scholarly journals Marginal Zone B Cell Depletion Prevents Factor VIII Inhibitor Development in Model of Hemophilia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1068-1068
Author(s):  
Courtney Cox ◽  
Patricia Zerra ◽  
Connie Authur ◽  
Seema Patel ◽  
Shannon Meeks ◽  
...  
Keyword(s):  
Immune Response ◽  
B Cells ◽  
Factor Viii ◽  
B Cell ◽  
Antibody Formation ◽  
Marginal Zone ◽  
Hemophilia A ◽  
Critical Role ◽  
Cell Depletion ◽  
B Cell Depletion

Abstract Background While Factor VIII (fVIII) therapy can provide a life saving intervention in patients with hemophilia A, patients receiving fVIII can develop anti-fVIII antibodies. These antibodies, otherwise known as fVIII inhibitors, often prevent the therapeutic impact of fVIII. As a result, hemophilia A patients with fVIII inhibitors often experience increased morbidity and mortality. Given the unique role of the splenic marginal sinus in the immune response to blood borne antigens, we hypothesized that key immunological constituents within the marginal zone (MZ) likely play a critical role in the initiation and development of anti-fVIII antibodies in hemophilia A recipients. Methods: To deplete MZ B-cells, complete F8 gene knockout mice (TKO mice) received two initial 100 µg intraperitoneal injections of MZ B-cell depleting antibodies (anti-CD11a and anti-CD49d) or isotype controls on days -4, -2, 10 and 20. Additional mice that received MZ B cell depleting antibodies were examined for MZ B cell depletion efficacy and specificity on days 0, 7, 14, 21 and 28 by staining splenocytes with CD3, CD4, CD8, CD11b, CD11c, NK1.1, B220, CD21 and CD23. Starting on day 0, mice received 4 weekly retro-orbital injections of 2 µg of B-domain deleted human fVIII (BDD hfVIII). At 6 weeks, mice received a 4 µg "boost" dose of BDD hfVIII. At eight weeks, mice were then re-challenged with 4 weekly doses of 2 µg of BDD hfVIII. At each interval (pre-boost, post-boost, and after re-challenge) mice were bled and ELISA and inhibitor titers were determined. Results: As the development of detectable anti-fVIII antibodies often requires repeat fVIII exposure, we examined whether repeat injection of MZ B cell depleting antibodies could sustain MZ B cell depletion. Injection of MZ B cell depleting antibodies on days -4, -2, 10 and 20 completely prevented MZ B cell localization within the spleen for 28 days, while failing to alter follicular B cell, NK cell, CD4 T cell, dendritic cell and macrophage numbers. Injection of isotype controls failed to alter MZ B cell numbers when evaluated in parallel. Weekly injection of BDD hfVIII during the first four weeks completely failed to induce an anti-fVIII antibody response in MZ B cell depleted recipients (p<0.0015, Mann Whitney), while isotype control treated recipients developed inhibitors at the same rate as non-treated controls. However, following MZ B cell reconstitution at day 35, recipients previously treated with the MZ B cell depletion regiment developed an immune response that closely resembles naive hemophilia A recipients. Conclusion: These results demonstrate that marginal zone constituents, in particular MZ B cells, play a critical role in the initiation and development of fVIII inhibitors. While MZ B cell depleted recipients failed to generate anti-fVIII antibodies following fVIII exposure, MZ B cell reconstitution after fVIII exposure readily induced anti-fVIII antibodies, suggesting that transient removal of MZ B cell may provide a unique mechanism to prevent fVIII antibody formation without permanently altering host immunity. As a result, MZ B cells and additional unique targets within the marginal sinus may be used to develop specific strategies to prevent anti-fVIII antibody formation in patients with hemophilia A. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Effect of B-Cell Depletion On Inhibitor Antibody Formation and Immune Tolerance Induction to Human Factor VIII in Hemophilia A Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2137-2137
Author(s):  
Ai-Hong Allan Zhang ◽  
Jonathan Skupsky ◽  
David W. Scott
Keyword(s):  
Immune Response ◽  
B Cells ◽  
B Cell ◽  
Immune Tolerance ◽  
Hemophilia A ◽  
Tolerance Induction ◽  
Cell Depletion ◽  
High Dose ◽  
B Cell Depletion ◽  
Immune Tolerance Induction

Abstract Abstract 2137 Poster Board II-114 B-cell depletion using anti-human CD20 monoclonal antibodies has been reported to be effective in autoimmunity and in temporarily eliminating inhibitory antibodies in hemophilia A patients. In the current study, we examined the effect of anti-murine CD20 (αCD20) depletion on the immune response to factor VIII (FVIII) and its influence on an immune tolerance induction (ITI) protocol. Previous studies have shown that IgG subclasses of anti-murine CD20 monoclonal antibody (αCD20) have differential effects on B-cell depletion in the mouse. Thus, IgG1 αCD20 selectively depletes follicular B cells, while sparing marginal zone (MZ) B cells. Combined with evidence that MZ B cells may be tolerogenic antigen-presenting cells, we tested the hypothesis that follicular B-cell depletion using αCD20 IgG1 might favor tolerance induction to human FVIII. Hemophilic (FVIII knockout) mice were primed with physiological doses of recombinant human FVIII by weekly IV injection, followed by αCD20 IgG1 or control IgG1 treatment. Ten days after the αCD20 treatment, the mice were treated with daily high dose (2μg) FVIII IV injections to model ITI in hemophilia A patients. After 4 weekly injections, 70% of the mice developed titers of anti-FVIII IgG as high as 1:12,800. Unlike whole B-cell depletion, subsequent follicular B-cell depletion did not significantly decrease the anti-FVIII IgG titer, compared with mice receiving control IgG1. Repeated high dose FVIII injections to mimic ITI significantly increased the anti-FVIII IgG titer in both groups. However, in the mice that received αCD20 IgG1 treatment, the increase of anti-FVIII IgG levels were significantly lower than that in control IgG1 treated mice. In conclusion, we found that follicular B-cell depletion by αCD20 IgG1 antibody in hemophilia A mice did not switch the immune response to tolerance, but it diminished the immunogenicity of human FVIII in vivo in hemophilic mice. (Supported by NIH R01 HL061883) Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effect of B-cell depletion using anti-CD20 therapy on inhibitory antibody formation to human FVIII in hemophilia A mice

Blood ◽  
2011 ◽  
Vol 117 (7) ◽  
pp. 2223-2226 ◽  
Author(s):  
Ai-Hong Zhang ◽  
Jonathan Skupsky ◽  
David W. Scott
Keyword(s):  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Hemophilia A ◽  
Tolerance Induction ◽  
Cell Depletion ◽  
High Dose ◽  
B Cell Depletion ◽  
Marginal Zone B Cells ◽  
Anti Cd20

Abstract We herein tested the effect of B-cell depletion on tolerance induction to factor VIII (FVIII) in a mouse model of hemophilia A. Two subclasses of anti–mouse CD20 monoclonal antibodies with differential depletion effects were used. Thus, IgG1 anti-CD20 selectively depleted follicular B cells and spared marginal zone B cells, whereas IgG2a anti-CD20 efficiently depleted both. In FVIII primed mice, a single dose of either IgG1 or IgG2a anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice by subsequent daily, high-dose FVIII intravenous injection as a model for immune tolerance induction. However, the IgG1, but not the IgG2a, anti-CD20 pretreatment led to a significant increase of regulatory T cells in the spleen. Importantly, 3 months after the partial B-cell depletion with IgG1 anti-CD20, the FVIII-specific hyporesponsive state remained. We suggest a tolerogenic role of the remaining marginal zone B cells as a potential mechanism for anti-CD20 therapy.

B-Cell Depletion Using IgG1 Anti-CD20 Spares MZ B Cells and Promotes Tolerance to Human FVIII In a Murine Model of Hemophilia A

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2206-2206
Author(s):  
Ai-Hong Zhang ◽  
Jonathan Skupsky ◽  
David W. Scott
Keyword(s):  
B Cells ◽  
B Cell ◽  
Hemophilia A ◽  
Tolerance Induction ◽  
Cell Depletion ◽  
High Dose ◽  
Marginal Effect ◽  
B Cell Depletion ◽  
Significant Difference ◽  
Anti Cd20

Abstract Abstract 2206 Preventing and reversing inhibitor formation remains one of the major challenges for hemophilia A therapy. Anti-CD20 mAb (Rituximab) has been reported to be beneficial for hemophilia A patients who failed immune tolerance induction (ITI). However, the evaluation of anti-CD20 therapy often is complicated in the clinical setting by concomitant use of other immune modulating drugs, such as hydrocortisone and IVIG. In this study, we tested the effect of B-cell depletion per se on tolerance induction to FVIII in a mouse model of hemophilia A. Two subclasses of anti-mouse CD20 monoclonal antibodies with differential effects were used. We previously showed that IgG1 anti-CD20 selectively depleted follicular (FO) B cells and spared marginal zone (MZ) B cells, while IgG2a anti-CD20 efficiently depleted both. In FVIII primed mice (inhibitor titer = 30.7 ± 4.8 BU/ml), a single dose of IgG1 anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice given daily, high dose FVIII i.v. injection as a model for ITI. Surprisingly, only a marginal effect was achieved when we repeated the same protocol using IgG2a anti-CD20 for B-cell depletion, which efficiently depletes both FO and MZ B cells. To examine tolerance to FVIII, we re-challenged the treated mice with 2 μg FVIII intraperitoneally three months after the initiation of B cell depletion using IgG1 anti-CD20 when the number of peripheral B cells had recovered 60 % or more. The inhibitor titers remained significantly lower in the IgG1 anti-CD20 group after this FVIII boost injection (60.9 ± 33.2 versus 190.3 ± 33.5 BU/ml in control IgG1 group; p = 0.02). Importantly, after the mice were subcutaneously challenged with an unrelated antigen, OVA in CFA, there was no significant difference in anti-OVA IgG titers between the two groups. Taken together, these results suggested that selectively depletion of FO B cells by IgG1 subtype anti-CD20 mAb treatment may facilitate the tolerance induction to FVIII. (Supported by NIH R01 HL061883 and a fellowship from the American Heart Association) Disclosures: No relevant conflicts of interest to declare.

scholarly journals Toll-like Receptor 9 Activation Accelerates Inhibitor Formation in Response to Factor VIII

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1113-1113
Author(s):  
Radoslaw Kaczmarek ◽  
Alexandra Sherman ◽  
Moanaro Biswas ◽  
Roland W Herzog
Keyword(s):  
Immune Response ◽  
B Cells ◽  
Factor Viii ◽  
Germinal Center ◽  
Antibody Formation ◽  
Hemophilia A ◽  
Innate Immune ◽  
Toll Like Receptor ◽  
Tlr9 Agonist

Inhibitor (neutralizing anti-drug antibody) formation against factor VIII (FVIII) is currently the most serious complication of FVIII replacement therapy for hemophilia A. The role of innate immune signals in this adaptive immune response is unclear. Lipopolysaccharide (LPS, the major component of the outer membrane in Gram-negative bacteria), which activates the innate immune sensor toll-like receptor 4 (TLR4), enhances inhibitor formation in hemophilia A mice. Intriguingly, earlier data suggested that activation of the endosomal DNA sensor TLR9 may actually suppress inhibitor formation. However, we demonstrated that a TLR9 agonist induced inhibitor formation against a factor IX transgene product in gene therapy for hemophilia B, which resulted from activation of monocyte-derived dendritic cells (moDCs) and enhancement of T follicular helper (Tfh) cell responses. Tfh cells drive germinal center (GC) formation and T cell help-dependent antibody formation. The aim of this study was to elucidate the role of TLR9 signaling in FVIII inhibitor formation in hemophilia A mice. Hemophilia A (F8e16-/-) B6/129 mice were co-injected IV (n=4) with FVIII (1.5 IU) and ODN-1826 (a class B CpG oligodeoxynucleotide, 50 µg), which is a TLR9 agonist, or injected on two consecutive days (n=3), first with ODN-1826 and then with FVIII on the next day. Control mice received FVIII only (n=5). Injections were performed once weekly for 4 weeks. Blood samples, spleens and subiliac (superficial inguinal) lymph nodes were collected for ELISA and Bethesda assays, and flow cytometry analysis. In our analysis, Tfh were defined as CD4+CXCR5+PD1+Bcl6- cells, while GC B cells were defined as CD19+GL7+CD95+. Tfh cell response and GC formation in the spleen were robustly enhanced by the TLR9 agonist compared to mice injected with FVIII only (2-fold for Tfh frequencies and 6-fold for GC B cells when co-injected; P< 0.05). As a result, inhibitor titers increased >400-fold (on average from 6.4 to 2746.5 BU/ml). In contrast, anti-FVIII IgG1 levels increased only 2.5-fold. The differences between mice injected on two consecutive days and the FVIII-only group were not statistically significant. A time-course experiment was also carried out to monitor progress of immune response to FVIII (1.5 IU administered once weekly) in the absence of TLR9 agonist over time: 4 (n=4), 6 (n=4) and 8 weeks (n=5) after the initial antigen challenge. Inhibitor titers continued to rise beyond the fourth week of antigen challenges, reaching 46-fold higher values at eighth week (from 3 BU/ml in the fourth week to 138 BU/ml in the eighth week, p<0.05), while anti-FVIII IgG1 levels increased 2.5-fold (ns). These results suggest that the TLR9 agonist sped up the neutralizing response to FVIII, which otherwise progressed in the same manner (towards more neutralizing versus total anti-FVIII) but at a slower rate. Overall, TLR9 activation enhanced GC formation and accelerated neutralizing immune response to FVIII. Similar results were obtained in hemophilia A mice on a different strain background (F8e16-/- BALB/c). We propose that signaling via the innate immune receptor TLR9 leads to a more targeted immune response by reinforcing Tfh activation, likely through moDC activation, which results in enhanced germinal center formation and thus accelerated development of neutralizing antibodies. Disclosures No relevant conflicts of interest to declare.

Anti-CD20 to Control Antibody Formation Against FVIII In Gene and Protein Replacement Therapy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 711-711
Author(s):  
Brandon Sack ◽  
Babak Moghimi ◽  
Barry Byrne ◽  
Roland W Herzog
Keyword(s):  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Neutralizing Antibodies ◽  
Hemophilia A ◽  
Major Complication ◽  
Cell Depletion ◽  
Clotting Factor ◽  
B Cell Depletion ◽  
Anti Cd20

Abstract Abstract 711 Neutralizing antibodies to FVIII (“inhibitors”) following delivery of exogenous clotting factor represents a major complication in the treatment of hemophilia A. Current treatment via immune tolerance induction (ITI) is effective but expensive, protracted, and imprecise. Recently, the drug rituximab—an anti-CD20 antibody used to deplete B cells—has been used with some success to treat existing inhibitors in patients. However, the potential for this drug to prevent inhibitor development has not been addressed. We used an IgG2a monoclonal anti-murine CD20 (kindly provided by Biogen Idec) to investigate the effect of B cell depletion on the immune response to human FVIII in hemophilia A mice (exon 16 deletion, C57BL6/129) and to determine the possibility of using B cell depletion to augment gene therapy. To deplete CD20+ B cells, mice were given two i.v. doses of 10mg/kg anti-CD20 (αCD20) two weeks apart. Depletion of peripheral blood B cells was confirmed by flow cytometry of blood, and depletion of lymph node and splenic B cells was tracked in control animals given the same regimen. Following administration of a single treatment of αCD20, peripheral blood B cells were depleted to <1% of total lymphocytes compared to 10–30% in control animals treated with isotype control antibody. One group of these mice (n=5, “αCD20+AAV8”) was given 1011vg/mouse of an AAV8 vector expressing hFVIII under a liver-specific promoter (AAV8-FVIII) one week after the first αCD20 injection. Another group (n=5, “AAV8-only”) was given AAV8-FVIII but not αCD20. Ten weeks after AAV8-FVIII treatment (8 weeks after last αCD20 treatment), both AAV8-only and αCD20+AAV8 groups were “challenged” with weekly i.v. injections of hFVIII at 1 IU/mouse for 4 weeks. Animals treated with αCD20+AAV8 showed somewhat better improvements in clotting times compared with animals treated only with AAV8, albeit that no anti-FVIII was detected in either group. However, the two groups differed more dramatically in their response to subsequent FVIII challenge. Mice in the AAV8-only group developed a mean anti-FVIII IgG1 titer of 7001 (±867) ng/mL and an average Bethesda titer of 336 (±88) BU, similar to the anti-hFVIII response seen normally in mice of this strain. In contrast, mice given αCD20+AAV8 were hypo-responsive with an anti-FVIII IgG1 titer of 1609 (±868) ng/mL and 22 (±11) BU. A third group of mice (n=5, “αCD20-only”) did not receive gene transfer and were challenged similarly, starting at 4 weeks after the last αCD20 treatment, i.e. when peripheral B cells were rebounding. Two weeks later, only 1 of 5 mice had a detectable antibody response with an IgG1 titer of 513 ng/mL and a Bethesda titer of 3.6 BU. Seven weeks after the end of the initial challenge, two of these mice were again challenged with another 4-week FVIII injection cycle. One animal again had no detectable anti-FVIII, while the other had 3478 ng/mL anti-FVIII IgG1 and 126 BU, still below the normal response. Together, these data suggest that antigen exposure upon transient depletion of B cells with anti-CD20 induces significant hypo-responsiveness to hFVIII. We have now generated human CD20 transgenic hemophilia A mice to test Rituximab for this purpose. Disclosures: Herzog: Genzyme Corp: Patents & Royalties.

scholarly journals Morphologic and Functional Effects of Gamma Secretase Inhibition on Splenic Marginal Zone B Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Maria Cristina de Vera Mudry ◽  
Franziska Regenass-Lechner ◽  
Laurence Ozmen ◽  
Bernd Altmann ◽  
Matthias Festag ◽  
...  
Keyword(s):  
Immune Response ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Nuclear Translocation ◽  
Recovery Period ◽  
Functional Characterization ◽  
Notch Receptor ◽  
Marginal Zone B Cells ◽  
Cell Fate Decisions

Theγ-secretase complex is a promising target in Alzheimer’s disease because of its role in the amyloidogenic processing ofβ-amyloid precursor protein. This enzyme also catalyzes the cleavage of Notch receptor, resulting in the nuclear translocation of intracellular Notch where it modulates gene transcription. Notch signaling is essential in cell fate decisions during embryogenesis, neuronal differentiation, hematopoiesis, and development of T and B cells, including splenic marginal zone (MZ) B cells. This B cell compartment participates in the early phases of the immune response to blood-borne bacteria and viruses. Chronic treatment with the oralγ-secretase inhibitor RO4929097 resulted in dose-dependent decreased cellularity (atrophy) of the MZ of rats and mice. Significant decreases in relative MZ B-cell numbers of RO4929097-treated animals were confirmed by flow cytometry. Numbers of MZ B cells reverted to normal after a sufficient RO4929097-free recovery period. Functional characterization of the immune response in relation to RO4929097-related MZ B cell decrease was assessed in mice vaccinated with inactivated vesicular stomatitis virus (VSV). Compared with the immunosuppressant cyclosporin A, RO4929097 caused only mild and reversible delayed early neutralizing IgM and IgG responses to VSV. Thus, the functional consequence of MZ B cell decrease on host defense is comparatively mild.

scholarly journals B cell depletion with anti-CD20 mAb exacerbates anti-donor CD4+ T cell responses in highly sensitized transplant recipients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Asuka Tanaka ◽  
Kentaro Ide ◽  
Yuka Tanaka ◽  
Masahiro Ohira ◽  
Hiroyuki Tahara ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Responses ◽  
Cell Depletion ◽  
B Cell Depletion ◽  
Transplant Recipients ◽  
Cell Responses ◽  
Anti Cd20

AbstractPretransplant desensitization with rituximab has been applied to preformed donor-specific anti-human leukocyte antigen antibody (DSA)-positive recipients for elimination of preformed DSA. We investigated the impact of pretransplant desensitization with rituximab on anti-donor T cell responses in DSA-positive transplant recipients. To monitor the patients’ immune status, mixed lymphocyte reaction (MLR) assays were performed before and after desensitization with rituximab. Two weeks after rituximab administration, the stimulation index (SI) of anti-donor CD4+ T cells was significantly higher in the DSA-positive recipients than in the DSA-negative recipients. To investigate the mechanisms of anti-donor hyper responses of CD4+ T cells after B cell depletion, highly sensitized mice models were injected with anti-CD20 mAb to eliminate B cells. Consistent with clinical observations, the SI values of anti-donor CD4+ T cells were significantly increased after anti-CD20 mAb injection in the sensitized mice models. Adding B cells isolated from untreated sensitized mice to MLR significantly inhibited the enhancement of anti-donor CD4+ T cell response. The depletion of the CD5+ B cell subset, which exclusively included IL-10-positive cells, from the additive B cells abrogated such inhibitory effects. These findings demonstrate that IL-10+ CD5+ B cells suppress the excessive response of anti-donor CD4+ T cells responses in sensitized recipients.

Marginal Zone B Cells Mediate a CD4 T Cell Dependent Extrafollicular Antibody Response Following RBC Transfusion in Mice

Blood ◽  
2021 ◽  
Author(s):  
Patricia E Zerra ◽  
Seema R Patel ◽  
Ryan Philip Jajosky ◽  
Connie M Arthur ◽  
James W McCoy ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Antibody Response ◽  
B Cell ◽  
T Cell Activation ◽  
Antibody Formation ◽  
Marginal Zone ◽  
Cd4 T Cell ◽  
Marginal Zone B Cells ◽  
Rbc Transfusion

Red blood cell (RBC) transfusions can result in alloimmunization toward RBC alloantigens that can increase the probability of complications following subsequent transfusion. An improved understanding of the immune mechanisms that underlie RBC alloimmunization is critical if future strategies capable of preventing or even reducing this process are to be realized. Using the HOD (hen egg lysozyme and ovalbumin fused to human Duffy) model system, we aimed to identify initiating immune factors that may govern early anti-HOD alloantibody formation. Our findings demonstrate that HOD RBCs continuously localize to the marginal sinus following transfusion, where they co-localize with marginal zone (MZ) B cells. Depletion of MZ B cells inhibited IgM and IgG anti-HOD antibody formation, while CD4 T cell depletion only prevented IgG anti-HOD antibody development. HOD-specific CD4 T cells displayed similar proliferation and activation following transfusion of HOD RBCs into wild type or MZ B cell deficient recipients, suggesting that IgG formation is not dependent on MZ B cell-mediated CD4 T cell activation. Moreover, depletion of follicular B cells failed to substantially impact the anti-HOD antibody response and no increase in antigen specific germinal center B cells was detected following HOD RBC transfusion, suggesting that antibody formation is not dependent on the splenic follicle. Despite this, anti-HOD antibodies persisted for several months following HOD RBC transfusion. Overall, these data suggest MZ B cells can initiate and then contribute to RBC alloantibody formation, highlighting a unique immune pathway that can be engaged following RBC transfusion.

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