Gamma delta T-cell differentiation and effector function programming, TCR signal strength, when and how much?
Introduction
Since their serendipitous discovery some thirty years ago, γδ T-cells have emerged as an evolutionarily conserved lymphocyte subset with great functional range, varying based on the species, tissue, and immunological milieu [1], [2], [3], [4], [5], [6], [7]. The differential contribution to immunity by these cells is best illustrated by their variable abundance based on species and disease state. To this end, γδ T-cells can comprise between 60–80% of circulating CD3+ cells in livestock such as cattle, pigs, and sheep [8], and in humans γδ T-cells can rise from 2% to 60% of total CD3+ lymphocytes based on the immunological challenge [9]. In mice, γδ T-cells are shown to play a role in pathogen clearance, tissue repair, tumor surveillance, and immunoregulation, as well as in autoimmunity, allergy and carcinogenesis [4], [6], [10], [11], [12].
Perhaps the best-studied tissue that displays the contribution of γδ T-cells to both health and disease is the skin. To this end, γδ T-cells are known to be key players in down-regulating inflammation and mediating wound repair in the skin [11], [13], [14], [15] but have also been shown to exacerbate disease under different conditions such as experimentally-induced psoriasis, in which diminished accumulation of IL-17-producing γδ T-cells (γδ17) in the skin results in a decrease in inflammatory symptoms [16], [17], [18], [19]. Further deleterious contributions of γδ T-cells is observed in models of ischemic brain injury, experimental autoimmune encephalomyelitis (EAE), and collagen induced arthritis (CIA), where γδ17 cells are known to be major contributors of inflammation and associated disease pathology [20], [21], [22], [23], [24]. In addition to contributing to autoimmunity, γδ T-cells can also be deleterious in some models of breast and ovarian cancers, as their contribution to the inflammatory milieu and recruitment of other immune cells (such as small peritoneal macrophages in an ovarian cancer model), works directly against cancer immunosurveillance [25], [26]. Conversely, immune regulation by lung resident γδ T-cells is crucial for down regulating inflammation and inducing tissue repair in different models of infection or stress induced emphysema [27], [28], [29]. Finally, there exists extensive evidence of γδ17 involvement in mounting an effective immune response against pathogens including Staphylococcal aureus, Listeria monocytogenes, Escherichia coli, Bacillus subtilis, and Mycobacterium tuberculosis [20], [30], [31], [32], [33], [34]. Thus, it is clear γδ T-cells can serve as important players in both immunity and tissue homeostasis, but also contribute to immune dysfunction and inflammatory pathogenesis. With this in mind, it is important to better understand how γδ T-cells are first generated and how their differentiation within the thymus affects their final effector function.
Both γδ and αβ T-cells develop from a bipotent progenitor in the thymus following a process that involves the rearrangement of their namesake receptors through V(D)J recombination [35], [36]. In the mouse thymus, these CD4−, CD8− double negative (DN) progenitors first progress from a DN2 (CD44+ CD25+) towards a DN3 (CD44− CD25+) stage of development while undergoing events that lead to γδ or αβ lineage fate determination. Early explanations of the divergent development of these cells arose from an observation that γδ T-cells appeared in the mouse thymus prior to αβ T-cells, hence suggesting that the progenitor was poised to preferentially give rise to a γδ T-cell [37]. This timing dependent or stochastic model was directly opposed by the discovery of a γ gene silencer that resulted in normal αβ T-cell frequencies in γδ T-cell transgenic mice [38]. As the absence of this silencer led to reduced αβ T-cell frequencies, a second instructional model was proposed whereby the uninhibited expression of γδ TCR or pre-TCR (pTα/TCRβ) would directly result in development along the γδ or αβ lineage [38], [39]. Subsequent studies investigating this theory at a single cell level showed that DN3 progenitors expressing pre-TCR or γδ TCR retained the potential to progress towards either lineage, hence discounting the original instructional model [35], [40]. These findings among others lend direct support to a quantitative signal strength model, whereby the amount of signal accumulated downstream of the TCR would drive the differentiation of progenitors along either lineage [36], [41], [42], [43]. More specifically, the model suggests that a strong γδ TCR signal would result in higher activation of the ERK-MAPK pathway, leading to a higher induction of the Early Growth Response (EGR1, EGR3) transcription factors and their target Inhibitor of DNA binding 3 (Id3) [44], [45], [46]. Id proteins are direct inhibitors of E2A, a helix-loop-helix protein, which serves an important role in thymocyte development as a checkpoint regulator [47]. As proposed by the signal strength hypothesis, the higher accumulation of Id3 results in stronger inhibition of E2A, and thus expression of γδ T-cell hallmark genes. Conversely, weaker signals through the Notch receptors and pre-TCR would result in more modest accumulation of Id3, and hence weaker inhibition of E2A leading to further development along the αβ lineage [36].
Although it has been proposed that in some instances, the simple expression of the γδ TCR is sufficient to initiate the differentiation signals [48], [49], more recent work by our group and others points the possibility of an active role for the γδ TCR – ligand interaction during the development of at least some subsets with known ligands [40], [50]. Studies making use of RAG2−/− progenitors transduced with the transgenic KN6 γδ TCR suggest bipotency based on the strength of ligand engagement for DN3 progenitors expressing the same γδ TCR. KN6 is an example of a γδ T-cell that specifically binds non-classical MHC-I like molecules such that include T10 and T22 [51], [52], [53]. More specifically, following interaction with TCR weak ligand T10, a fair proportion of the transgenic KN6 γδ T-cells were shown to develop towards the αβ lineage, whereas provision of a strong T22 ligand, resulted in a higher proportion of cells maturing as DN, CD24lo, CD73hi γδ T-cells [50]. In what can be deemed as further proof for a cumulative signal strength hypothesis, KN6 γδ T-cells that also expressed pre-TCR, were more adept at maturing along the γδ T-cell lineage when presented with T10, in comparison to progenitors only expressing the γδ TCR, suggesting that the signals through both the γδ and pre-TCR combine to deliver a stronger signal [50]. It can therefore be concluded that differences in signal strength dictate αβ versus γδ lineage choice through modulation of lineage specific transcription factors. It is then also likely that within these distinct lineages, the graded signals downstream of each TCR results in differential regulation of transcription factors essential for the functional maturation of effector subtypes (Fig. 1).
γδ T-cells are largely associated with the innate arm of the immune system thanks to their rapid cytokine response and their preferred persistence in mucosal tissues. While this association has often called into question the functionality of the γδ TCR, recent findings provide support for the relevance of ligand engagement in the functional maturation and activity of at least a group of these cells. Among other works, this is supported by evidence showing that differential signal accumulation downstream of the TCR can directly affect IFNγ production by in vitro-derived KN6 γδ T-cells [50]. This association is further supported by earlier publications that show Id3 induction is required for cytokine secretion, lineage specific gene expression, and overall functional competence of several γδ T-cell subtypes.
Arguably, the most widely accepted and clear surface marker to delineate γδ T-cell cytokine profile in both mouse and human subsets has been the TNF superfamily member CD27, with CD27hi cells representing IFNγ producers, and CD27lo cells containing the γδ17 T-cell subsets [54], [55], [56], [57]. Although the low expression of this marker is commonly associated with antigen naïve cells, γδ T-cells that express IL-17 following TCR engagement of the model antigen phycoerythrin (PE), are found to be CD27− CD44+ CD62lo, hence necessitating further clarification of the link between CD27 expression, TCR signals, and cytokine production [58], [59]. More recently, Wencker et al. showed that dampening of the γδ TCR signaling components led to a near depletion of the CD27− γδ17 population in both adult and neonates, insinuating that the γδ TCR is at the very least essential to the development, if not effector function, of the γδ17 subset [60]. These findings are contradictory to earlier work by Jensen et al., who analyzed T22-tetramer binding γδ T-cells from wild type or β2m−/− (MHC-Ib ligand deficient) mice, and concluded that only antigen-naïve T22-tetramer binding γδ T-cells from β2m−/− mice were able to produce IL-17, whereas antigen-experienced γδ T-cells produced IFNγ [61]. While this work further suggests the importance of γδ TCR signal strength in determining functional differentiation for IFNγ producing γδ T-cells, it does not rule out the possibility of other environmental cues leading to the differentiation of γδ17 cells in the abnormal β2m−/− thymic environment. Therefore, a controlled system whereby the binding strength of the γδ TCR ligand can be directly modulated and measured would serve as an ideal model for understanding the role of the γδ TCR in determining their functional differentiation. This system would also help elucidate the role of γδ TCR signal strength in IL-4 production, a topic for which most of the current literature focuses on NKT like γδ T-cell subsets that paradoxically require TCR cross-linking but not Id3 expression for IL-4 production [62], [63]. While one means of achieving this is using the KN6 transgenic model for which a weak and strong ligand have been defined, another method may involve mutating the CDR3 region of a γδ TCR with a known ligand, and assessing effector maturation as a function of binding affinity. These experiments will be important in defining a mechanism of how differential TCR signals are translated and integrated into the effector programming of γδ T-cell subsets.
The transcriptional programs that enable γδ T-cells to recognize pathogens and respond to them in a context-specific manner are set during intrathymic development, resulting in the generation of IFNγ or IL-17 producing γδ T cells. These functional programs can be linked to expression of different Vγ TCR chains. The transcription factors Sox13 and RORγt (Rorc) are essential for IL-17 producing γδ T-cells, including Vγ6+ and some Vγ4+ T cells, while Eomes and Tbet are hallmarks of IFNγ producing subtypes, such as Vγ5+ dendritic epidermal T-cells [20], [64], [65]. A recent study showed that thymic mature Vγ4+ T cells from Sox13-deficient mice exhibit a block in IL-17 production and RORγt expression [65], suggesting that Sox13 is upstream of RORγt in the gene network that drives IL-17 expression. However, despite recent studies that provided insights into the transcriptional network that drives installment of functional programs in γδ T-cell subsets [65], [66], [67], there is still much to be understood.
Current models suggest that interplay of Notch signals and TCR signals drives development as well as functional programming of γδ T-cells. Specifically, a combination of strong Notch signals and weak TCR signals leads to the development of IL-17 producing cells, whereas strong TCR signals result in the development of IFNγ producing γδ T-cells [68], [69], [70]. One of the results of strong TCR signaling is the activation of RAS-ERK-MAP kinase cascade, which ultimately leads to up-regulation of Id3, which then acts as the dominant negative inhibitor of the activity of E proteins E2A, HEBCan and HEBAlt [71]. E protein activity is necessary to turn on RORγt, a master regulator of IL-17 production in αβ lineage Th17 cells, and may be involved in γδ T-cell RORγt regulation as well [72]. Furthermore, recent studies have provided evidence that E proteins and Id factors are involved in directing precursors towards expression of certain Vγ chains and functional phenotypes associated with them [73]. Specifically, temporal rearrangement of TCRγ genes during fetal and adult life has been shown to be regulated by E2A and Id factors [46], [74]. E2A deficient mice have decreased Vγ4 and increased Vγ5 gene arrangement in adult thymus, suggesting that E2A activates Vγ4 rearrangement and inhibits Vγ5 rearrangement during adult life [74]. Id3 deficient mice, by contrast, have an increased number of Vγ1+ T-cells, which are double producers of IFNγ and IL-4 [46]. These studies together suggest that E proteins may play a role in directing precursors towards IL-17 producing γδ T-cells and Id factors in driving them towards IFNγ producers both by directing TCR expression and by modulating the evens that occur downstream of the TCR signals. Therefore, studies that directly investigate the role of E proteins and Id factors in functional programming of γδ T-cells will provide further insights into the complex network of TCR signaling and transcription factors that allow these cells to acquire and exert their functions.
Section snippets
Restricted temporal developmental of γδ17 T-cells?
γδ T-cells with associated Vγ usages develop in coordinated waves beginning from fetal life. In mice, Vγ5+ cells constitute the first wave, arising from approximately embryonic day 12 (E12) to E16, followed by Vγ6+ cells from E14 to birth, Vγ4+ cells from E16 onward, and Vγ1+ cells from E18 onward [75]. The tissue and functional specificities associated with these Vγ subsets, potentiates the theory that functional subsets of γδ T cells similarly develop in coordinated waves during fetal life.
Concluding remarks
In sum, it can be said that effective functional differentiation of γδ T-cells is fine-tuned by the integration of signals from the γδ-TCR, and those arising from other environmental cues, such as cytokine and Notch receptor signals. Noting that the functional differentiation of these cells is the product of multiple environmental and intrinsic cues, which are in turn potentially under temporal control, an in vitro system enables an ideal setting where the function of individual contributors of
Acknowledgments
We are thankful to Dr. Geneve Awong for her expert flow cytometry assistance. This work was supported by the Canadian Institutes of Health Research (MOP# 42387) and the National Institutes of Health Grant P01AI102853. JCZP is supported by a Canada Research Chair in Developmental Immunology. PZ is supported by an Ontario Graduate Scholarship.
References (79)
Structure, organization, and somatic rearrangement of T cell gamma genes
Cell
(1985)Pivotal role of dermal IL-17-producing gammadelta T cells in skin inflammation
Immunity
(2011)Interleukin-1 and IL-23 induce innate IL-17 production from gammadelta T cells, amplifying Th17 responses and autoimmunity
Immunity
(2009)Gammadelta T cells enhance autoimmunity by restraining regulatory T cell responses via an interleukin-23-dependent mechanism
Immunity
(2010)Tumor-infiltrating gammadelta T cells suppress T and dendritic cell function via mechanisms controlled by a unique toll-like receptor signaling pathway
Immunity
(2007)Stage-specific and differential notch dependency at the alphabeta and gammadelta T lineage bifurcation
Immunity
(2006)- et al.
Gammadelta and alphabeta T cell lineage choice: resolution by a stronger sense of being
Semin. Immunol.
(2010) Attenuation of gammadeltaTCR signaling efficiently diverts thymocytes to the alphabeta lineage
Immunity
(2005)- et al.
Distinct structure and signaling potential of the gamma delta TCR complex
Immunity
(2002) - et al.
TCR signal strength influences alphabeta/gammadelta lineage fate
Immunity
(2005)
Marked induction of the helix-loop-helix protein Id3 promotes the gammadelta T cell fate and renders their functional maturation Notch independent
Immunity
GammadeltaTCR ligands and lineage commitment
Semin. Immunol.
Gammadelta T cells recognize a microbial encoded B cell antigen to initiate a rapid antigen-specific interleukin-17 response
Immunity
Thymic selection determines gammadelta T cell effector fate: antigen-naive cells make interleukin-17 and antigen-experienced cells make interferon gamma
Immunity
Notch-Hes1 pathway is required for the development of IL-17-producing gammadelta T cells
Blood
Skint-1 identifies a common molecular mechanism for the development of interferon-gamma-secreting versus interleukin-17-secreting gammadelta T cells
Immunity
Interplay between RORgammat, Egr3, and E proteins controls proliferation in response to pre-TCR signals
Immunity
Development of interleukin-17-producing gammadelta T cells is restricted to a functional embryonic wave
Immunity
The natural and the inducible: interleukin (IL)-17-producing gammadelta T cells
Trends Immunol.
Complete primary structure of a heterodimeric T-cell receptor deduced from cDNA sequences
Nature
Immunosurveillance by gammadelta T cells: focus on the murine system
Chem. Immunol. Allergy
Six-of-the-best: unique contributions of gammadelta T cells to immunology
Nat. Rev. Immunol.
Molecular mechanisms of differentiation of murine pro-inflammatory gammadelta T Cell subsets
Front. Immunol.
Gammadelta T cells: first line of defense and beyond
Annu. Rev. Immunol.
Evolutionary implications of a third lymphocyte lineage in lampreys
Nature
The bovine model for elucidating the role of gammadelta T cells in controlling infectious diseases of importance to cattle and humans
Mol. Immunol.
Nonpeptide antigens, presentation mechanisms, and immunological memory of human Vgamma2Vdelta2 T cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens
Immunol. Rev.
Gammadelta T cell effector functions: a blend of innate programming and acquired plasticity
Nat. Rev. Immunol.
Skin gammadelta T-cell functions in homeostasis and wound healing
Immunol. Rev.
Gammadelta T-cell receptors: functional correlations
Immunol. Rev.
Gammadelta T cell-induced hyaluronan production by epithelial cells regulates inflammation
J. Exp. Med.
A keratinocyte-responsive gamma delta TCR is necessary for dendritic epidermal T cell activation by damaged keratinocytes and maintenance in the epidermis
J. Immunol.
A role for human skin-resident T cells in wound healing
J. Exp. Med.
Differential developmental requirement and peripheral regulation for dermal Vgamma4 and Vgamma6T17 cells in health and inflammation
Nat. Commun.
Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis
J. Immunol.
Rorgammat+ innate lymphocytes and gammadelta T cells initiate psoriasiform plaque formation in mice
J. Clin. Invest.
Pivotal role of cerebral interleukin-17-producing gammadeltaT cells in the delayed phase of ischemic brain injury
Nat. Med.
Exacerbation of collagen-induced arthritis by oligoclonal, IL-17-producing gamma delta T cells
J. Immunol.
Gamma/delta T cells are the predominant source of interleukin-17 in affected joints in collagen-induced arthritis, but not in rheumatoid arthritis
Arthritis Rheum.
Cited by (33)
Development of γδ T cells in the thymus – A human perspective
2022, Seminars in ImmunologyCitation Excerpt :To further develop these technologies, a detailed understanding of human γδ T cell differentiation and maturation is crucial. Many exceptional reviews on the development of γδ T cells in the mouse thymus have been published [11–20], however, insights into this process in the human context are still more limited. This gap is not surprising given the experimental limitations in studying human samples and the elegant ways in which mouse models and perturbation approaches have been able to advance the field.
Ontogenic timing, T cell receptor signal strength, and Notch signaling direct γδ T cell functional differentiation in vivo
2021, Cell ReportsCitation Excerpt :T cell commitment occurs at the CD4−CD8− double-negative (DN) 3 stage of T cell development, when cells bifurcate into the αβ or γδ lineage (Ciofani et al., 2006; Wong and Zúñiga-Pflücker, 2010). Both T cell lineages can secrete interferon γ (IFNγ), interleukin-4 (IL-4), or IL-17 (In et al., 2017; Parker and Ciofani, 2020; Zarin et al., 2015). However, γδ T cells are distinct in that ontogenic timing is thought to influence the generation of specific Vγ repertoires and functional subsets (Carding and Egan, 2002; Prinz et al., 2013).
γδTCR-independent origin of neonatal γδ T cells prewired for IL-17 production
2019, Current Opinion in ImmunologyCitation Excerpt :If the stochastic model is correct, precision in terminology was expected to come from the identification of the actual deterministic molecular process, which may be probabilistic or directed. To what extent TCR type or different strength of TCR signaling dictates T cell lineage specification in otherwise homogeneous progenitors continues to be debated [2,3]. But this question is predominantly considered from a framework where data from TCR signaling studies have been interpreted to demonstrate a deterministic role in γδ versus αβ T cell lineage commitment and effector subset specification.
Homeostatic γδ T Cell Contents Are Preserved by Granulocyte Colony-Stimulating Factor Priming and Correlate with the Early Recovery of γδ T Cell Subsets after Haploidentical Hematopoietic Stem Cell Transplantation
2018, Biology of Blood and Marrow TransplantationCitation Excerpt :γδ T cells are the first identified functional population of peripheral T lymphocytes that normally account for 1% to 10% of circulating T cells in humans. Because of the MHC-independent effector feature, γδ T cells are so-called innate-like immune cells that can directly kill target cells, produce molecules required for pathogen clearance, and release immunomodulatory cytokines [9,10]. IFN-γ and IL-17 are the 2 major cytokines secreted by γδ T cells for immune responses [11,12].
Nano-technology based carriers for nitrogen-containing bisphosphonates delivery as sensitisers of γδ T cells for anticancer immunotherapy
2017, Advanced Drug Delivery ReviewsCitation Excerpt :They are implicated in the immune response to infectious diseases, autoimmune disease and tumour surveillance [80]. They are associated with the innate function of the immune system as they have a rapid cytokine response and mainly reside in mucosal tissues [86]. They share effector functions with alpha beta (αβ) T cells and natural killer (NK) cells [87,88].
Thymic Determinants of γδ T Cell Differentiation
2017, Trends in Immunology