Mechanism of Thrombocytopenia Induced by Anti-CD40 Ligand Immune Complexes and the Prevalence of CD40 Ligand Autoantibodies in Patients with Thrombotic Autoimmune Disorders

Anti-CD40 ligand (anti-CD40L) immunotherapy in patients with systemic lupus erythematosus (SLE, a chronic inflammatory autoimmune disease) resulted in unexpected thromboembolic fatalities. In our laboratory, previous in vitro mechanistic (flow cytometry, aggregation, and dense granule release) studies have shown that monoclonal anti-CD40L immune complexes potently activate platelets via the IgG receptor (FcγRIIa). The data suggested this activity was also dependent on the CD40L receptor (CD40), which is constitutively expressed on resting and activated platelets. This raised the possibility that autoantibodies against CD40L maybe present in patients with thrombotic autoimmune diseases such as SLE and anti-phospholipid syndrome (APS) and possibly contribute to the pathogenesis of thrombosis in such patients. We hypothesized that

1. monoclonal anti-CD40L immune complexes (anti-CD40L IC) should exhibit prothrombotic effects in animals via IC-induced platelet activation, and

2. CD40 ligand autoantibodies may be prevalent in patients with thrombotic auto-immune disorders.

Mouse platelets, however, do not carry FcγRIIa. Therefore, to study anti-CD40L IC-induced platelet activation in vivo, we used mice transgenic for human FcγRIIa (“hFcR” mice). Immune complexes consisting of the anti-CD40L monoclonal antibody, M90, plus recombinant soluble CD40L (M90+sCD40L), or control reagents were injected intravenously (tail vein) into wild type (WT) or hFcR mice. Platelets were counted from 10–60 minutes thereafter. Additionally, plasma samples from patients with SLE (n=54), APS, (n=8), idiopathic thrombosis (n=34), and control subjects (n=86) were tested for the presence of IgG-type anti-CD40L autoantibodies using a highly optimized in-house ELISA. The injection of M90+CD40L IC (100–500 nM) produced symptoms consistent with thrombotic shock and induced severe thrombocytopenia (10–30% of basal platelet count) in hFcR (n=10–20) but not WT (n=5) mice—indicating that IC-induced thrombocytopenia was mediated via platelet FcγRIIa, as was found in vitro. Platelet priming by subaggregatory amounts of ADP greatly increased the sensitivity of hFcR mice to anti-CD40L IC (≥ eight-fold—as low as 12.5 nM). Furthermore, sequential injections of sCD40L followed by M90 in hFcR mice caused similar effects, indicating that ICs can also form while circulating. Injections of M90 or sCD40L alone were inactive in all animals. The prevalence of CD40L autoantibodies was notably higher in patients with SLE or APS compared to control subjects [13/54 (24%) or 3/12 (25%) vs. 5/86 (6%), P=0.002 and P=0.09 respectively]. Although CD40L autoantibodies were also more prevalent in patients with SLE and APS than in those with idiopathic thrombosis [2/34 (6%)], this difference was not statistically significant (P=0.058 and 0.2 respectively). Our findings demonstrate that the platelet activation caused by of anti-CD40L IC can be reproduced in mice, but only in those transgenic for the human IgG receptor (Fcγ RIIa). These in vivo findings may shed light on the thromboembolic complications associated with CD40L immunotherapy. Furthermore, our hFcR mouse model is a promising approach for assessing the hemostatic safety of CD40L—and possibly other—therapeutic antibodies. Our results also show that autoantibodies to CD40L occur at relatively high frequency in patients with SLE and APS. While a causal relationship between such antibodies and thrombotic risk remains unidentified, our in vivo studies suggest further investigation is warranted.