Autoimmune thyroid disease (AITD) is a common organ specific disorder characterized by immune reactivity against self-thyroid antigens [1]. In addition, these patients show abnormal levels and function of different lymphocyte subsets, including T regulatory (Treg) cells, Th17/Th22 lymphocytes and NK cells [2,3,4,5,6]. In this regard, and as it has been widely described, Graves’ disease (GD), is an autoimmune disorder that mainly affects the thyroid gland and that is characterized by an excessive synthesis of thyroid hormones. The pathogenesis of GD involves the breakdown of immune tolerance towards thyroid self-antigens, leading to the activation of B lymphocytes and the development of a humoral autoimmune response. The thyroid stimulating hormone receptor (TSHR) is the main target antigen of the immune system in GD, which induces the synthesis of autoantibodies against it, stimulating the secretion of thyroid hormones and leading to gland hyperplasia and hyperthyroidism [1]. In contrast, Hashimoto’s thyroiditis (HT) is mainly characterized by the induction of a cellular autoimmune response directed against different thyroid antigens and mainly mediated by Th1 and Th17 lymphocytes. This autoimmune phenomenon leads to a destructive inflammatory process in the gland, causing hypothyroidism. However, one additional important feature of HT patients is the presence of increased levels of autoantibodies directed against thyroperoxidase (anti-TPO Ab) and thyroglobulin (anti-TG Ab), indicating that a humoral autoimmune response and the synthesis of autoantibodies also participate in the pathogenesis of this condition [1, 7].

Follicular T helper cells (Tfh) are a subset of CD4+ T lymphocytes mainly located in the germinal centers of secondary lymphoid organs (SLO), which are formed mostly by B lymphocytes [8]. Tfh cells play a key role in providing help for naïve B cells undergoing their differentiation into plasmablasts or memory B cells [8, 9]. Thus, Tfh cells induce the maturation and proliferation of B cells, increasing antibody production. Accordingly, different studies strongly suggest that Tfh lymphocytes may have a relevant role in autoimmune diseases mediated by auto-antibodies, including AITD [10,11,12,13,14].

Several reports have contributed to define the phenotype of Tfh cells. In this regard, it has been informed the expression of the chemokine receptor CXCR5 by these cells, which directs their migration towards the germinal center [9, 15]. Furthermore, the expression of the programmed death protein 1 (PD-1, CD279) as well as the inducible T cell co-stimulator (ICOS, CD278) by these cells have been also reported [16]. In addition, Tfh cells are characterized by the production of interleukin 21 (IL-21), a cytokine that plays a pivotal role in B cell differentiation into plasma cells and the formation and maintenance of germinal centers into secondary lymphoid tissues [16, 17]. According to this, it is now feasible to accurately detect and quantify Tfh cells by using multiparametric flow cytometry.

An additional subset of CD4+ T cells closely related to Tfh lymphocytes, and also showing the ability to provide significant help to B cells, has been described in the peripheral blood and synovial fluid from patients with rheumatoid arthritis [18]. These Tph or peripheral helper T cells, in contrast to Tfh cells, do not express the chemokine receptor CXCR5; however, the CCR2, CCR5 and CX3CR1 receptors are detected on their cell surface [18, 19]. Furthermore, these cells, as their counterparts located into the germinal centers, synthesize large amounts of IL-21 [20]. In this regard, several phenotypes have been employed for the flow cytometry analysis of this lymphocyte subset under different conditions. Accordingly, Rao et al. defined the Tph cell phenotype as CD4+CXCR5-PD-1hiCD278(ICOS)+ plus the expression of CD38 and CD69 [18]. However, other groups have employed only three markers (CD4+CXCR5-PD-1hi) for its analysis, mainly in samples from patients with autoimmune diseases [11, 20, 21]. In addition, in a recent study, these cells were simply defined as CD4+CXCR5-PD-1+, without considering the level of expression of PD-1 [22]. Although several studies have assessed the levels of Tfh cells in samples from patients with AITD [10, 12], to our best knowledge Tph cells have not been hitherto analyzed in this condition.

In this study, we analyzed the levels of Tph, Tfh and plasmablast cells in the peripheral blood of patients with AITD and healthy controls. Furthermore, we evaluated the possible association between the levels of these cells and clinical parameters. We detected high levels of Tfh, Tph and plasmablast cells in AITD, which were associated with the titers of autoantibodies in patients with GD and HT. These data suggest that Tph lymphocytes are involved in the pathogenesis of thyroid autoimmunity.