PF4/H Complexes Induce Germinal Centers in Vivo

Abstract 269 Mice injected with ultra-large complexes (ULCs) of platelet factor-4 (PF4) and heparin (PF4/H) exhibit a strong immune response which mimics that seen in patients developing Heparin induced thrombocytopenia (HIT)(Suvarna, Blood 2005). Previous studies with this murine immunization model have shown that murine (m) PF4/H ULCs potently activate dendritic cells (DCs) (Chudasama, Blood 2010) and initiate a T-cell dependent immune response (Suvarna, Blood 2005). To examine cellular/anatomic basis of PF4/H antibody formation, we examined the role of the spleen and germinal center formation in mice injected with mPF4/H complexes. To evaluate the role of the spleen, we immunized splenectomized mice (SPL) (n=15) and wild type (WT) mice (n=15) retro-orbitally with 100 mg/mL mPF4 and 5 U/mL Hep for 5 days. Antibodies to mPF4/H complexes were detected by an ELISA developed in our laboratory (Suvarna, Blood 2005; Suvarana, Blood 2007). As shown in [Figure 1][1], SPL animals showed a marked reduction in Ab formation compared to WT animals (mean A450nm ± SD; SPL 0.206 ± 0.253 vs. WT 0.76 ± 0.788, p<0.003). We next asked if antibody responses to PF4/H were associated with germinal center (GC) formation. The GC is a hallmark of T-cell dependent (TD) immune responses and is considered vital for development of high-affinity and isotype-switched antibodies. For these studies, mice were injected with a high dose of PF4/H (400 ug/mL PF4: 10 U/mL heparin) (n=2), standard protocol dose of PF4/H (100 ug/mL: 5 U/mL) and or buffer (Hank's Balanced Salt Solution, n=3) daily for 5 days via retro-orbital injection. As a positive control for GC formation, we injected two mice with a conventional antigen, nitrophenyl conjugated chicken gamma-globulin (NP-CGG). Mice expressing high antibody titers on Day 16 (mean A450nm± SD; high dose- 1.112 ± 0.5767; standard dose- 1.186 ± 0.7853) were sacrificed, spleens harvested and frozen in an embedding medium. Splenic sections (5m, longitudinal) were obtained as frozen sections and stained with immunohistochemical markers for GCs (GL-7 FITC; 1:400, counter-stain- FITC Alexa Fluor 488; 1:200), T-cells (TCR-β PE; 1:200) and B-cell follicles (B220 Biotin; 1:400, counter-stain- Streptavidin Alexa Fluor 350; 1:200). Images were obtained using Carl Zeiss AxioVert 200M microscope and analyzed with AxioVision Rel 4.6 software. Fluorescent images were processed with Adobe photoshop CS5.1. As shown in [Figure 2][2], we noted that mice injected with NP-CGG, had ∼ 22 GC's, mice injected with buffer had 2 GC's and mice injected with high and standard dose of PF4/H, had ∼20–22 and 15–17 GC's respectively. These studies show that mPF4/H complexes elicit GC formation in association with secretion of mPF4/H Abs and that GC formation is comparable to that seen with conventional antigen. ![Figure][3] ![Figure][3] Disclosures: No relevant conflicts of interest to declare. [1]: #F1 [2]: #F2 [3]: pending:yes