Review
Human gamma delta T cells: Evolution and ligand recognition

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Highlights

  • We review work on the evolution of primate V, D and J gene segments of γδ T cells.

  • We also review CD1d-lipid recognition of Vδ1+ γδ and αβ T cells.

  • Lastly we review recent work on phosphoantigen modulation of Vγ9Vδ2 T cells.

Abstract

The γδ T cell lineage in humans remains much of an enigma due to the low number of defined antigens, the non-canonical ways in which these cells respond to their environment and difficulty in tracking this population in vivo. In this review, we survey a comparative evolutionary analysis of the primate V, D and J gene segments and contrast these findings with recent progress in defining antigen recognition by different populations of γδ T cells in humans. Signatures of both purifying and diversifying selection at the Vδ and Vγ gene loci are placed into context of Vδ1+ γδ T cell recognition of CD1d presenting different lipids, and Vγ 9Vδ2 T cell modulation by pyrophosphate-based phosphoantigens through the butyrophilins BTN3A. From this comparison, it is clear that co-evolution between γδ TCRs and these ligands is likely occurring, but the diversity inherent in these recombined receptors is an important feature in ligand surveillance.

Introduction

There are three main lineages of lymphocytes in jawed vertebrates that use genetically recombined receptors to survey their environment and mediate host defense against disease: B cells, αβ T cells and γδ T cells. Similar, analogous lineages have been found in jawless vertebrates that use an entirely different family of receptors for surveillance [1]. The best studied of the T cell lineages are those expressing an αβ TCR; within this lineage we know most about those αβ T cells that are often described as “conventional”; those that express either CD4 or CD8 co-receptors and interact with the classical Major Histocompatibility Complex (MHC) molecules presenting peptides (MHCp). These T cells are generally categorized as either “helper CD4+ T cells”, secreting cytokines to modulate other immune cells, or are “killer CD8+ T cells”, exhibiting cytotoxicity towards the target cell(s). Less well understood are the specialized populations of αβ T cells that recognize non-peptide presenting MHC molecules and are often found at high frequencies in particular tissues or organs. Many of these αβ T cells express T cell receptors that have low variation, using only a small sampling of the variable gene segments available during somatic rearrangement. These semi-invariant populations include invariant Natural Killer T (iNKT) cells and Mucosal Associated Invariant T (MAIT) cells.

Even more enigmatic are the cells that express a γδ TCR; despite decades of research we still have little information regarding how many defined γδ populations exist, what antigens/ligands they respond to and what roles they play in host defense and homeostasis. It is unclear whether we can extrapolate what we know from the study of αβ T cell populations to that of the γδ lineage; are there “classical” αβ T cell equivalents that recognize diverse antigens in polymorphic antigen-presenting molecules? Are there semi-invariant γδ T cell populations restricted to non-polymorphic antigen-presenting molecules? Do γδ T cells use their recombined receptors to recognize antigen in an antibody-like fashion? This review focuses on the evolutionary pressures that have shaped the diversity of the γ and δ gene segments in primates (in relation to humans) and extrapolates this to the limited information we have on the molecular recognition of Vδ1+ and Vδ2+ human γδ T cells.

In humans, γδ T cells have a small repertoire of V gene segments to select from when undergoing chain rearrangement in comparison with those available for Vα (43–45 [2]), Vβ (40–48 [3]), Ig light (IgVκ 34–38 [4], IgVλ 29–33 [5]), or Ig heavy (38–46 [6]) chain rearrangement . Three main Vδ gene segments, Vδ1, Vδ2 and Vδ3, are most frequently used in rearrangement of the δ chain; less commonly used are the five V segments that have both Vδ and Vα designation (Vδ4/TRAV14, Vδ5/TRAV29, Vδ6/TRAV23, Vδ7/TRAV36 and Vδ8/TRAV38) [7]). This designation is in part due to the location of the δ locus within the α locus on chromosome 14 in humans. Seven functional Vγ gene segments, Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9 and Vγ11, located within the γ locus on chromosome 7 in humans, are used for rearrangement of the γ chain. Several Vγ pseudogenes are also found in the γ locus in humans and are not used in productively arranged γδ TCRs: Vγ1, Vγ5P, Vγ6, Vγ7 and Vγ10. As discussed in more detail below, these do not all appear to be pseudogenes in other higher primates and at least one (Vγ10) has been found in productively rearranged γ chain transcripts in the chimpanzee [8]. The restricted repertoire of Vδ and Vγ gene segments available for rearrangement has led to speculation that these TCRs recognize conserved self-proteins of low variability [9]. This is further supported by the observation of particular Vδ and Vγ pairing requirements in the mouse [10] although pairing biases have not been experimentally observed in humans. This idea of limited γδ TCR diversity was confounded by the discovery that δ chain rearrangement allows for the incorporation of multiple Dδ segments (in forward and reverse direction) [11], [12] such that the loop encoded by this rearrangement, the CDR3δ, is theoretically the most diverse, in sequence and in length, CDR3 loop of all the rearranged receptors [13]. Indeed, more recent discussion has compared this feature of the CDR3δ loop to antibodies [14], along with the demonstration of “antibody-like” recognition of foreign proteins by some γδ TCRs [15].

Comparisons between the δ and γ V, D and J gene segments within the primate species has revealed an interesting pattern of how these loci have evolved [16] (Fig. 1, upper left panel). The gene segments of the δ locus have not changed substantially in the primate lineage from humans to marmosets; the gene order, overall, is conserved and most of the genes remain functional with few duplications or deletions. This holds true for the δ locus V gene segments as well as the D and J gene segments, which are located between the Vδ2 and Vδ3 gene segments. This can be further visualized through dot-plot comparisons of the entire locus; where the full length genomic sequences are compared across species and black dots indicate regions of high sequence identity (>90%) and gray, slightly lower (>80%) and displayed on a dot-matrix [16]. For the δ locus, the dot matrix reveals a prominent diagonal line, indicating high correlation between the gene organization and sequence from human to marmoset (Fig. 1, lower panel). Regions of deletion or insertion are visualized as small breaks or gaps in the diagonal line.

In contrast, the pattern of gene organization across primate species is quite different at the γ locus. Three Vγ gene segments, Vγ9, Vγ10 and Vγ11 (these were previously designated as groups II, III and IV, respectively [17]) are found in highly conserved positions in each of the primate genomic sequences examined, with the exception of Vγ11, which is a pseudogene in macaque and is missing from the marmoset genome [16]. The remaining group 1 Vγ genes (Vγ1–Vγ8) cluster together. Surprisingly, the positioning and sequence homology of this group of genes quickly diverge in the genomic sequences of even the most closely related species to humans, the great apes (Fig. 1, upper right panel). For example, the Vγ5P pseudogene is only present in humans and, in the orangutan, it is difficult to assign homology to human Vγ5, Vγ3, Vγ4 and Vγ2 (they are thus designated Vγ3/5, Vγ5/3 and Vγ4/2). Dot plot analysis reveals the close sequence homology between the group 1 Vγ sequences, which is the product of the gene duplications, deletions and/or genetic exchange between them that has occurred recently in primate evolution [16] (Fig. 1, lower panel). Phylogenetic analysis of the sequences of the V gene segments [16] reflect the conclusions derived from the dot plot analysis: the Vδ gene segments group together with long branch lengths (reflecting evolutionary distance) and strong statistical support (bootstrapping analysis), in some cases including homologs from mouse (Fig. 2, left panel). The phylogenetic tree of the Vγ gene segments (Fig. 2, right panel) reflect the dichotomy observed in the genomic organization across species; the Vγ9, Vγ10 and Vγ11 sequences group together with well-supported, long branch lengths similar to those of Vδ, whereas the group 1 Vγ sequences form a bush-like structural grouping, containing subgroups within consisting of the Vγ1, Vγ2/4, Vγ3/5, Vγ6, Vγ7 and Vγ8 sequences with very short branch lengths.

Evolutionary comparisons such as these provide insight into the selective pressures that shape genes or gene loci. Gene duplication, early on, was recognized as an ideal form of adaptive evolution [18] and has been widely observed in genes that participate in an organism’s adaptation to a quickly changing environment. The highly polymorphic class I genes of the human MHC, HLA-A, -B and -C [19] have also been the product of frequent duplication and deletion, such that conservation of these genes is lost, similar to that of the group 1 Vγ gene segments, the further out in primate evolution one explores [20]. The question that arises, then, is what has driven the rapid evolution of these group 1 gene segments during primate evolution? Why is this region so dynamic, where as the Vγ9, Vγ10 and Vγ11 gene segments, located only ∼10 kilobases away, and the Vδ gene segments have remained so static? These intriguing patterns of evolution are most relevant when placed in the context of antigen recognition. While we are making progress on defining antigens for γδ T cells in humans (see [14] for a comprehensive review of known antigens), unfortunately only a few of these have been successfully explored at the structural level. Below we focus on two of the three major Vδ domains, Vδ1 and Vδ2, and the progress that has been made thus far in understanding antigen recognition by the T cells that utilize these domains in their TCRs. First, we will focus on recognition of the MHC-like protein CD1d by Vδ1+ T cells (both γδ and αβ [21]) and then will turn to the recent progress on understanding the modulation of the Vγ9Vδ2 T cell population by small pyrophosphate containing organic molecules called phosphoantigens.

Section snippets

Vδ1+ T cell recognition of CD1d

γδ T cells expressing a Vδ1 domain paired with various γ chains represent more than 50% of fetal blood γδ T cells at birth [22]. In adults, Vδ1+ γδ T cells constitute a minority of the blood γδ T cell population and instead mainly populate epithelial tissues, to a large extent the intestine [23], and are also found responsive to epithelial tumors [24], [25], [26] and lymphomas [27], [28]. Vδ1+ γδ T cells have been reported to recognize several different members of the MHC superfamily family [29]

Vγ9Vδ2 T cell activation by phosphoantigens

Vγ9Vδ2 T cells are characterized by the expression of a TCR comprised of a Vγ9 domain paired with a Vδ2 domain and reactivity to small, organic based pyrophosphate molecules [55], [56], [57], [58] (Fig. 5). They are the major subset of γδ T cells in human blood and can comprise between 1% and 10% of total blood T cells in healthy humans [59]. These cells are also found at high frequency in the gut, liver and other mucosal tissues [60], [61], [62], [63]. Vγ9Vδ2 T cells respond potently to

Acknowledgment

The authors would like to thank Caitlin Castro and Viola Nawrocka for helpful discussion and help with figures. This work was supported by the National Institutes of Health Grants to E.J. Adams: R01_AI073922 and R01_AI115471.

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