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

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.