4.1.1. The Role of Foxp3 Protein in IPEX Development

The occurrence of mutations in the *FOXP3* gene is associated with the development of the IPEX disease syndrome and was described for the first time in 1982 by Powell et al. as a rare immunodeficiency syndrome with a genetic predisposition [176]. This disease is characterized by the presence of three specific clinical symptoms such as enteropathy with chronic diarrhea (most often acute, watery, and bloody diarrhea), endocrinopathy (insulin-dependent diabetes type I), and dermatitis (Figure 5) [35]. IPEX is a recessive disorder related to the X chromosome; therefore, it occurs only in males (in the first six months of life) and causes T lymphocyte activation, accompanied by the overproduction of cytokines, and leads to autoimmune disorders with the presence of various autoantibodies [177]. This results in the development of many serious diseases such as type 1 diabetes and autoimmune hemolytic anemia as well as hypopituitarism or thyroid gland disorders and numerous skin lesions such as eczema. In many patients, exacerbation of

the disease is also observed and caused by infections or food allergens. Subsequently, the main symptoms of the gastrointestinal tract or skin lesions are intensified or exacerbated as well as other disorders related to kidney, digestive, and immune system diseases (Figure 5) [163,178]. Due to such a wide range of symptoms, patients without appropriate treatment (immunosuppressants and bone marrow transplantation) die before reaching the age of 2 [176].

**Figure 5.** Symptoms of IPEX developed from [177].

The assessment of the presence of the IPEX syndrome requires the use of many extensive, basic, and specialized tests aimed at a correct diagnosis. Basic tests include a complete blood count with smear, determination of serum glucose concentration, thyroid function, and immunoglobulin levels as well as food hypersensitivity tests and the percentage of individual cells of the immune system, especially T and B lymphocytes (Table 5). Based on the obtained results of basic tests, advanced tests are ordered, including endoscopy with intestinal biopsy, skin biopsy, immunophenotyping of Treg lymphocytes, and sequencing of the *FOXP3* gene (Table 5) [177].

**Table 5.** Meanings of basic and specialized tests in the diagnosis of IPEX.



Genetic conditions are related to the mutation within the transcription factor Foxp3 which was found in nearly 60% of patients with IPEX syndrome. These include missense or frame alteration mutations and insertion or deletion mutations at splicing sites which result in the loss of function of this protein. The occurring mutations also have consequences regarding the quantitative and functional disorders of Treg cells in the body deficiencies which cause autoimmune disease. We can find a description in the literature of most cases where mutations occurring within the Foxp3 protein are hereditary, although this does not exclude the occurrence of mutations in a sporadic manner [185–187]. These mutations take place in almost every region encoding the Foxp3 protein which clearly indicates that each domain of this protein is functional and necessary for its proper functioning. Although mutations in the non-coding regions are also known, their frequency is much lower compared to the mutations in the coding regions, however, their occurrence may also be the cause of the development of IPEX [28,188]. Nevertheless, scientists have not shown a clear correlation between genotype and phenotype [181,189,190].

An important diagnostic and cognitive tool turned out to be the use of flow cytometry which makes it possible to assess the expression of Foxp3 on cells of the immune system. Although high levels of *FOXP3* expression are commonly attributed to CD4+CD25high Treg cells, it is also induced by TCR stimulation in naive CD4+CD25− T cells and may persist for up to several days in activated CD4+ T cells [191]. The belief of many scientists that subsets of CD4+ T cells expressed in different tissues have different patterns of CD25 and other Treg cell markers further complicates the task of distinguishing Treg *FOXP3*+ from *FOXP3*+ found on other cells of the immune system. Therefore, it is necessary to search for new diagnostic markers to allow their safe differentiation. One such marker is the IL-7 receptor (CD127) promoter which is the target of *FOXP3* mediated transcriptional repression. Thanks to this discovery, scientists were able to identify the most suppressive population of human Treg cells, expressing the phenotype CD4+CD25+CD127low [192,193]. This has been used in the diagnostic methodology of the IPEX team where cells identified as CD4+CD25+CD127low correlate well with the population of CD4+CD25+*FOXP3*+ cells. Of course, the two populations should not be considered identical since conventional CD4+CD25-CD127+ T (Tcon) cells rapidly decrease CD127 expression following IL-7 signaling and TCR stimulation [194]. Studies have shown that cells with the CD4+CD25+CD127low phenotype may also be present in some IPEX patients who suffer from decreased FOXP3 expression due to the occurrence of hypomorphic mutations [55,195]. It has also been demonstrated that patients with IPEX syndrome who have missense mutations and deletions in splicing sites do not have Treg CD4+CD25+Foxp3+ lymphocytes and have a more severe form of the disease. Additionally, the absence of CD4+CD25+ cells confirms the diagnosis [35]. IPEX also has high levels of IgE and IgA immunoglobulins, as well as eosinophilia, which proves that the transcription factor Foxp3 is strongly associated with the human immune response [179–182].

Due to the special role of the Foxp3 protein in the development and progression of IPEX teams, many scientists are considering using this molecule as a therapeutic agent in the treatment of this disease. The studies available in the literature also show that in the absence of antigenic stimulation, *FOXP3* expression is promoted in CD4+CD25− T cells by signaling cytokines, which include STAT5, IL-2, IL-7, and IL-15, on peripheral blood cells [35,196,197]. Additionally, it has been shown that cytokine treatment does not induce *FOXP3* expression in lymph node derived CD4+ T cells [35,197]. The use of flow cytometry analysis has shown that the expression level of the Foxp3 protein on TCD4+ lymphocytes from peripheral blood can vary up to 40-fold between individual cells. This explains that the strength and duration of *FOXP3* induction may influence discrepancies in cytokine activation-induced T cell suppression [35,198–201].

Another therapy using the role of the Foxp3 protein proposed in the literature is the use of hematopoietic stem cell transplantation. Currently, this therapy is the only treatment option for sick patients with IPEX. According to the study by Passerini et al. in 2013, the conversion of CD4+ Treg cells after lentivirus-mediated transfer of the *FOXP3* gene results in a population of CD4+*FOXP3*+ T cells which exhibit a stable phenotype and preserved suppressive function. In addition, their studies have shown that CD4+*FOXP3*+ T cells are stable during inflammation not only in vitro but also in in vivo models. Therefore, based on the above observations, the researchers proposed the use of *FOXP3* gene transfer therapy in the IPEX syndrome in order to restore immune tolerance [202]. From the description of clinical cases and literature data, we can conclude that there are also patients with symptoms that resemble IPEX syndrome, including enteropathy, autoimmune endocrinopathy, and dermatitis, but the age and gender of these patients are more varied (these symptoms are also present in women) [203]. From the study by Ochs and Torgerson on 100 patients with a phenotype corresponding to IPEX syndrome, nearly half of them did not have a mutation in the *FOXP3* transcription factor gene [186]. Such diseases include CD25 or IL-2RA deficiency, mutations within STAT5b, STAT1 or STAT3, Dedicator of Cytokinesis 8 (DOCK8) deficiency as well as infantile or eosinophilic enteropathies and severe combined immunodeficiency (SCID) (Figure 6). The underlying genetic defect of these syndromes is unknown and requires further intensive research.

#### 4.1.2. The Role of the Foxp3 Protein in the Development of CVID

Recent studies have also shown the role of the Foxp3 protein in the development of another PID disease, i.e., CVID. This disorder belongs to the humoral immunity deficiencies and is characterized by a relatively mild course. It is a type of polygenic disorder associated with the defective production of immunoglobulins, often accompanied by autoimmunity [204]. The epidemiology of this disorder is still difficult to define. Literature data show that the estimated prevalence of CVID in the population is 1 in 30,000 people. The diagnosis of CVID occurs in most patients between the age of 20 and 40 due to late symptoms [205,206]. Based on the analysis carried out by Grathman et al., in 2014, it was possible to establish the relationship between the clinical picture and the differences and effects of immunoglobulin treatment in 2212 patients from several European countries. The results of this review showed that patient survival depends on the time of diagnosis as well as the age when the first symptoms of the disease appeared [207]. The later the first symptoms appeared and the more delayed diagnosis of the disease, the greater the risk of death at any age (each year of delay in diagnosis increases the risk of death by about 4.5%) [207]. However, the etiology of CVID is not fully known, as only 20% of patients have the genetic cause identified. The most common form is sporadic cases with no family history of the disease (90%) [208]. They can be caused by a complex interaction of environmental and genetic components (multi-factor inheritance), but genes involved in the development and function of immune cells have now been shown to be the main cause [208,209]. As we know, the main role of the immune system is to defend against infections while protecting the body's own cells. Antibodies, also known as immunoglobulins, are proteins produced by B lymphocytes. In order for B lymphocytes to function effectively, they usually need the help of other immune cells such as T lymphocytes. Most people with CVID have a normal number of B cells, but this is characterized by a maturation disruption and thus a decrease

in antibody synthesis. These disorders can be caused by a lack of needed help from T cells to develop a normal immune response [210–212]. As a result, people suffering from CVID will differ in their ability to elicit effective antibody responses due to the lowered levels of immunoglobulin. We can distinguish three types of disorders here; the first one concerns the three main types of immunoglobulins (IgG, IgA, and IgM), the second one concerns disorders within IgG and IgA, and the third group concerns those in which only IgG is lowered [213,214]. A diagnosis of CVID is usually confirmed by abnormal blood tests and medical history.

**Figure 6.** Diseases classified into IPEX-like syndromes (based on [177]). Abbreviations: IL2RA—Interleukin 2 Receptor Subunit Alpha; STAT—signal transducer and activator of transcription protein; STAT5—Signal Transducer And Activator Of Transcription 5; STAT1—Signal transducer and activator of transcription 1; STAT3—Signal Transducer And Activator Of Transcription 3; CTLA4—cytotoxic T cell antigen 4; DOCK8—Dedicator of cytokinesis 8; SCID—severe combined immunodeficiency; NOMID—neonatal onset multisystemic disease; CINCA—chronic infantile neurological, cutaneous, and articular syndrome; ALPS—Autoimmune Lymphoproliferative Syndrome; APS-1—Autoimmune Polyglandular Syndrome Type 1; APECED—Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.

Due to the important role of T lymphocytes in stimulating the synthesis of antibodies by B lymphocytes, some scientists have started research to determine whether the Foxp3 protein may be involved in this process [215]. The research conducted by Horn et al. in 2009 shows that this protein may indirectly influence the development of CVID. They analyzed the percentage of CD4+ Treg lymphocytes among patients diagnosed with CVID from different cities/countries: Freiburg, London, and Sydney, and correlated it with clinical symptoms. The percentage of Treg cells defined as CD25+Foxp3+ and CD25+CD127lowFoxp3+ or CD25+CD127lowCD4+ was analyzed and the results were compared with data from healthy patients. They found that, regardless of the phenotype used to define them, patients with CVID experienced a significant decrease in the percentage of Treg cells which correlated with the development of autoimmune disease. This provided evidence that a reduction in the number of Treg cells in CVIDs may play a role in the development and progression of clinical symptoms and may also contribute to understanding the pathogenesis of CVID complications [216]. Other research by Genere et al. built on the work presented by previous researchers; they showed that patients diagnosed with CVID and autoimmune disease had a significantly reduced frequency of CD4+CD25HIGHFoxp3+ cells in the peripheral blood, accompanied by a reduced intensity of FOXP3 expression. Additionally, they found that although CVID patients with autoimmunity had a reduced frequency of CD4+CD25HIGHFoxp3+ cells, FOXP3 expression levels did not differ from those of healthy controls. Thanks to the obtained results, the researchers showed that CD4+CD25HIGHFoxp3+ cell homeostasis is disturbed in patients with CVID, especially in the presence of autoimmunity, which may indicate that Treg lymphocytes are involved in the pathological mechanisms of CVID [217]. The results of these studies were also confirmed by another research team (Arandi et al., 2013) which showed that the frequency of Treg was significantly lower in patients with CVID than in healthy subjects and that in patients with CVID, in whom autoimmunity was detected, the percentage of cells analyzed is significantly reduced compared to the cases without autoimmune diseases. There was also a significant difference in the expression level of the Foxp3 factor between patients with CVID and the control group [218]. The reduction of the expression level of the Foxp3 protein in patients with CVID was also analyzed and confirmed by Yu et al. [52]. They showed that the reduction in the levels of FoxP3, granzyme A, and pStat5 was significantly correlated with the degree of Treg dysfunction in CVID [52]. However, a full understanding of the role of Foxp3 in the development of CVID requires further extensive interdisciplinary research to understand its role in the pathogenesis of primary immunodeficiencies.
