What are invariant natural killer T (iNKT) and mucosal-associated invariant T (MAIT) cells?

Invariant natural killer T cells (iNKT cells) and mucosal-associated invariant T cells (MAIT) cells are T cell populations which recognize and respond to non-peptide antigens using a limited TCR repertoire. For a comprehensive understanding of iNKT and MAIT cells, refer to the detailed review by Mori, et al (Mori, Lepore, and De Libero in the 2016 Annual Review of Immunology).

HLA-like molecules restrict iNKT and MAIT cells

A cursory understanding of antigen presentation to T cells is necessary to understand iNKT and MAIT cells. Most T cells recognize peptide epitopes presented in the groove of human leukocyte antigen (HLA, also known as major histocompatibility complex, MHC). A subset of homologous, class I HLA-like proteins can also present non-peptide or peptide hybrid antigens. One such example is the CD1 family, which consists of CD1a-CD1e (CD1d1 and CD1d2 in mice). While CD1 associates with B₂-microglobulin like HLA class I molecules, it is targeted to vesicles like HLA class II molecules. CD1 family members have a hydrophobic channel that enables binding to hydrocarbon alkyl chains. By anchoring these alkyl chains inwards, CD1 molecules expose carbohydrate and hydrophilic groups of lipids to immune cells (see the table below).

Most T cells restricted by CD1 are thought to be CD4-CD8-, though some CD4+ T cells are known to bind CD1. Of these CD1-binding cells, a subset binds CD1d and its lipid antigens; these T cells express the natural killer cell marker KLRB1C and an invariant TCRα that can pair with multiple TCRβ chains. Hence, these T cells are called invariant natural killer T cells, or iNKT cells. iNKT cells are thought to comprise 0.01 to 0.1% of T cells in the blood.

MAIT cells were discovered initially in 1993 as a subset of CD4-CD8- TCRT cells expressing the CD8αα homodimer with an invariant TCRα V gene repertoire. MAIT cells typically comprise 5-10% of T cells in the blood, can express either CD4 or CD8, and can comprise 10-50% of T cells in the spleen, gut, lungs, skin, and other organs. The ligands of MAIT cells were unknown until 2012. A second HLA class I-like molecule, MHC-related protein 1 (MR1), is also capable of displaying non-classical ligands to T cells, such as folate derivatives and B vitamin metabolites produced by many microbial species. While initial work described MR1 as a potent activator of MAIT cells, the microbial metabolites presented by MR1 were identified instead by researchers attempting to co-crystallize a MAIT TCR and MR1. MR1 could only be successfully folded in media with B vitamins, but this 6-formyl pterin molecule that bound and stabilized MR1 could not activate MAIT cells. Instead, cultures from S. typhimurium and other bacteria both stabilized MR1 and activated MAIT cells. Recent efforts have led to the successful development of MR1 tetramers that can specifically identify antigen-specific MAIT populations.

Note that in addition to iNKT and MAIT cells, there are other T cell subtypes that are not restricted by HLA or MHC, such as germline-encoded mycolyl lipid-reactive (GEM), NKT, and LDN5-like T cells. Cell Ranger does not annotate these cell types.

The restricted diversity of the TRA chain and published CDR3α and CDR3β sequences can be used to identify MAIT/iNKT cells from full length annotated contigs in the 10x V(D)J data.

Users interested in knowing which MAIT and iNKT TCRs are used to annotate for junctional evidence can locate and request changes to our junction list here.

Note: In humans, MAIT cell frequencies dwarf those of iNKT cells (MAIT cell frequency in peripheral blood and certain tissues can be more than 100-fold greater than that of iNKT cells), while in laboratory mouse strains, the opposite is true. Due to the abundance of iNKT cells in mice and the earlier development of MAIT cell analysis reagents, iNKT cells have been more extensively studied in mice rather than humans compared to MAIT cells.

Cell Ranger labels cells as potential iNKT and MAIT cells based on a combination of 1) matching the appropriate V and J genes for the TCR alpha, 2) matching the appropriate V genes for the TCR beta, and 3) matching known V(D)J junctions (CDR3s) from the literature.

Consider the following example label for a given clonotype in the MAIT evidence column of clonotypes.csv: TRA:gene+junction;TRB:gene. This indicates that the TCR alpha genes for that clonotype match the known TRAV and TRAJ gene criteria for MAIT cells (TRA:gene), that the TCR alpha CDR3 matches that of a known MAIT TCR (TRA:junction), and that the TCR beta V gene matches the criterion for MAIT cells (TRB:gene).

These labels are stored in the Cell Ranger outs folder in clonotypes.csv. This filter is deliberately flexible to accommodate new CDR3 junctions that Cell Ranger users may discover. If you believe you have found new MAIT or iNKT junctions, please contact [email protected].