*5.2. Biostatistical Analysis*

For bioinformatic analysis, the amino acid sequences deposited in the UniProt database [219] were used. The identification numbers of the Foxp3 protein and its three isoforms, along with their amino acid sequences, are provided in Supplementary Materials Table S1. These sequences were used to carry out further bioinformatic analyses. The sequence length and molecular weight of individual proteins were pulled from the UniProt database. The determination of the isoelectric point of tested interleukins and their amino acid composition was carried out using the IPC isoelectric point calculator software available online [220]. The analysis of the second-order structure of interleukins was carried out using the NetSurfP-2.0 online program [221]. The amino acid sequences from the UniProt databases (Supplementary Materials Table S1) were used to analyze the identity of the amino acid sequences of the Foxp3 protein and its isoforms. The amino acid sequences of individual proteins were compared with each other using the Clustal Omega program available on their website [222]. The results of the analyses were presented as the percentage of identical amino acids in the analyzed amino acid sequences.

#### **6. Conclusions**

Numerous studies conducted in recent years have shown that Treg lymphocytes, which express Foxp3, appear in the human body immediately after birth and lead to the development of many inflammatory and autoimmune diseases after they are depleted. The Foxp3 protein has been shown to be necessary for lymphocytes in the thymus to differentiate into Treg lymphocytes. High expression of this transcription factor also guarantees their suppressive effect. The Foxp3 protein influences several cellular processes both directly and indirectly. In the process of cytokine production regulation, the Foxp3 protein interacts with numerous proteins and transcription factors such as NFAT, nuclear factor kappa B, and Runx1/AML1, and is involved in the process of histone acetylation in condensed chromatin. Thanks to their analyses and many experiments, scientists have shown that the similarity in the disturbance of the functioning of the FOXP3 gene in humans and mice is very similar. This allows for the conclusion that the process of dominant selftolerance in these organisms is similar to each other. Scientists' persistence in researching the Foxp3 protein has led to including this factor in one of the most reliable molecular markers of natural Treg lymphocytes. In addition, studies on the dysfunction of the Foxp3 transcription factor caused by the mutagenesis process have shown that it significantly affects disorders of the immune response as well as the development and progression of primary immunodeficiencies or autoimmune diseases.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jcm11040947/s1, Supplementary Materials Table S1: Amino acid sequences of the Foxp3 proprotein and its three isoforms from the UniProt database.

**Author Contributions:** Conceptualization, P.M., S.M., M.P. and E.G.; writing—original draft preparation, P.M., S.M. and M.P.; visualization, P.M.; methodology, P.M. and S.M.; software, P.M. and S.M.; formal analysis, P.M. and S.M.; investigation, P.M., S.M., M.P. and E.G.; writing—review and editing, E.G.; funding acquisition, E.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Research Grant No. DS640 of the Medical University of Lublin.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

