Antiphospholipid syndrome (APS) refers to an autoimmune disease characterized by multiple arterial or venous thrombosis, pregnancy complications, and/or thrombocytopenia with positivity for antiphospholipid antibodies (aPL), including lupus anticoagulants (LA), anticardiolipin antibodies (aCL), and anti-β2 glycoprotein-I (aβ2GPI) (1–4). The estimated incidence and prevalence of APS ranged between 1 and 2 cases per 100,000 and 40 and 50 cases per 100,000 respectively (5). Patients may also develop kidney disease, digestive tract ischemia, pulmonary manifestations, and cardiac and neuropsychiatric symptoms (6, 7). The most common complication of obstetric APS (OAPS) is recurrent miscarriage in the first trimester, which has been attributed to inhibition of the proliferation of trophoblastic cells (8). APS is classified into primary antiphospholipid syndrome (PAPS) and secondary antiphospholipid syndrome (SAPS) based on the presence or absence of other autoimmune diseases, mainly systemic lupus erythematosus (SLE), Sjögren’s syndrome (SS) and rheumatoid arthritis (RA) (1, 6). The concomitant presence of other autoimmune diseases complicates disease management. In addition, PAPS has several features not seen in SAPS, including low serum C3 and C4 levels, but elevated C3a-desArg and C4a levels, suggesting that complement activation is involved in the pathogenesis of PAPS (9). Other immunological similarities and differences between PAPS and SAPS, including those involving peripheral blood lymphocyte subsets, have not been investigated.

Several factors are involved in the development of APS, among which the inflammatory response is crucial. In APS, aPL activates Toll-like receptors (TLR) and, in turn, the innate immune pathway (10). aPL is an autoantibody produced by B cells, and the activation of both B cells and B cell subsets contributes to the development of autoimmune diseases, including APS (11, 12). However, few studies have compared changes in peripheral blood CD19+B cells between APS patients and healthy controls.

An association between APS and disorders of both cell and humoral immunity has been reported, including abnormalities in the number and function of T lymphocyte subgroups (4). Although T helper 1 (Th1) and T helper 2 (Th2) cells previously attracted the most research interest, in recent years the focus has been on the imbalance between T helper 17 (Th17) and T regulatory (Treg) cell subsets (13–16), especially with respect to rheumatoid diseases and cancer. For APS patients, overactivated CD4+T cells activate B cells, which then produce high levels of autoantibodies (17, 18). In this complex process, Th17 cells play an indispensable role by secreting inflammatory cytokines such as interleukin-IL (IL-17), which stimulates antibody production (19, 20). Conversely, Treg cells, a subset of regulatory CD4+T cells, inhibit Th17 and B cells activation, and thus negatively regulate antibody production (21, 22). Research on Tregs has revolutionized our understanding of immune control mechanisms and immune-mediated diseases, thereby opening up many avenues for the development of a new generation of therapies based on the regulation of Treg cell expression and function. Imbalances in Th17 and Treg subsets in various autoimmune diseases have been demonstrated, including SLE (23), RA (24) and inflammatory bowel disease (IBD) (25, 26). However, Alvarez-Rodriguez L and colleagues found no difference in the number and function of Treg subset between PAPS patients and healthy controls, and no increase in the Th17/Treg ratio (27). However, studies on Th cell subsets in APS patients are scarce (28, 29).

Therefore, based on advancements in cell-detection technology and its clinical application for determining absolute cell numbers in lymphocyte subpopulations, we investigated differences between PAPS and SAPS patients and healthy controls with respect to peripheral blood lymphocytes, CD4+T cell subsets, and cytokines. Our aim was to elucidate the role of CD4+T cell subsets, especially Th17 and Treg cell subsets, and cytokines in the pathogenesis of APS.

This study showed significant decreases in NK cells in PAPS patients, and in T, B, and CD4+T cells in SAPS patients, compared to a healthy control group. Among CD4+T cell subsets, similar trends were demonstrated in PAPS and SAPS patients, including increased Th1 cells and decreased Th2 cells. In addition, a decrease in Treg cells accounted for the increase in the Th17/Treg ratio in all APS patients compared to the healthy controls, but Th17 and Treg cell subsets were higher in PAPS than SAPS patients. Among APS patients, the levels of many cytokines were higher than in healthy controls and were associated with the aCL titre. Correlation analysis suggested that, for PAPS patients, the titres of two autoantibodies, aCL and aβ2GPI, correlated positively with the Th17/Treg ratio and negatively with both D-dimer levels and Treg subsets. In the SAPS group, serum complement C3 and C4 levels were elevated in ANA-negative patients. Taken together, these findings implicate Th17 and Treg cell subsets in the pathogenesis of APS, and in the different inflammatory profiles of PAPS and SAPS patients. As such, our study sheds light on the complex mechanisms of APS, which may improve its assessment and management.

Previous studies examining CD19+B cell numbers in the peripheral blood of APS patients yielded contradictory results. Dal Ben et al. reported decreases in the number of B cells in both PAPS (31) and APS secondary to SLE (32). Our results were consistent with those studies, as we determined a reduction in the number of peripheral blood B cells in PAPS and in SAPS patients. However, a 2018 study by Alvarez-Rodriguez L et al. (33) found no difference in the number and percentage of B cells in PAPS patients, with or without venous thrombosis, compared to healthy controls. The discrepancies may be related to the study population, the use of baseline drugs, and the timing of the study. The latter study also showed that PAPS patients had a lower proportion of naïve B cells and higher number of non-switched memory B cells, in contrast to the results of Simonin et al. (11). The difference in distribution of CD19+B cells in the peripheral blood of PAPS and SPAS patients is the current focus of our team’s research.

The imbalances in lymphocyte and T cell subsets in APS patients, and their role in APS pathogenesis and disease surveillance, have attracted increasing research attention in recent years (34, 35). In the above-mentioned study of Alvarez-Rodriguez et al. (27), it was concluded that Th cells did not play a direct role in PAPS; however, only PAPS, SLE, and healthy controls were compared; the study did not include SAPS patients. Simonin L et al. (11) investigated 22 PAPS patients with venous thromboembolism (VTE) and found disturbances in T and B cell homeostasis; however, they did not assess the atatus of Th subsets. A strength of our study was its full analysis of lymphocyte, Th subsets and cytokines in both PAPS and SAPS patients, and the comparison with a healthy control group. We were thus able to show reductions in of T, B, NK, and CD+T cells in PAPS and SAPS patients compared to healthy controls. While this result might be attributed to the use of immunosuppressants by APS patients, the finding that Treg cells were reduced in the PAPS and SAPS groups, whereas the Th17/Treg ratio was elevated, instead points to direct involvement of an imbalance in Th17 and Treg cell subsets in the pathogenesis of APS. This was also the conclusion of Jakiela et al. (29), but the main focus of their work was the effect of aPL-IgG titres on Th cell subsets. Treg and Th17 cells have a common progenitor and their differentiation, development and expression are regulated by transforming growth factor-β-mediated signaling (16, 36) as well as IL-2 (37–39). Th17 cells, a subset of pro-inflammatory cells, produce IL-17A, IL-17F, and IL-22 and participate in signal induction and dissemination (40, 41), whereas Treg cells down-regulate the activation and proliferation of self-reactive T cells and maintain immune tolerance (42, 43). Treg cells express the α chain of the IL-2 receptor on their surface (44); in response to IL-2 binding, they secrete negative immune regulatory cytokines such as IL-6 and IL-10 (45). Our study showed higher levels of these cytokines in APS patients than healthy controls, and their association with the aCL titre. We therefore speculate that, in APS patients, there is an imbalance in the number of Th17 and Treg subsets, as well as an impairment of the function of these cells.

Few studies have examined the differences between PAPS and SAPS patients. A cohort study of 148 APS patients in Egypt found that SAPS patients were more prone to systemic manifestations, venous thrombosis, cutaneous vasculitis, thrombocytopenia and hemolytic anemia (46). The authors also suggested different disease presentations depending on ethnicity. Unfortunately, our study does not reach the same conclusion. Other studies have showed that patients with SAPS are more likely to develop more severe diffuse alveolar hemorrhage (DAH) and non-infectious endocarditis, known as Libman-Sacks (LS) endocarditis, especially in APS secondary to SLE (47, 48). Only one other study reported differences in immune function between PAPS and SAPS patients, but the lower serum C3 and C4 levels in PAPS patients (9) were inconsistent with our results. Further studies are needed to determine the differences in immune function between PAPS and SAPS. Our analysis of peripheral blood lymphocyte and CD4+T cell subsets in PAPS and SAPS patients showed that the levels of T, B, NK, Th2, Th17 and Treg cells in PAPS were higher than those in SAPS, which may be related to the fact that most patients with SAPS received immunosuppressive therapy including DMARDs. The cytokine analysis indicated that the IL-10 level in PAPS patients was lower than that in SAPS patients, which may indicate that the immune regulation ability of PAPS patients is slightly weaker.

Recurrent thrombosis is an important feature of APS and complicates its management. APS patients are prone to a vicious cycle of thrombotic events (of which 50% are lower limb deep vein thromboses, with a high risk of pulmonary embolism) (49) that result in thrombocytopenia, which in turn increases the risk of thrombosis (50). Most APS patients are therefore prescribed anticoagulants, such as vitamin K antagonists and low molecular weight heparin. However, thrombus in APS is a complex process, involving oxidation of the domain 1 Arg 39 Arg 43 epitope and cross-linking of several surface receptors (51), such that anticoagulation therapy should be accompanied by immunological therapy. A 2021 study (52) suggested that Treg populations in the thrombus form stromal acid and cysteine-rich proteins (SPARC) to promote clot breakdown, by recruiting monocytes and promoting fibrinolytic activity to disrupt the fibrin network. According to this scenario, the balance of Treg cells plays an important role in thrombolysis and the prevention of recurrence. Our study also identified a negative correlation between D-dimer levels and Treg cell subsets in PAPS patients. As D-dimer is a sensitive indicator of hypercoagulability, this relationship would seem to implicate immune cells, especially Treg cell subsets, in thrombogenesis characteristic of APS, although the specific mechanism remains to be explored. In our study, due to the small number of blood samples in the PAPS group, we used logistics regression analysis to examine the correlation of clinical indicators with the presence of thrombosis in PAPS, but the result was not significant (data not shown).

ANA positive is mostly associated with adverse pregnancy outcomes (APO) in OAPS patients, as demonstrated in animal models (53) and clinical studies (54). In addition, high levels of C4a and C3b deposition were found in the placenta of OAPS women. Complement activation may be a key process in aPL-related thrombosis and fetal loss (55), and hypocomplement was shown to be an independent predictor of lower neonatal birth weight and premature delivery (56). A real-world prospective study concluded that high serum C3 levels in early pregnancy protect against obstetric complications in OAPS, whereas ANA titres > 1:160 are an independent risk factor (57). Similarly, our study identified higher serum C3 and C4 levels in ANA-negative SAPS patients. These results suggest that ANA also plays a role in the aPL-mediated activation of complement, leading to APO and thrombosis.

Although we are aware of the limitation regarding the possible effect of different therapies in SAPS, which were not considered as confounding factors. Patients with SAPS were treated with steroids, antimalarials, antiplatelet, and/or anticoagulant therapies. The effects of glucocorticoids on Th subsets, especially Th1 and Th17, have been studied and demonstrated (58, 59). And the immunosuppressive effects of hydroxychlorochin, a classic antimalarial drug, is well-known. Indeed, we did not find any effect of anticoagulants on lymphocytes. There is only one study (60) showing that prasugrel had an inhibitory effect on effector Th cells in vitro, which were induced to develop an inflammatory phenotype by the addition of platelets. Our patients did not receive prasugrel. These further confirms our findings that Th17/Treg responses are stronger in SPAS compared to healthy individuals, despite the inhibitory effect of treatment.

Despite its novel findings, our study also had several limitations. Frist, the sample size was small, due to the limited number of patients with a definitive diagnosis of APS, especially PAPS. Second, as it was a retrospective study, peripheral blood samples did not suffice for in vitro experiments. Third, direct evidence of a role of an imbalance between Th17 and Treg subsets in the pathogenesis of APS awaits confirmation by appropriate animal models. Fourth, the baseline medications in our APS patients differed, and neither ESR nor CRP was an accurate indicator of disease activity. Finally, the timing of thrombosis was not reported. Some of these issues may have had an important impact on peripheral blood lymphocyte subsets and cytokine levels.

Although it could not establish the precise mechanism by which Th17 and Treg cells contribute to APS, our study suggested that the increased Th17/Treg ratio resulting from decreased Treg cells is related to the occurrence of APS. If confirmed in further studies, this would support the use of immunomodulatory rather than immunosuppressive therapy in APS. Further studies are needed to examine the participation of Treg cells and Treg-derived cytokines in APS. In addition, we found immunological differences between PAPS and SAPS patients, but this may be related to the majority of SAPS patients receiving immunosuppressive therapy. More differences in immunity between the two are worth further exploration.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Ethics Committee of the Second Hospital of Shanxi Medical University. The patients/participants provided their written informed consent to participate in this study.

HY performed the data analyses and wrote the manuscript. BL participated in the collection of samples and clinical data. RS participated in the performance of the research and statistical analysis. CG and XL participated in the study design and revising of the manuscript. CW provided intellectual input and supervision throughout the study and made a substantial contribution to manuscript drafting. All authors contributed to the article and approved the submitted version.

This work was supported by National Natural Science Foundation of China (No. 81971543, No. 81471618); Key Research and Development (R&D) Projects of Shanxi Province (201803D31119).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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