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. 2015 Jan;21(1):133-9.
doi: 10.1111/hae.12522. Epub 2014 Nov 23.

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

B K Sack  1 , X Wang, A Sherman, G L Rogers, D M Markusic
Affiliations
Free PMC article

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

B K Sack et al. Haemophilia. 2015 Jan.
Free PMC article

Abstract

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 TH 1 (IFN-γ) and TH 2 (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 TH 2 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 TH 1 and TH 2 response (mainly TH 2). 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.

Keywords: BALBc; C3H/HeJ; anaphylaxis; factor IX; haemophilia B murine model; immune response.

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Figures

Fig. 1
Fig. 1
Genetic background impacts hypersensitivity and antibody responses against recombinant hFIX protein. (a) Kaplan–Meier survival plot for BALB/c F9−/Y (n = 9), C3H/HeJ/OuJ F9−/Y (n = 9), and C3H/HeJ F9−/Y (n = 16) mice. C3H/HeJ F9−/Y mice received antihistamine and PAF antagonist on the fourth, fifth and sixth exposures to hFIX protein. (b) Anti-hFIX IgG1 titres (ng mL−1) 1 week following last hFIX protein injection. (c) Anti-hFIX IgG2a titres (ng mL−1) 1 week following last hFIX protein injection. (d) Inhibitor (Bethesda) titres 1 week following last hFIX protein injection. Statistical analysis was performed using Student's t-test with P < 0.05 considered significant. Calculated P values are included on plots.
Fig. 2
Fig. 2
Comparison of B cell response against recombinant hFIX protein. Summary of B-cell ELISpot spot forming units (SFU) per 1 × 106 cells of anti-hFIX antibody secreting cells from spleen (a) and bone marrow cells (b) isolated from immunized mice. Statistical analysis was performed using Student's t-test with P < 0.05 considered significant. Calculated P values are included on plots.
Fig. 3
Fig. 3
Comparison of IgG1 (ng mL−1) and IgG2a antibody responses against a T-cell-dependent antibody-inducing antigen, keyhole limpet haemocyanin (KLH). BALB/c and C3H/HeJ mice (n = 5 per group) were i.v.-injected with 2 μg KLH and bled two and 4 weeks later to measure circulating anti-KLH (a) IgG1 and (b) IgG2a. Statistical analysis was performed using Student's t-test with P < 0.05 considered significant. Calculated P values are included on plots.
Fig. 4
Fig. 4
Comparison of T-cell responses against hFIX protein. IL-4 (a) and IFN-γ (b) ELISpot assays using splenocytes isolated from immunized mice of indicated strain. All samples were run in duplicate with unstimulated, 10 μg mL−1 hFIX, and SEB or PMA/Ionomycin stimulated. Data are presented as (SFUhFIX − SFUunstimulated per 1 × 106 cells). (c) Cytokine responses specific to hF.IX for BALB/c (white) and C3H/HeJ (grey) F9−/Y mice. Splenocytes (1 × 105 per well) were cultured in vitro without or with hFIX protein (10 mg mL−1) and harvested 48 h later for mRNA extraction and transcriptional analysis via qPCR array for indicated genes. Data are presented as fold change compared to unstimulated cells.
Fig. 5
Fig. 5
Verifying reconstitution of TLR4 signalling in C3H/HeJ/OuJ F9−/Y mice. 1 × 106 splenocytes from C3H/HeJ (n = 2), C3H/OuJ (n = 2), and C3H/HeJ/OuJ F9−/Y (n = 4) were cultured in vitro in triplicate for 48 h either unstimulated or stimulated with 10 μg mL−1 LPS-SM, a specific TLR4 activator. Cell culture media was collected, pooled and analysed for IL-6 (ng mL−1) secretion into media using a murine IL-6 ELISA kit.
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