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

Abstract The development of neutralizing anti-FVIII antibodies (FVIII inhibitors) in about 30% of patients with severe hemophilia A is the most serious complication in the treatment of hemophilia patients with FVIII products. Little information is available on the immunological mechanisms that regulate the development and maintenance of FVIII inhibitors. Memory B cells are a central component of humoral immunity. They drive the rapid anamnestic antibody response that occurs after re-exposure to antigen and seem to be important for replenishing the pool of long-lived plasma cells to maintain long-term antibody levels in the absence of antigen. Nothing is known about the dynamics of FVIII-specific memory B cells in patients with hemophilia A who develop FVIII inhibitors. Recently, Crotty et al. (J Immunol Methods, 2004) described an in vitro assay to quantify antigen-specific memory B cells in human blood. This assay utilizes a 6-day polyclonal stimulation of peripheral blood mononuclear cells (PBMC) followed by an antigen-specific ELISPOT for the detection of memory B cells that have differentiated into antibody-secreting plasma cells in vitro. We adapted this assay to human FVIII and used it to track FVIII-specific memory B cells in the blood of hemophilia A patients with and without FVIII inhibitors. Human serum albumin was used as a negative control and tetanus toxin as a positive control. The numbers of FVIII-specific, tetanus toxin-specific (positive control) and albumin-specific (negative control) memory B cells were calculated as percentage of total IgG memory B cells. So far, we have analyzed 14 patients with hemophilia A (age: 6–65 years). 8 were positive and 6 were negative for FVIII inhibitors. For comparison, we have analyzed 20 healthy individuals (age: 19–48 years). 2 out of 8 patients with inhibitors had detectable FVIII-specific memory B cells in their peripheral blood cells. However, none of the patients without inhibitors and none of the healthy individuals had any detectable FVIII-specific memory B cells in their circulation. The detection limit for FVIII-specific memory B cells in patients with inhibitors was about 0.2 % (percent of total IgG memory B cells). Current activities focus on further advancing the method with the aim to improve the detection limit for the detection of FVIII-specific memory B cells. All samples analyzed (including patients and healthy individuals) were negative for human serum albumin-specific memory B cells (negative control). Tetanus toxin-specific memory B cells (positive control) were found in both patients and healthy blood donors. The percentage of tetanus toxin-specific memory B cells in individuals who were vaccinated with tetanus toxoid was in the range of 0.25 – 0.58 % (percent of total IgG memory B cells). We conclude that the method described is suitable to track FVIII-specific memory B cells in the circulation. We are currently asking the question whether the presence of FVIII-specific memory B cells in the circulation correlates with the persistence of FVIII inhibitors. Furthermore, we will monitor patients with inhibitors during ITI therapy in order to find out whether the disappearance of FVIII-specific memory B cells in the circulation could be an early predictor of a successful ITI outcome.

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Preventing restimulation of memory B cells in hemophilia A: a potential new strategy for the treatment of antibody-dependent immune disorders

Blood ◽

10.1182/blood-2003-07-2456 ◽

2004 ◽

Vol 104 (1) ◽

pp. 115-122 ◽

Cited By ~ 52

Author(s):

Christina Hausl ◽

Rafi U. Ahmad ◽

Hans Peter Schwarz ◽

Eva M. Muchitsch ◽

Peter L. Turecek ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Hemophilia A ◽

Memory B Cells ◽

Third Party ◽

Activated T Cells ◽

Specific Memory ◽

Inducible Costimulator

Abstract Memory B cells are responsible for the rapidly emerging antibody response after antigen reexposure. The signals required for the restimulation of memory B cells have not been fully explained. We used a murine model of anti–factor VIII (FVIII) antibody responses in hemophilia A to study the requirements for the restimulation of FVIII-specific memory B cells and their differentiation into anti-FVIII antibody-producing cells. We were particularly interested in the significance of activated T cells and costimulatory interactions. Our results indicate that the restimulation of FVIII-specific memory B cells is strictly dependent on interactions with activated T cells. These activated T cells can be specific for either FVIII or third-party antigens. Restimulation by T cells specific for third-party antigens requires the presence of FVIII, indicating that signals induced by B-cell receptor (BCR) triggering and by interactions with activated T cells are important. The blockade of B7-1 or B7-2 as well as the blockade of CD40L inhibits the restimulation and differentiation of FVIII-specific memory B cells in vitro and in vivo. The interference with inducible costimulator–inducible costimulator ligand (ICOS-ICOSL) interactions, however, does not cause any modulation. As expected, the production of anti-FVIII antibodies by plasma cells is not dependent on any of the costimulatory interactions tested.

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

Blood ◽

10.1182/blood.v110.11.1155.1155 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1155-1155 ◽

Cited By ~ 1

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.

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Comparable Long-Term Persistence of Anti-FVIII Antibodies in Hemophilic E17 Mice on Different Genetic Backgrounds Despite Significant Differences in the Amplitude of the Immune Responses.

Blood ◽

10.1182/blood.v108.11.1027.1027 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1027-1027

Author(s):

Natalie Bauer ◽

Christina Hausl ◽

Rafi U. Ahmad ◽

Bernhard Baumgartner ◽

Hans Peter Schwarz ◽

...

Keyword(s):

Immune Response ◽

B Cells ◽

Genetic Background ◽

Neutralizing Antibodies ◽

Plasma Cells ◽

Memory B Cells ◽

Mouse Strains ◽

Igg Antibodies ◽

Specific Memory

Abstract About 30% of patients with severe hemophilia A develop neutralizing antibodies against FVIII (FVIII inhibitors) following replacement therapy. The type of FVIII gene mutation as well as other predisposing genetic factors contribute to the inhibitor phenotype. Based on these findings, we asked if the genetic background modulates the long-term persistence of anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells in the E17 murine hemophilia model. Furthermore, we asked if the recently described inhibition of memory-B-cell re-stimulation by high doses of FVIII is influenced by the genetic background of the murine model. E17 mice on two different genetic backgrounds (C57Bl/6J and Balb/c) were treated with four doses of 200 ng human FVIII at weekly intervals. Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were followed up to 12 months after the last dose of FVIII. Antibody titers and subclasses of antibodies (IgM, IgG1, IgG2a, IgG2b, IgG3) were measured by ELISA. Antibody secreting plasma cells in spleen and bone marrow were detected by ELISPOT as described (Hausl et al., Thromb Haemost 2002). The re-stimulation of FVIII-specific memory B cells was studied as described recently (Hausl et al., Blood 2005). Anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells were first detectable in E17 Balb/c mice. IgM antibodies in the circulation and IgM secreting plasma cells in the spleen were observed after the first dose of FVIII, IgG antibodies and IgG secreting plasma cells after the second dose. No anti-FVIII antibodies after the first dose of FVIII were observed in E17 C57BL/6J mice but both IgM and IgG antibodies as well as IgM and IgG producing plasma cells were detectable after the second dose of FVIII. The antibody response involved all IgG subclasses in both mouse strains. However, IgG1 was dominant in E17 Balb/c mice whereas IgG2a was dominant in E17 C57BL/6J mice. When the in vitro restimulation of FVIII-specific memory B cells was examined, similar patterns were observed for both mouse strains. Low concentrations of FVIII between 10 and 100 ng/ml FVIII restimulated memory B cells and induced their differentiation into antibody secreting plasma cells whereas high concentrations of FVIII between 1,000 and 20,000 ng/ml FVIII inhibited memory-B-cell-restimulation. These results indicate that the dose-dependent effect of FVIII on the restimulation of FVIII-specific memory B cells does not depend on the genetic background. The major difference between both hemophilic mouse strains was the amplitude of the anti-FVIII immune response. Peak titers of anti-FVIII antibodies and peak concentrations of anti-FVIII antibody secreting plasma cells in spleen and bone marrow were significantly higher in E17 C57BL/6J mice than in E17 Balb/c mice. Whether or not higher ELISA titers correlate with higher Bethesda titers of neutralizing antibodies is currently being investigated. Despite the substantial differences in the amplitude of the immune response, anti-FVIII antibodies and anti-FVIII antibody secreting plasma cells persisted for the whole observation period of 12 months after the last dose of FVIII in both mouse strains. We conclude that the amplitude of the anti-FVIII immune response in hemophilic mice is significantly different between E17 C57BL/6J and E17 Balb/c mice. However, the persistence of the immune response is comparable.

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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 ◽

10.1182/blood.v112.11.3382.3382 ◽

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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Decreasing the Humoral Response to Factor VIII By Targeted Deletion of Factor VIII - Specific B Cells

Blood ◽

10.1182/blood.v124.21.238.238 ◽

2014 ◽

Vol 124 (21) ◽

pp. 238-238 ◽

Cited By ~ 1

Author(s):

Rebecca C. Markovitz ◽

John F. Healey ◽

W. Hunter Baldwin ◽

Ernest T. Parker ◽

Shannon L. Meeks ◽

...

Keyword(s):

B Cells ◽

Factor Viii ◽

Hemophilia A ◽

Memory B Cells ◽

Full Length ◽

Treatment Groups ◽

Tail Vein ◽

Tail Vein Injection ◽

Specific Memory ◽

To Receive

Abstract The development of neutralizing anti-factor VIII (fVIII) antibodies (inhibitors) remains the most significant complication in the treatment of hemophilia A patients. Treatment of inhibitor patients consists of management of bleeding episodes using bypassing agents or porcine fVIII. Inhibitors can be eradicated by immune tolerance induction (ITI) using thrice-weekly administration of large doses of fVIII. However, ITI fails in approximately 30% of patients. Additionally, the median time to tolerance in successful cases is ~18 months, making ITI expensive and inconvenient. In the current study, we used a murine E16 hemophilia A model to test a novel approach to both prevent and eradicate fVIII inhibitors. We hypothesized that conjugation of fVIII to the toxin saporin, a Type I ribosome-inactivating protein, would target fVIII-specific cell surface immunoglobulin and selectively delete fVIII-specific naïve and memory B cells. Recombinant full-length fVIII was covalently linked to saporin using the heterobifunctional crosslinker N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). To test for eradication of existing fVIII inhibitors by fVIII-saporin, an adoptive transfer protocol was developed to measure fVIII-specific memory B cells. Hemophilia A donor mice were immunized with 2 μg of full-length fVIII by intravenous injection every other week for 8 weeks, followed by a final dose of 4 μg at ten weeks. Four weeks later, the mice were randomized into three treatment groups to receive equimolar doses of saporin, fVIII, or fVIII-saporin. Seven days after treatment, the mice were sacrificed and 4 x 106 plasma cell CD138+-depleted splenocytes were adoptively transferred as a source of fVIII-specific memory B cells into naïve recipient hemophilia A mice. At 24 hours, recipient mice were given a single injection of 0.5, 1.0 or 2.0 μg of recombinant full-length fVIII by tail vein injection. Anti-fVIII IgG antibodies in recipient mice were measured by ELISA 2 and 5 weeks following the fVIII injection. In the absence of fVIII-specific memory B cells from donor mice, naïve hemophilia A mice did not produce detectable anti-fVIII antibodies. Recipient hemophilia A mice receiving splenocytes from fVIII donor and saporin donor mice displayed a dose-dependent increase in anti-fVIII antibodies. In contrast, the slope of the anti-fVIII titer versus dose of fVIII was significantly decreased in recipient mice receiving splenocytes from fVIII-saporin donor mice. To test for prevention of fVIII inhibitor formation by fVIII-saporin, naïve hemophilia A mice were divided into three treatment groups to receive a single dose of saporin, fVIII, or fVIII-saporin by tail vein injection. Seven days after treatment, the mice were immunized by tail vein injection with 2 μg of full-length fVIII every other week for 10 weeks. Anti-fVIII IgG antibodies were measured 1 week after the fourth and sixth injections of fVIII. Anti-fVIII antibody titers were significantly lower in the fVIII-saporin group compared to the fVIII group (1,900 vs. 21,400 (p=0.027, n=4, Mann-Whitney test, see figure) after the fourth injection. After 6 injections, the average anti-fVIII titer of the fVIII group was 23,000 compared to 4,000 in the fVIII-saporin group (p=0.057, n=4, Mann-Whitney test, see figure). In conclusion, our results suggest that infusion of fVIII-saporin results in the depletion of both fVIII-specific naïve B cells and memory B cells. FVIII-saporin potentially could be used in the treatment of congenital hemophilia A patients with inhibitors and patients with acquired hemophilia A. In addition, fVIII-saporin potentially could be used in previously untreated patients with hemophilia A to prevent inhibitor development. Similar therapeutic strategies could be extended to other antigen-specific immune disorders. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Stimulation and inhibition of FVIII-specific memory B-cell responses by CpG-B (ODN 1826), a ligand for Toll-like receptor 9

Blood ◽

10.1182/blood-2010-06-289009 ◽

2011 ◽

Vol 117 (1) ◽

pp. 259-267 ◽

Cited By ~ 14

Author(s):

Peter Allacher ◽

Christina K. Baumgartner ◽

Aniko G. Pordes ◽

Rafi U. Ahmad ◽

Hans Peter Schwarz ◽

...

Keyword(s):

B Cells ◽

Memory B Cells ◽

Cpg Odn ◽

Specific Memory ◽

Cpg Oligodeoxynucleotide ◽

Low Concentrations ◽

Essential Components ◽

High Concentrations

Abstract Factor VIII (FVIII)–specific memory B cells are essential components for regulating anamnestic antibody responses against FVIII in hemophilia A with FVIII inhibitors. We asked how stimulation and inhibition of FVIII-specific memory B cells by low and high concentrations of FVIII, respectively, are affected by concurrent activation of the innate immune system. Using CD138− spleen cells from hemophilic mice treated with FVIII to study restimulation and differentiation of memory B cells in vitro, we tested modulating activities of agonists for Toll-like receptors (TLRs) 2, 3, 4, 5, 7, and 9. Ligands for TLR7 and 9 were most effective. They not only amplified FVIII-specific memory responses in the presence of stimulating concentrations of FVIII, but also countered inhibition in the presence of inhibitory concentrations of FVIII. Notably, CpG oligodeoxynucleotide (CpG-ODN), a ligand for TLR9, expressed biphasic effects. It amplified memory responses at low concentrations and inhibited memory responses at high concentrations, both in vitro and in vivo. Both stimulatory and inhibitory activities of CpG-ODN resulted from specific interactions with TLR9. Despite their strong immunomodulatory effects in the presence of FVIII, ligands for TLR induced negligible restimulation in the absence of FVIII in vitro and no restimulation in the absence of FVIII in vivo.

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An in Vitro Model of Differentiation of Memory B Cells Into Plasmablasts and Plasma Cells Including Detailed Phenotypic and Molecular Characterization.

Blood ◽

10.1182/blood.v114.22.1636.1636 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1636-1636

Author(s):

Michel Jourdan ◽

Anouk Caraux ◽

John De Vos ◽

Geneviève Fiol ◽

Marion Larroque ◽

...

Keyword(s):

B Cells ◽

In Vitro Model ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Memory B Cells ◽

Vitro Model ◽

Intermediate Cells

Abstract Abstract 1636 Poster Board I-662 Human plasma cells (PCs) and their precursors play an essential role in humoral immune response, but are rare and difficult to harvest. We report here i) the generation of human syndecan-1+ and immunoglobulin secreting PCs starting from memory B cells (MBCs) in a 3-step- and 10-day (D) culture, including a 6-fold cell amplification. ii) We report the detailed phenotypic and Affymetrix gene expression profiles of these in vitro PCs as well as of intermediate cells - activated B cells (actBCs) and plasmablasts (PBs) - compared to MBCs and bone marrow PCs, which is accessible through an open web ATLAS (http://amazonia.transcriptome.eu/). iii) We show this B cell to PC differentiation to involve IRF4 and AICDA expression in D4 actBCs, decrease of PAX5 and BCL6 expressions and increase in PRDM1 and XBP1 expressions in D7 PBs and D10 PCs. It involves downregulation of genes controlled by Pax5, induction of genes controlled by Blimp-1 and XBP1 (unfold protein response). iv) The phenotype of D10 PCs resembles that of peripheral blood PCs detected after immunization of healthy donors. This in vitro model will facilitate further studies in PC biology. It will likewise be helpful to study plasma-cell dyscrasias, including Multiple Myeloma. Disclosures No relevant conflicts of interest to declare.

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Detection of Factor VIII-Specific Memory B Cells in Patients with Hemophilia A and Factor VIII Inhibitors

37th Hemophilia Symposium ◽

10.1007/978-3-540-73535-9_4 ◽

2008 ◽

pp. 25-36 ◽

Cited By ~ 1

Author(s):

I. Lang ◽

J. Windyga ◽

A. Klukowska ◽

J. Ilas ◽

H. P. Schwarz ◽

...

Keyword(s):

B Cells ◽

Factor Viii ◽

Hemophilia A ◽

Memory B Cells ◽

Specific Memory

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MO251HIGHLY SENSITIVE FLOW CYTOMETRIC DETECTION OF RESIDUAL B-CELLS AFTER RITUXIMAB IN ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODY-ASSOCIATED VASCULITIS PATIENTS*

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab104.009 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Y K O Teng ◽

L Van Dam ◽

Jelle Oskam ◽

S W A Kamerling ◽

E J Arends ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Mononuclear Cells ◽

Plasma Cells ◽

Memory B Cells ◽

Cell Depletion ◽

B Cell Depletion ◽

Peripheral Blood Mononuclear

Abstract Background and Aims B-cell depletion with rituximab (RTX) is an effective treatment for anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients. Nevertheless, relapses are frequent after RTX, often preceded by B-cell repopulation suggesting that residual autoreactive B-cells persist despite therapy. Therefore, this study aimed to identify minimal residual autoimmunity (MRA) in the B-cell compartment of AAV patients treated with RTX. Method EuroFlow-based highly-sensitive flow cytometry (HSFC) was employed to study B-cell and plasma cell (PC) subsets in-depth in AAV patients before and after RTX treatment. Additionally, peripheral blood mononuclear cells (PBMCs) of these RTX-treated AAV patients were cultured and in vitro stimulated with CpG, IL-2, and IL-21 to induce antibody-secreting cells (ASC). (ANCA)-IgG was measured in these supernatants by ELISA. Results By employing EuroFlow-based HSFC, we detected circulating CD19+ B-cells at all timepoints after RTX treatment, in contrast to conventional low-sensitive flow cytometry. Pre-germinal center (Pre-GC) B-cells, memory B-cells and CD20+CD138− plasmablasts (PBs) were rapidly and strongly reduced, while CD20−CD138− PrePC and CD20-CD138+ mature (m)PCs were reduced slower and remained detectable. Both memory B-cells and CD20− PCs remained detectable after RTX. Serum ANCA-IgG decreased significantly upon RTX. Changes in ANCA levels strongly correlated with changes in naive, switched CD27+ and CD27− (double-negative) memory B-cells, but not with plasma cells. Lastly, we demonstrated in vitro ANCA production by AAV PBMCs, 24 and 48 weeks after RTX treatment reflecting MRA in the memory compartment of AAV patients. Conclusion We demonstrated that RTX induced strong reductions in circulating B-cells, but never resulted in complete B-cell depletion. Despite strongly reduced B-cell numbers after RTX, ANCA-specific memory B-cells were still detectable in AAV patients. Thus, MRA is identifiable in AAV and can provide a potential novel approach in personalizing RTX treatment in AAV patients.

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

Blood ◽

10.1182/blood.v104.11.38.38 ◽

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.

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