Inhibition of Factor VIII-Specific Memory B Cell Responses by Supra-Physiological Concentrations of Factor VIII.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 38-38
Author(s):  
Christina Hausl ◽  
Rafi U. Ahmad ◽  
Maria Sasgary ◽  
Christopher B. Doering ◽  
Pete S. Lollar ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Factor Viii ◽  
B Cell ◽  
Immune Tolerance ◽  
Hemophilia A ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Specific Memory ◽  
Stimulation Of

Abstract Inhibitory antibodies against factor VIII (FVIII) are the major complication experienced by hemophilia A patients treated with FVIII products. The most effective therapy to eradicate these antibodies is elevated doses of FVIII over a prolonged period. Despite clinical practice in using such protocols, nothing 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. Understanding the underlying mechanisms, however, would facilitate designing new therapeutic strategies. The re-stimulation of FVIII-specific memory responses after each dose of FVIII is probably the most important event in the maintenance of FVIII inhibitors in patients. Therefore, the eradication of these memory responses should be an essential step in the down-modulation of inhibitory antibodies and the induction of immune tolerance. We used a murine model of hemophilia A to answer the question whether FVIII-specific memory responses are sensitive to increasing doses of FVIII. In particular, we were interested in the differential effects of FVIII on memory-B-cell and memory-T-cell responses. For the analysis of FVIII-specific memory responses, we re-stimulated FVIII-specific memory B- and T-cells obtained from spleens of hemophilic mice treated with four doses of human FVIII or eight doses of murine FVIII as described (Sasgary et al.: Thromb Haemost2002; 87:266–72; Hausl et al.: Blood2004; 104:115–22). Our results show dose-dependent effects of FVIII on the re-stimulation of FVIII-specific memory B cells in vitro. Physiological concentrations of FVIII below 100 ng/ml re-stimulate memory B cells and induce their differentiation into anti-FVIII antibody-secreting plasma cells. Supra-physiological concentrations above 100 ng/ml, however, inhibit memory-B-cell re-stimulation. The inhibition of memory-B-cell re-stimulation is irreversible and seems to be due to an induction of apoptosis that is at least partly mediated by Fas-dependent mechanisms. Furthermore, the inhibition appears to be initiated by triggering the B-cell receptor (BCR) without the requirement of an excessive cross-linking of the BCR. The activation of FVIII-specific T cells is not affected by increasing doses of FVIII. We conclude that the induction of apoptosis in FVIII-specific memory B cells might be the first step in the induction of immune tolerance in hemophilia A patients with FVIII inhibitors who receive high doses of FVIII. The eradication of memory B cells would prevent their differentiation into antibody-secreting plasma cells and, moreover, might lead to a deficiency of effective antigen-presenting cells required for the re-stimulation of FVIII-specific memory T cells. The induction of regulatory T cells rather than effector T cells could be the consequence of this deficiency.

Toll-Like Receptor Triggering Modulates Factor VIII-Specific Immune Memory in Murine Hemophilia A with Factor VIII Inhibitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 214-214 ◽  
Author(s):  
Peter Allacher ◽  
Christina Hausl ◽  
Rafi U. Ahmad ◽  
Hans Peter Schwarz ◽  
Peter L. Turecek ◽  
...  
Keyword(s):  
B Cells ◽  
Factor Viii ◽  
B Cell ◽  
Hemophilia A ◽  
Memory B Cells ◽  
Immune Memory ◽  
Low Doses ◽  
Specific Memory ◽  
High Doses ◽  
Stimulation Of

Abstract The development of inhibitory antibodies against factor VIII (FVIII) is the major complication in the treatment of hemophilia A patients with FVIII products. Immune Tolerance Induction (ITI) therapy using long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and induce long-lasting immune tolerance. It is a common observation that infections, particularly central venous catheter infections during ITI cause a rise in anti-FVIII antibody titers that can prolong the course of ITI or possibly even lead to failure of ITI. Based on this observation, we asked the question whether microbial components derived from viruses or bacteria modulate the re-stimulation of FVIII-specific immune memory and disturb the recently described inhibition of memory-B-cell-re-stimulation by high doses of FVIII (Hausl et al.: Blood2005; in press). Microbial components are recognized by toll-like receptors (TLRs) that serve as an important link between innate and adaptive immunity. TLRs can discriminate various microbial components such as lipopeptides derived from bacteria or zymosan derived from yeast (recognized by TLR1/2 or TLR2/6), double-stranded RNA derived from viruses (recognized by TLR3), lipopolysaccharide (LPS) derived from gram-negative bacteria (recognized by TLR4), flagellin derived from bacterial flagella (recognized by TLR5), single-stranded RNA derived from viruses (recognized by TLR7/8) or bacterial DNA containing the unmethylated CpG motif (recognized by TLR9). We analyzed the re-stimulation of FVIII-specific memory-B cells using a murine model of hemophilia A as described previously (Hausl et al.: Blood2004; 104:115–22; Hausl et al.: Blood2005, in press). The following TLR ligands were tested: zymosan for TLR2 (0.1–10,000 ng/ml), poly I:C for TLR3 (1.0–50,000 ng/ml), LPS for TLR4 (0.1–10,000 ng/ml), Flagellin for TLR5 (0.01–1,000 ng/ml), Loxoribine for TLR7 (1.0–50,000 ng/ml) and CpG oligonucleotides for TLR9 (0.1–10,000 ng/ml). Our results indicate that none of the TLR ligands at the concentrations tested induced a significant re-stimulation of FVIII-specific memory B cells in the complete absence of either FVIII or T cells. However, ligands for TLR3, TLR4, TLR7 and TLR9 were able to disturb the inhibition of memory-B-cell-re-stimulation by high doses of FVIII and amplified the re-stimulation induced by low doses of FVIII substantially. We conclude that triggering of TLRs by microbial components that are present during infections amplify the re-stimulation of FVIII-specific memory B-cells induced by low doses of FVIII and disturb the inhibition induced by high doses of FVIII.

Single-Cell Analysis of FVIII-Specific Memory B Cells in Murine Hemophilia A.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1157-1157 ◽  
Author(s):  
Christina Hausl ◽  
Rafi U. Ahmad ◽  
Bernhard Baumgartner ◽  
Hans Peter Schwarz ◽  
Hartmut Ehrlich ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Cd4 T Cells ◽  
Single Cell Analysis ◽  
Memory B Cells ◽  
Cell Analysis ◽  
Memory B Cell ◽  
Specific Memory

Abstract The elimination of FVIII-specific memory B cells is an essential step in the design of new therapeutic strategies for the induction of immune tolerance in hemophilia A with FVIII inhibitors. Using a mouse model of hemophilia A we recently reported that low dose FVIII stimulates the differentiation of FVIII-specific memory B cells into antibody-secreting plasma cells whereas high dose FVIII inhibits this process. The inhibition of memory-B-cell re-stimulation is irreversible and seems to be due to an induction of apoptosis. Further understanding of the complex interactions that lead to either re-stimulation and differentiation of memory B cells or inhibition and eradication of these cells requires appropriate technologies for single-cell analysis and functional studies. We established a new technology for single-cell analysis and cell sorting of FVIII-specific murine memory B cells. A combination of magnetic bead separation and multi-color flow cytometry enabled us to analyze and purify FVIII-specific memory B cells obtained from hemophilic mice treated with FVIII. In a first step, we depleted undesirable cell populations (IgM+, IgD+, CD11c+, F4/80+, Gr1+ and CD49b+ cells) from total spleen cells by magnetic bead separation. In a second step, we used multicolor flow cytometry to exclude CD4+ T cells and analyze the FVIII-specific memory B cell compartment. This compartment was specified by staining the specific B-cell receptor with FVIII and anti-IgG antibodies. Frequencies of cells in this compartment ranged from 0.1–0.5% of total spleen cells in animals treated with 4 intravenous doses of FVIII, given at weekly intervals. We could not detect any FVIII-specific memory B cells in naïve mice. By means of single cell sorting we isolated FVIII-specific memory B cells for further functional studies. We were able to cultivate FVIII-specific memory B cells in microwell cultures in vitro and differentiate them into antibody-secreting plasma cells. The re-stimulation and differentiation of single-cell sorted memory B cells was strictly dependent on the presence of activated CD4+ T cells. CD4+ T cells obtained from naïve mice did not support the memory response. Furthermore, the re-stimulation and differentiation of memory B cells in the presence of activated CD4+ T cells did not require additional dendritic cells for antigen presentation. Obviously, memory B cells provide sufficient antigen presentation to CD4+ T cells to enable them to trigger the memory response. Our approach for single-cell analysis and purification of FVIII-specific memory B cells provides a new tool for tracking memory B cell populations in vivo and for directly analyzing the regulation of memory B cell function. It opens the field for future studies which should elucidate signals and molecules involved in activation or inhibition and eradication of FVIII-specific memory B cells. These activities will eventually lead to the identification of targets for the design of new treatment strategies for patients with FVIII inhibitors.

Deletion or Inhibition of Fc Gamma Receptor IIb (CD32) Prevents the Memory B Cell Response to Factor VIII in a Hemophilia A Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 204-204 ◽  
Author(s):  
Sonja Werwitzke ◽  
Marcus von Hornung ◽  
Katy Kalippke ◽  
Arne Trummer ◽  
Arnold Ganser ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Knockout Mice ◽  
Cell Response ◽  
Hemophilia A ◽  
Memory B Cells ◽  
Double Knockout ◽  
Memory B Cell ◽  
Specific Memory ◽  
Antibody Secreting Cells

Abstract Abstract 204 The formation of inhibitory antibodies to factor VIII (FVIII) is the foremost complication of replacement therapy in hemophilia A. Patients with inhibitors are treated with very high doses of FVIII, over prolonged periods of time, to induce immune tolerance. Studies in a hemophilia A mouse model demonstrated that very high doses of FVIII can induce apoptosis in FVIII-specific memory B cells and prevent their differentiation into antibody-secreting cells. The Fc gamma receptor IIb (FcgRIIb) is expressed on B cells and mediates inhibitory signals after crosslinking with the B cell receptor. Here, we studied the potential role of this receptor in the regulation of memory B cell response to FVIII. FVIII knockout mice (B6;129S4-F8tm2Kaz/J) were crossed with FcgRIIb knockout mice (B6;129S4-Fcgr2btm1Ttk/J). Comparing F8−/− mice and F8−/−/FcgR2b−/− double knockout mice, the initial anti-FVIII antibody formation was similar after intravenous exposure to 4 weekly doses of 80 or 400 IU/kg. Similar numbers of FVIII-specific antibody-secreting cells were detected in the spleen and bone marrow by ELISPOT. As previously shown, in vitro re-stimulation of memory B cells from spleens of immunized F8−/− mice at doses of 1 to 200 ng/ml induced their differentiation into antibody-secreting cells. Higher doses of 400 to 800 ng/ml prevented differentiation. In F8−/−/FcgR2b−/− double knockout mice, however, formation of antibody-secreting cells was completely inhibited across all FVIII doses tested. Addition of B220-depleted splenocytes from F8−/− mice did not restore memory B cell function in F8−/−/FcgR2b−/− double knockout mice, indicating that the observed effect was not due to dysfunction of follicular dendritic cells or other antigen-presenting cells. Inhibition of FcgRIIb using a monoclonal antibody prevented the FVIII-specific memory B cell response in splenocytes from immunized F8−/− mice. Staining with propidium iodide, annexin V, or anti-caspase 3 indicated increased rates of apoptosis when FcgRIIb was blocked during re-stimulation. In summary, FcgRIIb plays a crucial role for the differentiation of FVIII-specific splenic memory B cells into antibody-secreting cells. Inhibition of FcgRIIb appears to sensitize B cells for apoptosis during re-stimulation with FVIII. This mechanism could potentially facilitate the eradication of FVIII-specific memory B cells during ITI. Disclosures: No relevant conflicts of interest to declare.

Factor VIII-Specific Memory B-Cells in Patients with Hemophilia A.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1020-1020 ◽  
Author(s):  
Pauline M.W. van Helden ◽  
Paul H.P. Kaijen ◽  
H. Marijke van den Berg ◽  
Jan Voorberg
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Hemophilia A ◽  
Low Frequency ◽  
Memory B Cells ◽  
The Past ◽  
Specific Memory ◽  
Specific Igg ◽  
Inhibitory Antibodies

Abstract The quick anamnestic antibody response seen after recurrent exposure to antigen involves memory B- cells that, helped by T-cells, undergo rigid proliferation and subsequent differentiation into antibody producing cells. The presence of a pool of memory B-cells allows for a rapid response to antigens which quickly eliminates incoming pathogens. In the context of immune responses to therapeutic agents such as blood coagulation factor VIII (FVIII), antigenic re-stimulation of specific memory B-cells is undesirable. In approximately 25% of hemophilia A patients replacement therapy is hampered by inhibitory antibodies that bind to FVIII. Currently, the FVIII-specific memory B-cell compartment in patients with hemophilia A has remained poorly characterized. We have developed a protocol that allows for identification and quantification of circulating memory B-cells in patients with hemophilia A. CD19+ B-cells were sorted on a layer of irradiated EL4B5 thymoma cells expressing CD40L in the presence of the supernatant of mitogen-stimulated T-cells. These experimental conditions, that mimic the interaction of B-cells with an activating helper CD4+ T-cell, induce proliferation of memory B-cells and allow them to differentiate into antibody secreting cells (ASC) in an antigen-independent manner. After 9–10 days of culture, total IgG and FVIII-specific IgG was determined by ELISA and number of ASC was determined by ELISpot. We analyzed blood samples of five multi-transfused patients (>50 FVIII administrations) who never experienced any inhibitor episode, five patients who experienced inhibitory antibodies in the past but were successfully treated with immune tolerance induction and 6 patients with an inhibitor at the time of blood sampling. The ELISA set-up appeared to be more sensitive than ELISpot showing ASC producing anti-FVIII antibodies varying from 0.2–50 ng/ml. In contrast, ELISpot analysis only allowed for detection of B-cell clones producing over ~4 ng/ml of FVIII-specific IgG. Frequencies of FVIII-specific memory B-cells varied from 0–0.027% of total number of circulating peripheral B-cells. The relative amount of circulating memory B-cells did not correspond to inhibitor titers as measured in a Bethesda assay. The highest frequencies were observed in patients suffering from anamnestic response to FVIII suggesting the importance of antigenic stimulation for maintenance of memory B-cell levels. This is further supported by the low frequency that was observed in a high-titer inhibitor patient who had not been treated with FVIII for several months prior to blood sampling. Surprisingly, we detected FVIII-specific memory B-cells in two multi-transfused patients who did not experience any inhibitor episode in the past. These B-cells were present in a low frequency however and developed into ASC producing only limited amounts of anti-FVIII antibodies. These observations suggest that peripheral blood memory B-cells can develop in the absence of clinically relevant inhibitors.

T-Cell-Independent Re-Stimulation of FVIII-Specific Memory B Cells Requires Help from Activated Plasmacytoid Dendritic Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1155-1155 ◽  
Author(s):  
Aniko Ginta Pordes ◽  
Christina Hausl ◽  
Peter Allacher ◽  
Rafi U. Ahmad ◽  
Bernhard Baumgartner ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
B Cells ◽  
Cd4 T Cells ◽  
Plasma Cells ◽  
Hemophilia A ◽  
Plasmacytoid Dendritic Cells ◽  
Memory B Cells ◽  
Specific Memory ◽  
Stimulation Of

Abstract Memory B cells are essential for maintaining FVIII inhibitors in patients with hemophilia A. Using the murine E-17 model of hemophilia A, we showed previously that re-exposure to FVIII re-stimulates memory B cells very rapidly and drives their differentiation into antibody-producing plasma cells. Furthermore, we presented evidence that the re-stimulation of FVIII-specific memory B cells is regulated by the dose of FVIII used. Low doses re-stimulate memory B cells whereas high doses of FVIII inhibit this process and prevent the differentiation into anti-FVIII antibody-producing plasma cells. Both the re-stimulation and the inhibition can be modulated by triggering toll-like receptors (TLR) 7 and 9 with specific ligands that are typically found in microbial components derived from viruses or bacteria. Re-stimulation of FVIII-specific memory B cells in the presence of TLR ligands can even be observed in the absence of CD4+ helper T cells that are otherwise absolutely essential for this process. Based on these previous observations we asked whether the re-stimulation of FVIII-specific memory B cells in the absence of CD4+ helper T cells requires interaction with alternative “helper” cells that provide co-stimulatory signals to memory B cells. To address this question we used spleen cells obtained from hemophilic mice treated with FVIII to generate highly purified populations of memory B cells, CD4+ T cells and dendritic cells. The required purity of the different cell populations was achieved by a combination of magnetic bead separation and multi-color flow cytometric cell sorting. The memory B cell compartment was specified by the expression of CD19 together with surface IgG and the absence of surface IgM and IgD. Memory B cells were single-cell sorted and cultivated in micro-well cultures in the presence of FVIII to stimulate the in vitro differentiation into anti-FVIII antibody- producing plasma cells. Different combinations of CD4+ T cells, ligands for TLR 7 or 9 and dendritic cells were added to the micro-well cultures to find out which of the additives were required for the re-stimulation and differentiation of memory B cells. Neither FVIII alone nor any combination of FVIII and ligands for TLR 7 and 9 were able to re-stimulate highly purified memory B cells to differentiate into anti-FVIII antibody-producing plasma cells. The re-stimulation strictly depended on the presence of additional cells that could provide co-stimulation. These additional cells could be either activated CD4+ T cells or, alternatively, plasmacytoid dendritic cells activated by ligands for TLR 7 or 9. Some re-stimulation in the presence of activated plasmacytoid dendritic cells was even observed in the complete absence of FVIII. Based on our results we conclude that plasmacytoid dendritic cells that are activated by TLR ligands such as those expressed by infectious agents can replace CD4+ T cells in triggering the re-stimulation of memory B cells and their differentiation into antibody-producing plasma cells. Our findings provide important new insights into the regulation of memory-B-cell re-stimulation that need to be considered in the development of new therapeutic strategies for treating patients with FVIII inhibitors. Furthermore, our findings underscore the importance of environmental factors in the regulation of FVIII inhibitor development.

Requirements for Co-Stimulation in T-Cell Dependent and T-Cell Independent Re-Stimulation of FVIII-Specific Memory B Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 238-238 ◽  
Author(s):  
Aniko Ginta Pordes ◽  
Christina Hausl ◽  
Peter Allacher ◽  
Rafi Uddin Ahmad ◽  
Eva M Muchitsch ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Plasma Cells ◽  
Memory B Cells ◽  
Specific Memory ◽  
Stimulation Of

Abstract Memory B cells specific for factor VIII (FVIII) are critical for maintaining FVIII inhibitors in patients with hemophilia A. They are precursors of anti-FVIII antibody-producing plasma cells and are highly efficient antigen-presenting cells for the activation of T cells. The eradication of FVIII-specific memory B cells will be a prerequisite for any successful new approach to induce immune tolerance in patients with FVIII inhibitors. Little is known about the regulation of these cells. Previously we showed that ligands for toll-like receptors (TLR) 7 and 9 are able to re-stimulate FVIII-specific memory B cells in the absence of T-cell help. However, alternative “helper cells” such as dendritic cells are essential for providing help to memory B cells under such conditions. Based on these findings, we asked which co-stimulatory interactions are required for the restimulation of memory B cells in the presence of dendritic cells and ligands for TLR and whether these co-stimulatory interactions are the same as those required for the restimulation of memory B cells in the presence of activated T cells. We used spleen cells from hemophilic mice treated with human FVIII to generate highly purified populations of memory B cells, CD4+ T cells and dendritic cells. The required purity was achieved by a combination of magnetic bead separation and fluorescence-activated cell sorting. The memory B cell compartment was specified by the expression of CD19 together with IgG and the absence of surface IgM and IgD. Memory B cells were cultured in the presence of FVIII to stimulate their differentiation into anti-FVIII antibody-producing plasma cells. Different combinations of CD4+ T cells, ligands for TLR 7 and 9 and dendritic cells were added to the memory-B-cell cultures. Blocking antibodies and competitor proteins were used to specify the co-stimulatory interactions required for the re-stimulation of memory B cells in the presence of either CD4+ T cells or dendritic cells and ligands for TLR 7 and 9. Our results demonstrate that the blockade of B7-1 and B7-2 as well as the blockade of CD40L inhibit the re-stimulation of FVIII-specific memory B cells and their differentiation into anti-FVIII antibody-producing plasma cells in the presence of T-cell help. Similar requirements apply for the re-stimulation of memory B cells in the presence of dendritic cells and ligands for TLR 7 or 9. Dendritic cells in the absence of ligands for TLR are not able to provide help for the re-stimulation of memory B cells, which indicates that dendritic cells need to be activated. Furthermore, ligands for TLR 7 or 9 were not able to re-stimulate memory B cells in the complete absence of dendritic cells. Based on these results we conclude that dendritic cells activated by ligands for TLR 7 or 9 can substitute for activated CD4+ T cells in providing co-stimulatory help for memory-B-cell re-stimulation. CD40-CD40L interactions seem to be the most important co-stimulatory interactions for the re-stimulation of memory B cells, not only in the presence of activated CD4+ T cells but also in the presence of ligands for TLR and dendritic cells.

The Impact of VWF on FVIII Immune Responses in Hemophilia a Mice with Pre-Existing Anti-FVIII Immunity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 84-84
Author(s):  
Juan Chen ◽  
Jocelyn A. Schroeder ◽  
Xiaofeng Luo ◽  
Robert R. Montgomery ◽  
Qizhen Shi
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Specific Memory ◽  
The Impact

Abstract Development of inhibitory antibodies (inhibitors) against FVIII is the significant complication in protein replacement therapy for hemophilia A (HA). Currently, immune tolerance induction (ITI) with aggressive infusion of high-dose FVIII represents the only effective therapeutic approach for eradication of FVIII inhibitors and results in restoration of normal FVIII pharmacokinetics in inhibitor patients. Whether the use of FVIII products containing VWF will affect the efficacy of the ITI is still a debated issue in the treatment of inhibitor patients. In this study, we explored the impact of VWF on FVIII immune responses in HA with pre-existing anti-F8 immunity using both in vitro and in vivomodels. Since the FVIII immune response is CD4+ T cell dependent, we first investigated how VWF affects FVIII-primed CD4+ T cells in response to FVIII restimulation. To address this question, we used a T cell proliferation assay. FVIIInull (F8null) mice were immunized with recombinant human FVIII (rhF8) to induce inhibitor development. Splenocytes from primed mice were labeled with CellTrace™ Violet and cultured with rhF8 with or without rhVWF. Four days later, cells were analyzed by FACS to assess the daughter (proliferated) cell population. The percentage of daughter CD4+ T cells (14.0±7.5%) in the condition cultured with 1 U/ml of rhF8 was significantly higher than without rhF8 (3.7±1.7%, n=6). The daughter cells further increased to 21.5±10.3% when cells were incubated with 10 U/ml of rhF8. However, when rhVWF was added to the culture media in addition to rhF8, percentages of daughter CD4+ T cells were significantly decreased in both the 1 U/ml and 10 U/ml rhF8 treatment groups (10.4±7.1% and 15. 8±8.4%, respectively). To further explore how VWF affects the FVIII immune response, we analyzed cytokine profiles in T cell culture supernants using a multiplex ELISA assay. The levels of IFNg and IL10 in the groups cultured with rhF8 in the presence of rhVWF were significantly lower than in the groups cultured with rhF8 only. The levels of TNFa, IL4, IL5, and IL12 in the groups cultured with rhF8 together with rhVWF were not significantly different than those in rhF8 groups without VWF. These results demonstrated that VWF significantly suppresses rhF8-primed CD4+ T cell proliferation in response to rhF8 restimulation and the inhibition is via the Th1 pathway. In a setting of pre-existing anti-F8 immunity, how FVIII-specific memory B cells respond to FVIII-restimulation and mature to antibody secreting cells (ASCs) is the critical pathway in terms of the clinical efficacy of FVIII infusion. To investigate how VWF affects memory B cell maturation upon FVIII restimulation, we used ELISPOT-based assay. Splenocytes from rhF8-primed HA mice were used as the source to prepare F8-specific memory B cell pools. CD138+ cells were depleted and the remaining cells were used as a pool of memory B cells. To stimulate the maturation of F8-specific memory B cells into ASCs, memory B cell pools from primed F8null mice were cultured with rhF8 with or without rhVWF for 6 days. After culture, newly formed ASCs were assessed by the ELISPOT assay. There were 54.4±19.5 ASCs/106 cells when cells from memory B cell pool were cultured with 0.05 U/ml rhF8. In contrast, there was only 15.6±1.6 ASCs/106cells after the cells were cultured with rhF8 together with rhVWF, indicating that memory B cell maturation is suppressed in the presence of rhVWF. We then used an in vivo model to further evaluate the impact of VWF on the immunogenicity of FVIII in HA with pre-existing immunity. Since we are unable to mimic the human ITI in F8null mice, we transferred memory B cells from rhF8-primed F8null splenocytes into immunocompromised F8null mice followed by rhF8 immunization in the presence or absence of rhVWF. Blood samples were collected one week after immunization for analysis. The inhibitor titer in animals that received rhF8-primed memory B cell pool followed by rhF8 immunization was 45.9±63.0 BU/ml (n=11), which was significantly higher than the titer in animals immunized with rhF8 together with rhVWF (23.9±38.4, P<.01). These results demonstrate that VWF suppressed the anti-F8 memory response in vivo. In summary, our ex vivo and in vivo data demonstrated that VWF attenuates F8-primed CD4+T cells and memory B cells in response to rhF8 restimulation, suggesting that infusion of FVIII together with VWF might reduce anti-F8 memory responses in HA with inhibitors. Disclosures No relevant conflicts of interest to declare.

scholarly journals Potential anti-EBV effects associated with elevated interleukin-21 levels: a case report

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Kristian Assing ◽  
Christian Nielsen ◽  
Marianne Jakobsen ◽  
Charlotte B. Andersen ◽  
Kristin Skogstrand ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Cell Formation ◽  
Memory B Cells ◽  
Memory B Cell

Abstract Background Germinal center derived memory B cells and plasma cells constitute, in health and during EBV reactivation, the largest functional EBV reservoir. Hence, by reducing germinal center derived formation of memory B cells and plasma cells, EBV loads may be reduced. Animal and in-vitro models have shown that IL-21 can support memory B and plasma cell formation and thereby potentially contribute to EBV persistence. However, IL-21 also displays anti-viral effects, as mice models have shown that CD4+ T cell produced IL-21 is critical for the differentiation, function and survival of anti-viral CD8+ T cells able to contain chronic virus infections. Case presentation We present immunological work-up (flow-cytometry, ELISA and genetics) related to a patient suffering from a condition resembling B cell chronic active EBV infection, albeit with moderately elevated EBV copy numbers. No mutations in genes associated with EBV disease, common variable immunodeficiency or pertaining to the IL-21 signaling pathway (including hypermorphic IL-21 mutations) were found. Increased (> 5-fold increase 7 days post-vaccination) CD4+ T cell produced (p < 0.01) and extracellular IL-21 levels characterized our patient and coexisted with: CD8+ lymphopenia, B lymphopenia, hypogammaglobulinemia, compromised memory B cell differentiation, absent induction of B-cell lymphoma 6 protein (Bcl-6) dependent peripheral follicular helper T cells (pTFH, p = 0.01), reduced frequencies of peripheral CD4+ Bcl-6+ T cells (p = 0.05), compromised plasmablast differentiation (reduced protein vaccine responses (p < 0.001) as well as reduced Treg frequencies. Supporting IL-21 mediated suppression of pTFH formation, pTFH and CD4+ IL-21+ frequencies were strongly inversely correlated, prior to and after vaccination, in the patient and in controls, Spearman’s rho: − 0.86, p < 0.001. Conclusions To the best of our knowledge, this is the first report of elevated CD4+ IL-21+ T cell frequencies in human EBV disease. IL-21 overproduction may, apart from driving T cell mediated anti-EBV responses, disrupt germinal center derived memory B cell and plasma cell formation, and thereby contribute to EBV disease control.

Agonist for Toll-Like Receptor (TLR) 9 Amplifies as Well as Inhibits the Differentiation of FVIII-Specific Memory B Cells into Antibody-Producing Plasma Cells in Vitro and in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3382-3382
Author(s):  
Peter Allacher ◽  
Christina Hausl ◽  
Aniko Ginta Pordes ◽  
Rafi Uddin Ahmad ◽  
Hartmut J Ehrlich ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Cpg Dna ◽  
Cell Responses ◽  
Specific Memory ◽  
B Cell Responses

Abstract Memory B cells are essential for maintaining long-term antibody responses. They can persist for years even in the absence of antigen and are rapidly re-stimulated to differentiate into antibody-producing plasma cells when they encounter their specific antigen. Previously we demonstrated that ligands for TLR 7 and 9 amplify the differentiation of FVIII-specific memory B cells into anti-FVIII antibody-producing plasma cells at low concentrations of FVIII and prevent the inhibition of memory-B-cell differentiation at high concentrations of FVIII. The modulation of FVIII-specific memory-B-cell responses by agonists for TLR is highly relevant for the design of new immunotherapeutic approaches in patients with FVIII inhibitors because TLR are activated by a range of different viral and bacterial components. Specifically, TLR 7 is triggered by single-stranded RNA derived from viruses and TLR 9 is triggered by bacterial DNA containing unmethylated CpG motifs. We further explored the modulation of FVIII-specific memory-B-cell responses by agonists for TLRs by studying a broad range of concentrations of CpG DNA, a ligand for TLR 9, both in vitro and in vivo using the murine E17 model of hemophilia A. We used CpG-DNA in concentrations ranging from 0.1 to 10,000 ng/ml to study the modulation of FVIII-specific memory-B-cell responses in vitro and verified the specificity of the effects observed by including a blocking agent for TLR 9 and GpC-DNA, a non-stimulating negative control for CpG DNA. Furthermore, we used doses of CpG DNA ranging from 10 to 50,000 ng per dose to study the modulation of FVIII-specific memory-B-cell responses in vivo. E17 hemophilic mice were treated with a single intravenous dose of 200 ng FVIII to stimulate the generation of FVIII-specific memory B cells and were subsequently treated with another dose of FVIII that was given together with CpG DNA. We analyzed titers of anti-FVIII antibodies in the circulation of these mice one week after the second dose of FVIII. Previously we had shown that a single dose of 200 ng FVIII, given intravenously to E17 hemophilic mice, stimulates the formation of FVIII-specific memory B cells but is not sufficient to induce anti-FVIII antibodies that would be detectable in the circulation. Our results demonstrate a biphasic effect of CpG DNA on the re-stimulation of FVIII-specific memory B cells and their differentiation into antibody-producing plasma cells. Both in vitro and in vivo studies show that CpG DNA at high doses inhibits the re-stimulation and differentiation of FVIII-specific memory B cells. However, CpG DNA at low doses amplifies these processes. Amplification and inhibition of memory-B-cell responses are due to specific interactions of CpG DNA with TLR 9. Both effects are blocked by addition of a blocking agent for TLR 9 in vitro. We conclude that triggering of TLR 9 by bacterial DNA has a substantial influence on FVIII-specific memory-B-cell responses. The consequence of TLR 9 triggering can be inhibitory or stimulatory, depending on the actual concentration of the bacterial DNA. Our findings demonstrate the potential modulatory effects of bacterial infections on the regulation of FVIII inhibitor development.

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