GEM T cells: A newly identified class of restricted α-chain TCRα/β T cells

The diversity of the T cell repertoire allows for recognition of a wide diversity of pathogens. During T cell development, T cell receptors (TCRs) undergo genetic rearrangements of their V, D, and J segments, as well as random deletions and nontemplated additions of nucleotides.  Furthermore, major histocompatibility complex (MHC) class I and II molecules are highly polymorphic.  Thus, each person has a unique and highly diverse T cell-MHC repertoire.  In addition, there are two known classes of lymphocytes with restricted diversity of their TCR α-chains, and which bind to the non/rarely polymorphic antigen-presenting molecule families CD1 and MR1.  These are the invariant natural killer T cells (iNKT cells), and the mucosa-associated invariant T cells (MAIT cells), respectively.  In the June issue of Nature Immunology, Van Rhijn et al. identify a new class of T cells with restricted TCR α-chains, termed GEM T cells, that recognize the Mycobacterium tuberculosis (Mtb) lipid glucose monomycolate presented on CD1b.

To study the human TCR repertoire recognizing CD1b, Van Rhijn et al., utilized CD1b tetramers loaded with glucose monomycolate (GMM), to isolate and clone T cells from peripheral blood mononuclear cells (PBMC) of Mtb infected donors.  Two groups of T cell clones with differing avidity for CD1b-GMM were isolated from each patient, differentiated by intermediate (CD1bint) and high (CD1bhi) CD1b tetramer staining intensities.  CD1bint T cells were diverse in their TCR α-chain sequences.  TCR α-chains of CD1bhi T cells however, all utilized the same variable and joining sequences (TRAV1-2, and TRAJ9, respectively) with few nontemplated additions, resulting in a specific complementarity-determining region 3 (CDR3) consensus sequence.  Thus, these were termed “germline-encoded, mycolyl lipid–reactive” (GEM) T cells.  These TCR α-chain sequences furthermore had to be paired with specific TCR β-chain sequences in order to recognize CD1b-GMM complexes.

Other properties of these uniquely identified GEM T cells included expression of CD4 and production of IFNγ and TNFα upon activation, two cytokines important for anti-mycobacterial responses.  GEM T cells expressed various rates of CD161, a marker widely expressed by NKT cells and MAITs, and thus GEM T cells could not be defined by expression of CD161.  In addition, sorting of TRAV1-2+ CD4+ cells from two healthy donors followed by deep sequencing of the TCR α-chain revealed identification of the GEM-specific CDR3 sequence, demonstrating that GEM T cells were present in Mtb uninfected individuals in the naïve T cell repertoire.  However, these cells become clonally expanded in Mtb infected patients, and thus likely to contribute to anti-mycobacterial immune responses.

In conclusion, GEM T cells are a newly identified third class of CD1-recognizing T cells with restricted TCR α-chain sequences.  These cells arise via VDJ recombination, and indicate that special selection mechanisms exist to generate T cells bearing this specific TCR α-chain.  Although what CD1b-self antigen complex could positively select for these cells in the thymus is unknown.  Furthermore, the role these cells play during mycobacterial infections will be an interesting avenue for future studies.

Further Reading:

A conserved human T cell population targets mycobacterial antigens presented by CD1b.  Van Rhijn I, Kasmar A, de Jong A, Gras S, Bhati M, Doorenspleet ME, de Vries N, Godfrey DI, Altman JD, de Jager W, Rossjohn J, Moody DB. Nat Immunol. 2013 Jun 2;14(7):706-13.

A ‘GEM’ of a cell.  Mitchell Kronenberg & Dirk M Zajonc. Nature Immunology 14, 694–695 (2013) doi:10.1038/ni.2644. Published online 18 June 2013.

Types of immune cells present in human PBMC

When peripheral whole blood is drawn for human immune system studies, it is often processed to remove red blood cells by density gradient centrifugation. Most commonly this method uses Ficoll Paque, a solution of high molecular weight sucrose polymers, a product of GE Healthcare Ltd.  Ficoll separates whole blood into two fractions above and below the density of 1.077g/ml.

Peripheral blood mononuclear cells (PBMC) are the populations of immune cells that remain at the less dense, upper interface of the Ficoll layer, often referred to as the buffy coat and are the cells collected when the  Ficoll fractionation method is used.

Erythrocytes (red blood cells) and polymorphonuclear cells (PMNs) which include neutrophils and eosinophils are generally removed during this fractionation as they are denser then 1.077g/ml.  Basophils, however can be greater or less dense then 1.077g/ml and thus may be present to a small degree in the less dense PBMC fraction.

PBMCs include lymphocytes (T cellsB cells, and NK cells), monocytes, and dendritic cells.  In humans, the frequencies of these populations vary across individuals.  In my experience as well as that of others, lymphocytes are typically in the range of 70 – 90% of PBMCs, monocytes range from 10 – 30% of PBMCs, while dendritic cells are rare, being only 1 – 2% of PBMCs.  The frequencies of cell types within the lymphocyte population include 70 – 85% CD3+ T cells (45 – 70% of PBMC), 5 – 20% B cells (up to 15% of PBMC), and 5 – 20% NK cells (up to 15% of PBMC).

The CD3+ compartment is composed of CD4 (25 – 60% of PBMC) and CD8 T cells (5 – 30% of PBMC), in a roughly 2:1 ratio.  Both CD4 and CD8 T cells can be further subsetted into naïve, and the antigen-experienced central memory, effector memory, and effector subtypes that exist in resting or activated states.  Multiple markers can be used to identify these compartments to varying similarities and thus the frequencies reported by people using different markers may vary.

CD4 T cells are known as helper T cells and can be further classified into various functional subtypes based on the expression profiles of specific cytokines, surface markers, or transcription factors.  These include regulatory T cells, TH1, TH2, and TH17 cells as well as other described subpopulations such as TH9, follicular helper, and TR1 types.  These classifications however will certainly become more complex in the future, as recently the cytotoxic CD8 T cell compartment has been to shown to be extremely heterogenous in marker expression and function and may be comprised of roughly 200 functional phenotypes.

Dendritic cells 2

Circulating B cells include transitional, naïve, and memory subtypes as well as plasmablasts, all of which can be found at varying populations in peripheral blood.  Circulating dendritic cells include plasmacytoid dendritic cells as well as myeloid derived dendritic cells.  Circulating monocytes have been described as either being classical monocytes or nonclassical CD16+ proinflammatory monocytes, which comprise up to 10% of the monocytes in peripheral blood and have unique functions compared with classical monocytes.

Human immune system studies rely heavily on the phenotypic and functional assessments of PBMCs.  In order to take advantage of PBMCs for human immune studies, it is important to know what populations are represented in peripheral blood and how PBMC populations differ in distribution and function from tissue immune cells.  Finally it is critical to become familiar with the identifying surface and intracellular markers and the types of assays best suited for human PBMC studies.  The markers most suitable for identification of the major immune populations in human PBMC using flow cytometry will be the topic of the next blog.