Anti-CD40 antibodies in antiphospholipid syndrome and systemic lupus erythematosus

Abstract

Anti-beta2glycoprotein I (anti-beta2GPI) antibodies constitute the main autoantibody specificity in the sera of patients with antiphospholipid syndrome (APS). There is evidence that anti-beta2GPI antibodies induce the precoagulant activity of the endothelium by cross-linking the beta2 glycoprotein I (beta2GPI) on the cell surface. Since beta2GPI lacks intracellular domains, homology with other molecules such as CD40 that could initiate signaling, was extensively searched. A 86% homology between the amino acid position 239-245 of the CD40 and 7-13 of the beta2glycoprotein was found. The CD40 peptide corresponding to amino acids 239-245 of the CD40 molecule was synthesized and coupled to a multiple antigenic peptide carrier. Antibodies to CD40 peptide were found in 61.5% APS patients (n = 39), in 72.7% of systemic lupus erythematosus (SLE) positive for anti-beta2GPI antibodies (n = 11) and 31.6% of SLE negative for anti-beta2GPI antibodies (n = 19), but not in rheumatoid arthritis patients (n = 28) or controls (n = 36). Antibodies to CD40 peptide were associated with arterial thrombosis and/or brain microinfarcts. Affinity purified anti-CD40 peptide antibodies as well as affinity purified anti-beta2GPI antibodies recognized both, the beta2GPI and the CD40 peptide. The specificity of this recognition was confirmed with homologous and heterologous inhibition experiments. Confocal microscopy experiments demonstrated this cross-recognition of CD40 and beta2GPI molecules, by the purified anti-CD40 peptide antibodies, at the protein level. Thus, antibodies reacting with the beta2GPI can react and potentially activate different cells which express CD40 molecules at their surface.

Autoantibodies specific to a peptide of β2-glycoprotein I cross-react with TLR4, inducing a proinflammatory phenotype in endothelial cells and monocytes

“…Because of the lack of CD40 expression in both wild-type HEK293 and HEK293T4 cell lines, we can completely exclude the cross-reactivity between anti-␤ 2 GPI peptide Abs and CD40. [39][40][41] Furthermore, the phosphorylation of IRAK1, an adaptor protein involved in TLR signaling pathways, confirmed the specific TLR4 activation induced by anti-␤ 2 GPI peptide Abs in endothelial cells and monocytes. 39,[41][42][43] A previous study demonstrated the cross-reactivity between a subset of anti-transglutaminase Abs, typical serologic markers in patients with active celiac disease, with an epitope of the extracellular region of human TLR4 (aa 435-446).…”

“…39 The investigators suggested that Abs binding this peptide could react and potentially activate different cells expressing CD40 molecules on their surface. Nevertheless, our confocal microscopy experiments clearly demonstrated the specific binding of anti-␤ 2 GPI peptide Abs to TLR4.…”

Patient-derived anti-β2GP1 antibodies recognize a peptide motif pattern and not a specific sequence of residues

“…Vlachoyiannopoulos et al . 15 identified a consensus motif between CD40 and p3 capable of interacting with aPLA. Although this sequence had one additional nonpolar amino acid between the polar and phenylalanine (ϕxFfζζϕϕϕ) amino acids, it was strongly related to the initial motif ( Figure 3A ).…”

“…21 Another research group highlighted a sequence homology between residues 239–245 of CD40 and residues 26–32 of β2GP1. 15 Residues 26–32 of β2GP1 corresponded to p3 and belonged to the ϕϕϕζζ part of the ϕϕϕζζϕFxϕ motif ( Figure 3 and Figure 5 ). Moreover, the hexapeptide TLRVYK, described by Blank et al .…”

β2 Glycoprotein I (β2GPI) binds platelet factor 4 (PF4): implications for the pathogenesis of antiphospholipid syndrome

“…33 Briefly, polystyrene microtiter plates were coated overnight with 50 L of recombinant ␤2GPI (10 g/mL). The plates were washed, blocked, and washed again as described before.…”