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Immunosurveillance and Immunoregulation by γδ T Cells

  • Michael Girardi
    Correspondence
    Associate Professor and Residency Director, Department of Dermatology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520-8059, USA
    Affiliations
    Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut, USA
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      Whereas the vast majority of T cells express a T-cell receptor (TCR) composed of αβ heterodimers, a smaller population expresses a γδ TCR. In contrast to αβ T cells, γδ T cells show less TCR diversity, are particularly enriched at epithelial surfaces and appear to respond to self-molecules that signal potential danger or cellular stress. In addition, various subsets of γδ T cells have shown antitumor and immunoregulatory activities. This review considers what has been discovered about the important cutaneous functions of γδ T cells through the study of mutant mice and offers perspectives on the roles of γδ T cells in human disease.

      Abbreviations:

      CDR3
      complementarity-determining region 3
      D
      diversity (region)
      DETC
      dendritic epidermal T cell
      IEL
      intraepithelial lymphocyte
      J
      junctional (region)
      MHC
      major histocompatibility complex
      MICA
      major histocompatibility complex class I chain-related A
      MICB
      major histocompatibility complex class I chain-related B
      NK
      natural killer
      NKT
      natural killer T (cell)
      Rae-1
      retinoic acid early-1
      V
      variable (region).
      The coordinated responses of several different lymphoid subsets are critical to the protection of the host from various outside challenges. Epithelial surfaces, including the skin, serve as selective barriers where the immune system first encounters tissue-damaging radiation, toxins, mutagens, and various microorganisms. Studies in mice and humans have advanced the understanding of the integrated responses of T cells — including the major subsets defined by their TCR usage, αβ and γδ — and their relationship to other lymphoid cells, such as natural killer (NK) and natural killer T (NKT) cells. Parallel to the mechanisms that enable protective immune effects, misdirected and/or excessive activities of lymphoid cells may result in autoimmune or inflammatory disease states. Fortunately, cells with a regulatory function help to maintain immunologic balance. It is within this context that the crucial roles played by γδ T cells are increasingly being understood, an effort aided in large part by studies with mice in which the TCRδ locus has been genetically disrupted (that is, TCRδ−/− knockout mice) and no γδ T cells are present. Studies of skin cancer and contact dermatitis in these and other genetic variants have shed new light on the functions of γδ T cells, their relationship to other lymphoid cells, and the implications of such for the understanding of immune surveillance in human skin. Likewise, studies of murine γδ T cells have elucidated their similarities and differences relative to the more populous αβ T cells (Table 1).
      Table 1Comparison of αβ and γδ T cells in mice
      Featureαβ T cellsγδ T cells
      CD4 and CD8 phenotypeMajor subsetting based on CD4 or CD8 expressionPredominantly CD4CD8 (double-negative); murine intestinal IELs may be CD8αα+
      Antigen type and presentationPeptide antigen in the grooves of MHC-I or -II; primary responses require antigen-presenting cellIdentification of TCR ligands incomplete; β2 microglobulin independent; some subsets recognize MHC-Ib molecules
      T helper functionsPredominantly CD4+; T helper 1 and 2 cytokine profilesT helper 1 and 2 cytokine profiles
      T cytotoxic functionsPredominantly CD8+; e.g., perforin/granzyme production, Fas ligand-mediated, NKG2D-mediatedVarious subsets using the same mechanisms
      T regulatory functionsVarious T regulatory subsets including CD4+CD25+ cellsAttributable to various subsets, including murine Vγ5+ DETCs and human Vγ1+ peripheral cells
      TCR junctional diversityRelatively vastRelatively limited; especially limited for IEL populations
      Abbreviations: DETC, dendritic epidermal T cell; IEL, intraepithelial lymphocyte; MHC, major histocompatibility complex.
      For all four TCR loci (α, β, γ, and δ), the recombination of variable (V), diversity (D; for β and δ chains), and junctional (J) region sequence elements (Table 2) generates a vast degree of TCR diversity (reviewed in
      • Janeway C.A.
      • Travers P.
      • Walport M.
      • Schlomchick M.
      ), similar to the generation of B-cell antibody diversity via recombination of heavy and light chains. Within the area of the V(D)J joins — what is referred to as the complementarity-determining region 3 (CDR3) — additional variability may be introduced via random nucleotide insertion and/or deletion. It is the CDR3 that encodes the hypervariable TCR loops of antigen recognition. Though the basic heterodimeric structure of αβ and γδ TCRs is similar, there are several key differences in their array of genetic elements, the nature of their recombination, and their ultimate diversity. For example, there are far more V elements that may be utilized in α and β gene rearrangements; nevertheless, γδ TCRs have a greater potential diversity because of their capacity to use multiple tandem copies of their D elements. A detailed analysis of CDR3 length (
      • Rock E.P.
      • Sibbald P.R.
      • Davis M.M.
      • Chien Y.H.
      CDR3 length in antigen-specific immune receptors.
      ) revealed that TCR α- and β-chain distributions are highly constrained, doubtless because of structural requirements enabling binding and recognition of antigenic peptide within major histocompatibility complex (MHC) molecules. In contrast, TCRδ chains are highly variable in length, resembling antibody heavy chain in this manner. This finding is consistent with the understanding that γδ TCRs have antigen recognition properties fundamentally different from those of αβ TCRs and likely bind their respective antigens in a fashion similar to that of antibodies (that is, independent of MHC presentation, and in a fashion more dependent on conformational shape of intact protein or non-protein compounds) (Table 3).
      Table 2TCR V(D)J gene segments of αβ and γδ T cells
      Total number of genes that are available for recombination for both mouse and human TCRs.
      γδ Segmentsαβ Segments
      MouseHumanMouseHuman
      1132341
      236161
      24
      3130
      7522
      431414
      1 Total number of genes that are available for recombination for both mouse and human TCRs.
      Table 3Characteristics of γδ+ T-cell subsets as defined by TCR usage
      γ/δ Usage
      Vγ5Vδ1 is also known as Vγ3Vδ1; Vγ9Vδ2 is also known as Vγ2Vδ2 (for further clarification of TCRγδ nomenclature, see Kabelitz and Wesch, 2003).
      Subset characteristics
      See Huber et al., 2005; Dalton et al., 2004. Abbreviations: DETC, dendritic epidermal T cell; IEL, intraepithelial lymphocyte; MHC, major histocompatibility complex; MICA, MHC class I chain-related A.
      Murine subsets
      Vγ1Major peripheral recirculating γδ T cells of the blood and lymphatics
      Downregulatory function, such as autoimmune myocarditis (see also Vγ4)
      Subset capable of killing Listeri.-infected macrophages via Fas/Fas ligand, whereas other Vγ1 subset may be downregulatory
      Vγ2Recognizes non-classical MHC-Ib molecules, for example, T22, T10 (
      • Shin S.
      • El-Diwany R.
      • Schaffert S.
      • Adams E.J.
      • Garcia K.C.
      • Pereira P.
      • et al.
      Antigen recognition determinants of γδ T cell receptors.
      )
      Implicated in protection against lung mycobacteria
      Vγ4Pro-inflammatory functions, for example, autoimmune myocarditis
      Vγ5Vδ1
      Vγ5Vδ1 is also known as Vγ3Vδ1; Vγ9Vδ2 is also known as Vγ2Vδ2 (for further clarification of TCRγδ nomenclature, see Kabelitz and Wesch, 2003).
      Vast majority of the IELs of the skin, the prototypic DETCs
      First subset of T cells to emerge from the fetal thymus
      May lyse stressed/transformed keratinocytes via NKG2D ligands (for example, Rae-1)
      Downregulatory function for αβ T cell-mediated inflammation
      Vγ6Vδ1Vast majority of the IELs of the tongue and reproductive tract
      Vγ7Vast majority of the γδ+ IELs of the intestine (with Vδ4/Vδ5)
      Generated in the absence of a functional thymus
      Human subsets
      Vδ1Large component of the γδ+ IELs of the intestine
      Frequently found infiltrating tumors of epithelial origin
      May lyse stressed/transformed epithelial cells via recognition of NKG2D ligands (for example, MICA)
      Vγ9Vδ2
      Vγ5Vδ1 is also known as Vγ3Vδ1; Vγ9Vδ2 is also known as Vγ2Vδ2 (for further clarification of TCRγδ nomenclature, see Kabelitz and Wesch, 2003).
      Major peripheral recirculating γδ T cells of the blood and lymphatics
      Recognizes and expands in response to isoprenoids and alkylamines
      Has demonstrated cytotoxicity to a variety of cancer cell lines (for example, lymphoma, myeloma, prostate, renal)
      Kills both intracellular and extracellular Mycobacterium tuberculosi. organisms (
      • Dieli F.
      • Troye-Blomberg M.
      • Ivanyi J.
      • Fournie J.J.
      • Krensky A.M.
      • Bonneville M.
      • et al.
      Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by Vγ9/Vδ2 T lymphocytes.
      )
      May also function as professional antigen-presenting cells (
      • Brandes M.
      • Willimann K.
      • Moser B.
      Professional antigen-presentation function by human γδ T cells.
      )
      1 Vγ5Vδ1 is also known as Vγ3Vδ1; Vγ9Vδ2 is also known as Vγ2Vδ2 (for further clarification of TCRγδ nomenclature, see
      • Kabelitz D.
      • Wesch D.
      Features and functions of γδ T lymphocytes: focus on chemokines and their receptors.
      ).
      2 See
      • Huber S.A.
      • Born W.
      • O'Brien R.
      Dual functions of murine γδ cells in inflammation and autoimmunity in coxsackievirus B3-induced myocarditis: role of Vγ1+ and Vγ4+ cells.
      ;
      • Dalton J.E.
      • Howell G.
      • Pearson J.
      • Scott P.
      • Carding S.R.
      Fas-Fas ligand interactions are essential for the binding to and killing of activated macrophages by γδ T cells.
      . Abbreviations: DETC, dendritic epidermal T cell; IEL, intraepithelial lymphocyte; MHC, major histocompatibility complex; MICA, MHC class I chain-related A.

      Tissue Distribution and Major Subsets of γδ T Cells

      Most T cells express TCRαβ and either of the TCR-associated molecules CD4 and CD8 on their surface, but a minority (1–10%) demonstrates a TCRγδ+, predominantly CD4CD8 ‘double-negative’ phenotype. In the mouse, a substantial proportion of γδ T cells reside in the intraepithelial lymphocyte (IEL) compartments of skin, intestine, and genitourinary tract. Within the fetal thymus, precursors for these γδ T cells emerge in successive waves, guided to their respective tissues by the loss and gain of appropriate chemokine receptors. Vγ5Vδ1+ T cells are the first to leave the mouse fetal thymus and take residence in the suprabasal epidermis, forming a dendritic network that is unique among T cells but similar to that of Langerhans cells, the antigen-presenting cells of the epidermis. In physiologic states, these Vγ5+ dendritic epidermal T cells (DETCs) constitute more than 90% of the epidermal T cells, with virtually no TCR diversity (
      • Bergstresser P.R.
      • Sullivan S.
      • Streilein J.W.
      • Tigelaar R.E.
      Origin and function of Thy-1+ dendritic epidermal cells in mice.
      ). No phenotypically equivalent γδ+ IEL population has been identified in human epidermis, but γδ+ T cells of limited diversity and distinct from the peripheral blood γδ T-cell populations clearly reside within the dermis (
      • Holtmeier W.
      • Pfander M.
      • Hennemann A.
      • Zollner T.M.
      • Kaufmann R.
      • Caspary W.F.
      The TCR-δ repertoire in normal human skin is restricted and distinct from the TCR-δ repertoire in the peripheral blood.
      ). Characterization of these cells and their role in local immune responses awaits further investigation.
      Several studies have demonstrated that the TCR expressed by the IEL population directs the association with the particular epithelial tissue. For example, developing DETCs demonstrate a more diverse set of γδ TCRs in the neonatal period, and these become much more uniform over the first few weeks of life (
      • Tigelaar R.E.
      • Lewis J.M.
      Immunobiology of mouse dendritic epidermal T cells: a decade later, some answers, but still more questions.
      ). Eventually, in a manner analogous to that of Vγ5 DETCs, other γδ T-cell subsets of limited TCR diversity are restricted to other epithelial tissues (for example, Vγ6+ T cells in the genitourinary tract, Vγ7+ T cells in the intestinal tract) (
      • Itohara S.
      • Nakanishi N.
      • Kanagawa O.
      • Kubo R.
      • Tonegawa S.
      Monoclonal antibodies specific to native murine T-cell receptor γδ: analysis of γδ T cells during thymic ontogeny and in peripheral lymphoid organs.
      ;
      • Asarnow D.M.
      • Goodman T.
      • LeFrancois L.
      • Allison J.P.
      Distinct antigen receptor repertoires of two classes of murine epithelium-associated T cells.
      ). This strongly suggests that a set of corresponding self-ligands is present in fetal and/or neonatal epithelia. Although no such set of IEL TCR-specific epithelial ligands has yet been definitively identified, several investigators have suggested that they may be expressed within the fetal thymus (allowing positive selection), as well as within each fetal/neonatal epithelium (allowing homing, peripheral selection, and expansion), but subsequently only on stressed or dysregulated epithelial cells (
      • Janeway Jr, C.A.
      • Jones B.
      • Hayday A.
      Specificity and function of T cells bearing γδ receptors.
      ;
      • Allison J.P.
      • Havran W.L.
      The immunobiology of T cells with invariant gamma delta antigen receptors.
      ).
      This hypothesis would be consistent with the paradigm that γδ IELs play a large role in protection of epithelial barriers via monitoring and appropriate destruction of stressed (that is, dysregulated, metabolically compromised, and/or transformed) epithelial cells (
      • Pardoll D.M.
      Immunology. Stress, NK receptors, and immune surveillance.
      ). In the skin, DETCs may additionally promote wound healing, effectively protecting the barrier from microbial invasion and dissemination throughout the organism (
      • Jameson J.
      • Ugarte K.
      • Chen N.
      • Yachi P.
      • Fuchs E.
      • Boismenu R.
      • et al.
      A role for skin γδ T cells in wound repair.
      ). Additionally, γδ T cells may be important in helping to regulate inflammatory responses stimulated by αβ T cells, protecting against overly exuberant immune responses that might otherwise cause excessive tissue damage. Thus, an improved understanding of γδ T cells and their relationships to the skin has major implications for an improved understanding of cutaneous malignancy, infection, and inflammatory disease.
      In the human fetal thymus, the first γδ T cells to emerge use the Vδ1 chain (paired with various Vγ chains), and these will eventually preferentially populate epithelial tissues such as the intestine (
      • Hayday A.
      • Theodoridis E.
      • Ramsburg E.
      • Shires J.
      Intraepithelial lymphocytes: exploring the Third Way in immunology.
      ). Thus, whereas such Vδ1+ T cells constitute only a minor proportion of the γδ T cells present in human blood, they constitute a much larger proportion of the human IELs and have also been found to be enriched within various human epithelial tumors (for example, lung, kidney, and colon carcinomas) and lymphomas (
      • Fisch P.
      • Meuer E.
      • Pende D.
      • Rothenfusser S.
      • Viale O.
      • Kock S.
      • et al.
      Control of B cell lymphoma recognition via natural killer inhibitory receptors implies a role for human Vgamma9/Vdelta2 T cells in tumor immunity.
      ). Vδ1 T cells appear to recognize stressed cells via presentation of self-lipids by CD1 and/or expression of stress-induced MHC-Ib molecules. In contrast, Vγ9Vδ2 T cells (also referred to as Vγ2Vδ2; for nomenclature, see
      • Kabelitz D.
      • Wesch D.
      Features and functions of γδ T lymphocytes: focus on chemokines and their receptors.
      ) continually expand and take on a memory phenotype during childhood, presumably because of recurrent exposure to foreign agents, such that they eventually constitute approximately 80% of the γδ T cells of normal adult human blood. These cells will recognize, expand, and release cytokines in response to non-peptide compounds found across a spectrum of microbial pathogens as well as within mammalian cells.
      The immune system has classically been divided into two major arms: the more evolutionarily primitive innate immune system, where cell receptors and molecules may recognize and permit a rapid beneficial response to a variety of foreign agents, and the adaptive immune system, where B and T cells with antigen-specific receptors will produce a slower but more specific and coordinated initial immune response while also leading to expansion and a memory phenotype ready for subsequent challenges by the same antigen. One on hand, γδ T cells may be considered a component of the adaptive immune system in that they rearrange TCR genes to produce junctional diversity and will develop a memory phenotype. However, the various subsets may also be considered part of the innate immune system, where a restricted TCR may be used as a pattern recognition receptor. For example, according to this paradigm (
      • Kabelitz D.
      • Marischen L.
      • Oberg H.H.
      • Holtmeier W.
      • Wesch D.
      Epithelial defence by γδ T cells.
      ), large numbers of memory Vγ9Vδ2 T cells will respond within hours to common molecules produced by microbes, and highly restricted intraepithelial Vδ1 T cells will respond to stressed epithelial cells bearing sentinels of danger. Clearly, the complexity of γδ T-cell biology spans definitions of both innate and adaptive immune responses.

      Antimicrobial Immunosurveillance by γδ T Cells

      A range of studies have demonstrated a marked expansion of γδ T cells in the blood of systemically infected patients, including those with leprosy, tuberculosis, malaria, tularemia, salmonellosis, brucellosis, ehrlichiosis, or bacterial meningitis due to Haemophilus influenzae, Neisseria meningitidis, or Streptococcus pneumoniae (reviewed in
      • Chen Z.W.
      • Letvin N.L.
      Vγ2Vδ2+ T cells and anti-microbial immune responses.
      ). The broad recognition of the response may be the direct result of Vγ9Vδ2 stimulation by one of two major sets of shared non-peptide compounds: isopentenyl pyrophosphate and other intermediates of the mevalonate pathway; and alkylamines, non-phosphate compounds ubiquitously found in plants and bacteria (reviewed in
      • Kabelitz D.
      • Marischen L.
      • Oberg H.H.
      • Holtmeier W.
      • Wesch D.
      Epithelial defence by γδ T cells.
      ). In fact, the most potent natural stimulator of Vγ9Vδ2 T cells appears to be (E)-4-hydroxy-3-methylbut-2-enyl diphosphate, one of the precursors of isopentenyl pyrophosphate synthesis. Importantly, phosphoantigens are found expressed on many human tumor cells, possibly reflecting their state of raised metabolic stress, and will stimulate secretion of cytotoxic molecules by Vγ9Vδ2 cells (
      • Bonneville M.
      • Fournie J.J.
      Sensing cell stress and transformation through Vgamma9Vdelta2 T cell-mediated recognition of the isoprenoid pathway metabolites.
      ). Synthetic aminobisphosphonate compounds (for example, the drug pamidronate) also stimulate Vγ9Vδ2 cells, but this is more likely due to their stimulation of farnesyl diphosphate synthetase, which leads to accumulation of isopentenyl pyrophosphate. Studies are ongoing to identify potential immunostimulatory phosphoantigen drugs that might be therapeutic against malignancy (
      • Wilhelm M.
      • Kunzmann V.
      • Eckstein S.
      • Reimer P.
      • Weissinger F.
      • Ruediger T.
      • et al.
      γδ T cells for immune therapy of patients with lymphoid malignancies.
      ;
      • Lozupone F.
      • Pende D.
      • Burgio V.L.
      • Castelli C.
      • Spada M.
      • Venditti M.
      • et al.
      Effect of human natural killer and γδ T cells on the growth of human autologous melanoma xenografts in SCID mice.
      ;
      • Liu Z.
      • Guo B.L.
      • Gehrs B.C.
      • Nan L.
      • Lopez R.D.
      Ex vivo expanded human V[gamma]9V[delta]2+ [gamma][delta]-T cells mediate innate antitumor activity against human prostate cancer cells in vitro.
      ) and/or infection (
      • Wang L.
      • Kamath A.
      • Das H.
      • Li L.
      • Bukowski J.F.
      Antibacterial effect of human Vγ2Vδ2 T cells in vivo.
      ).
      Human peripheral Vγ9Vδ2 cells, after their exposure to foreign infectious agents or dying or metabolically stressed host cells (for example, in tumor states), may enhance other immune components as well. Through the rapid secretion of chemokines and T helper 1 cytokines such as IFN-γ, Vγ9Vδ2 cells may stimulate NK, NKT, and αβ T-cell functions (
      • Smith A.L.
      • Hayday A.C.
      An αβ T-cell-independent immunoprotective response towards gut coccidia is supported by γδ cells.
      ). Moreover,
      • Brandes M.
      • Willimann K.
      • Moser B.
      Professional antigen-presentation function by human γδ T cells.
      recently discovered that Vγ9Vδ2 cells can also function as professional antigen-presenting cells capable of ingesting, processing, and presenting peptide antigens to stimulate both CD4+ and CD8+ subsets of αβ T cells. These findings collectively describe a scenario whereby Vγ9Vδ2 cells may be very early responders to states of infection or host cellular dysregulation, providing direct cytotoxic effects, altering the local cytokine/chemokine milieu to facilitate other lymphoid cells and initiating antigen-specific αβ T-cell immune responses through their capacity to function as APCs much like dendritic cells (
      • Brandes M.
      • Willimann K.
      • Moser B.
      Professional antigen-presentation function by human γδ T cells.
      ).

      Antitumor Immunosurveillance by γδ T Cells

      Within human epithelial compartments, γδ T cells, most notably gut Vδ1+ T cells, may express surface NKG2D, a molecule found on two other major subsets of cells with cytotoxic potential, namely CD8+ αβ T cells and NK cells. Engagement of NKG2D by one of its several identified ligands, including MHC class I chain-related A and B (MICA and MICB) in humans (
      • Bauer S.
      • Groh V.
      • Wu J.
      • Steinle A.
      • Phillips J.H.
      • Lanier L.L.
      • et al.
      Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA.
      ) and retinoic acid early-1 (Rae-1) in mice (
      • Cerwenka A.
      • Bakker A.B.
      • McClanahan T.
      • Wagner J.
      • Wu J.
      • Phillips J.H.
      • et al.
      Retinoic acid early inducible genes define a ligand family for the activating NKG2D receptor in mice.
      ), provides a co-stimulatory function and targets cellular destruction. These molecules are upregulated under cellular stress and are expressed on a variety of tumor cells (
      • Groh V.
      • Rhinehart R.
      • Secrist H.
      • Bauer S.
      • Grabstein K.H.
      • Spies T.
      Broad tumor-associated expression and recognition by tumor-derived γδ T cells of MICA and MICB.
      ), including melanoma (
      • Vetter C.S.
      • Groh V.
      • thor Straten P.
      • Spies T.
      • Brocker E.B.
      • Becker J.C.
      Expression of stress-induced MHC class I related chain molecules on human melanoma.
      ). They may act as signals that target host cells for destruction by locally resident or infiltrating γδ T cells as well as other NKG2D+ lymphoid cells. When the prototypic γδ IELs of mouse skin, the Vγ5+ DETCs, are co-cultured with a keratinocyte tumor line expressing the NKG2D ligand Rae-1, the transformed cells are lysed, and such killing is dependent on TCR engagement (
      • Girardi M.
      • Oppenheim D.E.
      • Steele C.R.
      • Lewis J.M.
      • Glusac E.
      • Filler R.
      • et al.
      Regulation of cutaneous malignancy by γδ T cells.
      ).
      More recently, the relative contributions of αβ and γδ T cells to cutaneous tumor immunosurveillance have been systematically studied in mice (
      • Girardi M.
      • Oppenheim D.E.
      • Steele C.R.
      • Lewis J.M.
      • Glusac E.
      • Filler R.
      • et al.
      Regulation of cutaneous malignancy by γδ T cells.
      ;
      • Girardi M.
      • Glusac E.
      • Filler R.B.
      • Roberts S.J.
      • Propperova I.
      • Lewis J.
      • et al.
      The distinct contributions of murine T cell receptor (TCR)γδ+ and TCRαβ+ T cells to different stages of chemically induced skin cancer.
      ;
      • Gao Y.
      • Yang W.
      • Pan M.
      • Scully E.
      • Girardi M.
      • Augenlicht L.H.
      • et al.
      γδ T cells provide an early source of interferon γ in tumor immunity.
      ;
      • Girardi M.
      • Oppenheim D.
      • Lewis J.
      • Filler R.
      • Tigelaar R.E.
      • Hayday A.C.
      Characterizing the protective component of the αβ T cell response to transplantable squamous cell carcinoma.
      ). Intradermal injection of the mutagen methylcholanthrene can provoke the development of poorly differentiated fibrosarcomas and spindle-cell carcinomas. This occurs with shorter latency in TCRδ−/− mice. Similarly, tumors form more readily in γδ-deficient mice after injection of squamous-cell carcinoma (
      • Girardi M.
      • Oppenheim D.E.
      • Steele C.R.
      • Lewis J.M.
      • Glusac E.
      • Filler R.
      • et al.
      Regulation of cutaneous malignancy by γδ T cells.
      ) or melanoma (
      • Gao Y.
      • Yang W.
      • Pan M.
      • Scully E.
      • Girardi M.
      • Augenlicht L.H.
      • et al.
      γδ T cells provide an early source of interferon γ in tumor immunity.
      ) tumor cell lines. In addition to the potential of γδ T cells to directly lyse transformed cells in an NKG2D-dependent fashion, infiltrating γδ T cells have also been shown to produce IFN-γ early in tumor development (
      • Gao Y.
      • Yang W.
      • Pan M.
      • Scully E.
      • Girardi M.
      • Augenlicht L.H.
      • et al.
      γδ T cells provide an early source of interferon γ in tumor immunity.
      ).
      The concept that the different T-cell compartments may contribute to tumor surveillance in distinct fashions and at different stages of tumor growth has been further explored in the system of two-stage chemical carcinogenesis (where tumor development may be monitored after a single application of the mutagen 7,12-dimethylbenz[a]anthracene to the skin followed by repeated application of the tumor-promoting agent D12-O-tetradecanoylphorbol-13-acetate) (
      • Hennings H.
      • Glick A.B.
      • Lowry D.T.
      • Krsmanovic L.S.
      • Sly L.M.
      • Yuspa S.H.
      FVB/N mice: an inbred strain sensitive to the chemical induction of squamous cell carcinomas in the skin.
      ;
      • Owens D.M.
      • Wei S.
      • Smart R.C.
      A multihit, multistage model of chemical carcinogenesis.
      ). In this system, benign papillomas readily develop and can progress to squamous-cell carcinoma. In TCRδ−/− mice, both the development of papillomas and the progression to carcinoma were significantly increased (
      • Girardi M.
      • Oppenheim D.E.
      • Steele C.R.
      • Lewis J.M.
      • Glusac E.
      • Filler R.
      • et al.
      Regulation of cutaneous malignancy by γδ T cells.
      ). This result, revealing that γδ T cells not only inhibit the early stages of tumor development but also limit progression to carcinoma, is consistent with the use of two or more different antitumor mechanisms by γδ T cells.
      Further insight was provided by studies in TCRβ−/− mice, which lack αβ T cells. Under an intense regimen of chemical promotion, tumors were significantly less likely to progress to carcinoma than in normal controls. This indicated a paradoxical tumor-promoting effect attributable to αβ T cells, an observation made in several other experimental cancer systems (
      • Coussens L.M.
      • Werb Z.
      Inflammatory cells and cancer: think different!.
      ). The findings also suggested that γδ T cells may downregulate potentially tumor-promoting αβ T cells (see below). In summary, mouse studies have elucidated three different pathways by which γδ T cells may provide anticancer activities: (1) direct killing of transformed cells, (2) early IFN-γ production, and (3) a critical immunoregulatory mechanism. Thus, the antitumor role attributable to γδ T cells in the skin may be viewed as a component of a larger function of immunosurveillance and protection of the epidermal barrier (Figure 1).
      Figure thumbnail gr1
      Figure 1Immunosurveillance by γδ T cells in murine skin. According to this model, γδ T cells would recognize damaged keratinocytes (KCs) as those cells displaying the (yet unidentified) TCR ligand as well as ligands for NKG2D (for example, retinoic acid early-1 (Rae-1)). Such cells can be rapidly eliminated via secretion of granzymes and perforin. Concurrently, γδ T cells may promote ‘microscopic wound healing’ through the release of fibroblast growth factor-VII (FGF-VII; keratinocyte growth factor-1) and IGF-1 (
      • Sharp L.L.
      • Jameson J.M.
      • Cauvi G.
      • Havran W.L.
      Dendritic epidermal T cells regulate skin homeostasis through local production of insulin-like growth factor 1.
      ), stimulating lateral healthy keratinocytes to proliferate and fill in the voids. The secretion of anti-inflammatory compounds, as has been described for the lymphoid form of thymosin-β4 (L-Tβ4) (
      • Girardi M.
      • Sherling M.A.
      • Filler R.B.
      • Shires J.
      • Theodoridis E.
      • Hayday A.C.
      • et al.
      Anti-inflammatory effects in the skin of thymosin-β4 splice-variants.
      ), would also protect the epidermal barrier from the damaging effects of oxidative mediators released by neutrophils. Furthermore, the local secretion of IFN-γ by γδ T cells (
      • Gao Y.
      • Yang W.
      • Pan M.
      • Scully E.
      • Girardi M.
      • Augenlicht L.H.
      • et al.
      γδ T cells provide an early source of interferon γ in tumor immunity.
      ) would enhance the antimicrobial, antitumor, and other functional activities of natural killer (NK) and αβ T cells, thus maintaining epidermal integrity.

      T Regulatory Function by Dendritic Epidermal T Cells and Other γδ T Cells

      The observation that γδ T cells express certain chemokines and other regulatory molecules characteristic of T regulatory cells is consistent with the findings that immune responses to several pathogens (for example, intestinal Eimeria, Listeria monocytogenes, Mycobacterium tuberculosis, and Klebsiella) are not appropriately regulated in γδ-deficient mice (
      • Roberts S.J.
      • Smith A.L.
      • West A.B.
      • Wen L.
      • Findly R.C.
      • Owen M.J.
      • et al.
      T-cell αβ+ and γδ+ deficient mice display abnormal but distinct phenotypes toward a natural, widespread infection of the intestinal epithelium.
      ;
      • Mombaerts P.
      • Arnoldi J.
      • Russ F.
      • Tonegawa S.
      • Kaufmann S.H.
      Different roles of αβ and γδ T cells in immunity against an intracellular bacterial pathogen.
      ;
      • Mukasa A.
      • Yoshida H.
      • Kobayashi N.
      • Matsuzaki G.
      • Nomoto K.
      γδ T cells in infection-induced and autoimmune-induced testicular inflammation.
      ;
      • D'Souza C.D.
      • Cooper A.M.
      • Frank A.A.
      • Mazzaccaro R.J.
      • Bloom B.R.
      • Orme I.M.
      An anti-inflammatory role for gamma delta T lymphocytes in acquired immunity to Mycobacterium tuberculosis..
      ). TCRδ−/− mice have also shown accelerated immune responses in non-infectious settings, most notably the MRL/lpr model of lupus erythematosus (
      • Peng S.L.
      • Madaio M.P.
      • Hayday A.C.
      • Craft J.
      Propagation and regulation of systemic autoimmunity by gammadelta T cells.
      ) and an experimental model of epidermal autoimmunity mediated by αβ T cells (
      • Shiohara T.
      • Moriya N.
      • Hayakawa J.
      • Itohara S.
      • Ishikawa H.
      Resistance to cutaneous graft-vs.-host disease is not induced in T cell receptor delta gene-mutant mice.
      ). Strikingly, in the absence of γδ T cells, certain strains of mice will develop a spontaneous, αβ T cell-dependent dermatitis, as well as augmented responses to contact allergens and irritants (
      • Girardi M.
      • Lewis J.
      • Glusac E.
      • Filler R.B.
      • Geng L.
      • Hayday A.C.
      • et al.
      Resident skin-specific γδ T cells provide local, nonredundant regulation of cutaneous inflammation.
      ). The selective repopulation of TCRδ−/− mice with Vγ5+ DETCs by neonatal transfer with fetal thymic DETC precursors abrogates the augmented dermatitis, thus demonstrating that the resident γδ T cells provide a local T regulatory function. Additionally, these findings raise the possibility that IELs might exhibit a T regulatory function for systemic responses in other epithelial sites, including the intestine. This would be consistent with reports that IEL deficiencies are associated with human inflammatory bowel disease pathologies (
      • Van Damme N.
      • De Keyser F.
      • Demetter P.
      • Baeten D.
      • Mielants H.
      • Verbruggen G.
      • et al.
      The proportion of Th1 cells, which prevail in gut mucosa, is decreased in inflammatory bowel syndrome.
      ).
      The mechanisms by which IELs may downregulate inflammatory responses in epithelia are beginning to be elucidated (
      • Pennington D.J.
      • Vermijlen D.
      • Wise E.L.
      • Clarke S.L.
      • Tigelaar R.E.
      • Hayday A.C.
      The integration of conventional and unconventional T cells that characterizes cell-mediated responses.
      ). For example, γδ+ IELs and DETCs express high levels of thymosin-β4 (
      • Girardi M.
      • Sherling M.A.
      • Filler R.B.
      • Shires J.
      • Theodoridis E.
      • Hayday A.C.
      • et al.
      Anti-inflammatory effects in the skin of thymosin-β4 splice-variants.
      ). Thymosin-β4 is found within all cells, but, consistent with its function in actin sequestration, the oxidized form is a potent anti-inflammatory agent (
      • Young J.D.
      • Lawrence A.J.
      • MacLean A.G.
      • Leung B.P.
      • McInnes I.B.
      • Canas B.
      Thymosin β4 sulfoxide is an anti-inflammatory agent generated by monocytes in the presence of glucocorticoids.
      ). Moreover, activated γδ+ T cells express high levels of an alternate spliced version known as lymphoid thymosin-β4. Consistent with the observation that lymphoid thymosin-β4 contains an extra methionine that is readily oxidized, this splice variant was shown in vivo to be particularly anti-inflammatory in the skin (
      • Girardi M.
      • Sherling M.A.
      • Filler R.B.
      • Shires J.
      • Theodoridis E.
      • Hayday A.C.
      • et al.
      Anti-inflammatory effects in the skin of thymosin-β4 splice-variants.
      ). Nevertheless, a range of other possible activities may be operative, including expression of Fas ligand by γδ T cells, induction of Fas ligand on keratinocytes and secretion of cytokines such as transforming growth factor-β and IFN-γ (reviewed in
      • Pennington D.J.
      • Vermijlen D.
      • Wise E.L.
      • Clarke S.L.
      • Tigelaar R.E.
      • Hayday A.C.
      The integration of conventional and unconventional T cells that characterizes cell-mediated responses.
      ).

      γδ T Cells in Human Disease

      The study of γδ T cells in mice has increased our understanding of these cells in the human, including their propensity for epithelial surfaces and their multidimensional immune effects. The absence of a precise phenotypic DETC homologue in human skin suggests that functional equivalents must be operative. Whether these are provided by human dermal γδ T cells, infiltrating peripheral γδ T cells, αβ T cells, and/or NK cells is being explored. Nevertheless, studies of the ligand systems used by murine γδ T cells, and the exploration of their parallel activities mediated by human γδ T cells as well as other lymphoid populations, are likely to continue to provide insight into immunosurveillance and immune regulation in human disease.
      As discussed above, DETCs constitutively express NKG2D, which recognizes the MHC-Ib molecules Rae-1 and H60 that are induced by skin chemical carcinogens. Human NKG2D+ cells similarly recognize stress-induced MICA and MICB on a myriad of solid tumors (
      • Pardoll D.M.
      Immunology. Stress, NK receptors, and immune surveillance.
      ). The importance of NKG2D and its ligands in tumor recognition and elimination is increasingly being recognized. For example, the avoidance of recognition by NKG2D+ cells has been elucidated as a mechanism of tumor-driven immune evasion (
      • Groh V.
      • Wu J.
      • Yee C.
      • Spies T.
      Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation.
      ). Hence, it is crucial to clarify in humans which NKG2D+ cells actively mount an antitumor response, and to investigate potential means by which tumors may evade this attack (
      • Oppenheim D.
      • Roberts S.
      • Filler R.
      • Lewis J.
      • Shires J.
      • Barber D.
      • et al.
      Innate and adaptive immunoregulation and tumor susceptibility in NKG2D-ligand transgenic mice.
      ). Correspondingly, one major issue yet to be fully clarified in mouse γδ T-cell biology is the nature of the TCR ligands of DETCs and other γδ IELs, which represent a putative set of epithelium-specific danger signals. Once these TCR ligands are identified, the role of these molecules in epithelial homeostasis, tumor immunosurveillance, and regulation of inflammatory responses might be elucidated experimentally and correlated to human disease, in which novel therapeutic inroads may be made.
      Furthermore, important strides continue to be made to explore the potential utility of human γδ T-cell subsets in immunotherapy protocols. Dendritic cell-based strategies, for example, target the adaptive immune system, providing peptide-antigen presentation and co-stimulatory molecules that may induce antigen-specific antitumor immunity. However, this may lead to editing of the tumor population with selection of those tumor cells that have altered their expression of tumor antigens, antigen-processing machinery, or MHC-presenting molecules (
      • Dunn G.P.
      • Old L.J.
      • Schreiber R.D.
      The three Es of cancer immunoediting.
      ). Thus, complementary or integrative strategies are necessary. The phosphoantigen-mediated stimulation and expansion of Vγ9Vδ2 peripheral T cells represents a source of autologous cells with the capacity to kill a variety of tumor cells independently of MHC presentation of peptide antigen (
      • Morita C.T.
      • Lee H.K.
      • Wang H.
      • Li H.
      • Mariuzza R.A.
      • Tanaka Y.
      Structural features of nonpeptide prenyl pyrophosphates that determine their antigenicity for human γδ T cells.
      ;
      • Bonneville M.
      • Fournie J.J.
      Sensing cell stress and transformation through Vgamma9Vdelta2 T cell-mediated recognition of the isoprenoid pathway metabolites.
      ).
      As mentioned previously, γδ T cells have features of both innate and adaptive immunity and may serve critical roles in bridging these types of responses. The recent identification of the capacity of Vγ9Vδ2+ T cells to function as APCs (
      • Brandes M.
      • Willimann K.
      • Moser B.
      Professional antigen-presentation function by human γδ T cells.
      ) adds yet another layer of complexity to our understanding of γδ T-cell biology. Brandes and colleagues demonstrated that, like dendritic cells, Vγ9Vδ2+ T cells can express co-stimulatory molecules and present conventional peptide antigens for the primary stimulation of CD4+ and CD8+ αβ T-cell responses. Hence, Vγ9Vδ2+ T cells may provide the ultimate link of innate and adaptive immunity by rapid expansion and secretion of cytokines (for example, through recognition of isopentenyl pyrophosphate, IPP, and (E)-4-hydroxy-3-methylbut-2-enyl diphosphate, MHB-PP) within hours of any encounter with pathogenic organisms, and by processing and presentation of foreign peptide to prime antigen-specific αβ T cells and coordinate a dual, innate and adaptive, immune response. As a potential avenue of γδ T-cell immunotherapy, it may ultimately prove possible to use peripheral γδ T cells in antitumor protocols in which such cells are activated and exposed to tumor antigen and thus may serve a dual role of stimulating both γδ T cell-directed antitumor activity and tumor antigen-specific CD4 and CD8 αβ T-cell responses.

      CONFLICT OF INTEREST

      The author states no conflict of interest.

      ACKNOWLEDGMENTS

      I would like to thank the Dermatology Foundation and the National Cancer Institute for support, Dr Adrian Hayday and Dr Robert Tigelaar for invaluable advice and collaboration, and Renata Filler and Scott Roberts for their relentless efforts in the laboratory. This review is dedicated to the memory of my colleague and friend Jeffrey Schechner MD.

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