The function of natural autoantibodies (nAAbs) in maintaining immunological tolerance has been comprehensively explained; however, their function in pregnant patients dealing with autoimmune diseases has not been thoroughly investigated. As Hashimoto’s thyroiditis (HT) is the predominant organ-specific autoimmune condition of women of childbearing age, this study’s objective was to evaluate IgM and IgG nAAbs targeting mitochondrial citrate synthase (CS) and heat shock proteins (Hsp60 and Hsp70) in women diagnosed with HT who were pregnant (HTP). Serum samples collected from HTP and healthy pregnant (HP) women in the first and third trimesters were tested using in-house-developed enzyme-linked immunosorbent assays (ELISAs). Our findings indicate the stability of nAAbs against CS and Hsps throughout the pregnancies of both healthy women and those with HT. However, during both trimesters, HTP patients displayed elevated levels of IgM isotype nAAbs against Hsp60 and Hsp70 compared to HP women, suggesting a regulatory role of IgM nAAbs during the pregnancies of patients with HT. Nonetheless, levels of IgG isotype nAAbs against Hsps were lower solely in the third trimester among HTP patients, resulting in a higher IgM/IgG ratio, which indicates their importance in alterations of the nAAb network during pregnancy in patients with HT.

Calcium (Ca²⁺) flux acts as a central signaling pathway in B cells, and its alterations are associated with autoimmune dysregulation and B-cell malignancies. We standardized a flow-cytometry-based method using various stimuli to investigate the Ca²⁺ flux characteristics of circulating human B lymphocytes from healthy individuals. We found that different activating agents trigger distinct Ca²⁺ flux responses and that B-cell subsets show specific developmental-stage dependent Ca²⁺ flux response patterns. Naive B cells responded with a more substantial Ca²⁺ flux to B cell receptor (BCR) stimulation than memory B cells. Non-switched memory cells responded to anti-IgD stimulation with a naive-like Ca²⁺ flux pattern, whereas their anti-IgM response was memory-like. Peripheral antibody-secreting cells retained their IgG responsivity but showed reduced Ca²⁺ responses upon activation, indicating their loss of dependence on Ca²⁺ signaling. Ca²⁺ flux is a relevant functional test for B cells, and its alterations could provide insight into pathological B-cell activation development.