**5.1 General characteristics of regulatory T cells**

Regulatory T cells are considered as the effector cellular arm of immune tolerance. Since the first publication of Kojima (1976), there is a constantly growing interest towards Tregs – cellular properties and medical applications [69]. Along with their unique suppressive phenotype (CD25 + FoxP3 + CD4+ T cells) [68, 69], Tregs express broad range of molecules that mirror their affiliation to the population of T cells and are widely applied in research and medical practice. Similarly to conventional T cells, they may be differentiated as naïve and memory, based on the expression of CD45RA, recent thymic emigrants (CD31), activated (HLA-DR) etc. [70, 71]. Their trafficking is ensured by the expression of chemokine receptors [72, 73].

The hallmark of Tregs is the expression of the transcription factor FoxP3 [68]. It controls the transcription program of Tregs by regulating several genes – increases expression of *Il2ra* (CD25), *Ctla4* (CTLA-4), *Tnfrsf18* (GITR), but inhibits those of *Il2.* At the same time *Foxp3* is subject of a tight regulation, where STAT5 signaling pathway is probably of key importance [74–77]. One may say that this is as two step process, starting with the generation of CD25hi, but FoxP3− Tregs-precursors, followed by the induction of FoxP3 through cytokine/STAT5-dependant signals involving HDAC [74, 78, 79]. Blocking of JAK/STAT pathway downmodulates Foxp3 expression [80]. In addition, a group of studies indicate that the maintenance of Tregs suppressive function is dependent on the epigenetic regulation of foxp3 locus by the Polycomb repressive complex 2 (PRC2). PRC2 consists of four subunits, primarily of enhancer of zeste homolog 2 (EZH2), EED, SUZ12, and RbAp48, where EZH2 is of particular interest [81]. The function of EHZ2 differs among different T-cell populations, leading to variations in H3K27me3 levels and silenced genes. In FoxP3 negative cells, EZH2 deficiency is associated with autoimmune diseases, reduced number of Tregs and expansion of memory T cells [82, 83].

The origin of Tregs in periphery is still a hot topic. Clear evidences show that a subset of Tregs - thymic Tregs (tTregs), come directly from the thymus during the process of intrathymic maturation of T cells [84–86]. Their selection occurs predominantly in the medulla during the negative selection by the high-avidity interactions between mTECs and thymocytes [87, 88], although some studies indicate that the process starts earlier, in the cortex [89]. Under specific conditions, like increased concentration of TGF-β, hormonal changes or continuous antigenic stimulation, Tregs can arise from naïve CD4+ T cells in periphery – inducible Tregs (iTregs) [90–92].

#### **5.2 Tregs are armed by different suppressive mechanisms**

Independently of the origin, Tregs are powerful immune suppressors. Both subsets use several approaches to regulate the strength of the immune response. Roughly, they are based on the expression of particular molecules, secretion of cytokines and consumption of IL-2 and might be categorized as contact-dependent and contact-independent.

Early studies demonstrated that the contact-dependent way is effectuate by the constitutive expression of CD152 (CTLA-4) by Tregs [93, 94]. The engagement of CD80/CD86 pathway activates tryptophan catabolism and expression of indoleamin 2,3 dioxygenase (IDO) [95]. Another mechanism involves PD-1/PD-L1 [95]. It is effective both against autoreactive B cells [96] and T lymphocytes [97]. Dilek et al. using alloreactive human T cells and blocking antibodies, evidenced by live cell dynamic microscopy that CD28, CTLA-4, and PD-L1 differentially control velocity, motility and immune synapse formation in activated Teff versus Tregs [94]. Although natural, their expression on the Tregs surface is inducibly increased and ensures the negative regulation of different receptors mediated signaling cascades in the target cells [98, 99]. Thus, Tregs directly attenuate cellular proliferation and activation.

The second line is facilitated by the production of different soluble factors upon activation. Among them are the immunosuppressive cytokines IL-10, TGF-b [100, 101], IL-34 [102] and IL-35 [103, 104]; perforins and granzymes [105]. It should be also considered that Tregs are target of cytokines like the proinflammatory TNF-α. The exact effect needs to be precised because current data are
