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Keywords:

  • FVIII inhibitors;
  • haemophilia A;
  • immune modulation;
  • memory B cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Summary.  The development of inhibitory antibodies against factor VIII (FVIII) is the major complication in patients with haemophilia A who are treated with FVIII products. Memory B cells play an essential role in maintaining established antibody responses. Upon re-exposure to the same antigen, they are rapidly re-stimulated to proliferate and differentiate into antibody-secreting plasma cells (ASC) that secrete high-affinity antibodies. It is, therefore, reasonable to believe that memory B cells have to be eradicated or inactivated for immune tolerance induction therapy to be successful in patients with haemophilia A and FVIII inhibitors. The aim of our studies was the development of strategies to prevent FVIII-specific memory B cells from becoming re-stimulated. We established a 6-day in vitro culture system that enabled us to study the regulation of FVIII-specific murine memory-B-cell re-stimulation. We tested the impact of the blockade of co-stimulatory interactions, of different concentrations of FVIII and of ligands for toll-like receptors (TLR). The blockade of B7-CD28 and CD40-CD40 ligand interactions prevented FVIII-specific murine memory B cells from becoming re-stimulated by FVIII in vitro and in vivo. Furthermore, high concentrations of FVIII blocked re-stimulation of FVIII-specific murine memory B cells. Triggering of TLR7 amplified re-stimulation by low concentrations of FVIII and prevented blockade by high concentrations of FVIII. We conclude that we defined modulators that either amplify or inhibit the re-stimulation of FVIII-specific murine memory B cells. Currently, we are investigating whether the same modulators operate in patients with haemophilia A and FVIII inhibitors.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

The development of inhibitory antibodies against factor VIII (FVIII) is the major complication in patients with haemophilia A who are treated with FVIII products. Long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and induce long-lasting immune tolerance [1–4]. Although this therapeutic approach was introduced by Dr Brackmann and co-workers more than 30 years ago [1], little is known about the immunological mechanisms that cause the down-modulation of FVIII-specific immune responses and the induction of long-lasting immune tolerance against FVIII.

Memory B cells play an essential role in maintaining established antibody responses. Upon re-exposure to the same antigen, they are rapidly re-stimulated to proliferate and differentiate into antibody-secreting plasma cells (ASC) that secrete high-affinity antibodies [5–7]. Furthermore, memory B cells have the potential to act as very efficient antigen-presenting cells and stimulators of CD4+ T cells because of the expression of high-affinity antigen receptors, major histocompatibility complex class II and co-stimulatory molecules [8]. It is, therefore, reasonable to believe that memory B cells have to be eradicated or inactivated for immune tolerance induction (ITI) therapy to be successful in patients with haemophilia A and FVIII inhibitors. Over the past few years, we have established technologies that have enabled us to study the regulation of FVIII-specific memory B cells and potential approaches to interfere with the re-stimulation of these cells in vitro. We have used a murine model of haemophilia A that is characterized by complete deficiency of biologically active FVIII because of a targeted disruption of exon 17 of the FVIII gene [9,10]. Intravenous injection of human FVIII into these mice results in high titres of anti-FVIII antibodies that have similar characteristics to those of FVIII inhibitors in patients [11–14]. This article summarizes our most important findings in the haemophilic mouse model. Furthermore, it describes our first attempt to analyse FVIII-specific memory B cells in patients with haemophilia A and FVIII inhibitors.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Animals

The animals used in the study were haemophilic E-17 mice. Our colony of fully inbred haemophilic E-17 mice (characterized by a targeted disruption of exon 17 of the FVIII gene) was established with a breeding pair from the original colony [9,10] and crossed into the C57Bl/6J background as described [15]. All mice were male and aged 8–10 weeks at the beginning of the experiments. All studies were done in accordance with the Austrian federal law (Act BG 501, 1989) regulating animal experimentation.

Treatment of mice with human FVIII

Mice received four intravenous doses of 200 ng recombinant FVIII (approximately 80 U kg−1 FVIII), diluted in 200 μL of Dulbecco’s phosphate-buffered saline (DPBS; Sigma-Aldrich, Irvine, UK), at weekly intervals. The recombinant human FVIII used throughout the studies was albumin-free bulk material obtained from Baxter AG (Thousand Oaks, CA, USA).

Preparation of spleen cells from mice

Spleens were collected 7 days after the last dose of FVIII. All invasive procedures were done under anaesthesia with pentobarbital (Nembutal; Richter Pharm, Wels, Austria). Spleen cells were prepared as described [16,17].

Re-stimulation of murine memory B cells in vitro

Factor VIII-specific memory B cells were re-stimulated as described [17,18]. Briefly, spleen cells were depleted of CD138+ ASC using a monoclonal rat anti-mouse CD138 antibody (BD Pharmingen, San Diego, CA, USA) coupled to M-450 sheep anti-rat IgG Dynabeads (Invitrogen Dynal, Lofer, Austria). CD138 spleen cells were cultured at 1.5 × 106 cells mL−1. Different concentrations of FVIII were added to the cells on day 0 as indicated. Antibodies and proteins with potential modulating activities, isotype-matched negative control antibodies or ligands for toll-like receptors (TLR) were added together with FVIII on day 0 or at later time points as indicated. After 6 days of culture, newly formed ASC were detected by enzyme-linked immunospot (ELISPOT) assays as described [16–18]. The purity of CD138 spleen cells was analysed by flow-cytometry [17,18].

Antibodies and proteins for the blockade of co-stimulators

Blocking antibodies against the co-stimulatory molecules CD80 (B7.1, clone 16-10A1, hamster IgG), CD86 (B7.2, clone P03.1, rat IgG2b), CD40 ligand (CD40L, clone MR1, hamster IgG) and ICOS ligand (ICOSL, clone HK5.3, rat IgG2a) as well as the respective isotype controls were of functional grade and obtained from eBioscience (San Diego, CA, USA). Each antibody was added at 10 μg mL−1 to the in vitro cultures on day 0. Additionally, the importance of ICOS-ICOSL and B7-CD28 interactions were evaluated by using the recombinant competitor proteins murine ICOS/Fc and murine CTLA4/Fc (both are fusion proteins of the murine protein with the Fc-part of human IgG1 and were obtained from R&D Systems, Minneapolis, MN, USA). These proteins were used at a concentration of 10 μg mL−1. Murine ICOS/Fc blocks interactions between ICOS and ICOSL; murine CTLA4/Fc blocks interactions between CD80/CD86 and CD28.

Ligands for TLR

The following ligands for TLR were tested: zymosan for TLR2, poly I:C for TLR3, LPS for TLR4, Flagellin for TLR5, Loxoribine for TLR7 and CpG oligonucleotides for TLR9. All TLR ligands were received from InvivoGen (San Diego, CA, USA).

Depletion of T cells

T cells were depleted from CD138 spleen cells using mouse pan-T (Thy 1.2) Dynabeads (Invitrogen Dynal, Oslo, Norway) as described [17].

Cytokine analysis and proliferation assays

Cytokine analysis and proliferation assays were performed as described [18].

Patients with haemophilia A

Twelve patients with severe haemophilia A (8–43 years old) were investigated. Six of the patients had FVIII inhibitors (Table 1). All patients signed individual forms of consent. The study was approved by the Ethics Committee of the Institute of Hematology and Transfusion Medicine, Warsaw, Poland.

Table 1.   Characteristics of patients enrolled in the study in 2006 (this table was originally published in reference [20]).
PatientsAge (in years)Titre of FVIII inhibitors (BU mL−1) in 2006Last treatment with FVIIICurrent treatmentHistorical peak inhibitor titre (BU mL−1)First detection of FVIII inhibitors
WA01241251993Bypassing agents1891985
WA022762003Bypassing agents1501994
WA04331.22003No treatment121993
WA053410001995Bypassing agents43401984
WA11812006High dose FVIII and rFVIIa681999
WA121681992FEIBA591993
WA033002006FVIII0No inhibitors
WA061102006FVIII0No inhibitors
WA073302006FVIII0No inhibitors
WA084302006FVIII0No inhibitors
WA094002006FVIII0No inhibitors
WA131202006FVIII0No inhibitors

Analysis of neutralizing anti-FVIII antibodies (FVIII inhibitors) in patients

FVIII inhibitors were analysed at the central laboratory of the Medical University of Vienna, Vienna, Austria. The Bethesda assay was used as described [19].

Blood sampling and cell preparation from patients

Blood was collected and peripheral blood mononuclear cells (PBMC) were prepared using Vacutainer cell preparation tubes with sodium citrate (Becton Dickinson, Schwechat, Austria). Cell isolation was carried out by following the manufacturer’s instructions. DPBS (Sigma-Aldrich, St Louis, MO, USA) supplemented with 2% preselected foetal calf serum (FCS; Hyclone, Logan, UT, USA) was used as a washing solution.

Freshly prepared cells were frozen in RPMI-1640 (Life Technologies, Paisley, Scotland) supplemented with 40% FCS and 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO, USA) and stored in liquid nitrogen until further analysis.

In vitro re-stimulation and analysis of human circulating memory B cells

Memory B cells contained in PBMCs were re-stimulated to differentiate into ASC in vitro as described [20]. After 6 days of culture, newly differentiated ASC were analysed by ELISPOT technology [20]. The frequency of antigen-specific ASC was calculated as a percentage of total IgG-producing cells.

The limit of detection (LD) was found to be three spots per well. These three spots were used to calculate the LD as a percentage of total spots obtained for IgG-producing cells for each individual patient.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Re-stimulation of FVIII-specific memory B cell in vitro

We set up an in vitro culture system that is suitable for studying the regulation of FVIII-specific memory B cells [17,18]. For this purpose, we obtained spleen cells from haemophilic mice treated with human FVIII and depleted these spleen cells of CD138+ ASC. Thereby, we generated a CD138 spleen cell population that did not contain any anti-FVIII ASC (Fig. 1) but contained FVIII-specific memory B, T cells and other cells. When we stimulated this CD138 cell mixture with human FVIII, FVIII-specific memory B cells were re-stimulated and differentiated into anti-FVIII ASC that could be detected as soon as 3 days after re-stimulation (Fig. 1) [17]. The maximum of newly formed anti-FVIII ASC was observed 6 days after re-stimulation (Fig. 1) [17]. In further experiments, we found that the re-stimulation of FVIII-specific memory B cells in our in vitro culture system strictly depended on the presence of activated T cells [17]. Furthermore, a direct cell-to-cell contact between FVIII-specific memory B cells and activated T cells was required [17].

image

Figure 1.  Re-stimulation of murine FVIII-specific memory B cells in vitro. Spleen cells were obtained from haemophilic mice treated with four intravenous doses of 200 ng (80 U kg−1) FVIII and depleted of CD138+ antibody-secreting cells (ASC). The remaining CD138 cells were re-stimulated with 10 ng mL−1 FVIII and analysed for newly formed ASC after 1, 3 and 6 days of culture. ASC were analysed by enzyme-linked immunospot (ELISPOT) assays as described [16]. This research was originally published in Blood [17] © the American Society of Hematology.

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Re-stimulation of FVIII-specific memory B cells involves CD40-CD40L and B7-CD28 interactions but does not require ICOS-ICOSL interactions

Based on our finding that activated T cells are required to re-stimulate FVIII-specific memory B cells in our in vitro culture system, we wanted to identify the specific co-stimulatory interactions that would be necessary for this process. Furthermore, we were interested to find out whether blocking essential co-stimulatory interactions would prevent the re-stimulation of FVIII-specific memory B cells. We added blocking antibodies against CD40L, CD80 (B7-1), CD86 (B7-2), ICOSL or recombinant competitor proteins (mICOS/Fc, mCTLA-4/Fc) to the CD138 spleen cell cultures immediately before re-stimulation with FVIII to study the importance of the relevant ligand receptor pairs. The blockade of B7-CD28 or CD40-CD40L interactions significantly inhibited the re-stimulation of FVIII-specific memory B cells (Fig. 2) [17]. Both CD80 (B7-1) and CD86 (B7-2) contributed to the required co-stimulatory interactions with CD28. Blockade of both molecules prevented the re-stimulation of memory cells almost completely, whereas the blockade of only one of the two molecules resulted in a partial blockade (Fig. 2) [17]. The negative control antibodies and human IgG1 (negative control for mCTLA-4/Fc) did not show any effect. In contrast to CD40-CD40L and B7-CD28 interactions, ICOS-ICOSL interactions did not contribute to the re-stimulation of FVIII-specific memory cells. Neither the addition of a blocking antibody against ICOSL nor the use of a recombinant competitor protein (mICOS/Fc) resulted in a significant alteration in the re-stimulation of memory B cells (Fig. 2) [17]. In further experiments, we confirmed the specific requirements of co-stimulatory interactions for the re-stimulation of FVIII-specific memory B cells in in vivo studies using haemophilic mice [17].

image

Figure 2.  The antigen-specific re-stimulation of FVIII-specific murine memory B cells in vitro involves CD40-CD40L and B7-CD28 interactions but does not require ICOS-ICOSL interactions. CD138 spleen cells obtained from haemophilic mice treated with human FVIII were re-stimulated with 10 ng mL−1 FVIII. Anti-FVIII ASC were analysed after 6 days of culture using ELISPOT assays. To interfere with co-stimulatory interactions, blocking antibodies (α-CD80, α-CD86, α-ICOSL and α-CD40L as indicated) or competitor proteins (mCTLA-4/Fc, mICOS/Fc as indicated) were added to the cultures at a concentration of 10 μg mL−1 together with FVIII. Presented are ELISPOT data obtained in a representative experiment. This research was originally published in Blood [17] © the American Society of Hematology.

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Re-stimulation of FVIII-specific memory B cells is inhibited by high concentrations of FVIII

After specifying important co-stimulatory interactions required for the re-stimulation of FVIII-specific memory B cells, we were interested to study the potential impact of different concentrations of FVIII on this process. We tested a range of concentrations between 1 pg mL−1 and 100 μg mL−1 of FVIII (Fig. 3a) [18]. Re-stimulation of memory B cells could be detected at concentrations of FVIII that were as small as 100 pg mL−1 (Fig. 3a) [18]. Optimal re-stimulation was achieved at concentrations of 3–10 ng mL−1, which correspond to about 3–10% of the physiological plasma concentration (Fig. 3a) [18]. When we further increased the concentration of FVIII, inhibition of memory B-cell re-stimulation was observed. The inhibition started at a concentration of FVIII of 100–300 ng mL−1 with an almost complete inhibition at 1 μg mL−1 FVIII (Fig. 3a) [18].

image

Figure 3.  Concentration of FVIII determines the response of FVIII-specific murine memory B cells and FVIII-specific T cells. CD138 spleen cells were obtained from haemophilic mice treated with human FVIII. Cells were re-stimulated in vitro with human FVIII as indicated for 3 days (b) or 6 days (a, c). Newly formed anti-FVIII ASC were detected by ELISPOT assay (a). ELISPOTs represent the results obtained in a typical experiment. Cell proliferation (b) and cytokine secretion into cell culture supernatants (c) were analysed as described in Materials and methods. Presented are the mean values and standard deviations of triplicate cultures (b) or the medians (c) obtained in a typical experiment. This research was originally published in Blood [18] © the American Society of Hematology.

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The dose-response relation for T-cell re-stimulation was very different from the dose-response relation for memory B-cell re-stimulation. Optimal stimulation of FVIII-specific T cells was observed at concentrations of 10–30 μg mL−1 FVIII (Fig. 3b,c). Inhibition of T-cell stimulation was seen at concentrations of 100 μg mL−1 FVIII. Based on these results, we conclude that the concentration of FVIII required for inhibition of memory B-cell re-stimulation and the concentration required for inhibition of T-cell re-stimulation are very different (Fig. 3a–c), which makes it unlikely that the inhibition of memory B-cell re-stimulation is caused by an inhibition of T-cell stimulation.

The major T-cell cytokines found in culture supernatants after stimulation of spleen cells with FVIII were IL-10 and IFN-γ (Fig. 3c), which is consistent with findings we reported previously [13,21]. To further support these results, we analysed the frequency of FVIII-specific T cells by intracellular cytokine staining 3 days after re-stimulation of spleen cells. We compared concentrations of 10 ng mL−1, which re-stimulate, and 20 μg mL−1 FVIII which inhibit memory B-cell differentiation and observed a correlation between the frequency of FVIII-specific T cells producing IL-2, IL-10 or IFN-γ and the concentration of FVIII used for the re-stimulation (data not shown). We did not observe any inhibitory effects of 20 μg mL−1 of FVIII on T-cell stimulation despite the fact that this concentration of FVIII completely blocks the re-stimulation of FVIII-specific memory B cells [18].

Both re-stimulation and inhibition of FVIII-specific memory B cells are modulated by ligands for TLR

Infections, particularly infections from the central venous catheter inserted in patients with haemophilia A and FVIII inhibitors during ITI therapy, commonly cause a rise in anti-FVIII antibody titres [22]. Based on this observation, we asked whether components derived from pathogens such as viruses or bacteria modulate the re-stimulation of FVIII-specific immune memory and disturb the inhibition of memory B-cell re-stimulation by high doses of FVIII. Microbial components are recognized by specific TLR that serve as an important link between innate and adaptive immunity. We studied the modulation of FVIII-specific memory B cells by a range of different ligands for TLR (zymosan for TLR2, poly I:C for TLR3, LPS for TLR4, Flagellin for TLR5, Loxoribine for TLR7 and CpG oligonucleotides for TLR9) [23,24]. The most dramatic effects were seen with Loxoribine, a ligand for TLR7 (Fig. 4a) [23]. Loxoribine at 10 000 ng mL−1 amplified the re-stimulation of FVIII-specific memory B cells at 10 ng mL−1 FVIII and completely abolished the inhibition of memory B-cell re-stimulation at 1000 ng mL−1 FVIII (Fig. 4a) [23]. Furthermore, Loxoribine facilitated a re-stimulation of FVIII-specific memory B cells in the complete absence of T cells (Fig. 4b) and even induced some re-stimulation in the complete absence of FVIII (Fig. 4a,b).

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Figure 4.  Loxoribine modulates both re-stimulation and inhibition of FVIII-specific murine memory B cells. CD138 spleen cells were obtained from haemophilic mice treated with human FVIII. Cells were re-stimulated for 6 days with different concentrations of human FVIII and Loxoribine as indicated. Newly formed ASC were detected by ELISPOT assay. ELISPOTs represent the results obtained in a typical experiment. (a) Total CD138 spleen cells (b) CD138 spleen cells depleted of T cells

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Next, we wanted to know whether to induce modulation of memory B-cell re-stimulation the triggering of TLR7 by Loxoribine needed to be simultaneous with the re-stimulation by FVIII. To address this question, we started our in vitro culture in the presence of FVIII on day 0 and added Loxoribine at different time points during a 6-day culture. Our results indicated that triggering TLR7 by Loxoribine can be induced up to 2 days after re-stimulation with FVIII to achieve an amplification of memory B-cell re-stimulation and a prevention of memory B-cell inhibition in our 6-day in vitro culture (Fig. 5a).

image

Figure 5.  Time-dependent modulation of FVIII-specific memory B cells by Loxoribine. CD138 spleen cells were obtained from haemophilic mice treated with human FVIII. Cells were re-stimulated for 6 days with different concentrations of human FVIII and with 10 000 ng mL−1 Loxoribine. FVIII was added on day 0. Loxoribine was added on different days as indicated. Newly formed ASCs were detected by ELISPOT assay. ELISPOTs represent the results obtained in a typical experiment. (a) Addition of Loxoribine at different time points as indicated. (b) Addition of buffer (negative control) at different time points.

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Detection of FVIII-specific memory B cells in patients with haemophilia A

In the preceding sections, we described several mechanisms by which FVIII-specific memory responses in haemophilic mice can be modulated. The question arises whether these mechanisms also operate in patients with haemophilia A and FVIII inhibitors. In particular, it would be important to know whether any of these mechanisms could be targeted to develop new therapeutic approaches for either the eradication of FVIII-specific immune memory or the prevention of anamnestic immune responses against FVIII in patients. To address this question, it is important to develop technologies that are suitable for analysing FVIII-specific memory B cells in patients.

We adapted a method established by Crotty et al. [24] to track FVIII-specific memory B cells in PBMC of patients with haemophilia A and FVIII inhibitors. For this purpose, PBMCs were polyclonally stimulated to allow all memory B cells to differentiate into ASC. ASC specific for FVIII and human serum albumin (HSA) and the total number of IgG-secreting cells were then analysed by ELISPOT technology (Fig. 6). The number of specific ASC directly correlates with the initial number of specific memory B cells [24].

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Figure 6.  Tracking FVIII-specific memory B cells in patients with haemophilia A. The results of the detection of FVIII-specific memory B cells in the peripheral blood of six patients with severe haemophilia A and FVIII inhibitors (a) and six patients with severe haemophilia A without FVIII inhibitors (b) are shown. Human serum albumin (HSA) was used as negative control. Each spot represents a single newly differentiated anti-FVIII antibody-producing plasma cell. The percentage of antigen-specific cells related to total IgG-producing cells (% of total IgG) as also the limits of detection (LD) were calculated for each patient. This research was originally published in reference [20].

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We analysed PBMC of 12 patients with severe haemophilia A (Table 1) for the presence of memory B cells specific for human FVIII and HSA (negative control). Six patients had FVIII inhibitors with Bethesda titres between 1 and 1000 BU mL−1 (Table 1). Five of the patients with inhibitors were not being treated with FVIII products at that time but with bypassing products, all patients without inhibitors were being treated with FVIII products (Table 1). None of the patients showed detectable levels of memory B cells specific for HSA (negative control, Fig. 6a). FVIII-specific memory B cells were detected in the peripheral blood cells of one of the patients with inhibitors but not in any of the patients without inhibitors (Fig. 6a,b). The frequency of FVIII-specific memory B cells in the positive patient was 0.24% of total IgG memory B cells (Fig. 6a). The limit of detection for antigen-specific memory B cells was in the range between 0.02% and 0.28% of the total IgG memory B cells and varied considerably between individual patients (Fig. 6a,b).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

We studied the re-stimulation and differentiation of FVIII-specific memory B cells using an in vitro culture system that is based on CD138 spleen cells obtained from haemophilic mice treated with FVIII. CD138 spleen cells contain all spleen cells except CD138+ ASC. Because of the nature of this mixed cell population as a source for FVIIII-specific memory B cells, it is difficult to exactly define the cell-to-cell interactions that are required for the re-stimulation or inhibition of FVIII-specific memory B cells. Furthermore, it is not possible to specify signal transduction pathways that are involved in the re-stimulation or inhibition of these cells. Therefore, we have further developed this method and established an in vitro culture system that operates with highly purified memory B cells and highly purified CD4+ T cells [25,26]. Currently, we use this improved system to study the mechanisms that are responsible for the re-stimulation and inhibition of FVIII-specific memory B cells under the conditions described in this article.

Based on our findings, that the re-stimulation of FVIII-specific memory B cells requires direct cell-to-cell contact with activated T cells [17], we initiated experiments that focussed on the modulation of FVIII-specific memory B-cell responses by interfering with essential co-stimulatory interactions. Our results indicate that B7-1/B7-2-CD28 and CD40-CD40L interactions are essential for the re-stimulation of these cells. On the other hand, ICOS-ICOSL interactions are not important. The B7-1/B7-2-CD28/CTLA-4 pathway is one of the best characterized co-stimulatory pathways for T-cell activation and is also essential for T-cell tolerance [27,28]. Qian et al. [29] were able to show that B7-2, but not B7-1, was involved in the primary immune response against FVIII in haemophilic mice. Furthermore, injecting murine CTLA-4-Ig into haemophilic mice prevented a further increase in anti-FVIII antibody titres in haemophilic mice with an established anti-FVIII immune response, indicating that CTLA-4-Ig blocks the re-stimulation of FVIII-specific memory B cells. Comparing our results [17] with those published by Qian et al. [29], we conclude that B7-2, but not B7-1, is involved in primary anti-FVIII antibody responses in haemophilic mice and that both molecules are important for the memory-driven antibody response.

CD40-CD40L interactions are a key event in T-cell-dependent humoral immune responses [30]. The results from studies on the significance of these interactions for the differentiation of memory B cells into ASC, however, are in conflict. Several reports suggest that CD40 signalling is important for the terminal differentiation of B cells and for antibody secretion [31–34]. Other reports show that CD40 signalling prevents the terminal differentiation of B cells [35–39]. Our results indicate that the re-stimulation of FVIII-specific memory B cells and their subsequent differentiation into anti-FVIII ASC requires CD40-CD40L interaction. The blockade of these interactions prevented the formation of anti-FVIII ASC in vitro and reduced it significantly in vivo [17]. We believe that the blockade of CD40-CD40L interactions in our system downregulates T-cell activation and, more importantly, blocks the interaction between activated T cells and memory B cells.

Based on the successful use of high-dose FVIII for the induction of immune tolerance in patients with haemophilia A [1], we speculated on the issue of whether the re-stimulation of FVIII-specific memory B cells was affected in any significant manner by high concentrations of FVIII. Our results demonstrate that concentrations of FVIII that are below the physiological plasma concentration of 100 ng mL−1 (1 U mL−1) re-stimulate FVIII-specific memory B cells and induce their differentiation into ASC in vitro, whereas concentrations that are above the physiological plasma concentration inhibit this process. These results support the idea that the inhibition or eradication of FVIII-specific memory B cells might be an early event in the downregulation of established anti-FVIII antibody responses in patients. The eradication of memory B cells would prevent their differentiation into ASC and, moreover, may lead to a deficiency of effective antigen-presenting cells required for the re-stimulation of FVIII-specific T cells. The induction of regulatory T cells rather than effector T cells could be the consequence of this deficiency. Currently, it is not clear, however, whether high-dose FVIII ITI therapy in patients would lead to local FVIII concentrations that are comparable with the concentrations that we used in our in vitro experiments. Further studies are necessary to investigate this hypothesis.

Toll-like receptors recognize invading pathogens such as viruses and bacteria and serve as an important link between innate and adaptive immunity [40,41]. Given the importance of TLR for the regulation of adaptive immune responses, we speculated as to how the triggering of TLR would influence the regulation of FVIII-specific memory B cells. In particular, we were interested to know whether the triggering of TLR would prevent the inhibition of memory B-cell re-stimulation by high concentrations of FVIII. Our results clearly indicate that both the stimulation of memory responses by low doses of FVIII as well as the inhibition of memory responses by high doses of FVIII are modulated by TLR-triggering. Furthermore, the triggering of TLR re-stimulates memory responses in the complete absence of T cells, and to a certain degree, even in the absence of FVIII. The natural ligands of TLR7 were identified as single-stranded RNA (ssRNA) [42–44]. Mouse TLR7, human TLR8 and human TLR7 recognize ssRNA viruses such as the influenza [43,44], Sendai [45] and Coxsackie B [46] viruses. This recognition requires the internalization of the virus and its replication to release the viral RNA into endosomes, where TLR7 and TLR8 reside. The interaction between the ssRNA and TLR7/8 triggers the recruitment of the adapter molecule MyD88 leading to the activation of nuclear factor κB and other transcription factors and the production of pro-inflammatory cytokines and chemokines. Based on our results, it can therefore be expected that any infection with the indicated viruses could potentially modulate FVIII-specific immune memory in patients with FVIII inhibitors.

In the last part of this article, we present the first results of our attempts to identify FVIII-specific memory B cells in the peripheral blood of patients with haemophilia A. For this purpose, we adapted a technology that was recently described by Crotty et al. [24] to human FVIII. We studied 12 patients with haemophilia A, six of them had detectable titres of neutralizing anti-FVIII antibodies. We could detect FVIII-specific memory B cells in one of the patients with FVIII inhibitors. This was the patient who showed the highest titres of neutralizing anti-FVIII antibodies. The frequency of FVIII-specific memory B cells in this patient was 0.24% of the total pool of IgG memory B cells. The detection limit for FVIII-specific memory B cells was in the range between 0.02% and 0.28% of the total IgG memory B cells and showed considerable variations between individual patients. The lack of detectable FVIII-specific memory B cells in five out of the six patients with FVIII inhibitors might be because of one or a combination of the following reasons. Four out of the five patients had last received FVIII treatment between 4 and 14 years earlier. Bypassing agents that had been given recently might not have provided sufficient stimuli to keep the pool of FVIII-specific memory B cells in the circulation large enough to be detectable. Alternatively, the remaining FVIII-specific memory B cells might have been located in secondary lymphoid organs and might have only re-circulated after re-stimulation with FVIII. Another reason for the lack of detectable FVIII-specific memory B cells in five out of the six patients with FVIII inhibitors might have been the sensitivity of the assay. In its current state of development, this assay cannot detect FVIII-specific memory B cells with frequencies below 0.02% of the total IgG memory B cells. Therefore, a further improvement in the detection limit of the method might be necessary.

Summarizing our data, we conclude that FVIII-specific memory B cells are an important target for the development of new strategies to induce FVIII-specific immune tolerance in patients with haemophilia A and FVIII inhibitors. Therefore, future efforts should focus on studying the regulation of these cells both in preclinical animal models and in patients. However, the eradication of memory B cells can only be a first step in the induction of immune tolerance in patients with FVIII inhibitors. A second step will most likely be necessary to keep a stable immune tolerance and prevent the re-induction of anti-FVIII antibodies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

The authors are grateful to all team members within Global Preclinical R&D of Baxter BioScience who have supported us in our studies. The author would also like to thank Elise Langdon-Neuner for editing this manuscript.

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

B. M. Reipert, P. Allacher, I. Lang, J. Ilas, E. M. Muchitsch and H. P. Schwarz are employees of Baxter Innovations GmbH. A. G. Pordes' PhD research is funded by Baxter Innovations GmbH. The other authors stated that they had no interests which might be perceived as posing a conflict or bias.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
  • 1
    Brackmann HH, Gormsen J. Massive factor VIII infusion in haemophiliac with factor VIII inhibitor, high responder. Lancet 1977; 2: 993.
  • 2
    Brackmann HH, Oldenburg J, Schwaab R. Immune tolerance for the treatment of factor VIII inhibitors – twenty years’‘Bonn protocol’. Vox Sang 1996; 70(Suppl. 1): 305.
  • 3
    Mariani G, Siragusa S, Kroner B. Immune tolerance induction in hemophilia A. Biomed Prog 2003; 16: 526.
  • 4
    DiMichele DM, Kroner BL. The North American Immune Tolerance Registry: practices, outcomes, outcome predictors. Thromb Haemost 2002; 87: 527.
  • 5
    McHeyzer-Williams LJ, McHeyzer-Williams MG. Antigen-specific memory B cell development. Annu Rev Immunol 2005; 23: 487513.
  • 6
    McHeyzer-Williams LJ, Malherbe LP, McHeyzer-Williams MG. Helper T cell-regulated B cell immunity. CTMI 2006; 311: 5983.
  • 7
    Moser K, Tokoyoda K, Radbruch A, McLennan I, Manz RA. Stromal niches, plasma cell differentiation and survival. Curr Opin Immunol 2006; 18: 26570.
  • 8
    Bar-Or A, Oliveira EML, Amderson DE et al. Immunological memory: contribution of memory B cells expressing costimulatory molecules in the resting states. J Immunol 2001; 167: 566977.
  • 9
    Bi L, Lawler AM, Antonarakis SE, High KA, Gearhart JD, Kazazian HH. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet 1995; 10: 11921.
  • 10
    Bi L, Sarkar R, Naas T et al. Further characterization of factor VIII-deficient mice created by gene targeting: RNA and protein studies. Blood 1996; 88: 3446550.
  • 11
    Qian J, Borovok M, Bi L, Kazazian HH, Hoyer LW. Inhibitor development and T cell response to human factor VIII in murine haemophilia A. Thromb Haemost 1999; 81: 2404.
  • 12
    Reipert BM, Ahmad RU, Turecek PL, Schwarz HP. Characterization of antibodies induced by human factor VIII in a murine knockout model of hemophilia A. Thromb Haemost 2000; 84: 82632.
  • 13
    Reipert BM, Sasgary M, Ahmad RU, Auer W, Turecek PL, Schwarz HP. Blockade of CD40/CD40 ligand interactions prevents induction of factor VIII inhibitors in hemophilic mice but does not induce lasting immune tolerance. Thromb Haemost 2001; 86: 134552.
  • 14
    Wu H, Reding M, Qian J et al. Mechanism of the immune response to human factor VIII in murine haemophilia A. Thromb Haemost 2001; 85: 12533.
  • 15
    Muchitsch EM, Turecek PL, Zimmermann K et al. Phenotypic expression of murine hemophilia (letter). Thromb Haemost 1999; 82: 13713.
  • 16
    Hausl C, Maier E, Schwarz HP, Ahmad RU, Turecek PL, Reipert BM. Long-term persistence of anti-factor VIII antibody–secreting cells in hemophilic mice after treatment with human factor VIII. Thromb Haemost 2002; 87: 8405.
  • 17
    Hausl C, Ahmad RU, Schwarz HP et al. Preventing re-stimulation of memory B cells in hemophilia A: a potential new strategy for the treatment of antibody-dependent immune disorders. Blood 2004; 104: 11522.
  • 18
    Hausl C, Ahmad RU, Sasgary M et al. High-dose factor VIII inhibits factor VIII-specific memory B cells in hemophilia A with factor VIII inhibitors. Blood 2005; 106: 341522.
  • 19
    Kasper CK, Aledort LM, Counts RB. A more uniform measurement of factor VIII inhibitors. Thromb Diath Heamorr 1975; 34: 86972.
  • 20
    Lang I, Windyga J, Klukowska A, Ilas J, Schwarz HP, Reipert BM. Towards the detection of factor VIII-specific memory B cells in hemophilia A patients with factor VIII inhibitors. In: Scharrer I, Schramm W, eds. 35th Haemophilia Symposium Hamburg 2005. Berlin, Heidelberg, New York: Springer Verlag, 2006: 2536.
  • 21
    Sasgary M, Ahmad RU, Schwarz HP, Turecek PL, Reipert BM. Single cell analysis of factor VIII-specific T-cells in hemophilic mice after treatment with human factor VIII. Thromb Haemost 2002; 87: 26672.
  • 22
    Hay CR, Keegan J, Goldstone J et al. International immune tolerance (ITI) study: frequency of central venous line infections and effect on ITI outcome. Haemophilia 2006; 12(Suppl. 2): 14PO379.
  • 23
    Allacher P, Hausl C, Ahmad RU, Schwarz HP, Turecek PL, Reipert BM. Toll-like receptor triggering modulates factor VIII-specific immune memory in murine hemophilia A with factor VIII inhibitors. Blood 2005; 106: 214A.
  • 24
    Crotty S, Aubert RD, Glidewell J, Ahmed R. Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system. J Immunol Methods 2004; 286: 11122.
  • 25
    Pordes AG, Hausl C, Allacher P et al. T-cell-independent re-stimulation of FVIII-specific memory B cells requires help from activated plasmacytoid dendritic cells. Blood 2007; 110: 1155A.
  • 26
    Hausl C, Ahmad RU, Baumgartner B, Schwarz HP, Ehrlich HJ, Reipert B. Single-cell analysis of FVIII-specific memory B cells in murine hemophilia A. Blood 2007; 110: 1157A.
  • 27
    Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol 2002; 2: 11626.
  • 28
    Rudd CE, Schneider H. Unifying concepts in CD28, ICOS and CTLA4 co-receptor signalling. Nat Rev Immunol 2003; 3: 54456.
  • 29
    Qian J, Collins M, Sharpe AH, Hoyer LW. Prevention and treatment of factor VIII inhibitors in murine hemophilia A. Blood 2000; 95: 13249.
  • 30
    Foy TM, Aruffo A, Bajorath J, Buhlmann JE, Noelle RJ. Immune regulation by CD40 and its ligand GP39. Ann Rev Immunol 1996; 14: 591617.
  • 31
    Spriggs MK, Armitage RJ, Strockbine L et al. Recombinant human CD40 ligand stimulates B cell proliferation an immunoglobulin E secretion. J Exp Med 1992; 176: 154350.
  • 32
    Maliszewski CR, Grabstein K, Fanslow WC, Armitage R, Spriggs MK, Sato TA. Recombinant CD40 ligand stimulation and murine B cell growth and differentiation: cooperative effects of cytokines. Eur J Immunol 1992; 23: 10449.
  • 33
    Grabstein KH, Maliszewski CR, Shanebeck K et al. The regulation of T cell-dependent antibody formation in vitro by CD40 ligand and IL-2. J Immunol 1992; 150: 31417.
  • 34
    Soro PG, Morales-A P, Martinez-M JA et al. Differential involvement of the transcription factor Blimp-1 in T cell-independent and -dependent B cell differentiation to plasma cells. J Immunol 1999; 163: 6117.
  • 35
    Arpin C, Déchanet J, Van Kooten C et al. Generation of memory B cells and plasma cells in vitro. Science 1995; 268: 7202.
  • 36
    Callard RE, Herbert J, Smith SH, Armitage RJ, Costelloe KE. CD40 cross-linking inhibits specific antibody production by human B cells. Int Immunol 1995; 7: 180915.
  • 37
    Bergman MC, Attrep JF, Grammer AC, Lipsky PE. Ligation of CD40 influences the function of human Ig-secreting B cell hybridomas both positively and negatively. J Immunol 1996; 156: 311832.
  • 38
    Miyashiba T, McIlraith MJ, Grammer AC et al. Bidirectional regulation of human B cell responses by CD40-CD40 ligand interactions. J Immunol 1997; 158: 462033.
  • 39
    Randall TD, Heath AW, Santos-Argumedo L, Howard MC, Weissman IL, Lund FE. Arrest of B lymphocyte terminal differentiation by CD40 signaling: mechanism for lack of antibody-secreting cells in germinal centers. Immunity 1998; 8: 73342.
  • 40
    Takeda K, Kaisho T, Akira S. Toll-like receptors. Ann Rev Immunol 2003; 21: 33576.
  • 41
    Uematsu S, Akira S. The role of Toll-like receptors in immune disorders. Expert Opin Biol Ther 2006; 6: 20314.
  • 42
    Heil F, Hemmi H, Hochrein H et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004; 303: 15269.
  • 43
    Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004; 303: 152931.
  • 44
    Lund JM, Alexopoulou L, Sato A et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci USA 2004; 101: 5598603.
  • 45
    Melchjorsen J, Jensen SB, Malmgaard L et al. Activation of innate defense against a paramyxovirus is mediated by RIG-I and TLR7 and TLR8 in a cell-type-specific manner. J Virol 2005; 79: 1294451.
  • 46
    Triantafilou K, Orthopoulos G, Vakakis E et al. Human cardiac inflammatory responses triggered by Coxsackie B viruses are mainly Toll-like receptor (TLR) 8-dependent. Cell Microbiol 2005; 7: 111726.