Immune responses to human factor IX in haemophilia B mice of different genetic backgrounds are distinct and modified by TLR4

Our laboratory develops protocols to prevent or reverse ongoing anti-hFIX IgG inhibitors in haemophilia B mice with a F9 gene deletion on BALB/c and C3H/HeJ backgrounds. C3H/HeJ F9−/Y mice develop high titre anti-hFIX IgG1 inhibitors and anaphylaxis, whereas most BALB/c F9−/Y mice have mild anti-hFIX IgG1 inhibitors and no anaphylaxis. Our aim was to determine if hFIX-specific B- and T-cell responses in BALB/c and C3H/HeJ F9−/Y mice trigger the difference in anti-hFIX immune responses. BALB/c and C3H/HeJ F9−/Y mice were challenged weekly with recombinant hFIX protein. Humoral immune responses were determined by IgG1 and IgG2a anti-hFIX ELISA, Bethesda assay for inhibitors and B-cell ELISpot on bone marrow and spleen cells. T-cell studies measured the TH1 (IFN-γ) and TH2 (IL-4) cytokine responses in splenocytes at the mRNA and protein level in response to hFIX protein. Antibody responses were also measured in C3H/HeJ/OuJ F9−/Y mice with restored toll-like receptor 4 (TLR4) function. BALB/c F9−/Y mice have a TH2 skewed response and a reduction in anti-hFIX secreting plasma cells in the bone marrow. Independent antigen challenge revealed both strains generated equivalent IgG1 antibody titres to an intravenously delivered antigen. C3H/HeJ F9−/Y mice have a mixed TH1 and TH2 response (mainly TH2). Importantly, TLR4 signalling has a modulatory role in the C3H background on the levels of anti-hFIX IgG1 and incidence of anaphylaxis. The background strain strongly impacts the immune response to hFIX, which can be significantly impacted by mutations in innate immune sensors.


Introduction
Haemophilia B (HB) is an X-linked inherited coagulation disorder that results in the loss of functional coagulation factor IX (FIX). Patients are managed with intravenous (i.v.) injections of plasma derived or recombinant FIX protein. Those with severe disease (<1% residual coagulation factor) are at risk for developing anti-hFIX antibodies that impede coagulation, inhibitors, during treatment. Inhibitors occur in about 1.5-3% of HB patients. High titre inhibitor patients (>5 BU/mL) become unresponsive to coagulation factor protein therapy and have an increased risk of severe bleeds.
Typically patients with high titre inhibitors are managed on demand with bypassing agents, such as activated factor VIIa or FEIBA (Baxter Healthcare Corporation Deerfield, IL USA). These bypassing products are quite expensive and are not generally designated for extended use. Haemophilia A patients with inhibitors can undergo immune tolerance induction (ITI), a protocol that requires daily infusion of high levels of FVIII protein. This protocol can take months to years and is expensive. Unlike haemophilia A, haemophilia B patients with inhibitors are less responsive to ITI and may develop pathogenic antibody responses including anaphylaxis and nephrotic syndrome [1][2][3][4][5][6].
In most patients, inhibitor development typically occurs within 50 exposure days. Therefore the identification of genetic risk factors that promote inhibitor development would improve the treatment of newly diagnosed haemophilia B patients. For example, pre-clinical studies have suggested that prophylactic immune suppression during initial hFIX infusions may prevent inhibitor development [7][8][9]. These strategies might be particularly beneficial for patients with a high-risk profile for developing inhibitors.
One of the best predictors of inhibitors in haemophilia B is the underlying F9 mutation [10][11][12][13]. However, the fact that only an estimated 1.5-3% of haemophilia B patients develop inhibitors and not all patients with gene deletions develop inhibitors suggests that there are still unidentified susceptibility factors. Lozier et al. investigated the immune responses in different strains of wild-type mice immunized with an adenoviral vector expressing F9 or with hFIX protein [14]. Their study identified a linkage between the major histocompatibility complex II locus H-2 and markers on chromosomes 1 and 10, which are near immunomodulatory genes such as interleukin 10 and interferon-c [14]. To better model inhibitor development and risk for pathogenic antibody responses, as seen in treated haemophilia B patients, we compared immune responses to hFIX protein (40 IU kg À1 ) by i.v. delivery in two different strains with a targeted gene deletion for murine F9 (BALB/c F9 À/Y and C3H/HeJ F9 À/Y ).
Recent studies have indicated links between innate immunity and coagulation [15,16]. Absence of toll like receptor 4 (TLR4), an innate immune sensor of bacterial lipopolysaccharide (LPS), has been described as sensitizing mice to food antigen allergy and inducing anaphylaxis, whereas restoration of TLR4 provides protection [17]. C3H/HeJ F9 À/Y mice are deficient in TLR4 and are prone to developing fatal anaphylactic responses to intravenously delivered FIX protein [13,18,19]. The availability of a congenic stain, C3H/OuJ that has functional TLR4, allowed us to determine if restoring TLR4 function will have an impact on the severity and frequency of anaphylactic responses to intravenously delivered FIX protein.

Materials and methods
Animal studies BALB/c, C3H/HeJ and C3H/OuJ mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Haemophilia B mice with targeted deletion of murine F9 ('null mutation') have been bred on BALB/c and C3H/ HeJ backgrounds for >10 generations [20]. Crossing female C3H/HeJ F9 À/Y with male C3H/OuJ mice generated C3H/HeJ/OuJ F9 À/Y F1 mice in which all male mice were F9 À/Y and heterozygous for toll-like receptor 4 (TLR4). Animals were housed under special pathogen free conditions under institutional Animal and Care and Use Committee approved protocols. All animals were male and 6-8 weeks old at the onset of the experiments.
Immunization protocols for haemophilia B mice were performed as described unless otherwise stated [13]. Mice were immunized weekly with 1 IU recombinant hFIX protein starting with an intraperitoneal injection, followed by five weekly i.v. injections via the tail vein. When indicated C3H/HeJ F9 À/Y mice were given 150 lg antihistamine (triprolidine; Sigma, St Louis, MO USA) and 50 lg platelet activating factor (PAF) antagonist CV-3988 (Sigma) along with hFIX protein to prevent anaphylactic responses. Blood for plasma analyses from haemophilia B mice were collected in citrate buffer (0.38%) by tail transection [20]. One week following a final intravenous immunization, hFIX mice were killed and spleen and bone marrow cells were collected for analysis. Wild-type BALB/c and C3H/HeJ mice received 2 mg keyhole limpet hemocyanin (KLH) antigen (Sigma) by intravenous injection [21]. These BALB/c and C3H/HeJ mice were bled from the retro-orbital plexus using heparinized capillaries for plasma antibody analyses.

Antibody measurements
Inhibitory antibody titres were determined by Bethesda assay [20]. One Bethesda unit is the reciprocal of the dilution of plasma that neutralizes 50% of FIX in a normal plasma sample which it is mixed with in vitro. Immunocapture assays to determine titres of hFIX and KLH specific IgG1 and IgG2a were as described [21,22] using purified mouse IgG1 and IgG2a as standards.

B-and T-cell assays
B-cell ELISpots were performed on total splenocytes and bone marrow cells as previously described [13,23,24]. For in vitro restimulation studies, isolated splenocytes were cultured in RPMI 1640 media (containing 55 lM b-mercaptoethanol, glutamine and antibiotics) with or without 10 lg mL À1 hFIX for 48 h (at 37°C, 5% CO 2 ). Transcript levels of cytokines in these cells were measured by quantitative RT-PCR using an SA Bioscience array [13].

Statistics
All statistical analysis was carried out using Prism software using Student's two-tailed t-test. A P < 0.05 was considered statistically significant.

Results
Immune responses to intravenous challenge of hFIX protein in F9 À/Y mice is strain dependent C3H/HeJ F9 À/Y mice develop high titre IgG antibodies and fatal anaphylactic reactions in a dose-dependent manner after weekly intravenous injection of hFIX protein, whereas BALB/c F9 À/Y mice are hyporesponsive [13]. To investigate why the two strains respond differently to hFIX protein we conducted studies to compare B and T responses directed against hFIX protein. All mice were challenged with six weekly exposures of 1 IU hFIX protein (40 IU kg À1 ). C3H/ HeJ F9 À/Y mice developed fatal anaphylactic reactions starting after the third hFIX injection (Fig. 1a) and required co-administration of antihistamine and platelet activating factor antagonist CV3988 to survive the challenge protocol. Similarly to previously published studies on food allergy [25], none of the BALB/c F9 À/Y mice showed any signs of anaphylaxis and all mice survived the challenge protocol (Fig. 1a). To determine if this could be due a deviated immune response against hFIX protein in BALB/c F9 À/Y mice, we assayed for T H 1 (IgG2a) and T H 2 (IgG1)-associated anti-hFIX immunoglobulin (IgG). IgG1 subclass was predominantly formed by either strain, albeit at significantly lower titres in BALB/c F9 À/Y mice (Fig. 1b). C3H/HeJ F9 À/Y mice had significantly higher levels of IgG2a, indicating an additional contribution of T H 1 responses (Fig. 1b, c). All animals that had measurable IgG1 were also positive for inhibitors in a Bethesda assay with BALB/c F9 À/Y mice again developing significantly lower titres (Fig. 1d).
To compare the B-cell response between the strains, spleen and bone marrow cells were analysed by ELI-Spot for the presence of anti-hFIX IgG1 secreting B and plasma cells (PC). While we observed no significant difference in the frequencies of anti-hFIX IgG1 secreting cells in splenocytes (Fig. 2a), there was a significant elevation in hFIX antibody secreting cells in the bone marrow of C3H/HeJ F9 À/Y mice (Fig. 2b). As long-term PC survival is dependent on signalling in the bone marrow, these data suggest that BALB/c mice may lack the ability to generate long-term anti-hFIX secreting PC following intravenous hFIX protein challenge [26]. To test if BALB/c mice in general have a failure to form antibodies against an intravenously delivered antigen, we challenged wild type BALB/c and C3H/HeJ mice with keyhole limpet hemocyanin (KLH). Mice on both backgrounds produced equivalent levels of anti-KLH IgG1 antibodies (Fig. 3a), while BALB/c mice had a significant reduction (~10 fold) in IgG2a, indicative of a T H 2 biased immune response. Together these data suggest that the hyporesponsiveness of BALB/c mice towards hFIX protein does not reflect a general unresponsiveness to exogenous proteins.
No difference in T-cell responses to hFIX in the BALB/c and C3H/HeJ F9 À/Y mice As antibody responses against hFIX protein are T-cell dependent, we investigated if there were differences in the T-cell responses to hFIX protein. Splenocytes from hFIX-immunized BALB/c and C3H/HeJ F9 À/Y mice were stimulated with full-length hFIX protein and analysed for the production of T H 1 and T H 2-associated cytokines by ELISpot. While not reaching statistical significance, the data showed a trend towards higher IL-4 spot forming units in BALB/c F9 À/Y mice (Fig. 4a). The majority of BALB/c F9 À/Y mice were negative for IFN-c, whereas most of the C3H/HeJ F9 À/Y mice had some level of IFN-c (Fig. 4b). This skewing of T cell responses to T H 1 in C3H/HeJ F9 À/Y mice correlated with previous observations in the anti-hFIX antibody classes (Fig. 1b, c). In another set of experiments, splenocytes from immunized BALB/c and C3H/HeJ F9 À/Y mice were restimulated in vitro with hFIX protein and mRNA was extracted to assess changes in T H 1, T H 2, and Treg-related gene expression. In agreement with our IL-4 ELISpot data, both strains showed an up-regulation in IL-4 mRNA (Fig. 4c). BALB/c F9 À/Y mice had a significant up-regulation in IL-10 mRNA compared to C3H/HeJ F9 À/Y mice, but was below our threshold to be considered relevant. In summary, the data show no significant difference in the T-cell responses to hFIX protein between the two strains of F9 À/Y mice.

Role of TLR4 signalling in modulating pathogenic immune responses against recombinant hFIX protein therapy
Food allergy-induced anaphylaxis studies showed that C3H/HeJ mice are highly susceptible and C3H/OuJ mice are highly resistant [17]. Introducing a similar defective TLR4 allele into BALB/c mice, who are normally resistant, failed to promote anaphylaxis [17,27], suggesting that TLR4 only modulates hypersensitivity in a high-responder strain. To address the role of TLR4 signalling in hFIX-mediated anaphylaxis we bred female C3H/HeJ F9 À/Y with male C3H/OuJ mice to generate male mice with a gene deletion for F9 and heterozygous for TLR4. To determine if TLR4 signalling was restored in these F1 mice, we compared the secretion of IL-6 by splenocytes from wild-type C3H/ HeJ, C3H/OuJ and F1 C3H/HeJ/OuJ F9 À/Y mice following in vitro stimulation with a TLR4-specific LPS [28]. As expected C3H/HeJ splenocytes were unresponsive to LPS stimulation (Fig. 5). Both C3H/OuJ and C3H/HeJ/OuJ splenocytes secreted IL-6 only in the presence of LPS, with splenocytes from C3H/HeJ/OuJ mice secreting approximately one half the level of C3H/OuJ mice (Fig. 5) confirming partial restoration of TLR4 function in the F1 offspring. When challenged with hFIX protein, without antihistamine and PAF antagonist, only two of nine F1 C3H/HeJ/OuJ F9 À/Y mice died from anaphylaxis, which is a substantial reduction compared to C3H/HeJ F9 À/Y mice (Fig. 1a). Anti-hFIX IgG1, IgG2a and Bethesda titres were intermediate when compared with C3H/HeJ F9 À/Y and BALB/c F9 À/Y mice (Fig. 1b-d). Consistent with previous studies using C3H/HeJ and C3H/OuJ mice in food allergy models [29], we show that TLR4 is also critical in down-modulating IgG1, IgG2a and hypersensitivity responses directed against hFIX protein. Nonetheless, 70% of C3H F9 À/Y formed high-titre inhibitors (>5 BU) regardless of the TLR4 mutation, whereas only 1 of 8 BALB/c mice had >5 BU, indicating that additional genetic factors modulate the response.

Discussion
Identifying genetic susceptibility factors for inhibitor formation and pathogenic antibody responses against recombinant hFIX protein would be of significant clinical benefit. Because of the relatively low numbers of haemophilia B patients with inhibitors, it has been difficult to study contributing factors. Therefore, we and others have generated murine haemophilia B models with human F9 mutations to study the immunogenicity of hFIX protein [10,12,20]. Our group was the first to describe fatal anaphylaxis to intravenously delivered hFIX protein in null mutant (but not missense and late stop F9 mutations) C3H/HeJ F9 À/Y mice [7,13,18,19]. These mice provide a robust and consistent model of inhibitor and anaphylaxis development that is much higher than that observed IFN-c (b) ELISpot assays using splenocytes isolated from immunized mice of indicated strain. All samples were run in duplicate with unstimulated, 10 lg mL À1 hFIX, and SEB or PMA/Ionomycin stimulated. Data are presented as (SFU hFIX À SFU unstimulated per 1 9 10 6 cells). (c) Cytokine responses specific to hF.IX for BALB/c (white) and C3H/HeJ (grey) F9 À/Y mice. Splenocytes (1 9 10 5 per well) were cultured in vitro without or with hFIX protein (10 mg mL À1 ) and harvested 48 h later for mRNA extraction and transcriptional analysis via qPCR array for indicated genes. Data are presented as fold change compared to unstimulated cells. Fig. 5. Verifying reconstitution of TLR4 signalling in C3H/HeJ/OuJ F9 À/Y mice. 1 9 10 6 splenocytes from C3H/HeJ (n = 2), C3H/OuJ (n = 2), and C3H/HeJ/OuJ F9 À/Y (n = 4) were cultured in vitro in triplicate for 48 h either unstimulated or stimulated with 10 lg mL À1 LPS-SM, a specific TLR4 activator. Cell culture media was collected, pooled and analysed for IL-6 (ng mL À1 ) secretion into media using a murine IL-6 ELISA kit.
in human haemophilia B patients, which have heterogeneous F9 mutations and immune profiles, and thus provides an ideal platform to develop protocols that can prevent or eliminate inhibitors [13,30]. The formation of an antibody response against a recombinant protein, such as hFIX, is dependent on multiple factors. Antigen presenting cells can modulate the CD4 + T-cell response by presenting antigen in the context of varying co-stimulation during T-cell receptor (TCR) engagement [31]. CD4 + T cells can in turn provide help to B cells to stimulate their expansion and differentiation into antibody-secreting PC. In this study, we investigated if differences in B-and T-cell responses to hFIX protein contributed to either the low-responding BALB/c F9 À/Y or high-responding C3H/HeJ F9 À/Y mice.
Similar to previously published data on food allergy, we found anaphylactic responses to hFIX in C3H/HeJ F9 À/Y , but not BALB/c F9 À/Y mice [25]. The only significant difference in B-and T-cell responses was a reduction in antibody secreting PC in the bone marrow of BALB/c F9 À/Y mice. An inability to generate sufficient anti-hFIX producing PC would explain the reduced systemic anti-hFIX levels in the BALB/c strain. The underlying cause for the diminished B cell response is less clear. Consistent with immune responses to pathogens in the two strains, BALB/c mice showed a stronger T H 2 bias in their response to hFIX. Furthermore, IL-4 ELI-Spot data and cytokine gene expression profiles suggest a similar strength of T H 2 responses for both strains, so that the comparatively low formation of the T H 2dependent IgG1 anti-hFIX in BALB/c mice unlikely is due to lack of T help (although we cannot rule out reduced levels of cytokine secretion) [32].
A similar hypo-responsiveness against FVIII in haemophilia A BALB/c mice has been previously reported [8,32]. Taken together these studies suggest that BALB/c mice have a reduced response to intravenously delivered coagulation factors. However, the robust anti-KLH IgG1 response (Fig. 3a) suggests that these differential responses are antigen-dependent rather than reflecting a general defect in responding to intravenous proteins.
As C3H/HeJ mice lack functional TLR4, we investigated the role of TLR4 signalling on shaping the anti-hFIX immune response. Our F1 C3H/HeJ/OuJ F9 À/Y mice with partially restored TLR4 function (Fig. 5) had a significant reduction in anaphylaxis associated mortality: 78% survival versus 40% (Fig. 1a). The 40% survival rate for C3H/HeJ F9 À/Y mice was dependent on prophylactic intervention with antihistamine and PAF antagonist, whereas 78% of the C3H/HeJ/OuJ F9 À/Y mice survived the challenge protocol without any prophylaxis. This indicates that TLR4 acts as negative modulator of anamnestic responses against hFIX protein in C3H mice. TLR4 has originally been identified as an innate sensor of LPSs, components of the cell walls of Gram-negative bacteria. More recently, it has become clear that TLR4 is also a critical link between the innate immune and coagulation systems, serving as a receptor for fibrinogen bound to a Plasmodium metabolite and for blood-born adenovirus decorated with factor X [15,16]. Interestingly, TLR4 activation in B cells is not a signal for antibody formation [33]. The absence of TLR4 signalling in C3H/HeJ mice results in a reduced ability to clear Gram-negative bacterial infections, such as Klebsiella oxytoca [34,35], which resides in the gut of healthy mice with functional TLR4. Such lesions may act to prime the immune system, although further studies are required to examine this. Multiple food allergy studies show that functional TLR4 protects against IgEmediated anaphylaxis [17,29] and TLR4 deficiency exacerbates autoimmune disease in colitis and type 1 diabetes [36,37]. We have now extended these findings to hFIX replacement therapy. The mechanism of the allergic response to hFIX, characterized by an enhanced response in the absence of TLR4, is distinct from that in allergic airway disease, where sensing of proteolytic cleavage products of fibrinogen by TLR4 is required [16].

Relevance to human haemophilia B patients
The most relevant finding of our study was the role of TLR4 signalling on both the magnitude of anti-hFIX antibodies and hypersensitivity. It is not known if polymorphisms or mutations with reduced functional activity in TLR4 or other innate immune sensors have an impact on the risk of inhibitors and hypersensitivity in haemophilia B patients as compared to our murine model [38]. Nonetheless, these data provide novel links between genetic factors, environmental stimuli and inhibitor formation/anaphylaxis.

Conclusions
Genetic background plays a significant role in shaping the immune response against recombinant hFIX protein. Comparing BALB/c and C3H/HeJ mice with the same F9 gene deletion showed that BALB/c mice are hyporesponsive to hFIX protein, fail to produce bone marrow-resident PC and are biased towards generating a T H 2 skewed immune response. C3H/HeJ mice are hypersensitive to hFIX protein and show a mixed T H 1 and T H 2 immune response. TLR4 was identified as a negative modulator of hypersensitivity in the C3H background. We would offer caution in the design and interpretation of studies when conducted in strains with limited immune responses.
Author contribution DMM, BKS, XW and AS performed the research and conducted data analysis. DMM and GLR designed research and wrote the manuscript. DMM supervised the research project.