Immune responses to human factor IX in haemophilia B mice of different genetic backgrounds are distinct and modified by TLR4

Our laboratory develops protocols to prevent or reverse ongoing anti-hFIX IgG inhibitors in haemophilia B mice with a F9 gene deletion on BALB/c and C3H/HeJ backgrounds. C3H/HeJ F9^-/Y mice develop high titre anti-hFIX IgG1 inhibitors and anaphylaxis, whereas most BALB/c F9^-/Y mice have mild anti-hFIX IgG1 inhibitors and no anaphylaxis. Our aim was to determine if hFIX-specific B- and T-cell responses in BALB/c and C3H/HeJ F9^-/Y mice trigger the difference in anti-hFIX immune responses. BALB/c and C3H/HeJ F9^-/Y mice were challenged weekly with recombinant hFIX protein. Humoral immune responses were determined by IgG1 and IgG2a anti-hFIX ELISA, Bethesda assay for inhibitors and B-cell ELISpot on bone marrow and spleen cells. T-cell studies measured the T_H1 (IFN-γ) and T_H2 (IL-4) cytokine responses in splenocytes at the mRNA and protein level in response to hFIX protein. Antibody responses were also measured in C3H/HeJ/OuJ F9^-/Y mice with restored toll-like receptor 4 (TLR4) function. BALB/c F9^-/Y mice have a T_H2 skewed response and a reduction in anti-hFIX secreting plasma cells in the bone marrow. Independent antigen challenge revealed both strains generated equivalent IgG1 antibody titres to an intravenously delivered antigen. C3H/HeJ F9^-/Y mice have a mixed T_H1 and T_H2 response (mainly T_H2). Importantly, TLR4 signalling has a modulatory role in the C3H background on the levels of anti-hFIX IgG1 and incidence of anaphylaxis. The background strain strongly impacts the immune response to hFIX, which can be significantly impacted by mutations in innate immune sensors.