*3.1. The Occurrence of Foxp3 Protein on Cells of the Immune System and Its Functions in the Human Body*

Treg cells belong to the subpopulation of lymphocytes responsible for suppressing an overly increased or autoreactive immune response, which may be specific or nonspecific for a given antigen, without causing general immunodeficiency [51,52]. It is an extremely heterogeneous population with several subpopulations of cells with different levels of Foxp3 protein expression (Table 3) [53]. Treg cells expressing Foxp3+ can also be divided into two smaller subpopulations according to the origin of the cells. The first one is natural Treg lymphocytes (nTreg), which are formed in the thymus as a separate developmental line, while the second is induced (adoptive) Treg lymphocytes (iTreg or aTreg) [54,55]. The latter are Treg cells arising in peripheral tissues which initially do not express the Foxp3 factor but acquire it along with suppressive properties upon stimulation with an appropriate antigen. Natural nTreg lymphocytes express CTLA-4 (cytotoxic T cell antigen 4) [56], GITR (glucocorticoid-induced TNFR-related protein) [57], CCR4 (CC chemokine receptor type 4) [58,59], and CD62L (L-selectin) [60]. The formation of nTreg and iTreg lymphocytes requires the presence of interleukin 2 (IL-2) and transforming growth factor beta 1 (TGF-β) [61]. The similarities in the role of these cytokines in the maintenance and survival of both Treg lymphocyte populations are crucial. nTregs develop in response to contact with intrinsic antigens in the thymus and require high affinity between these antigens and MHC complexes [62,63]. This is probably because they arise from continuously proliferating precursor cells. They also require costimulation with CD28 (cluster of differentiation 28) which also plays an important role in the process of inducing the expression of the CTLA-4 molecule in T lymphocytes which can inhibit the CD28 signal and thus, is responsible for the negative feedback mechanism [64,65]. On the other hand, iTreg, through interactions with environmental antigens presented by dendritic cells in peripheral lymphoid organs and their conversion to iTreg CD25+Foxp3+, requires weaker, incomplete TCR stimulation [66]. Foxp3 protein is also known to convert naive T cells into Treg cells that are capable of suppression in vivo and in vitro, suggesting that Foxp3 can regulate the expression of suppression-mediating molecules [53]. Elucidation of Foxp3 gene targets may be crucial for understanding the Treg cell suppressive capacity [67–69].

**Table 3.** Treg lymphocyte subpopulations (based on [70]).


Another group of immune system cells in which the Foxp3 protein is expressed is NKT cells [71]. This is a group of innate lymphocytes capable of producing cytokines characteristic for a Th1, Th2, or Th17 response [72,73]. They have also been confirmed to influence adaptive immunity by exacerbating or suppressing a variety of immune disorders such as autoimmunity and allergy. As mentioned before, Treg cells are characterized by strong immunosuppressive properties and expression of the Foxp3 transcription factor and constitute a key element in maintaining immune homeostasis in the human body. Studies from recent years have shown that NKT cells, like Treg cells, contribute to the maintenance of immune tolerance and are also capable of the Foxp3 protein expression. From animal model studies, scientists were able to establish that NKT cells in the lymph nodes of αgalactoceramide-stimulated mice increased the ability to express Foxp3 in response to TGF-β. However, further research is needed to use Foxp3+ NKT cells for therapeutic purposes in the treatment of immune response disorders [74,75].

In addition to T cells, expression of the Foxp3 protein is also observed on B cells. Regulatory B cells, which are characterized by the production of anti-inflammatory cytokines (e.g., IL-10 and IL-35), also contribute to the enhancement of the immune homeostasis in the human body [74]. Recent research has shown that some B cells are also capable of expressing the Foxp3 protein [76]. They are usually observed in patients diagnosed with multiple sclerosis [77] or in patients with systemic lupus erythema where their increased amount correlates with disease progression [78,79]. A study by Slobodin et al. (2010) shows that the percentage of Breg cells in the peripheral blood of SLE patients is higher than that of healthy controls and that Bregs have been shown to be functionally impaired. Additionally, they showed that with the expansion of CD25highIL-10highFoxP3high B regulator cells, increased disease activity occurs [80]. However, the current literature reports do not provide important information regarding the exact role of the Foxp3 protein on B lymphocytes and its role in the regulation of inflammation [70,81]. The subject of Foxp3 protein expression on cells of the immune system is quite debatable and requires a lot of research to fully understand the importance and role of this protein in the human body.

#### *3.2. Interactions of Foxp3 Protein with Proteins and Transcription Factors*

The Foxp3 protein plays an important role in the regulation of cytokine production through interaction with numerous proteins and transcription factors such as NFAT, nuclear factor kappa B (NF-κB, kappa-light-chain-enhancer of activated B cells) [82], and Runx1/AML1 (Runt-related transcription factor 1/acute myeloid leukemia 1 protein), also known as acute myeloid leukemia 1 protein (AML1) [83] or alpha-core binding factor 2 (CBFA2) subunit [84]. Transcription factors NFAT and Runx1/AML1 are necessary for the production of IL-2 following TCR receptor stimulation. The association of the Foxp3 protein with these factors inhibits the expression of IL-2, IL-4, (which are pro-inflammatory cytokines), and IFN-γ [85]. In vivo, the transcription factor NFAT, which is required to bind to the proximal region of the promoter for IL-2, participates in direct interactions with the forkhead domain of the Foxp3 protein [82]. These interactions are also necessary to stimulate the expression of some Treg lymphocyte antigens, such as CD25 or CTLA-4 [82,86,87]. CD25 is the alpha chain of the interleukin 2 receptor and is transiently expressed on activated T and B lymphocytes and constitutively present on Treg cells. The presence of this protein was also found on dendritic cells, fibroblasts, and endothelial cells [88,89]. In contrast, CTLA-4 is a protein receptor that acts as a checkpoint for immune responses. This protein is constitutively expressed in Treg lymphocytes, but when activated, it is only up-regulated in conventional T lymphocytes; this phenomenon is particularly evident in the case of neoplastic disorders [86,90]. The interaction of the Foxp3 protein with the Runx1/AML1 factor (association between the "fork-head" domain and the leucine zipper) consists of the attachment of this complex to the IL-2 promoter region. It should also be noted that the Foxp3 protein also participates in interactions with other members of the FOX family where it forms complexes capable of inhibiting the expression of selected genes [91]. The formation of either homodimeric or heterodimeric Foxp3 protein complexes occurs when a

leucine zipper is used which associates with the IL-2 promoter in vivo. Scientific studies have also shown that the multimerization of the Foxp3 protein is extremely important for the proper functioning of Treg lymphocytes [92–95].

The N-terminus of the Foxp3 protein also has properties to inhibit the transcriptional activity of the NFAT factor and is necessary for the inhibition of IL-2 production by T cells. Studies have shown that the association of the Foxp3 protein with the IL-2 and IFN-γ promoters correlates with the process of hypoacetylation (low level of acetylation) of histones in condensed chromatin [4,96]. The binding of the Foxp3 protein to the CD25 and CTLA-4 promoter sequences, with the simultaneous hypoacetylation of condensed chromatin histones, suggests a direct mechanism of transcription activation [97–99].

In Treg cells, the Foxp3 protein is present as a part of a large complex that also includes histone acetyltransferases (HAT) and histone deacetylases (HDAC) [100,101]. The process of acetylation and deacetylation is one of the post-translational modifications of histones, involving lysine residues located at the N-terminus (protruding from the nucleosome core), which is an epigenetic mechanism for the control of gene expression. The enzymes catalyzing the reversible acetylation of histones are HAT and HDAC [102]. Research conducted in recent years has allowed us to establish that the former simultaneously act as coactivators of transcription, while the latter is its corepressors. Thus, it is possible to prove the relationship between the covalent modification of chromosomal proteins (acetylation of core histones) and gene expression. HAT and HDAC enzymes are responsible for the acetylation process of the Foxp3 protein and thus determine the functions of this transcription factor. Studies have shown that Foxp3 acetylation is related to its function in Treg cells [103–105]. Detailed analyzes have shown that the acetylation process of this protein is influenced by molecules such as KAT5 (TIP60), p300, HDAC6, HDAC7, HDAC9, HDAC10, and SIRT1 (Table 4).

Moreover, it has been shown that the stability of the Foxp3 protein is significantly higher in the presence of the enzyme p300 than in the presence of KAT5. After combining with the p300 protein, Foxp3 shows a higher affinity for DNA chromatin, while after interaction with KAT5, its affinity for the IL-2 promoter has significantly increased [106]. The importance and role of HDAC7 and HDAC9 in the acetylation of Foxp3 protein are poorly understood. The link is due to the fact that class II HDAC has no intrinsic functional catalytic activity and possibly, as the scientists suggest, works by recruiting class I HDAC into the complex. The mechanism of action of HDAC6, belonging to class II HDAC proteins, is based on the regulation of the acetylation process of many cellular proteins, among which α-tubulin and HSP90 deserve special attention [107,108]. Studies conducted by Zoeten et al., showed that the pharmacological inhibition or the use of a genetic knockout of the HDAC6 gene increased the level of acetylation of both Foxp3 and Hsp90 [107] which resulted in an increase in the immunosuppressive activity of Treg cells. According to the researchers, this process may be used in the future to suppress autoimmunity caused by Treg and be used in the prevention of transplant rejection [107,108]. The combination of KAT5 and HDAC7 is an essential mechanism in the inhibition of IL-2 transcription by the Foxp3 protein [100,109]. NAD-dependent sirtuin-1 deacetylase is considered a negative regulator of the acetylation process of the Foxp3 protein [55,110].

#### *3.3. The Importance of Post-Transcriptional and Post-Translational Modifications in the Function of the Foxp3 Protein*

The transcription factor Foxp3 is a major regulator of Treg cells' growth and suppression activity. As indicated in the literature, it is also subject to complex regulation by the participation of many posttranscriptional modifications as well as posttranslational modifications (PTM) which also indirectly affect Treg suppressor activity. The first modifications of the Foxp3 protein concern epigenetic changes related to the regulation of DNA methylation [111], histone modification [99], or nucleosome positioning [112]. In the literature, we can find reports on the regulation of the Foxp3 protein by conserved non-coding sequences 2 (CNS 2) [113,114] or the share of ubiquitin-specific peptidase 22

(USP22) [115,116]. Many factors also play an important role in regulating the conversion of Foxp3 precursor messenger RNA transcripts at the level of post-transcription modification. This concerns the participation of microRNAs (miRNAs), including miR-24, miR-31, and miR-210, the activity of which leads to the degradation of Foxp3 mRNA which consequently prevents the translation of Foxp3 [117,118]. PTM, which includes phosphorylation, O-GlcNAcylation, acetylation, ubiquitylation, or methylation, also significantly influences the activity of the Foxp3 protein [105,119]. It should be noted that most of the PTMs refer to enzymatic processes that are designed to change the protein after its synthesis. Moreover, all the induced modifications affect the characteristics of the protein, including its location and interactions. This is also the case with the Foxp3 protein, in which the occurring PTMs affect the very structure of the protein (its stabilization or degradation) and its interactions with other proteins as well as reducing or increasing the activity of suppressive Treg lymphocytes [105]. Detailed information on the positive and negative contribution of individual PTMs to the suppressor functions of Treg cells is presented in Figure 3.

**Table 4.** Effect of enzymes on the acetylation process of the Foxp3 transcription factor.




**Figure 3.** Contribution of PTM to the functioning of the Foxp3 protein and the suppressor functions of Treg lymphocytes. Modifications that positively affect the suppressor functions of Treg lymphocytes are marked in green while those that have a negative effect are in red; based on [105].
