A Guide to Naïve T Cell Markers

There are numerous classes and subclasses of T cells, including helper and cytotoxic T cells, as well as different stages of differentiation. Naïve T cells, defined by their co-receptor markers, are generally found among the helper or cytotoxic class but have not yet been activated to differentiate into memory or effector cells. Naïve T cells remain on standby in order to respond to new pathogens or substances. Thus, the maintenance of their population is critical for the overall function of the immune system. Techniques like flow cytometry are the workhorse of immune cell characterization, with a multiparameter labeling strategy being effective at distinguishing distinct T cell classes and maturation states. This guide summarizes markers commonly used to identify naïve T cells and distinguish them from other T cell maturation states.

T Cell Maturation

Developing in the thymus from progenitor cells, most immature T cells express the αβ form of the T cell receptor (TCR) along with the co-receptors CD4 and CD8. As they mature, T cells continue to express either CD4 or CD8, classifying them as CD4 or CD8 T cells. In addition, a small population of T cells (~1% of total) expressing neither CD4 nor CD8 exist in circulation, referred to as double negative (DN) T cells.¹ The different T cells play critical roles in various aspects of immunity, such as tumor surveillance, defense against pathogens, and immune response regulation. 

This diagram highlights common markers for naive T cells prior to activation into mature CD4 and CD8 T cells.

CD4 and CD8 T cells differentiate from the naïve state into their helper and effector lineages. While the maturation process differs slightly between CD4 and CD8, the complements of surface, nuclear, and secreted proteins change as they mature from naïve T cells into stem cell memory, central memory, effector memory, and effector cells.^1–3 

CD4 T cells—also known as helper T cells—orchestrate adaptive immunity by releasing cytokines that coordinate the immune response. CD4 T cells have TCRs that mature with interactions with major histocompatibility complex (MHC) class II.^4,5 CD8 T cells, on the other hand, are cytotoxic with direct cell-killing capability and have TCRs that mature with MHC class I interactions.^4,5 

Numerous subclasses of CD4 and CD8 T cells have been discovered in recent years, including the CD4 T cell subtypes Th1, Th2, Th9, Th17, Th22; CD8 T cell subtypes Tc1, Tc2, Tc9, Tc17; and CD4 or CD8 regulatory T cells (Tregs).^1,2 These subclasses have distinct roles in immunity, including tissue localization, homing capability, lifespan, and specific pathogen defense.^1,2,5 CD4 and CD8 naïve T cells await activation, at which point they are poised to differentiate into these specific subclasses depending on the cytokine environment. 

Markers of Naïve T Cells

T cells are identified primarily by the presence of the TCR, CD3, and either CD4 or CD8. A combination strategy is needed to distinguish naïve T cells from other T cell types, using the presence and absence of specific markers. Transcription factors, such as FOXP1 are involved in maintaining the naïve state, and as such can be used as positive markers for naïve T cells.^1,6

While intracellular proteins such as FOXP1, ZEB2, and TBX21 can be used as markers,¹ workflows in immunology typically use flow cytometry with surface protein labeling to simultaneously identify multiple populations of T cells. 

During maturation, T cells lose expression of certain proteins, such as CD27. Combinations of positive markers have been used to take advantage of this, such as using the presence of CD45RA, CCR7, CD27, and CD28 on CD8 T cells to identify naïve cells, with the presence of fewer than all four of these markers to identify more mature cells.³ Approaches using positive and negative markers are more common,^1,2 such as using a CD27⁺/CD45RO^- combination to identify naïve CD4 and CD8 T cells.⁷ Naïve CD4 and CD8 T cells have also been identified using the combinations of CD45RA⁺/CD127⁺/CD25^-/CD45RO^- and CD45RA⁺/CD62L⁺/CCR7⁺/CD45RO^-, respectively.²

Table of Naïve T Cell Markers

The table below lists T cell markers used to distinguish naïve T cells based on review literature. The list includes largely cell surface proteins, but also intracellular proteins and secreted proteins that can be detected by using exocytosis inhibitors such as brefeldin A or monensin. Identifying naïve T cells requires a combination approach and cannot be reliably performed using a single marker. Accompanying each marker are links to relevant antibodies and ELISA kits that can be used to detect naïve T cells in vitro and in vivo. The associated products are offered by a variety of manufacturers and can serve as a useful reference for T cell characterization.

Note: *Some markers are protein isoforms or multi-subunit protein complexes. Information on Protein Type and Localization obtained from UniProt.org (for human genes only). 

References

1. Mousset CM, Hobo W, Woestenenk R, Preijers F, Dolstra H, van der Waart AB. Comprehensive Phenotyping of T Cells Using Flow Cytometry. Cytometry A. 2019;95(6):647-654. doi:10.1002/cyto.a.23724

2. Golubovskaya V, Wu L. Different subsets of T cells, memory, effector functions, and CAR-T immunotherapy. Cancers (Basel). 2016;8(3):36. doi:10.3390/cancers8030036

3. Vrisekoop N, den Braber I, de Boer AB, et al. Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool. Proc Natl Acad Sci U S A. 2008;105(16):6115-6120. doi:10.1073/pnas.0709713105

4. Hutten TJA, Norde WJ, Woestenenk R, et al. Increased coexpression of PD-1, TIGIT, and KLRG-1 on tumor-reactive CD8+ T cells during relapse after allogeneic stem cell transplantation. Biology of Blood and Marrow Transplantation. 2018;24(4):666-677. doi:10.1016/j.bbmt.2017.11.027

6. Durek P, Nordström K, Gasparoni G, et al. Epigenomic profiling of human CD4+ T cells supports a linear differentiation model and highlights molecular regulators of memory development. Immunity. 2016;45(5):1148-1161. doi:10.1016/j.immuni.2016.10.022

7. Kumar BV, Connors T, Farber DL. Human T cell development, localization, and function throughout life. Immunity. 2018;48(2):202-213. doi:10.1016/j.immuni.2018.01.007