Dangerous B-cell clones

In this issue of Blood, Wang et al¹ demonstrate that the severe adverse effect vaccine-induced immune thrombotic thrombocytopenia (VITT) is caused by surprisingly structurally similar antibodies. Their findings indicate that VITT is caused by a single or only a few B-cell clones that undergo rapid clonal expansion, resulting in the production of just a few closely related antibodies. Moreover, the gene encoding the variable region of the immunoglobulin G (IgG) light chain seems to show the same polymorphism (IGLV3-21∗02) in all investigated patients. Most hematologists are familiar with the role of gene polymorphisms within the human leukocyte antigen (HLA) system, predisposing to some forms of autoimmunity.² The HLA system is important for T-cell receptor recognition of peptide–HLA complexes. In contrast, the polymorphisms in the variable regions of IgG are relevant for the structure of the antigen-binding site of IgG and the B-cell receptor.

Vaccination against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an important countermeasure to fight the ongoing COVID-19 pandemic. Two adenoviral vector-based COVID-19 vaccines (ChAdOx1-S [AstraZeneca; Vaxzevria] and Ad26.COV2.S [Janssen]) are known to induce VITT, a disorder caused by high-titer IgG antibodies directed against the platelet chemokine PF4 (platelet factor 4). These antibodies activate platelets via platelet FcγIIa receptors (FcγRIIa). VITT is characterized by thrombocytopenia, strongly elevated d-dimer concentrations, and often thrombosis at unusual sites, such as cerebral venous sinus thrombosis or splanchnic vein thrombosis. VITT occurs in otherwise healthy individuals 5 to 20 days after vaccination with an adenoviral vector-based vaccine. Production of high-titer IgG antibodies within 14 days indicates that VITT is a secondary immune response.³ 

PF4 opsonizes negatively-charged surfaces of microbial pathogens, facilitating the binding of anti-PF4 antibodies. This is likely an evolutionary ancient immune defense mechanism, as anti-PF4–producing B cells can be found in nearly all individuals.⁴ However, a strong anti-PF4 antibody response, when misdirected, underlies the thromboembolic disorder heparin-induced thrombocytopenia (HIT) and its most severe presentation, autoimmune HIT, in which the antibodies activate platelets even in the absence of heparin. VITT closely mimics autoimmune HIT both clinically and serologically. A major risk factor for forming anti-PF4 antibodies and for developing HIT is inflammation and/or tissue trauma. These stress factors provide an immunologic “danger signal” (trigger for B-cell activation) that increases the likelihood and intensity of forming an immune response against PF4. These danger signals also increase the likelihood of class-switching preexisting IgM responses to more pathogenic IgG isotypes, which can engage the full spectrum of activating Fcγ receptors (FcγRs). FcγRs bind to the IgG constant domain and are broadly expressed on platelets (but only FcγRIIa) and innate immune effector cells, such as neutrophils and macrophages.⁵ Furthermore, these danger signals may alter the IgG subclass and glycosylation pattern of anti-PF4 antibodies, which may result in additional enhancement of FcγR binding.

The exact mechanism leading to the production of high-titer platelet-activating anti-PF4 antibodies in both HIT and VITT is still uncertain. A better understanding of the underlying mechanisms is of great importance for future vaccination programs. Although high-income countries will likely avoid adenovirus vector-based vaccines after the experience of VITT in 2021, most parts of the world can afford only these vaccines.

The findings of Wang and colleagues may lead to a better understanding of the underlying pathogenesis of the misdirected anti-PF4 immune response seen in VITT. The authors build on previous findings indicating that the immune response to PF4 in patients with VITT is oligoclonal⁶ (ie, caused by very few B cells that rapidly expand after COVID-19 vaccination).

To further characterize the antibodies, Wang and colleagues first affinity-purified anti-PF4 antibodies from sera from patients with VITT and then assessed the amino acid sequences of the variable regions of the IgG heavy chain and the corresponding light chain, which form the antigen recognition region of an antibody (ie, the fragment antibody binding, or F[ab], region). Surprisingly, the anti-PF4 IgG antibodies in all 5 investigated sera showed striking similarities, especially with respect to the antibody light chain. In all 5 patients investigated, the antibody light chain variable region seemed to be derived from the same light chain variable gene segment, IGLV3-21∗02 (see figure). In addition, overlapping amino acid motifs and an identical length of the third complementarity determining region of the heavy and light chain (HCDR3 and LCDR3), which play a major role in antigen recognition, support the notion that a highly specific, antigen-driven oligoclonal B-cell response led to the formation of these highly pathogenic anti-PF4 antibodies in all 5 patients. Of note, the usage of V-gene segments in the IgG heavy chains was more diverse; however, in line with the light chain, a similar CDR3 length and overlapping amino acid motifs were also identified. These observations may eventually help to identify individuals at risk for developing a misdirected anti-PF4 response via the unique and highly specific usage of this λ light chain V-gene segment and the conserved CDR3 length and amino acid composition.

The binding site of VITT antibodies on PF4 has been characterized,⁷ and a monoclonal antibody that specifically blocks the binding of VITT antibodies⁸ to PF4 is now available. This finding of a characteristic PF4 antigen target site, representing a VITT epitope, has also been observed in a patient with recurrent severe thrombotic complications⁹ independent of COVID-19 vaccination; this patient had a monoclonal gammopathy, and the paraprotein bound PF4 at the VITT epitope site. Indeed, paraproteins have previously been implicated as a potential cause of autoimmune HIT. Monoclonal immunoglobulins, typically of the IgM class, have also been described in a variety of other disorders, including acquired von Willebrand disease. However, a typical feature of anti-PF4 antibodies in VITT and HIT is the transience of detectable antibodies, which is obviously not the case in most autoimmune disorders or in monoclonal paraproteinemia-associated disorders.

The observations of Wang and colleagues are intriguing but are based on only 5 patients. The findings should prompt further investigation of the potential restriction of the genes encoding the hypervariable region of IgG in additional patients with VITT and patients with other forms of autoimmunity. A more in-depth characterization of the IgG subclass and glycosylation patterns may provide further insights into immune pathways driving PF4-specific immune responses, allowing for an even better correlation with antibody pathogenicity.

Conflict-of-interest disclosure: A.G. reports grants and nonfinancial support from Aspen, Boehringer, Ingelheim, MSD, Bristol Myers Squibb (BMS), Paringenix, Bayer Healthcare, Gore Inc., Rovi, Sagent, and Biomarin/Prosensa; personal fees from Aspen, Boehringer Ingelheim, MSD, Macopharma, BMS, Chromatec, and Instrumentation Laboratory; and nonfinancial support from Boehringer Ingelheim, Portola, Ergomed, and GTH e.V. outside the submitted work. F.N. declares no competing financial interests.