Factor IX Delivery to the Skin Primes Inhibitor Formation and Sensitizes Hemophilia B Mice to Systemic Factor IX Administration

Inhibitor formation is the most serious complication of factor (F)IX replacement therapy for hemophilia B, exacerbated by anaphylactoid reactions occurring in up to 50% of inhibitor patients. Low success rates and a high burden of immune tolerance induction (ITI) therapy necessitate the search for novel immune tolerance therapies. Skin-associated lymphoid tissues (SALT) have been successfully targeted in allergen-specific immunotherapies. In this study, we aimed to develop a prophylactic immune tolerance protocol based on intradermal (ID) administration of rFIX. In a dose-finding experiment, hemophilia B mice on C3H/HeJ genetic background (C3H/HeJ-F9tm1Dws) received twice weekly ID injections of 0.01, 0.1 or 1 IU rFIX only for four weeks, followed by one intraperitoneal (IP) and four weekly intravenous (IV) injections of 1 IU FIX co-injected with anti-allergic agents (triprolidine and ABT-491). Control animals received the IP/IV doses only. One week after the last injection, plasma and/or lymph nodes (LNs) were collected for Bethesda assay, ELISA and flow cytometry analyses. Unexpectedly, all animals developed significantly (8.9-17.6-fold, p Inhibitor formation following ID treatment seemed to be driven by T follicular helper (PD1 +CXCR5 +CD4 +) and Germinal Center B cell (GL7 +CD95 +CD19 +) responses in inguinal LNs, the frequencies of which were 1.75-fold and 4-fold (p Notably, none of the ID treated mice died of anaphylaxis despite receiving eight ID injections of FIX without anti-allergic agents. IV administration of FIX alone in C3H/HeJ-F9tm1Dws hemophilia B mice results in fatal IgE-dependent anaphylaxis beginning after fourth injection with ~20% mortality and rising with subsequent injections in the surviving mice, which necessitates the use of anti-allergic agents. Interestingly, the ID treated mice had elevated levels of anti-FIX IgE antibodies. Animals that received ID injections only or followed by IP/IV treatment had 1.5- and 3.3-fold higher (p In conclusion, ID administration of FIX primes or produces strong inhibitor responses in a wide range of doses and sensitizes hemophilia B mice to systemic delivery of FIX, suggesting that the skin-associated lymphoid tissue may not be amenable to FIX tolerance induction. Disclosures No relevant conflicts of interest to declare.