Depending on generation, Foxp3
+ Trges are further categorized as naturally occurring thymus-derived Treg (tTreg) cells and Trges developed from conventional CD4+ T cells in the periphery (pTreg). These cells possess immunosuppressive functions and maintain peripheral tolerance [
61,
62]. tTregs are produced by the thymus at an early stage after birth and maintain tolerance toward self-antigens [
63,
64]. TGF-β1 directly enhances the Foxp3 promoter and encourages the generation of tTregs [
65]. Besides, exposure of naive T-cells to its cognate-antigen leads to the differentiation of pTregs under tolerogenic conditions [
66,
67,
68], and differentiation of pTregs is facilitated by the higher concentrations of TGF-β and higher levels of Foxp3 [
69,
70,
71]. Therefore, TGF-β1 is a key cytokine and plays a crucial role in the differentiation of both subsets of Trges. The induction of Foxp3 in peripheral naive T cells is achieved by a higher concentration of TGF-β, retinoic acid, and CD28 co-stimulation [
70,
72,
73,
74]. Both, tTreg and pTregs show similar expression levels of FoxP3, CD25, CTLA-4, GITR, ICOS, CD103, CD127 and a broad T-cell receptor (TCR) repertoire to deploy various suppressive mechanisms to control effector cells [
8,
59,
75,
76]. Foxp3
+ Trges are also known to secrete IL-10, TGF-β, and IL-35 [
77,
78] along with granzyme A and B [
79,
80,
81]. Furthermore, tTregs express higher levels of neuropilin-1(Nrp1), TF Ikzf2 (Helios), PD-1, and ecto nucleotidase CD73 than pTregs [
82,
83]. Helios and Nrp1 are considered as markers for tTregs since their greater expression is seen in tTregs as compared to pTregs [
83,
84,
85]. Interestingly, under
in-vivo conditions, pTregs could express helios [
86], and a fraction of the human tTreg population did not express helios [
87]. Moreover, tTregs are not differentiated based on helios and Nrp1 expression in mice [
88]. Treg specific demethylated region (TSDR) is highly demthylated in tTregs, and partially demethylated along with an unstable expression of Foxp3 and CD25 in pTregs [
69,
89,
90]. Apart from TSDR, Ig superfamily surface protein GPA33along with other Treg cell markers was recently used to identify Trges of thymus origin since this molecule is stably expressed on tTregs [
91].