Novel Correlation between B Cell Survival Cytokines and Inhibitors in Hemophilia A

The most significant complication of factor VIII (FVIII) replacement in hemophilia A (HA) is the development of neutralizing antibodies (inhibitors) in 20-30% of severe HA patients. While several studies have investigated links between polymorphisms in cytokine regulatory elements and inhibitors, very few have measured patients' serum cytokine levels to understand their contribution. As an antibody-mediated process, inhibitor development must rely on the maturation and proliferation of FVIII-specific B cells, but details of this response remain poorly understood. One of the principal mechanisms of B cell tolerance is regulated at the transitional (TR) B cell stage when newly emerging B cells from the bone marrow remain susceptible to clonal deletion. TR B cell survival is highly dependent upon the abundance of the cytokine BAFF whereas BCMA, another member of the same family, is key to plasma cell survival (Parsons et al, Transplant Rev 2010). Inherited elevated levels of BAFF increase the risk of multiple sclerosis and lupus, where levels of 2 ng/mL (~2x normal) are associated with poor outcome (Steri et al, NEJM 2017, Cheema et al, Arthritis Rheum 2001). BAFF is expressed by dendritic cells, monocytes and macrophages and stimulates B cell proliferation, differentiation and survival (Litinskiy et al, Nature Immunology 2002). High levels of BAFF have been observed during immune reconstitution after rituximab therapy in allograft transplant recipients and may potentiate alloreactive B-cell immunity to promote rejection (Rickert et al, Transplant Rev 2010). We hypothesized that BAFF and/or BCMA may contribute to 1) the development of inhibitors in HA and 2) persistence of inhibitors in refractory patients. Here we analyze correlation of BAFF, BCMA, and 10 other cytokines with inhibitors in human HA samples and BAFF levels in murine inhibitor models.

Samples from HA patients were collected longitudinally on an IRB approved protocol. Plasma BAFF and BCMA levels were measured by multiplex ELISA (R&D Systems). Nijmegen-modified Bethesda assays for inhibitor titer and anti-FVIII IgG subclasses were measured as described previously (Hofbauer et al, Blood 2015). A cohort of samples was analyzed for additional cytokines (IL2, IL4, IL5, IL6, IL10, IL12p70, IL17A, IL21, IFNγ, and TNFα) via a customized Luminex based multiplex microbead array (Millipore). C57Bl/6 HA mice were immunized with human FVIII 1 mcg weekly for 4 weeks and Bethesda assays tested at 8 weeks. Plasma from mice with high titer inhibitors (BU > 5, n=8) and non-inhibitor controls (n=2) was analyzed for murine BAFF via ELISA (R&D Systems). Data was analyzed with Prism using ANOVA, χ² for categorical or t-test for continuous variables and are reported as mean ± SEM.

Fifty-seven HA patients, 24 with and 33 without inhibitors, were enrolled with no difference in age, disease severity, or race (p = 0.13, 0.08 and 0.39, respectively by χ²); 2 were treated with rituximab. Patients with inhibitors had a higher frequency of large deletions or inversions. Excluding rituximab patients, BAFF levels were higher in patients undergoing immune tolerance induction (ITI) (1.53 ± 0.16) versus non-inhibitor patients (1.11 ± 0.09), and normalized in those who tolerized (0.98 ± 0.14 ng/mL, p = 0.024). BCMA levels were higher in patients with inhibitors (22.56 ± 1.95 vs 17.59 ± 0.59 ng/mL, p = 0.005) and correlated with Bethesda titer (r = 0.202, p = 0.024). Notably, none of the additional cytokines tested were associated with inhibitors. Consistent with allograft rejection models, BAFF levels rose 4-5 fold from baseline post-rituximab and did not normalize until anti-FVIII IgG was undetectable in rituximab-exposed patients. Consistent with human data, HA mice with inhibitors had higher BAFF levels than non-inhibitor mice (6.96 ± 0.37 vs 5.76 ± 0.08 ng/mL, p = 0.015, Welch's t-test).

Here we uncover a novel underlying mechanism for modulation of inhibitor formation. BAFF may allow for the persistence of anti-FVIII antibodies during ITI and after rituximab therapy in HA inhibitor patients. Soluble BCMA levels correlate with inhibitor titer, suggesting it plays a role in anti-FVIII plasma cell survival. BAFF levels also increase in HA mice with inhibitor formation. Preclinical testing of antibody or cellular therapies against these cytokines may facilitate translational studies with FDA approved anti-BAFF therapies and enhance tolerance induction in HA.