How To Differentiate T-Regulatory Cells (Tregs) By Flow Cytometry

T-regulatory cells (Tregs), formerly known as T suppressor cells, are a T cell subset with direct roles in both autoimmunity and responses to pathogens.

Tregs decrease inflammation via the secretion of immunosuppressive cytokines (IL-10, TGF-b) and also through direct suppression of inflammatory effector T cells (such as Th1 and Th17 cells).

Tregs control and likely prevent autoimmune diseases by contributing to the maintenance of tolerance to self-antigens. The therapeutic benefit of Treg transfer is well established in animal models and efforts to begin human Treg therapies are underway for transplantation and Type 1 diabetes patients.

Given the importance of this unique T cell subset in so many immune responses, many investigators feel remiss if they immunophenotype their cell populations of interest without including a Treg measurement in the mix. But quantifying Tregs can be complicated.

For example—What are the best markers to use?  How do you know for sure that you are measuring true suppressor T cells?

Gating Strategies For Defining Tregs By Flow Cytometry

The standard Treg gating strategy for both mouse and human samples (after first gating out doublets and gating on live cells) includes the antigens CD3, CD4, CD25, FOXP3, and CD127.

When looking solely at antigen expression, Tregs are often defined as CD3+, CD4+, CD25hi, FOXP3+, and CD127lo (shown in the figure below as Option 1). Using these markers, a clear population is often visible from samples such as mouse splenocytes and human PBMC.

However, activated T cells often up-regulate CD25, and FOXP3 expression has been found on ‘effector’ (non-suppressive) T cell lineages. Therefore, when relying on flow cytometry phenotyping alone to define Tregs, inflammatory T cells could be a wolf in sheep’s (Treg’s) clothing and lead to incorrect data interpretation.

A cell may look like a duck, but does it quack? Measurement of the effector functions of your possible Treg population will greatly help elucidate the accuracy of your flow gating strategy. In order to determine if the cells you are defining as Tregs functionally resemble them, Option 2 (see below) includes omitting FOXP3 from your panel, sorting CD3+, CD4+, CD25hi, CD127lo cells, then determining the functions of your ‘Treg’ population via cytokine analysis and/or suppression co-culture assays with non-Treg T cells (CD3+ CD4+ CD25-, CD127hi). Typically FOXP3 cannot be included in panels where viable cells are required post-sort as intracellular staining is required.

tregs by flow cytometry - t-regulatory cells gating

Defining The Increasing Variety Of Treg Subsets

There are many flavors of Tregs, including tTregs, pTregs, and iTregs.

For example, tTregs (also known as nTregs) are generated in the thymus and have a TcR repertoire that is biased towards self-peptides. Another flavor, known as pTregs, are generated in the periphery, and iTregs are induced in culture via TGF-b.

There are makers associated with these various Treg subsets and they should be considered for inclusion in a Treg anti body panel if subsetting them is of interest. For instance, in humans, CD39 is considered a reliable tTreg marker. Also, in both mice and humans, Helios has been found to reliably distinguish tTregs from the p, and iTreg subsets.

Defining A Single Cell As A Treg—Is It Possible?

A major limitation in the Treg field is the lack of a single cell suppression assay.

Defining an individual T cell as a member of a distinct memory lineage, such as Th1, Th2, or Th17, can be accomplished via analyte analysis with single cell resolution such as intracellular cytokine staining, as these cells are primarily if not exclusively defined by what cytokines they produce.

However, to show a single cell is a Treg we ideally want to be able to quantify that this one chosen cell can suppress the function of effector T cells (or other cell subsets) in co-culture. Currently the only way to test Treg suppressive function is in a bulk culture, where one can conclude that some (but not all, possibly not even most) of the cells designated as Tregs are suppressive.

treg cell chart

Thinking again of the potential ‘effector T cell’ wolves in sheep’s clothing, we just don’t know how many non-suppressive, even inflammatory cells are hiding in our Treg gating strategy. Using flow cytometry to first gate on and sort viable cells with markers consistent with Tregs, then functionally testing to see if, as a group, the cells defined by your gating strategy actually act like Tregs, is currently the best way to quantify Tregs in your sample.

By executing the right gating strategies for defining Tregs by flow cytometry and accounting for the growing number of Treg subsets, you can tease out your Treg populations of interest. The key is to test these populations’ functionally after identifying them because currently it is difficult if not impossible to define a single cell as a Treg. However, advances are being made daily and eventually labeling single Treg cells correctly will be possible.

To learn more about analyzing T cells and other cell types by flow cytometry, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

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Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

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