Skip to content

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

Written by Jennifer Snyder-Cappione, PhD

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.

Tim Bushnell, PhD

BOOKS

Advanced Microscopy

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.
flow cytometry tablet eBook cover

Modern Flow Cytometry

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more. 

Advanced 4-10 Color Compensation

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.

Top 40 Networking Scripts For PhDs

If you want to get replies from top employers and recruiters, this ebook is for you. These networking scripts will show you the exact words ...

Informational Interviews For PhDs

If you want to learn how to set up and execute informational interviews with PhDs working in industry, this ebook is for you. Here, you ...

Industry Resume Guide For PhDs

If you have a PhD and want to create the perfect industry resume to attract employers, this ebook is for you. Here, you will get ...

Top 20 Industry Jobs For PhDs

If you want to learn about the top 20 industry careers for PhDs regardless of your PhD background, this ebook is for you. Here, you ...

Salary Negotiation For PhDs

If you have a PhD and want to learn advanced salary negotiation strategies, this ebook is for you. Here, you will learn how to set ...

Top 20 Transferable Skills For PhDs

If you want to learn the top 20 transferable skills the industry employers ranked as most important for PhDs to include on their resumes and ...

Related Posts You Might Like

6 Areas Of Consideration For Flow Cytometry Cell Cycle Analysis

Written by Tim Bushnell, PhD As discussed previously, cell cycle assays require optimization of fixation and dye concentrations, but that is just the beginning. There ...
Read More

Why Cell Cycle Analysis Details Are Critical In Flow Cytometry

Written by Tim Bushnell, PhD The lifecycle of a cell can be described in stages. In diploid cells, much of the time they exist in ...
Read More

How To Choose The Correct Antibody For Accurate Flow Cytometry Results

Written by Tim Bushnell, PhD Next to the flow cytometer itself, the most important component of a flow cytometry experiment comes down to the antibodies. ...
Read More

5 Essential Beads For Flow Cytometry Experiments

Written by Tim Bushnell, PhD Flow cytometry is designed to measure physical and biochemical characteristics of cells and cell-like particles using fluorescence. Fundamentally, any single-particle ...
Read More

How To Use Flow Cytometry To Measure Apoptosis, Necrosis, and Autophagy

Written by Tim Bushnell, PhD “If one approaches a problem with order and method there should be no difficulty in solving it — none whatever.” ...
Read More

Flow Cytometry Protocols To Prevent Sample Clumping

Written By Tim Bushnell, PhD Good flow cytometry depends on a high-quality, single cell suspension. If the cells put through the instrument are not of ...
Read More