become less responsive, and even resistant to high doses of bypass agents. Immune tolerance induction (ITI) protocols, based on daily high dose infusion of factor, can eradicate the inhibitor but may cost >$1 million for an individual patient, presenting a substantial cost to the health care system. Moreover, the success rate for ITI in hemophilia B has been estimated to be ≤30%, and ITI is often terminated because of allergic reactions or nephrotic syndrome.3 In summary, current options of FIX inhibitor management are very limited, especially in patients with the anaphylactoid phenotype, which are predominantly patients with F9 gene deletions or similarly severe mutations. An alternative approach to prevent or treat FIX inhibitors is highly desirable. B cells are not only antibody producers but also play an important role in antigen presentation and immune regulation.4,5 Interestingly, gene-modified autologous primary B cells can induce tolerance to the expressed transgene product upon transplant via processing and major histocompability complex II presentation of the antigen to CD4+ T cells combined with negative costimulation and expression of immune suppressive cytokines such as IL-10.6,7

Correspondence: Xiaomei Wang, University of Florida, Cancer and Genetics Research Complex, 2033 Mowry Road, Gainesville, Florida 32610, USA. E-mail: [email protected] Or Roland W Herzog, University of Florida, Cancer and Genetics Research Complex, 2033 Mowry Road, Gainesville, FL 32610, E-mail: [email protected] Molecular Therapy  vol. 22 no. 6, 1139–1150 jun. 2014

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TLR9 Signaling Delineates between Tolerogenic and Immunogenic B Cells

While not strictly required, expression of the protein antigen as a fusion with immunoglobulin enhances tolerance induction. Ex vivo retrovirally transduced B cells induced tolerance in several murine models of autoimmune diseases including type 1 diabetes, experimental autoimmune encephalomyelitis, uveitis, and the genetic disease hemophilia A.8,9 Limited data are available on alternative vector systems or the impact of innate immune sensing of gene transfer vectors by B cells and its potential effect on tolerance induction. Using an animal model that recapitulates inhibitor formation and anaphylaxis in FIX replacement therapy,10 we sought to develop a tolerogenic B cell approach for hemophilia B. Upon transfer of lipopolysaccharide (LPS)-activated B cells (retrovirally transduced with IgG-FIX fusion gene, which we found to elicit minimal innate responses in B cells), inhibitor formation against FIX and anaphylaxis was entirely prevented. Furthermore, inhibitors were reversed to low-titer in mice with preexisting immune response, and animals were successfully desensitized. In parallel, we tested our recently optimized protocol for plasmid DNA gene transfer to primary B cells,11 since this nonviral method could be used to employ site-specific integration systems in the future and thus minimize risks of insertional mutagenesis, which are a concern for retroviral vectors. However, nucleofected B cells were immunogenic, increasing anti-FIX responses in a toll-like receptor 9 (TLR9)-dependent manner. TLR9-MyD88 signaling in response to plasmid DNA activated the classical NF-κB pathway and induced expression of the proinflammatory cytokine IL-6 and adaptor protein 3 (AP3) dependent expression of IFN-I. Hence, whether expression of the IgG fusion protein is tolerogenic depends on the context, and TLR9 activation in B cells has to be avoided as it provides a signal for antibody formation.

RESULTS Prevention of FIX inhibitor development and fatal anaphylaxis reaction in hemophilia B mice In this study, we applied a B-cell based gene therapy approach for tolerance induction to block or reverse inhibitor formation in a C3H/HeJ mouse model of hemophilia B (F9 gene deletion), which is unique in not only developing higher-titer inhibitors but also life-threatening anaphylactic reactions to FIX administration, therefore representing the clinical situation of hemophilia B patients with inhibitors more closely than other strains.10,12 C3H/HeJ F9−/− mice form IgG1 and IgE upon repeated exposure to recombinant human FIX protein and show fatal anaphylaxis.10,12 LPS-activated B cell blasts were transduced with retroviral vector containing either full-length mature (IgGFIX1) or a truncated version of hFIX (IgG-FIX2; to decrease the transgene length and increase the retroviral titers) in frame with a murine IgG1. Transduced cells were injected into hemophilia B C3H/HeJ mice (1 × 107 cells/mouse), followed by challenge with 1 IU of hFIX as outlined in Figure 1a. Because C3H/HeJ mice lack functional TLR4,13 the receptor for LPS, primary B cells derived from C3H/HeOuJ mice were used for gene transfer and subsequent transplant. B cells transduced with IgG retrovirus were used in control group. All vectors, containing a green fluorescent protein (GFP) reporter in addition to the IgG transgene, 1140

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transduced murine B cells at 15–30% with high cell viability of ~90% as published (data not shown).11 As shown in Figure 1b,c, a single injection of IgG-FIX transduced B cells resulted in complete lack of inhibitor formation (<1 BU) upon subsequent FIX replacement therapy, with Bethesda titers reduced by 24- to 30-fold compared to controls at the 8-week time point (Figure 1b). Consistent with these results, IgG1 formation against FIX was reduced by 35- to 37-fold at that time point for both versions of IgG-FIX (Figure 1c). Challenge with FIX protein was initially perform by intraperitoneal (i.p.) injection to minimize death from anaphylaxis. Subsequent injections were via the clinically more relevant intravenous route. Consequently, 50% of control mice died after 7 weeks of FIX challenge due to anaphylaxis, while mice receiving IgG-FIX transduced B cells lacked anaphylaxis and showed 100% survival (Figure 1d). In order to test whether lower B cell doses may be effective, 5 × 106, 1 × 106, or 5 × 105 IgG-FIX2 transduced cells were transplanted into hemophilia B mice prior to treatment with FIX. IgGFIX2 vector with truncated FIX sequence was used for these and all subsequent experiments as it was as effective as full length but consistently yielded higher-titer virus. As shown in Figure 1e,f, suppression of inhibitor formation was dose dependent, with 1 × 106 cells showing intermediate level of suppression, while 5 × 105 cells only marginally suppressed inhibitor formation. However, inhibitor formation in these suboptimally treated mice was reversible by a second, high cell dose (Figure 1g,h).

Suppression of inhibitor formation in FIX primed hemophilia B mice Next, we wanted to test whether tolerogenic B cell therapy would be effective in animals with established inhibitor formation, which would indicate that this method could be used in place of current ITI protocols (which are not effective in the majority of FIX inhibitor patients). As outlined in Figure 2a, C3H/HeJ F9−/− mice were first given FIX protein therapy for 5 weeks, and antihistamine was given at indicated time points to prevent fatal anaphylaxis. The mice were then randomly divided into two groups (receiving either IgG or IgG-FIX transduced B cells). As shown in Figure 2b,c (time point 2), animals receiving IgG-FIX2 fusion expressing B cells (referred to as “desensitization experiment” from here on) had markedly decreased inhibitor and anti-FIX IgG1 titers; whereas animals receiving B cells expressing IgG control had insignificant changes of inhibitor/antibody levels. Both groups of mice were further challenged with FIX protein therapy for up to 6 weeks (without antihistamine treatment). In the IgG-transduced control group, 8 out of 11 mice died during this time, apparently because of anaphylaxis. In contrast, the animals in the IgG-FIX2 transduced group had no overt signs of anaphylactic reactions, and only 1 out of 10 mice was lost during this time (Figure 2d). Consistent with lack of anaphylaxis, anti-FIX IgE levels were significantly reduced by tolerogenic B cell therapy (Figure 2e). Inhibitor titers in IgG-FIX2 transduced mice remained low-titer (2.4 ± 1 BU); in contrast, the three surviving mice in the control group had developed 21.2 ± 4.3 BU (Figure 2b, time point 3). The anti-FIX IgG1 titers were also significantly suppressed by IgG-FIX transduced B cells (Figure 2c, time point 3). Similarly, frequencies of anti-FIX secreting cells were substantially reduced in both bone www.moleculartherapy.org  vol. 22 no. 6 jun. 2014

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TLR9 Signaling Delineates between Tolerogenic and Immunogenic B Cells

marrow and spleens (Figure 2f). Taken together, these results indicate that B cell-based gene therapy is a promising strategy not only for prevention but also treatment of FIX inhibitors. To address whether the tolerance mechanism involves active suppression, we first determined the frequency of CD25+FoxP3+ cells among CD4+ T cells following B cell transplant and challenge with FIX protein. Mice that received retroviral transduced B cell blast therapy (naive or with preexisting inhibitor) showed a minor, not statistically significant increase in overall Treg frequency (Figure 2g). However, antigen-specific Treg typically exist in low numbers. Therefore, we tested for FIX-specific Treg induction using adoptive transfer. CD4−, CD4+CD25−, and CD4+CD25+ spenocytes were purified by magnetic sorting, followed by transfer into naive strain-matched mice and immunization with FIX in adjuvant. Substantial suppression of anti-FIX formation was obtained by transfer of CD4+CD25+ cells from mice that had received

a hF.IX IV (once/week)

Transduced B cell injection Week: 0

1

2

3

4

5

1 6

7

2 8

hF.IX IP (once/week)

F.IX inhibitor titer (BU/ml)

b

retroviral transduced B cells prior to or after exposure to FIX protein. CD4+CD25+ cells from naive control or from mice that had received plasmid-nucleofected B cells showed marginal or no suppression, respectively. CD4− or CD4+CD25− cells consistently failed to suppress. Interestingly, CD4+CD25− cells from the nucleofection group, likely containing FIX-specific effector T cells, increased antibody formation (although not reaching statistical significance).

Nucleofection mediated IgG-FIX gene transfer to B cells increased immune responses against FIX Given concerns about insertional mutagenesis with retroviral vectors, alternative gene transfer methods for primary B cells are desirable. We recently optimized plasmid gene transfer using nucleofection, resulting in transgene expression in 65–75% of LPSactivated B cells while retaining ~80% cell viability.11 This protocol was used to nucleofect C3H/HeOuJ B cells with the plasmid

30

IgG IgG-F.IX 1 IgG-F.IX 2

20

P < 0.01 P < 0.01

P < 0.01

10

P < 0.01

0 8 Weeks

6 Weeks

d

25,000

15,000

P < 0.01

10,000

P < 0.01

P < 0.01

P < 0.01

5,000

75 50 25 0

IgG-F.IX 2

IgG-F.IX 1

IgG

6 Weeks

8 Weeks

IgG

IgG-F.IX 1

IgG IgG-F.IX 2

15,000 P < 0.05

10,000 5,000

P < 0.01 P < 0.01

0

IgG

5

5×

10

6

6

1×

10

5×

10

7

10

Fix inhibitor titer (BU/ml)

20,000

20 10

P < 0.01 P < 0.01 P < 0.01

0

IgG

IgG-F.IX 2

g

25,000

30

8 Weeks

5

5×

10

6

1×

10

6

5×

10

7

10

IgG-F.IX 2

IgG

h 20 5 × 105 1 × 106

15 10 5

P < 0.05 P < 0.05

0 First therapy

Second therapy

IgG1 anti-FIX (ng/ml)

6 Weeks

IgG1 anti-FIX (ng/ml)

F.IX inhibitor titer (BU/ml)

20,000

0

f

e 100

Survival (%)

IgG1 Anti-F.IX (ng/ml)

c

25,000

5 × 105 1 × 106

20,000 15,000 10,000 5,000 0

P < 0.05

P < 0.05

First therapy Second therapy

Figure 1 Retrovirally transduced B cells expressing IgG-FIX fusions completely prevent inhibitor formation and anaphylaxis in hemophilia B mice (C3H/HeJ with targeted F9 gene deletion) receiving hFIX replacement therapy. (a) Experimental outline and time course. Numbers in circles indicate time points for blood draws 1 and 2. IgG-FIX1 represents the retrovirus containing the IgG fusion with full length mature FIX; IgG-FIX2 represents the retrovirus containing the IgG fusion with truncated FIX. (b) Inhibitor titers at weeks 6 and 8 of the experiment. (c) IgG1 anti-FIX titers compared to control mice that received IgG-transduced primary B cells from C3H/HeOuJ mice. Data are average ± SEM. (d) Survival of experimental groups (n = 6/group except for IgG-FIX1, which was n = 3; all animals were treated with 107 B cells). (e,f) Tolerance induction as a function of B cell dose. Inhibitor titers (e) and IgG1 anti-FIX titers (f) at week 8 in mice injected with different doses of IgG-FIX2 transduced B cell blasts (5 × 105–5 × 106, n = 4/group; for comparison, mice from panels b and c for treatment with 107 cells were shown again for comparison). (g) Inhibitor titers and (h) anti-FIX titers 1 month after secondary B cell therapy of IgG-FIX2 transduced 5 × 106 cells (and additional IV challenge with FIX) in those mice that had had developed inhibitors after initially receiving suboptimal doses of 5 × 105–1 × 106 B cells. FIX, coagulation factor IX.

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TLR9 Signaling Delineates between Tolerogenic and Immunogenic B Cells

hFIX IP boost hFIX IV with anti-histamine (once/week)

50

3

2

10

10 0

0 Weeks: 4

9

9

4

14

40,000

40,000

30,000

30,000

20,000

20,000

1

3

2

10,000 0 Weeks: 4

0 14

2

4

9

14

10

10

4

4

10

3

10

7.82%

20

4

10

6

5

k

10 0

Bone marrow

9.39%

1.66%

5

k

P < 0.01

4

10

Spleen

12 9

+

10

2.05%

5

W ee

P < 0.01

30

Desensitization

NF plasmid

11.36%

1.38%

5

IgG-FIX 2

IgG IgG-FIX 2

40

IgG IgG-FIX 2

8.35%

2.03%

4

0

f

1

k

W ee

10

IgG

10,000

9

10

3

4

10

5

10

10

2

1

2

3

–10 10 10

4

10

10

0

5

Co

D

N

CD25

l

ro

nt

10

n

2

–10 10

io

1

5

10

id

4

10

m

3

at

10

tiz

2

3

si

1

–10 10

1.65%

en

5

10

87.31%

es

4

10

−10

2

3

10

2.32%

IX

2

87.81%

as

1

–10 10 10

2

10 2 −10

2.74%

pl

2

2

84.52%

6

F

87.77%

3

10

-F

2

10 2 1.86% −10

2

3

G

10

Ig

3

10

+

Foxp3

P < 0.01

20

6

50,000

IgG

10

30

IgG1 anti-FIX SFU/10

IgG1 anti-FIX (ng/ml)

50,000

g 10

40

3

IgG-FIX 2

5

14

IgG

IgG-FIX 2

c

e

IgE anti-FIX (ng/ml)

1

W ee

2 1

W ee

20

1

20

0

3

k

FIX inhibitor titer (BU/ml)

30

IgG-FIX 2 IgG

25

b 30

P < 0.01

75

3 15

2 9

8

CD25 FOXP3 /CD4 (%)

7

3

6

+

1 5

4

k

3

W ee

2

2

1

k

0

100

Survival %

Week:

d

hFIX IV (once/week)

Transduced B cell injection

hFIX IP

W ee

a

h

P < 0.01

P < 0.01

IgG1 anti-FIX (ng/ml)

60,000

P < 0.05

P < 0.05

50,000

P < 0.001

40,000 P < 0.05

30,000

P < 0.05 Control

P < 0.01

IgG-FIX 2 20,000

NF plasmid

10,000

Desensitization

0 –

+

–

4

25

25

CD

+

4

CD

CD

+

4

CD

CD

–

25

25

+

4

CD

CD

+

4

CD

CD

–

–

+

–

4

CD

+

4

CD

–

+

25

4

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25

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CD

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CD

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CD

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4

CD

CD

Figure 2 Retrovirally transduced B cells expressing IgG-hFIX2 fusion reverse inhibitor formation and desensitize hemophilia B mice with preexisting response to FIX. (a) Experimental outline and time course. Numbers in circles indicate time points for blood draws 1–3. (b) Inhibitor titers at weeks 5, 9, and 15 of the experiment compared to control mice that received IgG-transduced B cells. (c) IgG1 anti-hFIX titers. Each line in a–c represents individual animal (n = 10–11/group; all animals were treated with 107 B cells). (d) Survival of experimental groups as a function of the number of weekly hFIX administrations. (e) Serum IgE anti-hFIX titers at time point 3. (f) B cell ELISpot for IgG1 anti-hFIX secreting cells in bone marrow and spleens of experimental and surviving control mice. (e–f) Data are average ± SEM. (g) The frequency of CD25+FoxP3+ cells gated on CD4 in splenocytes of control mice, IgG-FIX2 retroviral transduced B cell treated mice (IgG-FIX2), plasmid-nucleofected B cell treated mice (NF plasmid), or primed mice treated with IgG-FIX2 retroviral transduced B cell therapy for desensitization (n = 4–6/group, all animals were treated with 107 B cells). (h) Plasma levels of IgG1 anti-hFIX 3 weeks after immunologic challenge by subcutaneous administration of 1 IU FIX formulated in adjuvant in C3H/ HeJ mice that had received adoptive transfer of CD4−, CD4+CD25−, and CD4+CD25+ cells from naive (control), IgG-FIX2 retroviral transduced B cell therapy mice (IgG-FIX2), NF plasmid B cell blasts treatment mice (NF plasmid), or already primed mice treated with IgG-FIX2 retroviral transduced B cell therapy (desensitization). FIX, coagulation factor IX.

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TLR9 Signaling Delineates between Tolerogenic and Immunogenic B Cells

Among the 26 genes tested (including innate immunity receptors, B cell costimulatory markers, inflammatory, or immune regulatory cytokines; see Table 1), IL-6 and type I interferon (IFN-α/β) levels were highly upregulated in nucleofected B cells compared with retroviral infected B cells by quantitative reverse transcription-PCR analysis (Table  1 and Figure 3c) and by cytokine enzyme-linked immunosorbent assay (ELISA; Figure 3d). Mock nucleofection produced only a minimal levels of IL-6 expression and no IFN-I, indicating that the plasmid DNA was required for the response. LPS activation by itself (which occurs via TLR4 signaling) had no effect. Marked induction of IL-6 expression in response to plasmid nucleofection was also observed in primary human B cells (Figure 3e,f). In contrast to murine cells, human B cells secreted low levels of IFN-α, and IFN-β expression was undetectable for both mRNA and protein. It is known that plasmid DNA is sensed by the endosomal receptor TLR9, which can result in proinflammatory and in

encoding the retroviral MSCV-IgG-FIX2 expression cassette. Since plasmid vectors are episomal and thus likely to be lost in proliferating B cells, we suspected that transplanted B cells might fail to induce tolerance in C3H/HeJ FIX−/− mice. To our surprise, antiFIX formation following subsequent treatment with FIX protein was markedly increased compared to control mice (Figure 3a), indicating that B cells expressing IgG-FIX from the plasmid vector had sensitized the hemophilic mice to FIX. Western blot analysis showed that FIX-IgG expression was similar in plasmid-nucleofected B cells and retrovirally transduced B cells (Figure 3b).

Nucleofected B cell blasts show TLR9-dependent expression of IL-6 and IFN-I To delineate the underlining mechanisms of the different immunological outcomes between retroviral and plasmid gene transfer, we performed explorative reverse transcription-PCR array analysis.

a IgG1 Anti-FIX (ng/ml)

30,000

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plgG-FIX 2

25,000

Control

20,000

Retrovirus

15,000

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225 200 175 150 125 100 75 50 25 0

Cytokine level (pg/ml)

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Week 6

IFNα

0

IFNβ

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P < 0.01 LPS NF mock NF plasmid

150 100

P < 0.05 50 0

0 IL-6

IFNα2

IFNα4

IL-6

IFNα

Figure 3 B cells nucleofected with plasmid vector containing the retroviral IgG-hFIX expression cassette are immunogenic. (a). IgG-hFIX2 nucleofected B cells increase the antibody response against hFIX after subsequent replacement therapy (5 weekly injections of hFIX protein; n = 3 per group). (b) Western blot for hFIX and β-actin of B cells retrovirally transduced or nucleofected with plasmid. (c–d) Compared to LPS only stimulated or retrovirally transduced or mock-nucleofected mice B cells, plasmid-nucleofected mice B cells show robust induction of mRNA (c) and protein (d) for cytokines IL-6, IFN-α, and IFN-β. (e–f) Plasmid-nucleofected human B cells show robust induction of mRNA (e) and protein (f) for IL-6, modest induction of IFN-α compared to LPS only stimulated or retrovirally transduced or mock-nucleofected human B cells. Cytokine analyses were performed after 48 hours of B cell culture. All experiments were performed in triplicate. Data are average ± SEM. Mice B cells had been isolated from C3H/HeOuJ mice. FIX, coagulation factor IX.

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Table 1 Gene expression profile in retroviral transduced and nucleofection transfected B cell blasts Gene

Retrovirus

NF mock

TLR1

1.93

1.49

NF plasmid

1.16

TLR2

1.52

1.29

2.09

TLR3

1.44

2.23

6.29

TLR4

1.39

1.89

2.14

TLR5

nd

nd

nd

TLR6

1.63

1.30

1.24

TLR7

2.37

2.12

3.62

TLR8

nd

nd

nd

TLR9

1.49

1.82

1.48

Myd88

1.9

1.11

1.11

CD25

3.89

2.60

4.25

CD40

2.52

1.75

1.5

CD80

1.12

2.17

3.74

CD86

3.4

1.78

3.76

MHC class II

1.33

1.07

1.12

IL-2

nd

nd

Nd

IL-4

nd

nd

nd

IL-6

1.61

7.87

40.58

IL-10

2.09

2.30

9.48

IL-13

nd

nd

nd

IL-17a

nd

nd

nd

IFNα2

0.31

1.64

173.4

IFNα4

1.05

1.76

92.05

IFN-β1

1.93

1.97

149.81

IFN-γ

nd

nd

nd

TGF-β

0.84

1.06

0.99

MHC, major histocompability complex; nd, not detected (transcript levels at or below limit of sensitivity). Numbers in bold indicated greater than twofold upregulation compared to LPS only treatment.

type I IFN expression.14 Further, TLR9 agonists were reported to interfere with regulatory B cells.15 To test the hypothesis that the response to nucleofection of plasmid DNA is TLR9-dependent, we conucleofected an oligodeoxynucleotide (ODN) inhibitor of TLR9 (ODN 2088) or passive control ODN (20 μmol/l) with the pMSCV-FIX-IgG-2 plasmid vector. As shown in Figure 4a,b, IL-6 and type 1 IFN mRNA and protein levels were significantly suppressed by TLR9 inhibition. Furthermore, B cells from TLR9−/− mice failed completely to produce these cytokines in response to plasmid nucleofection (Figure 4c,d).

TLR9 mediates the immunogenicity of nucleofected B cell blasts In order to further investigate the role of TLR9 in the response to FIX in vivo, 100 μg ODN control or ODN 2088 were coadministrated intravenously with nucleofected B cell therapy, followed by weekly challenge with FIX protein (Figure 5a). Compared to mice that received FIX therapy but no B cells, plasmid-nucleofected B cells again robustly increased anti-FIX formation by ~sixfold in 1144

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the absence of TLR9 inhibition (Figure 5b,c). However, TLR9 inhibition effectively suppressed antibody/inhibitor formation to FIX, which was reduced to a level similar to those in mice that received FIX but no B cells. Frequencies of anti-FIX antibody forming cells in bone marrow and spleens were similarly reduced by TLR9 inhibition (Figure 5d). Therefore, blockage of TLR9 signaling abrogated the immunogenic phenotype of B cells nucleofected with plasmid DNA (which, however, still failed to suppress inhibitor titers below the level seen in control mice).

Nucleofection of plasmid DNA triggers the TLR9 signaling pathway, resulting in NF-κB activation and AP3-dependent expression of IFN-I Our data thus far show that the TLR9 pathway mediates an immunogenic B cell phenotype upon nucleofection with plasmid DNA. However, neither expression of TLR9 nor of its adaptor MyD88 changed significantly in nucleofected compared to retrovirally transduced B cell blasts (Table 1; Figure 6a). We performed coimmunoprecipitation to determine the association between TLR9 and MyD88, which would indicate TLR9 activation.16,17 As shown in Figure 6a, the interaction between TLR9 and MyD88 was indeed markedly increased in nucleofected plasmid B cell blasts compared with the other groups. It is known that downstream of TLR9-MyD88, there is a bifurcation of the signaling pathway. One path leads to the transcriptional activation of proinflammatory cytokine production through NF-κB; the other leads to the activation of type I IFN gene following AP-3 mediated translocation to a different endosomal compartment.18 We found that both pathways were activated in plasmid treated B cells. Phosphorylation of NF-κB p65 was substantially upregulated (Figure 6b). In contrast to wild-type mice, B cells derived from AP3−/− mice failed to upregulate IFN-I expression upon plasmid nucleofection, while induction of IL-6 expression was comparable to wild-type mice, indicating that AP-3 was required to connect to the IFN signaling pathway (Figure 6d,e). Interestingly, B cell activation with CpG ODN, a commonly used ligand for TLR9, induced IL-6 but not IFN-I in wild-type mice (Figure 6e), indicating that CpG ODN and plasmid nucleofection activate TLR9 differently. To further define the role of CpG, we compared the response to nucleofection of a CpG-rich and a matched CpG-free plasmid (same elements but with all CpG motifs edited out). The plasmid lacking CpG failed to induce IL-6 but showed only a minor reduction in the IFN-I response (Figure 6f,g). Therefore, induction of IL-6 but not IFN-I is CpG dependent.

DISCUSSION Gene modified B cell therapy: a potentially superior ITI for hemophilia B Through their dual function as professional antigen presenting cells and producers of immunoglobulin, B lymphocytes play a key role in antibody formation against therapeutic proteins used to treat genetic disease. Therefore, complete or partial B cell depletion can be useful to control antibody formation, albeit at the cost of transient or prolonged immune deficiency, depending on the protocol.19–21 However, B cells also have important immune regulatory functions. Thus, transplant of retrovirally gene modified primary B cells resulted in complete prevention of inhibitor formation and of www.moleculartherapy.org  vol. 22 no. 6 jun. 2014

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Figure 4 Inflammatory and IFN-I cytokine responses to plasmid nucleofection in B cells is TLR9 dependent. (a,b) Conucleofection of ODN 2088 (which is inhibitory to TLR9 signaling) significantly reduces the cytokine response to plasmid DNA in primary B cells. (a) Induction of IL-6, IFN-α, and IFN-β mRNA. (b) Induction of IL-6, IFN-α, and IFN-β protein. (c,d) Cytokine responses (c: mRNA; d: protein) are absent in TLR9-deficient mice. Cytokine analyses were performed after 48 hours of B cell culture. All experiments were performed in triplicate. Data are average ± SEM. Wild-type (WT) and TLR9−/− mice were C57BL/6. FIX, coagulation factor IX; ODN, oligodeoxynucleotide; TLR, toll-like receptor.

anaphylaxis against FIX in a murine model of severe hemophilia B due to F9 gene deletion. Moreover, the protocol successfully desensitized animals that had formed inhibitors and IgE after treatment with FIX and thus prone to fatal anaphylactic reactions. Therefore, the approach represents a potentially superior alternative to current clinical ITI for the treatment of FIX inhibitors. The method of retroviral transfer of an IgG fusion to B cells for tolerance induction was pioneered by David Scott’s group.8,22 Originally designed for cell-mediated autoimmune disease, this approach has been shown to control inhibitor formation against F.VIII in hemophilia A mice.23 Peptide-IgG processing of the IgG fusion protein in late endosomes followed by major histocompability complex II presentation under CTLA-4 mediated negative costimulation leads to induction of CD4+CD25+FoxP3+ Treg and deletion of effector T cells.7,24,25 FIX is a vitamin K-dependent glycoprotein. During normal biosynthesis, the N-terminal prepropeptide sequence is cleaved off prior to secretion, resulting in a circulating mature protein of 415 amino acids. While T cell epitopes have not yet been mapped in humans, the dominant CD4+ T cell epitope in three different strains of mice, including C3H/ HeJ, cluster in the catalytic domain between residues 222 and 283.26,27 Both our IgG fusion constructs (complete mature FIX or truncated at the N-terminus) include these epitopes. Prior studies showed Treg-dependent tolerance induction and prolonged interactions between the tolerogenic B cells and Treg.28 In addition, Molecular Therapy  vol. 22 no. 6 jun. 2014

IgG molecules contain epitopes that stimulate Treg.29 Our adoptive transfer experiments demonstrate B cell-mediated induction of Treg in prevention and reversal models, resulting in active suppression of antibody formation. It appears that after establishment of tolerance with transplanted B cells, suppression of antibody formation was mostly performed by induced Treg, which remained functional after transfer to mice that lacked transduced B cells.

Retroviral gene transfer elicits minimal innate responses in B cells In contrast to interactions with T cells, data on innate immune signaling during genetic modification of B cells or its impact on adaptive responses are limited. Certainly, innate immune pathways can modulate B cell maturation and function, including antibody production against coagulation factors.15,30,31 Retroviral gene transfer to LPS-stimulated B cells caused only subtle changes in the expression of genes related to innate responses with few genes being upregulated to twofold to fourfold. These data support the interpretation that limited innate immune responses, resulting in a lack of activation signals, allow the IgG fusion antigen to be presented in a tolerogenic fashion. Our results on modest (twofold) increase in expression of the immune regulatory cytokine IL-10 in retrovirally transduced B cells are consistent with data by others, who found colocalization of TLR2 and the cellular receptor for the retrovirus, thereby triggering TLR2 activation, which in 1145

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Figure 5 Conucleofection with TLR9-inhibitory ODN eliminates immunogenicity of plasmid vector in B cell transplant. (a) Experimental outline and time course. Number in circle indicates time point for blood draw. Hemophilia B mice (C3H/HeJ FIX−/−; n = 3–4/ experimental group) received PBS or 107 nucleofected C3H/HeOuJ B cells on day 0. Nucleofection was performed using the plasmid encoding the IgG-FIX2 expression cassette and a control ODN or ODN 2088 (which inhibits TLR9). Anti-hFIX formation upon subsequent hFIX replacement therapy was measured by (b) IgG ELISA and (c) Bethesda assay. B cell ELISpot for IgG1 anti-hFIX secreting cells in bone marrow and spleen. Data are average ± SD. ELISA, enzyme-linked immunosorbent assay; FIX, coagulation factor IX; ODN, oligodeoxynucleotide; PBS, phosphate-buffered saline; TLR, toll-like receptor.

turn caused an epigenic modification of the IL-10 promoter.32 The exact role of IL-10 in this model is under debate.33,34

TLR9 signaling in response to plasmid DNA confers an immunogenic phenotype to B cells via NF-κB activation and AP-3 dependent IFN-I induction TLRs recognize pathogen associated molecular patterns.35 For example TLR4 binds to lipopolysaccharides of gram- bacteria but may also recognize blood-born viral particles.36,37 TLR2 is a receptor for a diverse set of microbial (bacterial, viral, and fungal) 1146

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substances. Yet, the B cell gene transfer experiments show that stimulation of neither increases immune responses or negatively effects the ability to induce tolerance to FIX, eliciting only minimal innate responses in B cells. Hence, signaling through TLRs does not generally render B cells immunogenic. Others found that B cells activated with the TLR9 ligand CpG, in contrast to LPS (a TLR4 ligand) activated B cells, failed to induce tolerance to the retrovirally encoded transgene product.28 Moreover, B cells expressing IgG-FIX fusion from plasmid vector after nucleofection not only failed to tolerize but heightened antibody formation against FIX, which was TLR9 dependent. Therefore, whether expression of IgG fusion in B cells is tolerogenic is context dependent, as TLR9 activation can drive immunogenic antigen presentation. The spectrum of responses is dependent on how TLR9 is activated. Nucleofection of plasmid vector yielded a response pattern distinct from CpG ODN, which failed to induce IFN-I expression. Data with a CpG-depleted molecule reveal that plasmid DNA vectors induce TLR9- and CpG-dependent IL-6 expression as well as TLR9-dependent but CpG independent IFN-I expression in murine B cells. Therefore, a different property of the plasmid DNA molecule is responsible for signaling through TLR9 to the IFN response pathway. TLR9 resides in endoplasmic reticulum endosomes and detects unmethylated cytosine guanine dinucleotide motifs that are typically present in viral or bacterial DNA.38 The pivotal function of TLRs is the induction of immune responses via intracellular signaling pathways including the NF-κB and interferon regulatory factor transcription factors, resulting in secretion of proinflammatory cytokines or type I IFN or both.39,40 IFN-I is a potent inducer of antiviral innate immunity but also plays an important role in adaptive responses, including antibody formation and CD8+ T cell functionality. Our data show that TLR9 sensing of the plasmid vector in B cells activated the NF-κB pathway and induced AP-3 dependent expression of IFN-I (Figure 7). This was entirely due to binding of existing TLR9 to its adaptor MyD88 without increase in expression of either. Downstream signaling reflects bifurcation of the pathway. While activation of the NF-κB pathway by endosomal TLR9 causes the induction of proinflammatory cytokines, AP-3 can redirect TLR9 to the LAMP2+ lysosome-related organelle compartment, resulting in interferon regulatory factor 7 activation and transcription of IFN-I genes.18,39 In contrast to nucleofected plasmid vector, ligation of CpG ODN failed to direct TLR9 into this second arm. Interestingly, plasmacytoid dendritic cells (pDC) produce IFN-I in response to activation by CpG ODN, indicating that this ligand has different effects in different antigen presenting cells.41 Use of plasmid DNA should be helpful in studying the role of TLR9-induced IFN-I in B cells. It is known that IFN-I can enhance antibody production and IgG class switching in vivo and thus likely contributed to the increased anti-FIX response.42 Interestingly, expression of the immune suppressive cytokine IL-10 was more robustly induced by the plasmid vector than the retrovirus, which however failed to downregulate antibody formation, likely because of overriding effects by the very highly induced IL-6 and IFN expression. IL-6 is a potent proinflammatory cytokine produced by macrophages and other cell types and is involved in innate/acute phase www.moleculartherapy.org  vol. 22 no. 6 jun. 2014

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Figure 6 Plasmid nucleofection induces TLR9 binding to MyD88 and activation of NF-κB pathway. Protein extracts were prepared from B cells that had been LPS stimulated only (control), retrovirally transduced, or nucleofected (mock or with plasmid DNA). (a) Western immunoblot (IB) for MyD88 or TLR9 following immunoprecipitation (IP) with anti-TLR9, anti-MyD88, or control IgG. (b) Western blot for phosphorylated and total p65. (c–d) Cytokine responses (c: mRNA; d: protein) in AP-3 deficient mice. Cytokine analyses were performed after 48 hours of B cell culture. Wild-type (WT) and AP-3−/− mice were C57BL/6. (e) Cytokine mRNA levels in B cells upon activation with CpG ODN (after 48 hours of culture). (f,g) Cytokine (f: mRNA, g: protein) levels in B cells nucleofected with mock, CpG-rich plasmid, or CpG-free plasmid. All experiments were performed in triplicate. Data are average ± SEM. AP, adaptor protein; ODN, oligodeoxynucleotide; TLR, toll-like receptor.

responses. Furthermore, IL-6 secreted by DCs can make antigenspecific T cells refractory to suppression by Treg.43,44 IL-6 can also increase antibody formation and may be produced by Th2 cells. For example, we found IL-6 to be a component of the response to F.VIII in some strains of hemophilia A mice.20,45 Further studies may identify additional proinflammatory cytokines, not included in our array, that are produced by B cells in response to TLR9 activation by plasmid DNA.

FUTURE DIRECTIONS Going forward toward translational studies for tolerance induction in hemophilia B or other inherited protein deficiencies, Molecular Therapy  vol. 22 no. 6 jun. 2014

development of methods for ex vivo gene transfer to B cells should be done in a fashion that limits innate immune responses, with particular emphasis on avoiding TLR9 activation. Given that proinflammatory cytokine expression in response to plasmid DNA was CpG dependent and that human B cells produced weaker IFN-I responses, it should be feasible to design CpG-depleted tolerogenic DNA vectors, which may incorporate site-specific integration technology. Alternatively, retroviral vectors with reduced genotoxicity or lentiviral vectors targeting human B cells could be optimized. As a safety feature, a suicide gene could be incorporated to eliminate transplanted gene modified B cells, which can persist at low frequency for long periods of time.46 Our 1147

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Figure 7 Model for TLR9 signaling in primary B cells in response to plasmid nucleofection. Plasmid molecules are sensed by the endosomal DNA receptor TLR9. The resulting conformational change in TLR9 recruits the cytoplasmic adapter MyD88, resulting in induction of proinflammatory cytokine gene expression via the NF-κB pathway. The AP-3 complex interacts with activated TLR9 and facilitates its trafficking to the IRF7 endosome, whereby TLR9/MyD88 is able to activate interferon response factors that induce transcription of type I IFN genes. AP, adaptor protein; IRF, interferon regulatory factor; TLR, toll-like receptor.

data support the utility of repeated cell dosing, which may be required for complete reversal of inhibitors.

MATERIAL AND METHODS Mice. C3H/HeOuJ, C57BL/6 and B6Pin.C3-Ap3b1pe/J mice were pur-

chased from Jackson Laboratories (Bar Harbor, ME). Hemophilia B C3H/ HeJ mice (F9 gene deletion) were generated previously.27 C57BL/6 TLR9−/− mice were obtained from Dr Daniel Muruve (University of Calgary, Calgary, Canada).47 Animals used were 6–8 week old males and housed in accordance with guidelines from the National Research Council’s Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committees of the University of Florida, Gainesville, FL. Cell culture and reagents. C3H/HeOuJ mouse spleens were harvested to

make single cell suspension via 70-nm cell strainer in phosphate-buffered saline (PBS) supplemented with 1% fetal bovine serum and 2 mmol/l ethylenediaminetetraacetic acid. B cells were isolated using a negative selection method (Miltenyi Biotech, Auburn, CA) according to the manufacturer’s protocol. The purity of B cells was above 90% as determined by CD19 staining and flow cytometry. B cells were cultured in RPMI1640 containing 2 mmol/l l-glutamine, 50 μmol/l 2-mercaptoethanol, 100 μmol/l nonessential amino acids, 1 mmol/l sodium pyruvate, and 200 μmol/l ITS. Before gene transfer, B cells were activated with 20 μg/ml LPS (E. coli 055:B5) for 1 day. Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Primary B cells derived from healthy humans were purchased from Allcells (Alameda, CA). Cell culture reagents were purchased from Invitrogen (Grand Island, NY). Recombinant human FIX (Benefix) was produced by Pfizer (New York, NY). ODN2088, ODN control, CpG rich and free plasmids (pCpGrich-mcs and pCpGfree-mcs) were purchased from InvivoGen (San Diego, CA). Other chemical and reagents were purchased from Sigma (St Louis, MO).

Retroviral vector generation and B cell transduction. The full (1.4-kb) or a

truncated version (0.9-kb lacking the N terminus) of the human FIX coding sequence was PCR amplified from AAV2-EF1α-FIX vector and subcloned into BSSK-IgG plasmid (kindly provided by Dr. David Scott, Uniformed

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Services University of the Health Sciences) with NotI/XhoI sites and thereby fused with a full-length secretory murine IgG1. The IgG-FIX was then inserted into a bicistronic murine stem cell virus MSCV-IRES-GFP vector (Clontech, Mountain View, CA). The truncated version retained all known T cell epitopes in mice, including the dominant CD4+ T cell epitope for C3H/HeJ mice.26,27 Correct in-frame fusion was confirmed by DNA sequencing. MSCV-IgG was produced as a control. It contains all the other elements of IgG-FIX1 and IgG-FIX2 except for the FIX sequence. Ecotropic retrovirus were generated in packaging cell line GPE-86 or EcoPack 2–293 cell line (Clontech) using standard calcium phosphate precipitation method. The supernatants were collected 48 hours later, concentrated by ultracentrifugation, and stored at −80 °C. Retrovirus was tittered in NIH3T3 cells. Briefly, a variety of retrovirus stock dilutions were added into NIH3T3 cells. After 72 hours, GFP positive cells were determined by flow cytometry and the titer was calculated by Poisson analysis. Concentrated virus titer were above 5 × 107 colony-forming units/ml. Isolated splenic B cells were stimulated with LPS for 36 hours and either cocultured with irradiated viral packaging cells, or viral supernatant at multiplicity of infection 3–5, in the presence of 6 μg/ml polybrene and LPS for 24 hours. Centrifugation (300g, 60 minutes at 32 °C) was used to improve retroviral transduction. B cell transduction efficiency reached ~20% as determined by flow cytometry. Total vector treated B cells were transferred into C3H/HeJ F9−/− mice by tail vein injected at doses up to 107 cells/mouse. Nucleofection. Nucleofection was performed with 2 μg of plasmid DNA

per 106 cells using Amaxa mouse B cell nucleofection kit (Lonza Group, Walkersville, MD) according to the manufacturer’s protocol.11 Flow cytometry. Spleens were harvested from mice and processed to

produce single cell suspensions, which were then subjected to surface staining for CD3-Perp, CD4-eFluor 450, and CD25-PE at 4 °C for 30 minutes in PBS, followed by viability dye eFluor 506 stain at 4 °C for 30 minutes in PBS. Fixation and Foxp3 Alexa Fluor 647 stain was performed using the transcriptional factor staining buffer set. All kits and antibodies were purchased form eBiosciences (San Diego, CA). Controls for all stains included isotype control, single positive, and unstained cells. Flow cytometry was performed on a LSR II system (BD Bioscience, San Jose, CA), and data were analyzed with FCSExpress software (De Novo Software, Los Angeles, CA).

Gene expression profiling. Purified mouse B cells were subjected to dif-

ferent treatment including control LPS treatment, retroviral transduction, mock nucleofection transfection, or plasmid nucleofection transfection. After 24 hours, mRNA was extracted from 2 × 106 cells using the RNeasy kit (QIAGEN, Valencia, CA), and first stranded cDNA was generated via SABiosciences kit (SABiosciences “First Strand cDNA” kit, Frederick, MD). Quantitative PCR was performed in duplicate with SYBR-Green PCR master mix, using primer precoated 96-well plates (SABiosciences) according to manufactures protocols. A MyIQ icycler fluorescent detection system with iQ5 operating software Version 2.0 (Bio-Rad Laboratories, Hercules, CA) was used to generate and analyze data. All gene expression was compared with that of glyceraldehyde-3-phosphate dehydrogenase. Fold-change was calculated using the 2ΔΔCt quantification method.47

Quantitative reverse transcription-PCR and cytokine ELISAs. IL-6, IFNα2, IFN-α4, and IFN-β1 mRNA levels were further quantified using RT2 qPCR primer assay from SABiosciences according to manufacturer’s protocols. Mouse IL-6 and IFN-α ELISA kit were purchased from eBiosciences. Mouse IFN-β ELISA kit was purchased from Thermo Scientific (Waltham, MA). Purified mouse B cells were subjected to different treatment as indicated. After 48 hours, the culture supernatant was harvested and ELISA was performed according to the manufacturer’s protocol. Coimmunoprecipitation and immunoblot analysis. Coimmu­ noprecipitation and immunoblot analysis were performed as previously described.48 β-actin and anti-hFIX antibody were purchased from Sigma. www.moleculartherapy.org  vol. 22 no. 6 jun. 2014

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Anti-MyD88 and anti-TLR9 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). NF-κB pathway antibodies as well as antimouse and antirabbit antibodies conjugated to HRP were purchased from Cell Signaling (Beverly, MA). Antibody assays and B cell ELISpot. Levels of anti-hFIX IgG1 and IgE were determined by ELISA as previously described.10,49,50 Activated partial thromboplastin time (aPTT) was measured using a fibrometer (BBL Fibrosystems, Cockeysville, MD), and Bethesda assays were as previously described.27 By definition, 1 BU indicates the degree of inhibition of 50% residual coagulation activity. For B cell ELISpot, single cell suspensions of splenocytes and bone marrow cells were made using a 70-nm cell strainer. Red blood cells were removed with red blood cell lysis buffer (eBiosciences). Cells (1 × 106 or 5 × 105) were seeded in duplicate into ELISPOT plate (Millipore, Billerica, MA) precoated with FIX (20 μg/ml coating buffer). The plate was also preblocked with RPMI with 10% fetal bovine serum for 1 hour. After culture for 1 day, the plate was washed with PBS three times and incubated for 1 hour at room temperature with rat antimouse IgG1 HRP (1:1,000, AbD Serotec, Kidlington, UK) in RPMI 1640 with 10% fetal bovine serum, followed by three PBS washes. Color development of spots was carried out using 3-amino-9-ethylcarazole substrate (BD Pharmingen, San Jose, CA). Anti-FIX secreting cells were counted with the CTL-ImmunoSpotH S5 UV analyzer (Cellular Technology, Shaker Heights, OH).50 Adoptive T cell transfer. For adoptive Treg transfer experiments, CD4−,

CD4+CD25−, and CD4+CD25+ cells were isolated from pooled single cell suspension of splenocytes from different groups of mice using Treg isolation kit (Miltenyi Biotech) according to the manufacturer’s protocol. Purified cells were washed with PBS and counted using a hemacytometer and trypan blue. Live cells (1 × 106 per mouse) were adoptive transferred into naive strain-matched mice via tail vein injection. After 24 hours, recipient mice were subcutaneously injected with 1 IU hFIX formulated in Sigma Adjuvant System. Blood was collected 3 weeks later, and anti-FIX titers in plasma were measured by ELISA. Statistical analysis. The differences between two experimental groups

were compared by unpaired two tail Student’s t-test. The differences among several groups were compared by two-way ANOVA. The difference of survival rates was examined by the long-rank test. All analyses were performed with SAS version 9.3 and were considered significant at P < 0.05.

ACKNOWLEDGMENTS The authors thank Dr David Scott for IgG fusion vector and retroviral gene transfer protocols. The authors also thank Dr Daniel Muruve for TLR9-deficient mice and Dr Lixin Wang for technical assistance. This work was supported by National Institutes of Health grant P01 HD078810 (R.W.H.), a Scientist Development Grant by the American Heart Association (O.C.), and by Pfizer ASPIRE-hemophilia grant WI181128 (X.W.). X.W., I.Z., B.M., and O.C. performed experiments. X.W., B.M., O.C., and R.W.H. designed experiments. X.W., B.M., L.M.M., O.C., and R.W.H. interpreted data. O.C. and R.W.H. supervised and coordinated the study. X.W., L.M.M., and R.W.H. wrote the manuscript.

References

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