The HLA complex (18) refers to a group of genes of 700 kilobases located on chromosome 6p21.3 encoding the proteins presenting the antigen, on the cell surface.

Certains HLA show a greater propensity to develop autoimmune disorders (e.g. DR/DQ allelic combination); others exhibit a protective mechanism for the self. In the case of HT, the polymorphism of the DQ locus is frequently involved. Each individual with HLA-DQ isoform can present different antigens to T cells, which can be mature and stimulate the production of antibodies by B cells. However, when the tolerance breaks up, the autoimmune process can start. In fact, there is strong evidence on how the DQ alleles are the most important susceptibility markers for the development of autoimmune diseases through their ability to modify the configuration of the antigenic binding site. HLA-A * 02: 07 and HLA-DRB4 have been associated with susceptibility to Hashimoto’s thyroiditis, while HLA-A * 33: 03-C * 14: 03-B * 44: 03-DRB1 * 13: 02 -DQB1 * 06: 04-DPB1 * 04: 01 were identified as protective genes. Specifically, APS2 is associated with HLA-DQB1 * 0201 and DQB1 * 0302 or with haplotype HLA-DRB1 * 03-DQA1 * 0501-DQB1 * 0201.

FOXP3 has provided relevant information on the generation and maintenance of Tregs. Its transcription is induced by T cell receptor signalling (TCR); in particular, temporally persistent TCR signals activate FOXP3 transcription in autoreactive thymocytes (19). Upon its expression, a self-regulating transcriptional circuit stabilizes the expression of FOXP3 to consolidate the differentiation of Tregs and activate the suppressive function. However, these molecular mechanisms are not yet fully understood. The analysis about the genetic expression of FOXP3 ⁺ T and FOXP3^- T cells also suggested that many specific genes of Tregs are independent of FOXP3 (20). These results change the paradigm of FOXP3 as the only factor required for the development of Treg cell phenotype.

However, determination of Treg cell fate may be influenced by FOXP3 interaction with elements related to TCR-mediated T cell activation, such as IL-2 (Interleukin-2) and TGF-β (Transforming growth factor-β) signalling pathways (21). TGF-β induces FOXP3 and by the binding to its receptor I (TGF-βRI), stimulates the differentiation of Tregs. Experts also suggest a possible anti-apoptotic role for TGF-β that would improve the survival of nTregs (natural Tregs) and thus contribute to their stability (22). In the study by B. Kristensen et al. (23), it was shown that IL-6 (Intereukin-6) and TGF-β are important for human Th17 differentiation; perhaps, patients with HT have a higher basal production of IL-6 and TGF-β1 than healthy donors. Indeed, Tregs can also be reprogrammed into Th17s via IL-6- and IL-1β-dependent signalling. This process is mediated by the activation of STAT3 (Signal transducer and activator of transcription 3), RORγt (Retinoic acid-related orphan receptor gamma t) and RORα (Retinoic acid-related Orphan Receptor alfa) which downregulate the expression of FOXP3 and promote the conversion of Tregs into Th17s ( Figure 1 ). Actually, in Kristensen’s study, the basal expression of FOXP3 mRNA show an attenuation in patients with HT, but this expression is quantitatively very similar between patients and healthy controls.

In pathogenesis of autoimmunity, the concept of an insufficiency of CD4⁺FOXP3⁺ Tregs is opposed by a theory postulating their functional deficit, rather than a numerical defect of them. In fact in HT, as well as in other autoimmune diseases, the self-reactive T cells would be resistant to suppression, not so much due to the low number of Tregs, but due to their regulatory inability. In support to this theory, a gene splicing variant FOXP3Δ2, devoid of exon 2, unable to inhibit Th17 differentiation, compared to the full-length form of FOXP3 has been documented. Indeed, in Kristensen’s study, the baseline expression of the mRNA encoding FOXP3 is similar in HT patients and healthy donors; however, HT patients show a higher constitutive expression of the FOXP3Δ2 splice variant (59-67% % vs approximately 30%) (23). Therefore, the increase of IL-6 and TGF-β1 in the microenvironment and the increased expression of FOXP3Δ2 may contribute to the shift towards Th17s in patients with HT.

Full-length FOXP3 is known to suppress RORγT, RORα, activated T cell nuclear factor (NFAT) and nuclear factor-kappa B (NF-κB) signalling. Conversely, FOXP3Δ2 is incapable of these trophic effects (23). Due to RORγt, the essential transcription factor for Th17s and RORα, highly expressed in Th17s (induced by TGF-β/IL-6), the relative overexpression of FOXP3Δ2 in persons with HT may compromise the inhibition of Th17 differentiation. On the other hand, the interaction of FOXP3Δ2 with GATA3 (GATA binding protein 3), can cause a reciprocal increase in its expression; this interaction is also involved in the control of Th2s (T-helper 2 lymphocytes) responses by Tregs. Instead, in the environmental presence of IFN-γ, Tregs promote their own expression of T-bet (T-box transcription factor, pivotal to the development of Th1s) (24). Conversely, this factor induces the upregulation of CXCR3 (C-X-C Motif Chemokine Receptor 3), that allow Tregs to migrate into inflamed tissues and inhibit Th1 responses (25). In this regard, in a study by Tokić et al. (26), a similar upregulation of FOXP3 and T-bet was shown in HT patients compared to healthy subjects. Specifically, there was also an increased expression in hypothyroid as compared to euthyroid HT patients. Actually, these data are correlated with the stage of disease, rather than the thyroid hormone levels.

In violation of immunological tolerance, changes in microenvironmental signals disrupt the transcriptional and epigenetic regulation of FOXP3 (19), resulting in reduced Treg cell generation and suppressive function. In fact, the activity of FOXP3 is influenced by possible post-translational modifications such as acetylation. Acetylation of specific FOXP3 lysine residues increases the stability of FOXP3 and its ability to bind DNA and activate specific effector functions. A key molecule involved in the acetylation process is TAZ (transcriptional coactivator PDZ-binding) which plays a vital role in driving Th17 cell differentiation and inhibiting Treg cell development. Indeed, TAZ is a co-activator of RORγT and limits the differentiation of Tregs by decreasing FOXP3 acetylation. Impaired FOXP3 acetylation has been associated with the pathogenesis of several autoimmune diseases, such as HT.

These results indicate that the regulation of FOXP3 and the subsequent function of Tregs are based on different molecular processes; pharmacological manipulation of these pathways could pave the way for novel immunological tools to control Treg cell function and immunological tolerance in immune-related disorders.

The major disorder associated with loss of Treg cell function is an immune-dysregulation syndrome, poly-endocrinopathies, X-linked enteropathy (IPEX) (27), characterized by germline mutations of FOXP3, in both non-coding and coding sequences. Thus, IPEX subjects with mutations express a functionally defective FOXP3 protein, resulting in reduced suppressive activity of Tregs. These findings underscore the importance of FOXP3 in controlling the function of Treg cell and how its related defects contribute to pathogenesis and progression of autoimmunity.

BACH2 (located on chromosome 6q15) is a gene recently studied in the pathogenesis of several autoimmune diseases. Historically known in monocytes and neurons, further studies have shown its expression in B and T cells (28). The exact molecular mechanisms by which BACH2 protects hosts from the development of autoimmunity are not completely yet understood.

BACH2 works within innate and adaptive lines to control immune responses. It is highly expressed in B cells of the germinal centers (GCs), where it promotes the change of antibody class and suppresses the differentiation of plasmacells. BACH2 is also expressed by T cells and directs T-helper cell differentiation, homeostasis and effector functions (29). In fact, it limits the complete differentiation of Th1, Th2 and Th17 cells and generally keeps Th cells in a naive state by suppressing memory-related effector genes ( Figure 2 ). BACH2 also promotes FOXP3 ⁺ regulatory T cell differentiation and enhances Treg-mediated immunity. Moreover, BACH2 suppresses the differentiation of Th2s, through the down-regulation of GATA-3, the main transcription factor of this cell subtype.

Some studies also show that the ablation of BACH2 on T cells is associated with the expansion of follicular helper T cells (Tfhs) and abnormal GCs with consequent humoral autoimmunity (29). Tfhs are generally considered to be a separate subset of Th cells and derived from naïve CD4 ⁺ T cells after sequential steps in response to certain antigens. Naive CD4 ⁺ T cells triggered by dendritic cells upregulate the chemokine receptor CXCR5 (C-X-C chemokine receptor type 5), which allows them to migrate into B-cell follicles to further differentiate into Tfhs. However, Tfhs can also be generated by the conversion of other effector T cells under conditions of chronic and sustained antigenic stimulation. The differentiation of Tfhs is controlled by specific molecules, including the transcription factor c-Maf (Musculoaponeurotic fibrosarcoma), CXCR5 (suppressed by BACH2) and BCL6 (B-cell lymphoma 6) that downregulates BACH2. Tfhs, with loss of BACH2 in differentiation, are diverted to a subset cell that produces IL-4 (Interleukin-4), inducing switching of the IgG1 (Immunoglobuline Gamma G1) and IgE (Immunoglobuline E) histotypes of B cells.

Within GCs, Tfhs interact with B cells via co-stimulatory and co-inhibitory molecules, including programmed cell death protein 1 (PD-1), to facilitate selection and maturation of B cells with high affinity. However, excessive Tfhs impair positive selection by reducing competition between B cells and thus resulting in a lower threshold for selection which allows for the emergence of autoreactive clones (29).

Genetic polymorphisms of BACH2 are associated with numerous autoimmune and allergic diseases in humans.

Deficient BACH2 mice exhibit Treg cell depletion, decreased FOXP3 expression, and at the same time increased Th2-related cytokine production, leading to the development of fatal inflammatory lung disease (30). In humans, BACH2 haplo-insufficiency results in a syndrome with defective lymphocyte maturation that causes immunodeficiency, recurrent broncho-pulmonary infections and intestinal inflammatory processes (31).

Polymorphisms of BACH2 have been found to be associated with several autoimmune conditions, including vitiligo, rheumatoid arthritis and systemic lupus erythematosus; in particular, the rs3757247 variant has been associated with APS. For this reason, even with regard to HT, the main studies are currently aimed at cases of polyglandular autoimmunity (28). A Polish population study clearly demonstrated an association of BACH2 polymorphism rs3757247 with APS (31). These results and other limited studies indicate BACH2 as one of the universal genetic markers of autoimmunity. However, establishing a reliable set of polymorphisms to predict a possible multiple autoimmunity is a hypothetical subject of further analysis.

The search for variants of BACH2 could be a part of the early diagnosis panel that identifies individuals prone to develope a polyglandular autoimmunity, including those already diagnosed with an autoimmune disorder or in first-degree relatives of patients with APS.