Inhibitor Development In Hemophilia Research Articles

Unearthing the genotype-inhibitor phenotype association in severe haemophilia A: A north Indian cohort study.

Various risk factors for inhibitor development in haemophilia A (HA) have been described but Indian data remains scanty. We aimed to evaluate the genetic changes in Indian HA-patients that are associated with the development of inhibitors. All HA-patients with inhibitors who availed coagulation-laboratory services from January-2015 till December-2021 and had their samples preserved for DNA extraction were included in this study. An equal number of severity-matched HA patients without inhibitors were also included as controls. Intron 22 and intron 1 inversions in Factor VIII gene were identified using inverse-shifting-PCR. Inversion-negative patients were further assessed by targeted NGS, MLPA. Thirty HA-patients with inhibitors were identified. All had severe-HA. Thirty severe-HA-patients without inhibitors were also included as controls. Intron 22 inversion (63.3%) and large deletions (15%) were the commonest variants identified. There was no difference in genetic variants in patients with low and high titre inhibitors. A3, A2 and C2 were the most common domains involved in inversion-negative patients with inhibitors. However, there was no significant difference in domain involvement among inversion-negative patients with and without inhibitors. Seven novel-variants were identified, including three large deletions, one large duplication and two nonsense variants in inhibitor-positive patients, and one frameshift variant in inhibitor-negative patient. After adjusting for clinical risk-factors, large deletions were independently associated with the presence of inhibitors [aOR:6.1 (1.41-56.3)]. Intron 22 inversions are the commonest variant in Indian patients with severe-HA. Large deletions predispose to inhibitor development independent of clinical risk factors.

The Tfh/Tfr Pathway Is Pivotal in FVIII Inhibitor Development in Mice

The development of neutralizing anti-FVIII antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. Our previous study demonstrated that the induction of activated follicular helper T (Tfh) cells in FVIII null mice is critical for FVIII inhibitor development. Follicular regulatory T (Tfr) cells are a specialized subset of Foxp3 + T cells identified in the germinal center (GC) that can modulate Tfh cell activation of B cells and antibody development. However, the role of Tfr cells in controlling anti-FVIII inhibitor development in vivo is still unclear. Here, we explored the activation and induction of Tfr cells and their functions in anti-FVIII inhibitor development in FVIII-immunized mice. We first characterized the features of activated Tfr cells in the spleen of immunized FVIII null mice by flow cytometry analysis. Activated Tfr cells were defined as CD4 +Foxp3 +CD44 +CD62L -CXCR5 +PD-1 +. Tfr cells express a high level of transcription factor BCL6 and costimulatory receptor ICOS. Activated Tfr cells also express a high level of costimulatory receptor CTLA4, which is similar to non-Tfr effector Treg cells, but CD25 expression is low on Tfr cells. FVIII immunization increases the frequency and the absolute number of activated Tfr cells in the spleen of mice that produce inhibitors. The percentage of activated CXCR5 hiPD-1 hi Tfr cells within the CD4 population significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (0.88 ± 0.2 %, 0.33 ± 0.16 %, and 0.36 ± 0.2%, respectively). The Tfh/Tfr ratio also significantly increased in FVIII inhibitor-producing mice, suggesting a preferential expansion of Tfh cells relative to Tfr cells in inhibitor-producing mice. Interestingly, similar to Tfh cells as we reported before, the emergence of Tfr cells correlated with titers of anti-FVIII inhibitors in FVIII immunized mice. This indicates that a functional relationship exists between Tfr cell activation and FVIII inhibitor production after FVIII immunization. Using Tfr deficient Foxp3-Cre Bcl6 fl/fl (BCL6FC) mice, we determined the role of Tfr cells in inhibitor generation in vivo. Loss of Tfr cells led to significantly decreased FVIII inhibitor levels, with FVIII inhibitor titers in BCL6FC mice markedly lower than in wild-type (WT) mice upon recombinant human full-length FVIII (rhFVIII, Kogenate 200 U/kg/week IV x4) immunization (24 ± 16 and 131 ± 114 BU/ml, respectively). However, the total anti-FVIII IgG titers were similar between the BCL6FC and WT groups, suggesting that Tfr cells help generate neutralizing anti-FVIII antibodies. FVIII immunization could still efficiently induce Tfh cell activation in BCL6FC mice. However, both GC Tfh cells and GC B cells were reduced in FVIII immunized BCL6FC mice, suggesting that Tfr cells promote FVIII-specific GC responses. To further investigate the regulatory function of Tfr cells on FVIII immune responses, mice with phosphatase Pten deficiency in Treg cells (Foxp3-Cre Pten fl/fl [PtenFC]), a model with augmented Treg and Tfr cells, were immunized with rhFVIII and anti-FVIII antibody titers were measured at 1 week after the 4 th weekly immunization. Of note, FVIII inhibitor titers (8.06 ± 8.6 BU/ml) were significantly lower than those in WT controls under the same immunization protocol. There were significant positive correlations between the inhibitor titers and total anti-FVIII IgG in the WT and PtenFC groups but not in the BCL6FC group after rhFVIII immunization. To confirm the important role of the Tfh/Tfr pathway in FVIII inhibitor development, we used CD4-Cre Bcl6 fl/fl (CD4-Bcl6-cKO) mice, a model with Bcl6 deficiency in all CD4 + T cells. As expected, none of the CD4-Bcl6-cKO mice developed detectable levels of FVIII inhibitors, demonstrating that the transcription repressor BCL6 is pivotal for FVIII inhibitor development. In conclusion, we found that FVIII immunization induces Tfr cell activation and expansion. Tfr cells have a dual function in regulating FVIII immune responses in mice, and the Tfh/Tfr pathway is essential for FVIII inhibitor development in hemophilia A mice.

Development Of Inhibitors In Hemophilia A Patients Receiving Different Treatment Options

Introduction: Hemophilia A is the most common inherited bleeding disorder in Pakistan. Apart from frequent bleeds and joint deformities, the formation of inhibitors is the major complication of hemophilia. Inhibitors are antibodies formed against the coagulation factor. The purpose of this study is to determine the incidence of inhibitor development in hemophilia A patient receiving different treatment modalities namely fresh frozen plasma (FFP)/cryoprecipitate, plasma derived FVIII concentrates and long-acting recombinant FVIII concentrates. Aims & Objectives: To assess the development of inhibitors in hemophilia A patients receiving treatment modalities. Place and Duration of Study: The study was conducted at Chughtai Institute of Pathology from November 2021 to November 2023. Material &Methods: A total of 75 registered patients of the Hemophilia Patient Welfare Society, Lahore and Sundas Foundation were initially screened for FVIII inhibitors. Those who were found negative for these inhibitors were included in the study and then divided into three equal groups of 25 patients each. One group received only FFP/cryoprecipitate for treatment, the other group received plasma derived FVIII concentrates and the last group received recombinant factor VIII concentrates. These patients were observed for two years i.e after 15-20 exposures and following this period their inhibitors screening was done again to establish the effect of different treatment modalities on inhibitor development. The data collected was entered and analyzed by SPSS-21, a p-value of ?0.05 was considered significant. Results: Our study showed that only 3 patients out of 25, receiving FFP as treatment modality developed inhibitors. Whereas, none of the patients receiving plasma derived FVIII concentrates and recombinant factor VIII concentrates developed inhibitors. The P value calculated is 0.044 which is significant. Conclusion: Plasma derived FVIII concentrates and recombinant FVIII concentrates are safer as compared to FFP/Cryoprecipitate and have lesser risk for inhibitor development.

Factor IX administration in the skin primes inhibitor formation and sensitizes hemophilia B mice to systemic factor IX administration

BackgroundFactor IX inhibitor formation is the most serious complication of replacement therapy for the bleeding disorder hemophilia B, exacerbated by severe allergic reactions occurring in up to 60% of patients with inhibitors. Low success rates of immune tolerance induction therapy in hemophilia B necessitate the search for novel immune tolerance therapies. Skin-associated lymphoid tissues have been successfully targeted in allergen-specific immunotherapy. ObjectivesWe aimed to develop a prophylactic immune tolerance protocol based on intradermal administration of FIX that would prevent inhibitor formation and/or anaphylaxis in response to replacement therapy. MethodsWe measured FIX inhibitor, anti-FIX immunoglobulin G1, and immunoglobulin E titers using the Bethesda assay and enzyme-linked immunosorbent assay after 4 weeks of twice-weekly intradermal FIX or FIX-Fc administration followed by 5 to 6 weeks of weekly systemic FIX injections in C3H/HeJ hemophilia B mice. We also measured skin antigen-presenting, follicular helper T, and germinal center B cell frequencies in skin-draining lymph nodes after a single or repeat intradermal FIX administration. ResultsIntradermal administration enhanced FIX inhibitor formation in response to systemic administration. We further found that intradermal administration alone triggers inhibitor formation, even at a low dose of 0.4 IU/kg, which is 100-fold lower than the intravenous dose of 40 IU/kg typically required to induce inhibitor development in hemophilia B mice. Also, intradermal administration triggered germinal center formation in skin-draining lymph nodes and sensitized mice to systemic administration. Factor IX–Fc fusion protein did not modulate inhibitor formation. ConclusionIntradermal FIX administration is highly immunogenic, suggesting that the skin compartment is not amenable to immune tolerance induction or therapeutic delivery of clotting factors.

OC 50.2 Low Dose FVIII Injections as a Potential Treatment to Prevent Inhibitor Development in Hemophilia a - an in Vivo Study

OC 50.2 Low Dose FVIII Injections as a Potential Treatment to Prevent Inhibitor Development in Hemophilia a - an in Vivo Study

Impact of ABO blood group antigens on residual factor VIII levels and risk of inhibitor development in hemophilia A

The clinical phenotype of hemophilia A (HA) does not always correlate with severity. Similarly, the presence of inhibitors does not necessarily increase the risk of bleeding. This paradox between clinical and laboratory findings may be partially attributed to non-modifiable factors, such as blood group, which is known to influence FVIII levels in healthy individuals. Our aim was to assess the effect of ABO blood group antigens on FVIII levels across the severity spectrum of HA and risk of inhibitor development. Data of consecutive patients with HA who visited the coagulation unit of a northern Indian tertiary care hospital between 2010‒2021 were reviewed. Patients with missing blood group data, transfusion histories, or baseline FVIII levels were excluded. Mild, moderate, and severe HA was present in 41 (6.9%), 72 (12.2%), and 479 (80.9%) patients, respectively. There were no differences in the FVIII levels among the various blood groups across the HA severity spectrum. Inhibitors were administered to 35 patients (5.9%). In the multivariate analysis, blood group A was an independent risk factor for the development of inhibitors (adjusted odds ratio 2.70, P=0.04) after adjusting for age at onset of bleeding, FVIII transfusion, age at first FVIII transfusion, and severity of HA. Unlike what is observed in healthy individuals, blood group did not influence residual FVIII levels across the severity spectrum of HA. Patients in group A had a higher risk of developing inhibitors.

F9 mutations causing deletions beyond the serine protease domain confer higher risk for inhibitor development in hemophilia B

F9 mutations causing deletions beyond the serine protease domain confer higher risk for inhibitor development in hemophilia B

Genetic Risk Factors for Inhibitor Development in Hemophilia A

Genetic Risk Factors for Inhibitor Development in Hemophilia A

A Novel Molecular Indicator for Inhibitor Development in Haemophilia A

A Novel Molecular Indicator for Inhibitor Development in Haemophilia A

Structure of blood coagulation factor VIII in complex with an anti–C1 domain pathogenic antibody inhibitor

AbstractAntibody inhibitor development in hemophilia A represents the most significant complication resulting from factor VIII (fVIII) replacement therapy. Recent studies have demonstrated that epitopes present in the C1 domain contribute to a pathogenic inhibitor response. In this study, we report the structure of a group A anti–C1 domain inhibitor, termed 2A9, in complex with a B domain–deleted, bioengineered fVIII construct (ET3i). The 2A9 epitope forms direct contacts to the C1 domain at 3 different surface loops consisting of Lys2065-Trp2070, Arg2150-Tyr2156, and Lys2110-Trp2112. Additional contacts are observed between 2A9 and the A3 domain, including the Phe1743-Tyr1748 loop and the N-linked glycosylation at Asn1810. Most of the C1 domain loops in the 2A9 epitope also represent a putative interface between fVIII and von Willebrand factor. Lastly, the C2 domain in the ET3i:2A9 complex adopts a large, novel conformational change, translocating outward from the structure of fVIII by 20 Å. This study reports the first structure of an anti–C1 domain antibody inhibitor and the first fVIII:inhibitor complex with a therapeutically active fVIII construct. Further structural understanding of fVIII immunogenicity may result in the development of more effective and safe fVIII replacement therapies.

ABO Blood Group and Inhibitor Risk in Severe Hemophilia A Patients: A Study from the Italian Association of Hemophilia Centers.

Considering the profound influence exerted by the ABO blood group system on hemostasis, mainly through the von Willebrand factor and factor VIII (FVIII) complex, we have conducted a study evaluating the possible role of blood type on the risk of inhibitor development in hemophilia A. A total of 287 consecutive Caucasian patients with severe hemophilia A (202 without FVIII inhibitors and 85 with FVIII inhibitors) followed at seven Italian Hemophilia Treatment Centers belonging to the Italian Association of Hemophilia Centers (AICE) were included in the study. A higher prevalence of O blood group was detected in patients without inhibitors as compared in inhibitor patients (55 vs. 30.6%; p < 0.001). Among the other variables analyzed (age, F8 mutation, type and intensity of treatment and treatment regimen), F8 mutation class (high-risk vs. low-risk), and treatment regimen (on-demand vs. prophylaxis) were significantly correlated with inhibitor development. However, on a multivariate analysis, only the effects of F8 mutation and ABO blood type were independent of other covariates, being that non-O blood type is associated with a 2.89-fold increased risk of inhibitor development. In conclusion, our study supports the protective effect of O blood type on inhibitor risk in severely affected hemophilia A patients.

Risk of Hypertension in Hemophilia Patients with a History of Inhibitors

Hemophilia is an X-linked bleeding disorder characterized by deficiencies of Factor VIII or IX. Hypertension in persons with hemophilia (PWH) appears to be more prevalent compared to the general population. A major complication of hemophilia remains the development of neutralizing antibodies (inhibitors) against infused clotting factor. It tends to occur within the first 50 exposure days and therefore mostly in children with severe hemophilia. Since inhibitor development is associated with high mortality and morbidity, their eradication with high dose clotting factor concentrates is critical and successful in the majority of patients. Recently a cohort study showed a positive association between presence of inhibitors in elderly patients and cardiovascular disease, although a small number of patients with inhibitors were included in that study (Sood SL, Blood Advances 2018). To date, the link between a history of inhibitors and hypertension has not been established. The primary purpose of our study was to evaluate the relationship between hypertension in PWH and a history of inhibitors, with the additional goals of investigating the association between hypertension and hemophilia type and viral status. In a retrospective review, clinical records were extracted for PWH aged 18 to 79 between 2003 and 2014 from four Hemophilia Treatment Centers in North America: Los Angeles Orthopaedic Hospital, University of California San Diego, Tulane University and University of British Columbia. We extracted demographic information on age and ethnicity, weight, height, hemophilia type and severity, inhibitor history, Hepatitis C (HCV; by serology) and Human Immunodeficiency Virus (HIV) status or reported history thereof and medication history. Primary outcomes were prevalence of hypertension stratified by inhibitor status, hemophilia type, and presence of viral coinfection. Blood pressure measurements were taken for the three clinic visits closest to the evaluation date. We fitted generalized additive models with a non-parametric spline function for age. The outcomes were hypertension (with a binary logit), systolic blood pressure (SBP) and diastolic BP (DBP). The models were adjusted for logBMI, type of hemophilia, severity, race/ethnicity, HCV and HIV. We plotted standardized curves generated by the models to compare trends in each outcome. A total of 691 PWH were included. Forty-four (6%) had a history of inhibitors, 533 had hemophilia type A (77%) and 157 had hemophilia type B (23%). HCV was positive in 419 patients (61%) and HIV in 128 patients (19%). There was no difference in the risk of hypertension between patients with or without a history of inhibitors. SBPs and DBSs were only analyzed for patients not on anti-hypertensives, and were not associated with a history of inhibitors (Fig. 1A). However, risk of hypertension was considerably higher among patients with Hemophilia B compared to patients with Hemophilia A (Fig. 1B). Hemophilia B patients were more likely to be categorized as hypertensive because both SBPs and DBPs were higher, especially in patients between 40 and 60 years of age. Finally, risk of hypertension was higher among those coinfected with both HCV and HIV (Fig. 1C). This large-scale study found no evidence that inhibitor history influenced the risk for hypertension or elevated BPs in PWH. Interestingly, Hemophilia B was associated with a higher risk of hypertension and higher BPs, similar to prior studies showing Hemophilia B had 1.87-fold higher odds of hypertension compared to Hemophilia A (von Drygalski A, Hypertension, 2013). Inhibitor development in Hemophilia B is very rare, which provides additional support that factors other than inhibitors play a role. Finally, our study showed coinfection with HIV and HCV was associated with higher hypertension risk, adding clarity to inconsistent findings on the association between hypertension and blood transmitted viral diseases in PWH. While there was no evidence that patients with a history of inhibitors have a higher risk of hypertension, our observations add to the continued efforts to elucidate the etiology of the hypertension in hemophilia and confirm that patients with Hemophilia B and/or viral infections may be at higher risk. These findings continue to inform medical care and also pave the way for additional studies to address physiological factors inherent to hemostasis and vascular pathology. Disclosures Kruse-Jarres: NovoNordisk: Other; CSLBehring and Genentech/Roche: Research Funding; Biomarin, Chugai, CSLBehring, and Genentech/Roche: Consultancy. Quon:Orthopaedic Institute for Children: Current Employment; Bayer: Honoraria; Shire/Takeda: Speakers Bureau; Genentech, Inc./F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Octapharma: Honoraria; Novo Nordisk: Honoraria, Speakers Bureau; Bioverativ/Sanofi: Honoraria, Speakers Bureau; Biomarin: Honoraria, Speakers Bureau. Jackson:Octapharma, Takeda, Bayer, Roche and Pfizer: Honoraria; Bayer: Research Funding; Sanofi: Research Funding. von Drygalski:Hematherix Inc: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder; superFVa; Joint Activity and Damage Examination (JADE) Ultrasound measurement tool: Patents &amp; Royalties; Biomarin, Bioverativ/Sanofi-Genzyme, Novo Nordisk, Pfizer, Uniqure, Takeda: Consultancy.

Rituximab for treating inhibitors in people with inherited severe hemophilia.

Hemophilia A and B are inherited coagulation disorders characterized by a reduced or absent level of factor VIII or factor IX respectively. The severe form is characterized by a factor level less than 0.01 international units (IU) per milliliter. The development of inhibitors in hemophilia is the main complication of treatment, because the presence of these antibodies, reduces or even nullifies the efficacy of replacement therapy, making it very difficult to control the bleeding. People with inhibitors continue to have significantly higher risks of morbidity and mortality, with considerable treatment costs. Given the wide 'off-label' use of rituximab for treating people with hemophilia and inhibitors, its efficacy and safety need to be evaluated. This is an update of a previously published Cochrane Review. To assess the efficacy and safety of rituximab for treating inhibitors in people with inherited severe hemophilia A or B. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Coagulopathies Trials Register, complied from electronic database searches and handsearching of journals and conference abstract books. We searched the reference lists of relevant articles and reviews and also searched for ongoing or unpublished studies. We also undertook further searches of other bibliographic databases and trial registries. Date of last search of the Cochrane Cystic Fibrosis and Genetic Disorders Group's Coagulopathies Trials Register: 19 March 2020. Randomized controlled trials and controlled clinical trials investigating the efficacy and safety of rituximab for treating inhibitors in people with hemophilia. No randomized controlled trials matching the selection criteria were eligible for inclusion. No randomized controlled trials on rituximab for treating inhibitors in people with hemophilia were identified. We were unable to identify any relevant trials on the efficacy and safety of rituximab for treating inhibitors in people with hemophilia. The research evidence available is from case reports and case series. Randomized controlled trials are needed to evaluate the efficacy and safety of rituximab for this condition. However, prior to the publication of any possible future randomized controlled trials, meta-analysis of case reports and case series may provide some evidence.

Is the Detection of Factor IX Inhibitors in Hemophilia B Orphan than Factor VIII Inhibitors in Hemophilia A? A Concise, Systematic Review.

Development of inhibitors in hemophilia A and B comprise significant challenge for patients, hematologists, and health provider systems. It has recommended by the World Federation of Hemophilia (WFH) to check inhibitors every 3-4 months. The incidence of inhibitor in hemophilia B is lower than hemophilia A. Here, it tried to unravel whether the detection of inhibitors in hemophilia B neglected compared to hemophilia A or not? A comprehensive review carried out using six international and local medical search engines on published contributions about inhibitors in hemophilia A and B in Iran. From 699 titles, 12 relevant papers were selected. The mean of factor VIII inhibitors in hemophilia A was 14.8%. The mean of factor IX inhibitors in hemophilia B was 6%. The minimum and maximum reported percentages of factor VIII inhibitors were 4% and 19.6%, while the minimum and maximum of reported percentages of factor IX inhibitors were 0% and 11.8%, respectively. The inhibitors in hemophilia A had reported in 6 papers. One paper had covered the inhibitors in hemophilia B. There were five papers on inhibitors in both hemophilia A and B. The comparison between the reported patients showed that 3020 patients with hemophilia A and 314 patients with hemophilia B had studied. Consistent with the lower frequency of hemophilia B and the lower development of inhibitors in hemophilia B compared to hemophilia A, it was concluded that hemophilia B had not neglected in Iran. It seems to be rational that each country, check rates of detection of inhibitors in hemophilia B to identify whether it has neglected or not.

Molecular Mechanisms of Inhibitor Development in Hemophilia.

The development of neutralizing antibodies in hemophilia is a serious complication of factor replacement therapy. These antibodies, also known as “inhibitors”, significantly increase morbidity within the hemophilia population and lower the quality of life for these patients. People with severe hemophilia A have an overall 25–40% lifetime risk of inhibitor development, compared to that of 5–15% lifetime risk in those with moderate/mild hemophilia A. The risk is lower in hemophilia B population (about 1–5%) and occurrence of inhibitors is almost only seen in patients with severe hemophilia B. The understanding of the pathophysiological mechanism leading to the development of inhibitors in patients with hemophilia has improved considerably over the last 2 decades. Identification of early biomarkers which predict inhibitor development in previously untreated patients with hemophilia will assist in risk identification and possible early intervention strategies. In this review, we aim to summarize the molecular mechanisms of inhibitor development in hemophilia and to identify potential areas in need of further investigation.

Mixing-based inhibitor screening in haemophilia A: challenges in interpretation.

: Inhibitor development in haemophilia A patients is a dreaded complication of factor VIII (FVIII) replacement therapy. With increasing use of FVIII replacement therapy, there is an imperative need for cost-effective and standardized screening. To evaluate the efficacy of mixing-based inhibitor screening (MBIS) in the detection of FVIII inhibitors and to assess the best cut-off values for MBIS. Forty inhibitor positive and 40 inhibitor negative haemophilia A patients, diagnosed by standard criteria, with detailed clinical, haematological and on-demand treatment records were included. MBIS was evaluated in all 80 cases and a classical Bethesda assay and Nijmegen modification of Bethesda assay (NBA) were used as gold standards for inhibitor diagnosis. Classical Bethesda assay missed eight cases, most with low titres, which were confirmed by NBA. A systematic analysis of cut-offs for MBIS using a receiver operating characteristic curve fixed the cut-off at more than 5 s. MBIS detected 36 out of 40 inhibitor positive haemophilia A patients with a sensitivity, specificity, PPV and NPV of 90.0, 95, 94.7, 90.5%, respectively, whereas at the conventional cut-off of more than 10 s, MBIS detected only 25 of 40 cases with a low sensitivity of 62.5%. The likelihood ratio of a positive test was 11. The false-negative haemophilia A patients had low titres from 1.6 to 4.2 BU/ml. MBIS at a cut-off of 5 s can be considered as an effective screening test in low-resource situations. In clinical situations and in cases with clinical evidence of inhibitors we recommend that a direct NBA should be done.

Induction of Activated T Follicular Helper Cells Is Critical for Anti-FVIII Inhibitor Development in Hemophilia a Mice

The development of neutralizing anti-FVIII antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. While multiple lines of evidence indicate that the immune response against FVIII is CD4 T cell-dependent, the role of T follicular helper (TFH) cells, a newly identified subset of CD4 T cells, in FVIII inhibitor development is unknown. TFH cells are a critical player in germinal center formation, which provide cognate help to B cells and are fundamentally required for the generation of T cell-dependent B cell responses. Here we explored the activation and induction of TFH cells and their roles in anti-FVIII inhibitor production in FVIII-immunized mice. We first confirmed that inhibitor development depends on CD4 T cells as depletion of CD4 T cells during FVIII immunization in FVIIInull mice prevents inhibitor production. We then characterized the features of activated TFH cells in the spleen of immunized FVIIInull mice by flow cytometry analysis. Activated TFH cells were defined as CD4+Foxp3-CD44+CD62L-CXCR5+PD-1+. These cells express high levels of transcription factors BCL6, ICOS, and CD40L. We showed for the first time that intravenous FVIII immunization induced activation and accumulation and/or expansion of TFH cells in the spleen of FVIIInull mice. The percentage of CXCR5hiPD-1hi TFH cells within the effector CD4 population and the absolute numbers of activated TFH cells per spleen significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (19.6 ± 1.6 %, 8.1 ± 1.2%, and 7.6 ± 1.4%, respectively; 14.4 ± 2.0×104, 3.55 ± 0.8×104, and 3.5 ± 0.9×104, respectively). There was no TFH cell expansion observed in other secondary lymphoid organs (mesenteric and inguinal lymph nodes), suggesting that the spleen is the primary site for TFH activation when FVIII is administered intravenously. Our results showed that FVIII inhibitor-producing mice had increased GL-7+germinal center TFH cells together with increased germinal center formation in response to FVIII immunization and the emergence of TFH cells correlated with titers of anti-FVIII inhibitors. This indicates that a functional relationship exists between TFH cell activation and FVIII inhibitor production after FVIII immunization. To determine the extent to which the anti-FVIII inhibitor development is related to CXCR5+ TFH cells, we used a mixed BM chimera model consisting of a CD4 T cell-specific CXCR5 deficiency (CD4-CXCR5-/-). Compared with CD4-CXCR5+/+ control mice, there was a significant decrease in the inhibitor titer in CD4-CXCR5-/- mice after FVIII immunization (111.7 ± 46.2 vs 4.43 ± 3.53 BU/ml, respectively). Our results showed that CXCR5+/+ TFH cell-specific deletion impaired anti-FVIII inhibitor production, confirming the essential role of CXCR5+/+ TFH cells for the generation of FVIII-neutralizing antibodies. To examine specificity of TFH cell responses after FVIII immunization, we used an ex vivo T-cell proliferation assay. In vitro FVIII restimulation induced the antigen-specific proliferation of splenic CD4 T cells from FVIII-primed FVIIInull mice and those proliferating cells expressed the TFH hallmark transcription factor BCL6. These results demonstrated that antigen-specific TFH cells were induced after FVIII immunization and those cells could specifically respond to FVIII upon restimulation. To evaluate the FVIII-specific memory TFH cell responses in FVIIInull mice, matched pairs of FVIII-primed mice with similar inhibitor titers were rechallenged with FVIII antigen in vivo 2 months after the last immunization. The absolute numbers of TFH cells per spleen in FVIII rechallenged animals were significantly higher than those obtained from saline-injected primed mice (36.7 ± 11 ×104 vs 14.8 ± 3.8 ×104, respectively). These data suggest that memory FVIII-specific TFH cells are persistent which can induce efficient recall TFH responses after FVIII rechallenge. Together, our results demonstrate that the induction of activated TFH cells in FVIIInull mice is critical for FVIII inhibitor development, suggesting that inhibition of FVIII-specific TFH cell activation might be a promising strategy to prevent anti-FVIII inhibitor formation in hemophilia A patients. Disclosures No relevant conflicts of interest to declare.

Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice

AbstractThe development of neutralizing anti-FVIII antibodies (inhibitors) is a major complication of FVIII protein replacement therapy in patients with hemophilia A (HA). Although multiple lines of evidence indicate that the immune response against FVIII is CD4 T-cell–dependent and many FVIII-derived CD4 epitopes have already been discovered, the role of T follicular helper (TFH) cells in FVIII inhibitor development is unknown. TFH cells, a newly identified subset of CD4 T cells, are characterized by expression of the B-cell follicle-homing receptor CXCR5 and PD-1. In this study, we show for the first time that IV FVIII immunization induces activation and accumulation and/or expansion of PD-1+CXCR5+ TFH cells in the spleen of FVIII-deficient (FVIIInull) mice. FVIII inhibitor-producing mice showed increased germinal center (GC) formation and increased GC TFH cells in response to FVIII immunization. Emergence of TFH cells correlated with titers of anti-FVIII inhibitors. Rechallenge with FVIII antigen elicited recall responses of TFH cells. In vitro FVIII restimulation resulted in antigen-specific proliferation of splenic CD4+ T cells from FVIII-primed FVIIInull mice, and the proliferating cells expressed the TFH hallmark transcription factor BCL6. CXCR5+/+ TFH-cell–specific deletion impaired anti-FVIII inhibitor production, confirming the essential role of CXCR5+/+ TFH cells for the generation of FVIII-neutralizing antibodies. Together, our results demonstrate that the induction of activated TFH cells in FVIIInull mice is critical for FVIII inhibitor development, suggesting that inhibition of FVIII-specific TFH-cell activation may be a promising strategy for preventing anti-FVIII inhibitor formation in patients with HA.

The role of variant alleles of the mannose-binding lectin in the inhibitor development in severe hemophilia A

IntroductionThe administration of FVIII leads to inhibitors in up to 30% of patients with hemophilia A (HA), the most severe treatment complication. FVIII-mannosylation fosters the presentation of FVIII to CD4+-T-lymphocytes. Mannose as primary ligand for the mannose-binding lectin (MBL) activates the lectin pathway of complement. MBL2 single nucleotide polymorphisms (SNPs) lead to low peripheral MBL concentrations that may hamper the removal of mannosylated FVIII. ObjectiveInvestigation of the association between the inhibitor development in hemophilia A and MBL2-SNPs. MethodsIn a case-control study the MBL2-SNPs in exon 1 at codons 52, 54 and 57 (C, B, D-Alleles respectively) were determined in 237 patients with severe hemophilia A with and without inhibitors to FVIII (119 vs 118). The association of MBL2-SNPs and the -308 G>A TNF-α-polymorphism with the presence of inhibitors were determined. ResultsIn the inhibitor group higher frequencies of the B allele (codon 54) (OR: 1.77, P < 0.05) were present. Summarising the MBL2 SNPs (alleles B, C and D) as 0, the 0/0 type occurred only in the inhibitor group (frequencies: 0.08 vs 0, P = 0.003). Based on the genetic background a functional immune response phenotype was determined. 11.8% of patients with inhibitors were of the low MBL/high TNF-α phenotype vs 0.03% of the non-inhibitor patients (OR: 3.71). ConclusionData suggest an association of MBL2-SNPs alone or combined with the 308-TNF-α polymorphism in the inhibitor development. Investigations of components of all three complement pathways are required to comprehend their individual and overall contribution to the inhibitor development in HA.

Genetics of Factor VIII Inhibitor Development in Hemophilia Patients: Novel Statistical Approaches in the PATH Study

Here we apply state-of-the-art statistical genetic approaches toward investigating the genetic architecture of factor VIII (FVIII) inhibitor (FEI) development in Hemophilia A (HA). A total of 442 North American HA patients (237 Whites and 205 Blacks; 88% severely affected) enrolled in the PATH Study were: 1) ImmunoChip genotyped at ~167,000 single nucleotide polymorphisms (SNPs) in genes previously implicated in autoimmune disease risk; 2) Evaluated by DNA sequencing and assays for the recurrent intron (I)1 and I22 inversions to identify their causative F8 mutations; and 3) Tested with the Bethesda assay to determine their FEI status. The ImmunoChip genotypes were used to construct a genetic relationship matrix (GRM), denoted by K, following our previously published method,1 and the F8 sequence data along with results from the I1 and I22 inversion assays were used to construct a shared F8-mutation matrix, denoted by F. We analyzed a dichotomous FEI variable under the statistical genetic threshold/liability model (a probit regression in the fixed effects) in conjunction with a variance components model for the FEI liability phenotypic covariance matrix, denoted by P, to model potentially important random effects. For the latter, we specifically assumed independent additive genetic, F8-mutation, and residual environmental random effects. By the independence assumption, the covariance matrix is then decomposable as a sum of the additive genetic (Va), F8-mutation (Vf), and residual environmental (Ve) variances respectively structured by K, F, and the identity matrix I. The variance component model is given as: P = K*Va + F*Vf + I*Ve. Heritability, denoted by h2, is defined as the ratio of Va to the total phenotypic variance (Vp): h2 = Va / Vp. We can further speak of the total heritability given as: h2t = h2r + h2f + h2snp, where the subscripts t, r, f, and snp respectively denote total, residual additive genetic, F8-mutation-specific, and SNP heritabilities. Using eigenstructure methods,2 we can compute power under a simpler model in which Va and Vf are combined as a single variance component. We computed power to detect genetic association as measured by SNP-specific heritability for a set of 403 SNPs in or near 14 candidate immune response genes previously implicated in FEI risk. To account for multiple hypothesis testing, power was computed at the Bonferroni-adjusted significance level of 0.05/403 = 1.2 × 10-4. Under the simplified model, we computed the statistical power to detect causal SNPs for our sample and study design for the sample FEI prevalences, denoted by Kp, for Whites (22.5%) and Blacks (45%), across a range of total heritabilities, h2t = 15%, 35%, and 55%, where the lattermost total heritability was observed for FEI liability in the current study (Figure 1). It should be noted that because the liability heritability is known to be biased upward, we applied the Dempster-Lerner correction to both the total and SNP-specific heritabilities.3 Close inspection of Figure 1 reveals that varying h2t from 15% to 35% to 55% results in slight decreases in power due to the decreasing ratio of the SNP-specific heritability to the total heritability. However, as seen in all three panels, the more important determinant of power is clearly the FEI prevalence in that the power curve for a Kp of 45% is associated with greater power than the power curve for a Kp of 22.5% across the range of total heritabilities examined. As seen in Figure 1, we have adequate power to detect SNP heritabilities as low as 5% and 6%, respectively, for a Kp of 45% and 22.5%. As noted above, we observed a FEI liability total heritability of 55% consisting of a 47% residual additive genetic heritability (p = 0.019) and 8% F8-mutation specific heritability (p = 0.005). This is the first study to use a GRM based on genotype data and a shared causal F8 mutation matrix to model additive genetic and F8-mutation specific effects.Almeida M, Peralta J, Farook V, …, Blangero J. Pedigree-based random effect tests to screen gene pathways. BMC Proc. 2014; 8(Suppl 1 Genetic Analysis Workshop): S100.Blangero J, Diego VP, Dyer T, …, Göring H. A kernel of truth: statistical advances in polygenic variance component models for complex human pedigrees. Adv Genetics. 2013; 81: 1-31.Glahn D, Williams J, McKay D, …, Blangero J. Discovering schizophrenia endophenotypes in randomly ascertained pedigrees. Biol Psychiatry. 2015; 77(1): 75-83. [Display omitted] DisclosuresChitlur:Baxter, Bayer, Biogen Idec, and Pfizer: Honoraria; Novo Nordisk Inc: Consultancy. Dinh:Haplomics Biotechnology Corporation: Employment, Equity Ownership. Howard:Haplomics Biotechnology Corporation: Equity Ownership, Other: Chief Scientific Officer, Patents & Royalties: Patent applications and provisional patent applications ; CSL Behring: Research Funding.