Regulatory T Cell Markers

Thymic regulatory T (tTreg) cells

Around 80% of circulating Treg cells develop in the thymus during neonatal development, and these cells are central to the establishment of self-tolerance and prevention of autoimmunity.⁹ tTreg cells develop from CD4+ single positive thymocytes, and their differentiation into CD25+ Foxp3+ Treg cells requires a tripartite signal: stimulation of the T-cell receptor (TCR) by self-antigen, co-stimulation via CD28, and a third signal downstream of γ-chain cytokines such as interleukin (IL)-2 and IL-15.^3,9-11

Markers of tTreg cells include the transcription factor Helios (intracellular), neuropilin-1 (Nrp1), and several co-inhibitory receptors including CTLA-4, 4-1BB, ICOS, and GITR. Activated CD4+ effector T cells express the same inhibitory markers, but at lower levels; as such, relative expression levels as measured by flow cytometry is often used to distinguish these cell populations (Figure 3). Most tTreg cells retain stable expression of FoxP3 after leaving the thymus.¹¹

Figure 3: Expression of markers on human peripheral blood mononuclear cells by flow cytometry, comparing resting regulatory T (Treg) cells and conventional T (Tconv) cells.Data is presented both as bar graphs and contour plots. Percentages on the contour plots indicate the percent gated cells as positive for a given surface marker (blue = Treg, red = Tconv). Reproduced under Creative Commons 4.0 CC-BY from ²

Mature tTreg cells can recirculate back to the thymus, where they function to fine-tune the de novo production of tTreg cells, for example via consumption of IL-2.¹² Recirculating Treg cells have an activated phenotype (CD44^highCD62L^low), and the relative proportion of these cells in the thymus increases with age. Functional and phenotypic heterogeneity exists within recirculating Treg cells, including a population that expresses the IL-1 decoy receptor IL-1R2 and promotes tTreg cell development under inflammatory conditions by quenching IL-1.¹³

Figure 4: Flow cytometry analysis of Expi293 cells transfected with irrelevant protein (A) or human CTLA-4 (B) stained with Recombinant Anti-CTLA4 Antibody [DM51] (A318645).

Figure 5: IHC of human tonsil stained with Anti-CTLA4 Antibody [IHC064] (A324529).

Peripheral Regulatory T (pTreg) Cells

Peripheral Treg cells arise from conventional CD4+ T cells and convert to FoxP3+ suppressive Treg cells in vivo in response to stimulation of the TCR by sub-immunogenic concentrations of antigen.¹¹ The full array of signals that promote the generation of pTreg cells in vivo are yet to be defined. pTreg cells can be identified by expression FoxP3+CD4+ cells and absence of Helios and Nrp1.⁶ FoxP3 is not stably expressed in pTreg cells, which exhibit suppressive capacity only when the transcription factor is expressed.³ pTreg cells that lose expression of FoxP3 are known as “exFoxP3” or “exTreg” cells. ExFoxP3 express high levels of CD127, low levels of GITR, and no longer express CD25. Expression of folate receptor 4 (FR4), CTLA-4 and CD103 has also been described on these cells.¹⁴

Inducible Regulatory T (iTreg) Cells

Inducible regulatory T (iTreg) cells arise in vitro from tTreg cells in response to stimulation with the cytokines TGF-β and IL-2, which induce and stabilize the expression of FoxP3, respectively.^11,15 Although the term iTreg cell is predominantly used to describe in vitro-generated Treg cells, this term is sometimes also applied to cells that are induced to express FoxP3 and gain suppressive function in vivo (i.e. pTreg cells).

Subsets of Peripheral Treg Cells

Peripheral Treg cells in tissues that arise from conventional CD4+ T cells adapt to the inflammatory environment in that tissue. This results in Treg cell subpopulations that are tailored to suppress the activity of the predominant local CD4+ helper T (Th) cell population — Th1, Th2, Th17, Th1/17, or follicular T helper (Tfh) cells.^1,7

These functionally specialized Treg cell populations mirror the corresponding Th cell population in their transcription factor and cytokine expression profiles. For example, in the context of a predominant Th1 response, such as during viral infection, Treg cells secrete interferon (IFN)-γ and express the transcription factor T-bet, which is critical for their ability to suppress Th1-driven inflammation. These specialized CD4+CD25+FoxP3+ eTreg cell subsets can be distinguished by variable expression of lineage-determining transcription factors and chemokine receptors, as outlined below and in Table 2.^1,11

• Th1-type Treg cells (or T-bet+ Treg cells)
• Th2-type Treg cells
• Th17-type Treg cells
• Th1/17-type Treg cells
• Regulatory Tfh cells (or Tfr cells)
• Type 1 regulatory cells (Tr1)
• Th3-type Treg cells

Table 2: Effector Treg cell subsets and their transcriptional, phenotypic, and cytokine profiles. Generated using data from ^1,11.

CD8+ Treg cells

Although CD8+ T cells with suppressive function have been described in mice since the 1970s, so-called CD8+ Treg cells have been less well studied and characterized than their CD4+ counterparts,^3,19 particularly in humans. More recent studies have identified CD8+ Treg cells in mice under various inflammatory and disease contexts, including in models of transplantation and cancer.

CD8+ Treg cells arise in the periphery from mature conventional CD8+ T cells. CD8+ Treg cells differ from CD4+ Tregs cells in that most lack FoxP3 expression; instead, these cells have been shown to require the transcription factors Helios and Eomes for their suppressive capacity and homeostatic functions.^3,19,20

The most common phenotype used to identify CD8+ Treg cells in mice is CD122^highLy49+CD8+ but subsets of CD8+ Treg cells have been shown to express other markers. Surface markers of CD8+ Treg cells include (also see Table 1):³

• CD49d
• CD103
• CD122
• Ly49
• PD-1

As with CD4+ Treg cells, none of these markers is unique to CD8+ Treg cells, being expressed on conventional T cells and other immune cell subsets. Proposed subsets of CD8+ T cells and their functions are outlined in Table 3. A recent study identified a population of suppressive CD8+ cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) that are increased in people with a variety of autoimmune diseases (as well as COVID-19).^19,21 The authors proposed that these cells are the human equivalent of Ly49+CD8+ Treg cells in mice.

Table 3: CD8+ regulatory T cell subsets in mice. Adapted from ¹⁹.

References

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21. Li, J., et al. KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19. Science 376, eabi9591 (2022)