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How To Differentiate T Cell State With Flow Cytometry

Written by Jennifer Snyder-Cappione, PhD

Have you ever wondered what has gotten into your T cells?

Are they activated, resting, tolerant, senescent, anergic, exhausted, or maybe just having an off day?

During short-lived immune responses, such as responses to acute infections or vaccines, there are three classically defined T cell states:

  1. naïve,
  2. activated memory, and
  3. resting memory.

Here’s how to determine whether your T cells are naive, activated or resting memory, or exhausted, and how effector function plays into that.

1. Naïve cells.

Naïve T cells have not encountered an antigen and secrete IL-2 and some chemokines. However, antigen-experienced (memory) T cells, have differentiated into effector cells that can produce cytokines besides IL-2, such as IFN-g, IL-4, and IL-17.

Markers used to identify naïve T cells include CD45RA and CD62L in human and mouse samples, respectively, with CD45RO (human) and CD44 (mouse) present on memory T cell populations.

It is best to use functional profiling as the primary determinant of T cell state in these instances, with surface markers as supporting evidence. Therefore, defining naïve T cells by their inability to exert effector functions such as IFN-g, IL-4, and IL-17 in response to either specific antigens or other stimulations (e.g. mitogens, anti-CD3/28 antibodies) is recommended.

T Cell state differentation during chronic disease

2. Memory cells.

T cells that express CD45RO/CD44 (human/mouse) and do exert effector cytokines can be defined as memory cells. To distinguish between an activated and resting memory state, a key difference is their polyfunctionality ex vivo, with true resting memory cells typically able to secrete a panoply of cytokines, and activated cells not necessarily able to do the same.

Also, activated cells will likely express some activation markers (CD69, CD25, etc.) and show evidence of active proliferation (Ki67+, for example) that may be absent on a true resting memory T cell.

Flow cytometry T Cell differentation phenotype function

During chronic diseases, such as HIV infection, cancer, and autoimmunity, it becomes increasingly difficult to define the state or “mood” of the T cell.

T cells, (like human beings) are significantly influenced by their environment and, with the complexities of cytokine milieus and other dynamic influences in chronic inflammatory states, figuring out the exact state of the T cell can be challenging.

Lower functional capacity of T Cells

3. Exhausted cells and the “others”.

There is much ado of late in immunology about “exhausted” or “checkpoint inhibited” T cells.

Exhausted T cells are believed to have progressively lost their effector function capabilities due to prolonged antigenic exposure.

Exhausted cells have a distinct molecular signature from naïve and traditional effector cells, and they are often defined via hypo-responsiveness to stimulation ex vivo and expression of inhibitory receptors (IRs), such as PD-1, TIM-3, and CTLA-4, with individual T cells expressing multiple IRs possibly suggesting of a more exhausted cell.

However, activated T cells can also express IRs, so it’s best to define IR+ hypo-responsive cells as ‘bearing a phenotype resembling exhaustion’ and if one wants to define the exhaustion phenotype more definitively, comprehensive molecular signatures should likely be measured.

Other terms to define memory T cell states of lower functional capacity include anergy, tolerance, and senescence.

Anergic cells are classically defined as non-responsive due to insufficient priming in vitro. Similarly, tolerant T cells are also improperly primed as they are self-reactive.

Senescent T cells are associated with aging and characterized via shortened telomeres and expression of CD57.

To date, the lines distinguishing senescence from exhaustion are blurred.

In summary, it’s suggested that you define your T cell population of interest by its functional profile (1st), surface marker expression (2nd), and finally take into account what you know about the cells (for how long have they been in an inflammatory, antigen-rich environment?). From this combined information, speculate as best you can on the T cell population’s status. Until T cell differentiation and effector function regulation is better understood, we can only make reasonable guesses as to the state of many T cell populations.

To learn more about how to differentiate T Cell state with 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


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