*3.5. Regulation of Foxp3 Protein Expression as a Potential Therapeutic Strategy for Autoimmune and Neoplastic Diseases*

Treg lymphocytes, due to their extremely important functions in the human body related to the suppression of immune reactions, have now become an important research target for many scientists. Particularly noteworthy are those studies that concern the possibility of their introduction as a treatment for autoimmune diseases and cancers [137]. The results of research conducted mainly on animal models show that the use of compounds as inhibitors or modulators of Foxp3 protein expression may not only confirm the correctness of the hypotheses put forward by scientists but also discover these molecules with high clinical potential [138].

The first group of such compounds are inhibitors of HDAC enzymes which, as shown by studies, can enhance not only the expression of *FOXP3* but also participate in the process of increasing the number of Treg cells and their functions. Currently, these compounds have found their application in animal models as therapeutics used to regulate the activity of Treg cells in autoimmune diseases (e.g., colitis, prostatitis) [124,139] and organ transplantation processes [107] as well as in the treatment of certain neoplastic diseases (e.g., breast cancer, lymphoma) [140,141]. The studies available in the literature show that the use of HDAC inhibitors influences the induction of FOXP3 acetylation in Treg cells, which prompt changes in markers related to the activity and functioning of Treg cells themselves, including receptors for TNF-α, CTLA-4 or PD-1 and IL-10 [142]. Obviously, a full understanding of the mechanisms of action of HDAC inhibitors still requires a lot of intensive research before such treatment strategies can be introduced into widespread clinical use.

Another group of compounds with therapeutic potential are histone acetyltransferase inhibitors [100]. The available studies on animal models show that the use of smallmolecule allosteric compounds, which may interact with the cofactors of the Foxp3 protein, contributes to the regulation of the function of this protein [100]. The analyses conducted so far focus on two compounds, Tip60 and p300, for which allosteric modifiers were developed [143]. In the case of Tip60, these compounds reduce the process of histone acetylation and induce association with the Foxp3 protein which has been used to treat autoimmune diseases (mainly colitis and collagen-induced arthritis) [144–146]. There are also studies on the use of Tip60 and p300 inhibitors for antitumor therapy—mainly prostate cancer [137,147]. Tip60 inhibitors have been shown to be able to inhibit the cancer cells' growth by inducing the apoptosis process and to allow the reduction of Treg suppression without affecting the proliferation of T effector cells [148]. In the case of p300 inhibitors, their ability to suppress Treg function has been demonstrated [149].

In order to determine the best therapeutic strategy aimed at changing the level of Foxp3 protein expression many thorough studies should be carried out which will determine not only the mechanisms of action of selected allosteric molecules but also allow the evaluation of the effectiveness of such therapies in a clinical setting. However, therapies with the use of a targeted modification of the expression of the Foxp3 protein will certainly become a valuable tool in the future in the fight not only of autoimmune diseases but also of neoplastic diseases.

#### **4. The Role of Foxp3 Protein in the Development of PIDs**

PIDs are a group of genetically determined diseases that are characterized by the impairment of one or more mechanisms of innate or acquired immunity. Unlike secondary immunodeficiencies, the symptoms of PID occur throughout the patient's life. This disease has a heterogeneous course, characterized by a wide spectrum of symptoms with varying severity which may manifest themselves in childhood or even in adulthood [150]. PIDs are genetic disorders that may be inherited (most often autosomal recessive) or appear for the first time in a given patient. According to the literature data presented by the International Union of Immunological Societies (IUIS), by 2019 over 406 diseases called PIDs and ~430 genes (the damage of which may lead to their development) have been described [151]. Individual mutations underlie the malfunction or lack of one or more elements of the immune system, including B and T lymphocytes, NK cells, phagocytes, or components of the complement system [151,152]. According to general estimates, the incidence of PID is in 1:2000 to 1:3000 live births [153,154]. Due to the possible occurrence of symptoms later in life, it is very difficult to determine the prevalence of PID in adults. Several reports conducted in various countries around the world show that diseases classified as PID occur in the population ranging from 1:8500 to 1:100,000 [155]. In the current literature we find that PIDs are classified into nine classes which were proposed and updated by the IUIS in 2019 (Figure 4A) [156]. Currently, there is a view in the literature that some PIDs may have a multi-gene basis because only the presence of defects of several genes at the same time is clinically manifested. In addition, the presence of a PID phenocopy may also be the result of an autoimmune reaction against certain components of the immune system itself (e.g., against certain interferons) (Figure 4B) [157].

**Figure 4.** PID classification and division. (**A**) Updated PID classification by IUIS for 2019; (**B**) Division of PID based on the type of mono- and polygenic disorders with examples of diseases (prepared based on [156–158]). Abbreviations: APECED—Autoimmune polyendocrinopathy-candidiasisectodermal dystrophy; IPEX—immunodysregulation polyendocrinopathy enteropathy X-linked syndrome; C1q—complement component 1q; C4—Complement component 4; C2—Complement component 2; MBL—mannose-binding lectin; AID—Activation-induced cytidine deaminase; ALPS—Autoimmune Lymphoproliferative Syndrome; CVID—Common Variable Immunodeficiency; IgA—immunoglobulin A; CD40—cluster of differentiation 40; CD40L—cluster of differentiation 40 ligand.

The course of PIDs varies considerably which means that many clinical symptoms may be present from birth or may gradually worsen over time until the disease develops in childhood or adulthood. According to the literature, the mean time from the onset of symptoms to full PID diagnosis is on average ~5 years and largely depends not only on the type of deficiency and the patient's age but also on the patient's country of origin [159,160]. This is due to the lack of awareness and education in society as well as the lack of preparation in doctors to make accurate and quick diagnoses. Therefore, it is extremely important to search for diagnostic markers that allow for the shortening of time for accurate diagnosis as well as a comprehensive analysis of genetic disorders contributing to the development and progression of many diseases classified as PID [161,162] One such molecule is the Foxp3 protein in which mutation causes the immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) classified as a monogenic disorder [163]. Additionally, recent research sheds new light on the role of this protein in the development of CVID (common variable immunodeficiency) which is an example of a polygenic disorder. The *FOXP3* transcription factor is expressed on CD4+ Treg cells and is crucial for Treg function which is responsible for suppressing immune responses, especially at their early stages. The most important function of Foxp3 is its ability to confer suppressive activity on Treg cells, for example, by maintaining constitutive high expression of CTLA-4. It acts by preventing the activation and proliferation of B and T lymphocytes [164]. However, Foxp3 alone does not control all aspects of Treg biology and is not the initiating factor in Treg development [165,166]. The importance of Treg cells has been demonstrated in murine models—depletion of Foxp3+CD4+ Treg cells resulted in severe autoimmunity, allergy, and immunopathology (e.g., IPEX) in otherwise normal animals and those same diseases can be prevented by reconstituting Treg cells [167–169]. Moreover, it has also been demonstrated that Treg cells could prevent the progression of and even cure established autoimmune/inflammatory diseases [170,171]. They also play an important role in allergy prevention [172]. Foxp3+ Treg cells likewise depend on a lot of other suppressive molecules, such as IL-10, TGF-β, CD39, CD73, IL-35, and TIGIT, for their inhibitory function; most of which work by suppressing autoimmunity [173].

#### *4.1. The Role of Mutations within the Foxp3 Protein in PID Development*

The studies conducted so far indicate the presence of 63 identified mutations within the *FOXP3* gene which affect its proper functioning and contribute to the development of autoimmune diseases [174,175]. The vast majority of the discovered mutations concern the fork-head binding domain of the Foxp3 protein that influences the processes of nuclear import and DNA binding which are necessary for the suppressive activity of this protein. Some of the mutations were also found in the leucine zipper region which impaired the dimerization function of the Foxp3 protein; other mutations influenced the spatial change in the structure and position of the domains within the protein or led to a decrease in mRNA stability for the *FOXP3* gene [174–176].
