Blockade of Inhibitor Formation by B-Cell Delivered Tolerance to Immunodominant A2 and C2 Domains.

Abstract A major impediment in the treatment of hemophilia is the formation of inhibitory antibodies, which occurs in approximately 25–30% of Hemophilia A patients treated with therapeutic Factor VIII (fVIII). We have focused on the development of a gene therapy protocol for tolerance induction, with an emphasis on the elimination of inhibitor production. Our lab has demonstrated that LPS-stimulated B-cell blasts, transfected with a retrovirus encoding an IgG-peptide fusion protein, such as fVIII domains, are tolerogenic in both normal and primed recipients. Last year, we reported (http://www.abstracts-on-line.com/abstracts/hemphiladelphia03; Scott and Lei 2003) that specific tolerance to the immunodominant epitopes in the C2 domain of fVIII (a major target of inhibitors) could be induced by our protocol. However, the immune response to full length fVIII was only modestly affected. Most inhibitory antibodies are reactive with conformational epitopes on the exposed surfaces of the A2, as well as the C2, domain of fVIII. Therefore, in this study, we inserted residues S2173-Y2332 of the C2 domain and S373-R740 of the A2 domain onto the IgG heavy chain backbone, respectively, to induce tolerance in hemophilic mice. Specific tolerance to each domain was induced by this protocol. Importantly, a combination of A2-IgG and C2-IgG expressing B cells induced tolerance to the full length fVIII molecule, a result which supports the dominance of these domains in the immune response to fVIII. Tolerance was manifested in terms of ELISA, T-cell proliferation and especially Bethesda Unit titers (95% reduction). Similar results were obtained even when treatment was initiated after priming injections of fVIII. In conclusion, this protocol offers great promise for prevention and potential reversal of this serious complication of fVIII replacement therapy. (Supported by HL61883 and a Laboratory Grant from the National Hemophilia Foundation).

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Inhibitor Formation in Congenital Hemophilia A: an Immunological Perspective

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0038-1657777 ◽

2018 ◽

Vol 44 (06) ◽

pp. 517-530 ◽

Cited By ~ 5

Author(s):

Sandrine Delignat ◽

Julie Rayes ◽

Jules Russick ◽

Srinivas Kaveri ◽

Sebastien Lacroix-Desmazes ◽

...

Keyword(s):

Risk Factors ◽

Immune Response ◽

Immune Cells ◽

Hemophilia A ◽

Preclinical Models ◽

Societal Costs ◽

Clinical Challenge ◽

Inhibitory Antibodies ◽

Challenging Question ◽

Specific Tolerance

AbstractThe immunogenicity of therapeutic factor VIII (FVIII) in patients with hemophilia A has been puzzling scientific and clinical communities for more than 3 decades. Indeed, the development of inhibitory antibodies to FVIII remains a major clinical challenge and is associated with enormous societal costs. Thus, the reasons for which a presumably innocuous, short-lived, intravenously administered glycoprotein triggers such a deleterious, long-lasting neutralizing immune response is an enigma. This review does not pretend to bring an answer to this challenging question. It will however summarize the latest findings regarding the molecular interactions at play in the recognition of FVIII by the immune cells, the validity of the proposed risk factors for FVIII alloimmunization, and the different solutions that allow induction of FVIII-specific tolerance in preclinical models of hemophilia A.

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THE THYMUS AND RECOVERY FROM CYCLOPHOSPHAMIDE-INDUCED TOLERANCE TO SHEEP ERYTHROCYTES

Journal of Experimental Medicine ◽

10.1084/jem.128.1.35 ◽

1968 ◽

Vol 128 (1) ◽

pp. 35-46 ◽

Cited By ~ 38

Author(s):

Alan C. Aisenberg ◽

Caroline Davis

Keyword(s):

Proliferative Response ◽

Tolerance Induction ◽

Recovery Process ◽

Immunological Tolerance ◽

Base Line ◽

Drug Injection ◽

Sheep Erythrocytes ◽

Lymphoid Depletion ◽

Induced Tolerance ◽

Specific Tolerance

Recovery from specific immunological tolerance to sheep erythrocytes induced with the drug cyclophosphamide was studied with the hemolytic plaque technique of Jerne. The base line plaque (19S antibody-forming cell of the unstimulated spleen) and the proliferative response to antigen, both of which had disappeared during tolerance induction, returned with the recovery of specific immunological reactivity. When cyclophosphamide is injected without sheep cells there is temporary immunological unreactivity and lymphoid depletion of the spleen, but specific tolerance is not induced. Recovery is largely complete at the end of 2 wk and does not require the participation of the thymus. When cyclophosphamide is injected together with sheep cells, 18 days after drug injection, tolerance is still complete. In nonthymectomized mice there is rapid recovery during the next 10 wk, followed by much slower restoration over the remaining 20–30 wk of observation. The entire recovery process evidently takes 40–50 wk. In thymectomized CBA mice only minimal recovery takes place in the first 10 wk and no further restoration occurs thereafter. Thymectomy performed 18 days after tolerance is induced, when tolerance is complete, is equally effective in preventing this recovery.

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

Blood ◽

10.1182/blood.v114.22.2137.2137 ◽

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.

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Role of IgD in the immune response and tolerance. I. Anti-delta pretreatment facilitates tolerance induction in adult B cells in vitro.

Journal of Experimental Medicine ◽

10.1084/jem.146.6.1473 ◽

1977 ◽

Vol 146 (6) ◽

pp. 1473-1483 ◽

Cited By ~ 66

Author(s):

D W Scott ◽

J E Layton ◽

G J Nossal

Keyword(s):

Immune Response ◽

B Cells ◽

B Cell ◽

Tolerance Induction ◽

Spleen Cells ◽

Cell Compartment ◽

Immature B Cells

Adult spleen cells from C57BL.Ige mice, which generally are resistant to in vitro tolerance induction in the B-cell compartment, became hyporesponsive (tolerant) when cultured with antigen in the presence of an anti-allotype serum. Both antigen and anti-delta had to be present for this effect, which was hapten-specific and did not occur in C57BL/L mice, which lack the Ig5-1 allotype of the delta-chain detected in this system. Preculture with anti-mu serum plus antigen, in contrast, did not cause tolerance induction in adult spleen B cells of either strain. These results suggest that the surface IgD may act as a failsafe receptor to prevent tolerance induction in adult B cells. Tolerance studies with spleen cells from mice with markedly reduced numbers of IgD+ve cells, because of regimen of repeated injections of anti-delta serum beginning at birth (delta-suppressed mice), confirmed the importance of membrane IgD in preventing tolerance, because such delta-suppressed mice were hypersusceptible to tolerance by antigen alone. Inasmuch as immature B cells lack IgD on their surface, these studies suggest that acquisition of IgD is an important maturational step in the ability of murine B cells to discriminate tolerogenic and immunogenic signals.

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HAPTEN-SPECIFIC TOLERANCE

Journal of Experimental Medicine ◽

10.1084/jem.136.3.426 ◽

1972 ◽

Vol 136 (3) ◽

pp. 426-438 ◽

Cited By ~ 31

Author(s):

Joseph M. Davie ◽

William E. Paul ◽

David H. Katz ◽

Baruj Benacerraf

Keyword(s):

Immune Response ◽

Guinea Pig ◽

Individual Cell ◽

Tolerance Induction ◽

High Affinity ◽

Individual Plaque ◽

Specific Tolerance ◽

Marked Suppression ◽

Antibody Secreting Cells ◽

Induction Of Tolerance

The induction of tolerance in guinea pigs with a 2,4-dinitrophenyl (DNP) derivative of a copolymer of copolymer of D-glutamic acid and D-lysine (D-GL) leads to a preferential depression of the capacity to produce high affinity anti-DNP antibody in response to immunization with DNP-guinea pig albumin. Thus, immunization 2 wk after tolerance induction with 3 mg of DNP-D-GL results in an immune response in which individual plaque-forming cells (PFC) secreting high affinity anti-DNP antibody are absent and in which the affinity of circulating anti-DNP antibody is reduced. A similar, but less marked, suppression is seen when 0.3 mg of DNP-D-GL is used for tolerance induction. If immunization is delayed until 2 months after tolerance induction, then suppression is restricted to the highest avidity PFC group. Our data is consistent with a state of tolerance in the pool of precursors of anti-DNP antibody-secreting cells induced as a result of their interaction with DNP-D-GL in the absence of specific "helper" cells, which appear to be lacking for DNP-D-GL. In such a situation, the affinity of receptors on precursor cells for tolerogen and the concentration of tolerogen appear to be crucial determinants of whether an individual cell will become tolerant.

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PD-1/PD-L1 Interaction Maintains Allogeneic Immune Tolerance Induced by Administration of Ultraviolet B-Irradiated Immature Dendritic Cells

Journal of Immunology Research ◽

10.1155/2016/2419621 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11

Author(s):

Lanfang Zhang ◽

Chang-Qing Xia

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Immune Tolerance ◽

Tolerance Induction ◽

Ultraviolet B ◽

Immature Dendritic Cells ◽

Programmed Death ◽

Induced Tolerance ◽

Different Strains ◽

Specific Tolerance

Our previous study demonstrated that transfusion of ultraviolet B-irradiated immature dendritic cells (UVB-iDCs) induced alloantigen-specific tolerance between two different strains of mice. Programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) have been suggested to play an important role in maintaining immune tolerance. In the present study, we seek to address whether PD-1/PD-L1 plays a role in the maintenance of UVB-iDC-induced tolerance. We first observe that the UVB-iDC-induced alloantigen-specific tolerance can be maintained for over 6 weeks. Supporting this, at 6 weeks after tolerance induction completion, alloantigen-specific tolerance is still able to be transferred to syngeneic naïve mice through adoptive transfer of CD4+ T cells. Furthermore, skin transplantation study shows that the survival of allogeneic grafts is prolonged in those tolerant recipients. Further studies show that PD-1/PD-L1 interaction is essential for maintaining the induced tolerance as blockade of PD-1/PD-L1 by anti-PD-L1 antibodies largely breaks the tolerance at both cellular and humoral immunological levels. Importantly, we show that PD-1/PD-L1 interaction in tolerant mice is also essential for controlling alloantigen-responding T cells, which have never experienced alloantigens. The above findings suggest that PD-1/PD-L1 plays a crucial role in maintaining immune tolerance induced by UVB-iDCs, as well as in actively controlling effector T cells specific to alloantigens.

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The Diversity of Anti-Factor VIII B-Cell Epitopes in Hemophilia a Patients with Inhibitors

Blood ◽

10.1182/blood.v124.21.2837.2837 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2837-2837 ◽

Cited By ~ 1

Author(s):

Margaret A. Robinson ◽

Courtney Cox ◽

Wallace Hunter Baldwin ◽

Philip Zakas ◽

Shannon L Meeks

Keyword(s):

Immune Response ◽

Factor Viii ◽

B Cell ◽

Hemophilia A ◽

Conflicts Of Interest ◽

C2 Domain ◽

Plasma Samples ◽

C2 Domains ◽

Competition Elisa ◽

A2 Domain

Abstract The most significant complication in the management of patients with hemophilia A continues to be the development of anti-factor VIII antibodies in response to infusions of the fVIII protein. Patients with hemophilia A typically develop a polyclonal B-cell response to fVIII with antibodies to the A2 and C2 domains being the most prevalent. Within the A2 and C2 domains, we have identified non-overlapping epitopes with unique characteristics. There are 5 non-overlapping epitopes in the A2 domain and 3 non-overlapping epitopes in the C2 domain. Within the C2 domain, antibody epitope was more important than inhibitory titer in predicting response to fVIII treatment in a murine in vivo bleeding model. Similar findings were seen within the A2 domain. In previous studies we developed a competition ELISA that assessed the ability of patient plasma to compete with biotinylated monoclonal antibodies (MAbs) with known epitopes for binding to fVIII. Competition with at least 1 of 3 non-overlapping anti-fVIII C2 domain MAbs was seen in 19 of 26 (73%) patient plasmas. The high volume of plasma needed for each monoclonal antibody assessed restricts the use of this assay to map a limited number of epitopes from a single plasma sample. Given the number of non-overlapping epitopes identified on the fVIII protein, the goal of this study was to modify the previous competition ELISA to better define the diversity of the immune response to fVIII using a clinically feasible volume of plasma. Plasma samples and limited clinical data from hemophilia A patients with inhibitors who were HIV negative were obtained from the Biologic Specimen and Data Repository Information Center (BioLINCC) of the NHLBI. A single sample was available from 27 patients while 2 samples from different time periods were available from an additional 23 patients. Epitope mapping was performed using a modified competition ELISA utilizing a single biotinylated MAb concentration with patient plasma (~50 µl total for all experiments) diluted 1:40. The rate of MAb binding in the presence of patient plasma was compared to the rate of MAb binding in the presence of control severe hemophilia A plasma without inhibitors. Competition was considered present when the binding was reduced more than 2 standard deviations below control. The anti-fVIII MAbs in this study were 4 anti-A2 antibodies with non-overlapping epitopes (A2-A, A2-B, A2-D, A2-E) and 3 anti-C2 antibodies with non-overlapping epitopes(C2-A, C2-B, C2-C) as well as one inhibitory antibody from both the A3 and C1 domains. A modified Bethesda assay was performed on each sample which allowed us to group the plasmas into 3 inhibitory titer ranges: < 6 BU/ml, 6-16 BU/ml, and >16 BU/ml. Of the 73 plasma samples 20 showed competition with at least 1 MAb as shown in Table 1. Six of 36 plasmas (17%) with titers of <6 BU/ml showed competition with a single MAb. Three of 20 plasmas (15%) with titers of 6-16 BU/ml showed competition with either 1 or 2 MAbs. Eleven of 17 plasmas with titers of >16 BU/ml showed competition with between 1 and 9 MAbs. No competition was detected in the 6 patients with moderate or mild hemophilia A. In 12 of the 23 patients with 2 samples available, epitopes were detected in at least 1 sample but none of these patients had identical epitope spectra in both samples. Despite the lower overall detection rate compared to the previous assay, our results highlight the diversity of epitope spectra present in patient plasmas with high inhibitory titers. This diversity underscores the need to better understand the makeup of the immune response to fVIII in patients with hemophilia A at the individual epitope level. Given the success of detecting each of the 9 MAb epitopes in at least one patient plasma, work is ongoing to improve the sensitivity of this assay to further define differences in response between patients. Table 1 Table 1. Disclosures No relevant conflicts of interest to declare.

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Maternal IgG immune complexes induce food allergen–specific tolerance in offspring

Journal of Experimental Medicine ◽

10.1084/jem.20171163 ◽

2017 ◽

Vol 215 (1) ◽

pp. 91-113 ◽

Cited By ~ 41

Author(s):

Asa Ohsaki ◽

Nicholas Venturelli ◽

Tess M. Buccigrosso ◽

Stavroula K. Osganian ◽

John Lee ◽

...

Keyword(s):

Breast Milk ◽

Immune Complexes ◽

Tolerance Induction ◽

Specific Ige ◽

Human Breast Milk ◽

Maternal Supplementation ◽

Induced Tolerance ◽

And Control ◽

Specific Tolerance ◽

Maternal Igg

The role of maternal immune responses in tolerance induction is poorly understood. To study whether maternal allergen sensitization affects offspring susceptibility to food allergy, we epicutaneously sensitized female mice with ovalbumin (OVA) followed by epicutaneous sensitization and oral challenge of their offspring with OVA. Maternal OVA sensitization prevented food anaphylaxis, OVA-specific IgE production, and intestinal mast cell expansion in offspring. This protection was mediated by neonatal crystallizable fragment receptor (FcRn)–dependent transfer of maternal IgG and OVA immune complexes (IgG-IC) via breast milk and induction of allergen-specific regulatory T (T reg) cells in offspring. Breastfeeding by OVA-sensitized mothers or maternal supplementation with IgG-IC was sufficient to induce neonatal tolerance. FcRn-dependent antigen presentation by CD11c+ dendritic cells (DCs) in offspring was required for oral tolerance. Human breast milk containing OVA-IgG-IC induced tolerance in humanized FcRn mice. Collectively, we demonstrate that interactions of maternal IgG-IC and offspring FcRn are critical for induction of T reg cell responses and control of food-specific tolerance in neonates.

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