Factor VIII-Fc Activates Natural Killer Cells via Fc-Mediated Interactions With CD16.

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level <1%) hemophilia A (HA) patients and are a serious hindrance to effective management of HA. Consequently, strategies that can either prevent anti-FVIII inhibitors from developing or "tolerize" individuals who develop such antibodies represent a clinically important unmet need. One intervention for patients with high-titer inhibitors is immune tolerance induction (ITI) therapy. Although ITI therapy is the only clinically proven strategy to eradicate anti-FVIII inhibitors, mechanisms of inhibitor reduction remain unknown. Factor VIII Fc-fusion (rFVIIIFc) is an enhanced half-life antihemophilic factor used in replacement therapy for HA. Fc-fusion is a successful protein bio-engineering platform technology. In addition to enhancement of plasma half-life via neonatal Fc receptor (FcRn) binding, other Fc-mediated interactions, including engagement with Fc gamma receptors (FcγR), may have immunological consequences. Several case reports and retrospective analyses suggest that rFVIIIFc offers superior outcomes with respect to ITI compared to other FVIII products. Previously we and others demonstrated rFVIIIFc interactions with activating FcγRIIIA/CD16. Here, we investigated if rFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16 independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potently activated NK cells in a CD16-dependent fashion resulting in IFNγ secretion and cytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cell IFNγ secretion levels and the high-affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activated CD16+ NK cells were able to lyse a B-cell clone (BO2C11) bearing an anti-FVIII B-cell receptor in an antibody-dependent cellular cytotoxicity (ADCC) assay. These in vitro findings provide an underlying molecular mechanism that may help explain clinical case reports and retrospective studies suggesting rFVIIIFc may be more effective in tolerizing HA patients with anti-FVIII inhibitors compared to FVIII not linked to Fc. Our in vitro findings suggest a potential use of Fc-fusion proteins acting via NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.

Increased synapse formation obtained by T cell epitopes containing a CxxC motif in flanking residues convert CD4+ T cells into cytolytic effectors.

The nature of MHC class II-binding epitopes not only determines the specificity of T cell responses, but may also alter effector cell functions. Cytolytic CD4+ T cells have been observed primarily in anti-viral responses, but very little is known about the conditions under which they can be elicited. Their potential as regulators of immune responses, however, deserves investigations. We describe here that inclusion of a thiol-disulfide oxidoreductase motif within flanking residues of class II-restricted epitopes results, both in vitro and in vivo, in elicitation of antigen-specific cytolytic CD4+ T cells through increased synapse formation. We show that both naïve and polarized CD4+ T cells, including Th17 cells, can be converted by cognate recognition of such modified epitopes. Cytolytic CD4+ T cells induce apoptosis on APCs by Fas-FasL interaction. These findings potentially open the way towards a novel form of antigen-specific immunosuppression.

Regulatory CD4+ T cells in allergic asthma.

Active suppression by regulatory T cells (T(regs)) appears to play a key role in the downregulation of T-cell responses to foreign antigens. Several subtypes of T(regs) have been described but their mechanisms of action remain unclear. Recent data demonstrate that the suppressive capacity of natural T(regs) could be associated with cytotoxicity due to the release of granzymes, which are capable of apoptosis induction in target effector T lymphocytes and in antigen-presenting cells, such as dendritic cells. The mechanism of such nonspecific T(regs) is discussed. Peptide immunotherapy is thought to induce regulatory cells capable of suppressing autoimmune and allergic diseases. We have recently optimized a vaccination strategy by which cytotoxic antigen-specific adaptive T(regs) can be elicited towards allergens involved in allergic asthma. Such a strategy could be of value in the treatment of allergic asthma.

Factor VIII alloantibodies in hemophilia.

PURPOSE OF REVIEW: The development of an inhibitory response to factor VIII (FVIII) remains a puzzling challenge both for clinicians and scientists, not to mention the difficulties of maintaining hemostasis in patients producing inhibitors. RECENT FINDINGS: Three main research lines have been explored in recent months. The mechanisms by which an anti-FVIII antibody response is elicited in patients has been examined at both the B- and T-cell levels, with particular emphasis on the generation of specific B- and T-cell clones. The hemophilia A mouse model has served to confirm the main characteristics of the anti-FVIII immune response in terms of T-cell dependency and memorization of the response. Novel strategies for the prevention and downregulation of inhibitors have emerged, with special interest in antigen-specific approaches. SUMMARY: Although the ultimate goal, preventing or suppressing inhibitor formation in patients, is not yet achieved, the research activity developed over the past months brings us forward in that direction.

In vivo neutralization of a C2 domain-specific human anti-Factor VIII inhibitor by an anti-idiotypic antibody.

Factor VIII (FVIII) administration elicits specific inhibitory antibodies (Abs) in about 25% of patients with hemophilia A. The majority of such Abs reacts with FVIII C2 domain. mAbBO2C11 is a high-affinity human monoclonal antibody (mAb) directed toward the C2 domain, which is representative of a major class of human FVIII inhibitors. Anti-idiotypic Abs were raised to mAbBO2C11 to establish their neutralizing potential toward inhibitors. One mouse anti-idiotypic mAb, mAb14C12, specifically prevented mAbBO2C11 binding to FVIII C2 domain and fully neutralized mAbBO2C11 functional inhibitory properties. Modeling of the 3-D conformation of mAb14C12 VH and alignment with the 3-D structure of the C2 domain showed putative 31 surface-exposed amino acid residues either identical or homologous to the C2 domain. These included one C2 phospholipid-binding site, Leu2251-Leu2252, but not Met2199-Phe2200. Forty putative contact residues with mAbBO2C11 were identified. mAb14C12 dose-dependently neutralized mAbBO2C11 inhibitory activity in mice with hemophilia A reconstituted with human recombinant FVIII (rFVIII), allowing full expression of FVIII activity. It also neutralized in an immunoprecipitation assay approximately 50% of polyclonal anti-C2 Abs obtained from 3 of 6 unrelated patients. mAb14C12 is the first example of an anti-idiotypic Ab that fully restores FVIII activity in vivo in the presence of an anti-C2 inhibitor. The present results establish the in vitro and in vivo proof of concept for idiotype-mediated neutralization of a major class of FVIII inhibitors.

CD4+CD25+ T cells lyse antigen-presenting B cells by Fas-Fas ligand interaction in an epitope-specific manner.

Suppression by regulatory T cells is now acknowledged to play a key role in the down-regulation of T cell responses to foreign and self Ags. In addition to the naturally occurring CD4(+)CD25(+) population, several subtypes of induced regulatory cells have been reported, but their mechanisms of action remain unclear. Conversely, cytotoxic CD4(+) cells that lyse cells presenting their cognate peptide have been described, but their potential role in immunoregulation remains to be delineated. A CD4(+) T cell line derived from BALB/c mice immunized with peptide 21-35, containing a major T cell epitope of a common allergen, Dermatophagoides pteronyssinus group 2 allergen, was found to lyse the Ag-presenting WEHI cell line via Fas-Fas ligand and only in the presence of the cognate peptide. Cytolytic activity was likewise shown for other T cell lines and occurred even after a single cycle of in vitro stimulation. Moreover, T cells that efficiently lysed WEHI cells were unresponsive to stimulation with their cognate Ag and were dependent on IL-2 for growth and survival, which was reflected in a constitutive expression of CD25 independently of activation status. Proliferating B cells were also killed by the CTLs. By lysing Ag-presenting B cells in an epitope-specific manner, the nonproliferating CTLs were shown to down-regulate the proliferation of bystander T cells. These data demonstrate that cytotoxic CD4(+)CD25(+) T cells that lack proliferation capacities have the potential to down-regulate an immune response by killing Ag-presenting B cells. This could represent an important and specific down-regulatory mechanism of secondary immune responses in vivo.

The Dermatophagoides pteronyssinus group 2 allergen contains a universally immunogenic T cell epitope.

The use of T cell epitope-containing peptides for the induction of anergy in allergen sensitization is limited by genetic restriction that could be circumvented by using universally immunogenic epitopes. We attempted to identify such epitopes on Dermatophagoides pteronyssinus group 2 allergen (Der p 2), a major allergen of D. pteronyssinus T cells from BALB/c (H-2(d)), C57BL/6 (H-2(b)), C3H (H-2(k)), and SJL (H-2(s)) mice that were immunized with rDer p 2, recognized an immunodominant region encompassing residues 21-35. A synthetic 21-35 peptide (p21-35) induced strong dose-dependent in vitro T cell proliferation with cells of the four mouse strains and required processing for MHC class II presentation. Substitution of Ile(28) with Ala resulted in reduction of T cell proliferation in each strain. Ile(28) could represent an important MHC class II anchoring residue for T cell response to p21-35. An immunodominant T cell epitope of Der p 2 therefore behaves as a universal epitope and could be a suitable candidate for T cell anergy induction.