A Guide to Natural Killer T Cell Markers

Natural killer T (NKT) cells are a type of T cell that express both αβ T cell receptors (TCR) and natural killer (NK) cell surface markers and recognize peptides presented by CD1d, an MHC class I-like molecule. Through their secretion of cytokines, NKT cells play a critical role in several different immune responses, such as tumor surveillance and immunity, host defense against microbial pathogens, and the regulation of autoimmune disease. In addition, they can also promote either T helper 1 (Th1) responses or T helper 2 (Th2) responses.  Understanding this population of T cells, how different NKT cells cross-communicate, and which factors influence NKT-promoted Th1 or Th2 responses can help drive the development of targeted immunotherapies for a variety of conditions. Moreover, the non-polymorphic nature of CD1d means transferred NKT cells will not cause graft-versus-host disease. Several NKT subtypes exist and can be differentiated based on a combination of surface markers, transcription factors, and secreted cytokines. 

General Characteristics of NKTs

Similar to T cells, NKT cells develop from CD4-/CD8- T progenitor cells. In both humans and mice, NKT cells may be broadly subdivided into CD4+ and CD4- subsets; additionally, a population of CD4-/CD8- NKTs, called double negative (DN) cells have been identified. In both humans and mice, DN cells have higher NK cell potential. Four subsets of DN cells have been identified in mice: DN1 (CD44+/CD25-), DN2 (CD44+/CD25+), DN3 (CD44-/CD25-), and DN4 (CD44-/CD25+) which also differ in their level of NK1.1 expression (highest in DN1, lowest in DN4). A subset of rodent CD44+ DN NKTs also express CD3 and IL-7Rα.

Figure: This diagram highlights how NKT cells can be categorized into Type I or Type II subsets on the basis of CD1d antigen binding. Common markers associated with Type I NKT cell subtypes are also illustrated. 

NKT cells can be split into two types based on the ligands recognized by their TCR. Type I NKT cells, also known as invariant NKTs (iNKTs), express a semi-invariant TCR that recognizes the lipid α-galactosylceramide (α-GalCer). Meanwhile, the type II NKTs possess a diverse TCR that recognizes a variety of lipids. Type I NKTs have been more extensively studied than Type II NKTs and are very well described in the literature. Both types can be either CD4+ or DN. Surface markers for general NKT cell identification include NK1.1, CD122, Ly49G2, and CD49b in rodents, and CD69 and CXCR3 in both rodents and humans.

Markers of Type I NKTs (iNKTs)

Human iNKTs

In humans, iNKTs are identified by the semi-invariant Vα24-Jα18 TCR and may be further identified by the expression of the NK cell lineage marker CD161. The study of iNKTs from peripheral blood lymphocytes (PBLs) in healthy humans is difficult because these cells are present at less than one NKT cell per thousand PBLs; however, their successful isolation has been achieved with a method combining α-GalCer-loaded CD1d tetramers and anti-Vα24 monoclonal antibodies.

A variety of surface marker expression and cytokine secretion patterns have been observed across human CD4+ and DN iNKTs. CD4+ cells produce more IL-4 and IL-13 than DN cells, and the IL-2R alpha chain (CD25) is exclusively produced by CD4+ cells. Expression of the chemokine receptors CCR5, CCR6 and CXCR6 are higher on DN NKTs, while expression of CD49a is higher on CD4+ cells. NK lineage receptors are more often expressed by DN cells. 2B4 (CD244), CD94, and NKG2A are nearly exclusive to DN cells. CD161, while expressed by both, is much higher on DN cells.

There are some similarities between CD4+ and DN human NKTs as well. Both secrete similar levels of the Th1 cytokines TNF-α and IFN-γ and both express similar levels of the chemokine receptors and integrins CCR1 (low), CCR2 (low), CXCR3 (variable), CXCR4 (variable), CCR4 (high), CCR7 (low), CD49d (high), CD49e (high), and CD49f (high). The NK lineage marker CD56 is also highly expressed by both CD4+ and DN NKTs, as are the costimulatory receptors CD28, CD45RB, and CD69.

Human NKTs have also been observed to differentiate into follicular helper (FH) NKTs, which are CD4+ cells that express CXCR5, PD-1, and the transcription factor BCL-6.

Rodent iNKTs

In mice, iNKTs are identified by the semi-invariant Vα14-Jα18 TCR and may be further identified by the expression of the NK cell lineage markers NK1.1, CD49b, and CD56. In the thymus, rodent iNKTs differentiate in the thymus into three distinct subsets–NKT1, NKT2, and NKT17–depending on the type of immune response they elicit: Th1, Th2, and T helper 17 (Th17), respectively. Two additional iNKT subsets that appear to differentiate after exit from the thymus have also been described: NKT10 (adipose tissue) and NKTFH cells (spleen; also observed in human NKTs). Of note, different inbred strains of mice contain different ratios of iNKT subsets, which should be taken into account when designing experiments.

NKT1 cells may be either CD4+ or CD4- and have a cytokine profile similar to that of Th1 and group 1 innate lymphocyte cells (ILCs). They are characterized by their high expression of the transcription factor T-bet and secretion of IFN-γ, and, sometimes, IL-4, upon activation. Surface markers expressed by NKT1 cells include CD49a, CD122, CD27, and CXCR3.

NKT2 cells are CD4+ cells that resemble Th2 cells and can be identified by their expression of the transcription factors PLZF and GATA-3, expression of the surface markers ICOS and CD27, and secretion of IL-4, IL-5, and IL-13. A subset of CD4+ cells with a Th2-like cytokine secretion profile (IL-13 and other cytokines) and surface IL-17RB expression has also been observed.

NKT17 cells are CD4- cells with a cytokine secretion profile similar to that of Th17 cells (IL-17, IL-22, and TNF-α) and can be identified by expression of the transcription factors PLZF and RORγt and expression of the surface markers ICOS, Syndecan, CCR6, CD103, and NRP1.

NKT10 cells, as their name implies, secrete IL-10 (as well as IL-2) upon activation and can be identified by expression of the E4BP4 transcription factor. Like NKT17 cells, they also express the cell surface marker NRP1, in addition to PD1 and CD49d.

As in humans, rodent NKTFH cells are CD4+ cells marked by expression of the transcription factor BCL-6 and the surface markers CXCR5 and PD1. They also secrete IL-21.

Markers of Type II NKTs

While iNKTs are more frequent in mice, in humans type II NKTs are the more frequent cell population. Nevertheless, due to the lack of specific surface markers and the fact that ligand/CD1d tetramer reagents recognize only a small number of surface markers, the study of type II NKTs is difficult, limited to small subsets, and often done indirectly. One way to identify type II NKTs is by CD1d reactivity plus the absence of the Vα14 (rodent) or Vα24 (human) TCR α-chains. Studies have also shown that type II NKTs react to self-lipids, including sulfatide, as well as multiple ligands of microbial origin. Other studies have revealed similar surface marker expression patterns as different iNKT subsets, including PLZF, CD44, CD122, and CD69.

Table of NKT Markers

The table below lists human and rodent markers of NKTs as described by recent literature. Accompanying each marker are links to relevant antibodies and ELISA kits, as these immunodetection tools are routinely used in cell characterization studies via flow cytometry and immunostaining. The associated products are offered by a variety of manufacturers and can serve as a useful reference for isolating and differentiating NKTs.

Note: *Some markers are protein isoforms, multi-subunit protein complexes, or protein families composed of several distinct genes. **These markers are not individual proteins. Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org (for human genes only). 

References

1. Lee PT, Benlagha K, Teyton L et al. Distinct Functional Lineages of Human Vα24 Natural Killer T Cells. J Exp Med. 2002; 195(5):637-641. doi: 10.1084/jem.20011908

2. Crosby CM and Kronenberg M. Tissue-specific functions of invariant natural killer T cells. Nat Rev Immunol. 2018; 18(9):559-574. doi: 10.1038/s41577-018-0034-2

3. Singh AK, Rhost S, Löfbom L et al. Defining a novel subset of CD1d‐dependent type II natural killer T cells using natural killer cell‐associated markers. Scand J Immunol. 2019; 90(3):e12794. doi: 10.1111/sji.12794

4. Lee YJ, Holzapfel KL, Zhu J et al. Steady state production of IL-4 modulates immunity in different strains and is determined by lineage diversity of iNKT cells. Nat Immunol. 2013; 14(11):10.1038/ni.2731. doi: 10.1038/ni.2731

5. Terashima A, Watarai H, Inoue S et al. A novel subset of mouse NKT cells bearing the IL-17 receptor B responds to IL-25 and contributes to airway hyperreactivity. J Exp Med. 2008; 205(12):2727-2733. doi: 10.1084/jem.20080698

6. Hu Z, Gu W, Wei Y et al. NKT Cells in Mice Originate from Cytoplasmic CD3-Positive, CD4−CD8− Double-Negative Thymocytes that Express CD44 and IL-7Rα. Sci Rep. 2019; 9(1):1874. doi: 0.1038/s41598-018-37811-0

7. Coquet JM, Chakravarti S, Kyparissoudis K et al. Diverse cytokine production by NKT cell subsets and identification of an IL-17–producing CD4−NK1.1− NKT cell population. Proc Natl Acad Sci USA. 2008; 105(32):11287-11292. doi: 10.1073/pnas.0801631105

8. Chang PP, Barral P, Fitch J et al. Identification of Bcl-6-dependent follicular helper NKT cells that provide cognate help for B cell responses. Nat Immunol. 2011; 13(1):3-43. doi: doi: 10.1038/ni.2166

9. Singh AK, Tripathi P, and Cardell SL. Type II NKT Cells: An Elusive Population With Immunoregulatory Properties. Front Immunol. 2018;9:1969. doi:10.3389/fimmu.2018.01969