Uncovering the role of γδ T cells in adaptive immunity

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    Previously, when we’ve talked about T cells, we’ve focused on the most abundant type of T cells, the ɑβ T cells. These include CD4+ and CD8+ T cells and represent up to 95% of the T cells in the body. This article explores the different T-cell types and the role of the less common γδ T cells.

    The role of ɑβ T cells in the adaptive immune response is relatively well understood. Major histocompatibility complex (MHC) antigen presentation by antigen-presenting cells (APCs) causes clonal expansion and activation of CD4+ and CD8+ T cells. Activated CD8+ T cells play a role in the direct killing of infected cells, while CD4+ T cells promote CD8+ T-cell and B-cell activation.

    The role of γδ T cells in immunity is less well understood. These unconventional T cells exhibit features of both innate and adaptive immunity. Like CD8+ and CD4+ T cells, γδ T cells are implicated in the direct killing of infected cells and pathogens, as well as in B-cell activation. However, they also produce a range of cytokines that regulate other immune cells. (1,2)

    T-cell Types: Differences between ɑβ T cells and γδ T cells

    ɑβ T cells and γδ T cells are defined by the expression of either ɑβ or γδ T-cell receptors (TCRs), respectively. Both types of T cells come from a common precursor T cell (Figure 1); however, the factors that determine if a T-cell precursor becomes an ɑβ or a γδ T cell are currently unclear. (3)

    An image of T cells to depict the different T-cell types.

    Figure 1. Overview of the types of ɑβ and γδ T cells.

    One key distinction between γδ T cells and ɑβ T cells is the greater diversity of antigens that γδ T cells can potentially recognize. This greater diversity is because:

    1. The possible use of tandem D gene segments in the complementarity-determining region 3 (CDR3), means that γδ T cells theoretically have the potential for even greater sequence diversity than ɑβ T cells or even antibodies; and
    2. γδ T cells are not MHC restricted and can recognize a wide variety of non-peptide antigens such as lipids and metabolites. (4)

    The γδ T-cell repertoire can be extremely diverse or essentially invariant, depending on the subtype and location. (4)

    The importance of γδ T cells is becoming increasingly apparent as we gain more insight into the adaptive immune response from technologies such as immunosequencing. In particular, γδ T cells have been implicated in:

    • the response to infection, including to SARS-CoV-2 and M. tuberculosis; (1,5,6)
    • affecting outcomes in transplant patients; (7)
    • influencing disease outcomes in cancer patients. (8)

    Table 1 summarizes the differences and similarities between ɑβ and γδ T cells.

    Table 1: Comparison between ɑβ T cells and γδ T cells

    ɑβ T cells γδ T cells Reference
    Abundance (of total T-cell population)95%5% Fichtner et al. (1)
    MHC restricted?YesNo Fahl et al. (3)
    Antigens recognizedMHC-presented peptidesPeptides (not MHC restricted),
    metabolites, and lipids
    Fahl et al. (3)
    Receptor diversity1015–1018 potential TCR sequences>1018 potential TCR sequencesBorn et al. (9) Sewell (10)
    Tissue localizationBlood, lymphoid organs, and select
    tissues
    Epithelial surfaces (skin, blood, gut, lymph nodes, spleen) Fahl et al. (3)

    Monitoring the γδ T-cell repertoire with the immunoSEQ® Assay

    Advances in immunosequencing techniques have revealed the great diversity of the γδ T-cell repertoire. (1) To better understand the role of γδ T cells in infection and diseases, we need to gain further insight into the underlying dynamics of these repertoires.

    The immunoSEQ Assay is a powerful, highly sensitive immunosequencing platform for profiling T- and B-cell repertoires. The immunoSEQ Assay can comprehensively profile the adaptive immune system without the issue of amplification bias commonly associated with multiplex PCR. For more on how the immunoSEQ Assay works, view our blog post on immunosequencing methods.

    There are several immunoSEQ Assays to choose from, depending on your research goals. To profile the γδ T-cell repertoire, you have two choices: the immunoSEQ human TCRG and TCRA/D Assays.

    These assays are part of our immunoSEQ Service:

    • you send us your samples [e.g., sorted T cells, cultured cells, peripheral blood mononuclear cells, whole blood, bone marrow, fresh/frozen/formalin-fixed paraffin-embedded tissues, or even previously hematoxylin and eosin (H&E) stained slides];
    • we prepare and sequence your samples in-house;
    • you access your immunoSEQ Data via our user-friendly immunoSEQ Analyzer, which allows you to perform complex calculations and comparisons between samples and generate publication-ready figures.

    The role of γδ T cells in disease

    Infectious diseases

    Numerous studies have shown that γδ T cells expand upon viral, bacterial, or parasitic infection. Understanding how γδ T-cell repertoires change during infection can provide key insights into aspects of the immune response that are important for protecting against disease. (1)

    A protective role for γδ T cells in infection

    A 2006 study showed that γδ T cells might play a protective role during SARS-CoV-1 infection. Analysis of the γδ T-cell repertoire by flow cytometry found expansion of memory Vγ9Vδ2 T cells in patients who had recovered from SARS-CoV-1 infection, with no analogous expansion of other γδ T-cell subsets or ɑβ T cells (Figure 2). (11)

    An image of T cells to depict the different T-cell types.

    Figure 2. In a cohort of patients who recovered from SARS-CoV-1 infection, specific expansion of central and effector memory Vγ9Vδ2 T cells was observed. No analogous expansion was seen in other γδ subsets or ɑβ T cells. Adapted from Poccia et al. (11) CM: central memory; E: effector; EM: effector memory; N: naïve.

    More recently, exploratory research in individuals with SARS-CoV-2 infection has shown that γ9δ2 T cells are less abundant in the periphery of acutely infected individuals as well as in patients admitted to hospital compared with uninfected controls. (6,12) One explanation for this decrease is that γ9δ2 T cells, known to be cytotoxic, may move from the blood to the lungs to fight infection. (12)

    γδ T cells have also been shown to play a protective role in M. tuberculosis infection. (5) The immunoSEQ TCRA/D Assay was used to compare the peripheral and lung γδ T-cell repertoires in subjects with active Tuberculosis. As shown in Figure 3, there was little overlap in the γδ T-cell repertoires between the blood and lung or between the two lungs within a single subject. The authors suggested that this diversity may result from highly localized expansion of γδ T cells in the lung as they encounter antigens. (13)

    An image of T cells to depict the different T-cell types.

    Figure 3. Overlap of TCRD clonotypes detected in the blood and lung tissue of two subjects with Tuberculosis. Adapted from Ogongo et al. (13)

    While the TCRD repertoires were highly skewed toward the Vδ1 gene, there was significant heterogeneity at the CDR3 level. (13) The CDR3 is the most highly variable region of the TCR and is the focus of the immunoSEQ Assay. Therefore, the heterogeneity in γδ T-cell response would not have been captured by methods looking only at gene usage, thus highlighting the value of the immunoSEQ Assay.

    Transplant

    The role of T cells in transplant outcomes is well established. ɑβ T cells are a major contributor to graft-versus-host disease (GVHD) in hematopoietic stem cell transplant (HSCT). Less is known about how γδ T cells may affect outcomes. However, the γδ T-cell repertoire is known to reconstitute shortly after transplant and thus may play an essential role in protecting against infection. (14,15)

    Correlations between γδ T-cell repertoire and transplant outcomes

    Arruda et al. (14) used the immunoSEQ TCRG Assay to profile intragraft γδ T cells following HSCT in subjects with acute myeloid leukemia (AML). The authors did not find any correlation between intragraft γδ T-cell composition and outcomes such as relapse or GVHD.

    However, they did find higher overlap in the γδ T-cell repertoire among non-relapse subjects than among those who relapsed (Figure 4). They identified 12 frequently shared clones exclusively in the non-relapse patients as well as public clones found only in relapse patients. Thus, the immunoSEQ Assay can help identify key T-cell clones that warrant further investigation into their role in positive and negative outcomes and their potential as prognostic biomarkers. (14)

    An image of T cells to depict the different T-cell types.

    Figure 4. Overlap in TCRG repertoires among relapsers and non-relapsers (left) and subjects who experienced GVHD and those who did not (right). Adapted from Arruda et al. (14) aGVDH: acute GVDH; GVHD: graft-versus-host disease; TCRG: T-cell receptor gamma.

    Effects of donor cytomegalovirus status on γδ T cells in transplant patients

    Cytomegalovirus (CMV) is a ubiquitous virus that establishes a lifelong latent infection. While CMV infection is generally asymptomatic, it can be life-threatening in immunocompromised individuals, such as HSCT recipients. (16)

    Read more about CMV and T cells in our CMV blog post.

    Gaballa et al. (16) used the immunoSEQ TCRG Assay to compare the γδ T-cell repertoire in HSCT grafts from CMV positive (CMV+) and negative (CMV) donors to show how donor CMV status reshapes the recipient’s immune repertoire.

    The authors found that CMV+ grafts were less diverse and showed several single-clone expansions compared with CMV grafts. They also identified specific V/J pairings in γδ T cells that were more common in the CMV+ and CMV grafts. In particular, Vγ9 γδ T cells were more prevalent in CMV+ grafts and were differentiated to a terminal effector phenotype, suggesting that they play an adaptive role in responding to CMV. (15) Studies like these that add to the understanding of the role of γδ T cells in CMV immunity could facilitate the design of novel γδ T-cell-based therapies. (16)

    Cancer

    γδ T cells have been gaining attention as potentially important in cancer immunity. In both human and mouse models, γδ T cells have shown protective benefits and prognostic potential. In addition, they may offer benefits as cellular immunotherapies due to their lack of MHC restriction. (8)

    A unique γδ T-cell subset that correlates with outcomes

    In a recent study by Wu et al. (8) the immunoSEQ TCRA/D Assay was used to profile the γδ T-cell repertoire in individuals who had undergone surgery for triple-negative breast cancer (TNBC). They found a unique subset of γδ T cells in healthy breast tissue and tumors that mainly express the Vδ1 chain. The abundance of this T-cell subset, but not that of the Vδ2 or total γδ T cells, correlated with both progression-free survival (PFS) and overall survival (Figure 5). (8)

    An image of T cells to depict the different T-cell types.

    Figure 5. In women with TNBC, individuals with high numbers of Vδ1 T-cell abundance in the breast tissue showed improved PFS compared with those with low Vδ1 T-cell abundance (top left). Vδ1 and TCRA focusing among two subjects. Intratumor T cells showed a lack of focusing in Vδ1 compared with TCRA. Adapted from Wu et al. (8) PFS: progression-free survival.

    The authors also observed a lack of Vδ1 focusing in intratumor T cells, compared with the ɑβ T-cell repertoire (Figure 5). They proposed that this lack of focusing indicates an innate-like response and that the unique γδ T-cell compartment is associated with remission. (8)

    Using immunosequencing to understand the development of primary cutaneous γδ T-cell lymphoma

    In a separate study, the immunoSEQ Assay, in combination with other sequencing technologies, was used to profile the γδ T-cell repertoire in a series of primary cutaneous γδ T-cell lymphoma, a rare but aggressive type of cancer that originates from γδ T cells.

    The authors found that, among nine individuals, the cell of origin contained Vδ1 in five cases and Vδ2 in four cases. Vδ1 lymphomas originated in the epidermis and dermis, while Vδ2 lymphomas originated in the subcutaneous tissue. They showed that the cell of origin impacts the clinical and molecular features of the disease, which may have implications in how the two types of cells should be targeted therapeutically. (17)

    The immunoSEQ Assay also revealed the Vγ chain usage in each group. The most common Vγ in circulating cells was V9. However, Vδ2 lymphomas expressed only Vγ3, while V1 lymphomas primarily expressed Vγ3 or 5. The fact that both Vδ1 and Vδ2 lymphomas express similar Vγ chains suggests a common antigen in these diseases. The authors compared their immunoSEQ data with known γδ T-cell–antigen pairs and identified a putative antigen for Vδ1 and Vδ2 lymphomas. (17)

    Summary

    γδ T cells are a rare yet crucial T-cell type whose role in infection and disease is just beginning to be elucidated. Owing to their roles as mediators of both innate and adaptive immunity and their ability to recognize a diverse range of antigens, understanding the γδ T-cell repertoire can yield key insights on immune-related markers of disease and treatment response, as well as new T-cell-based therapies.

    γδ T cells have been implicated in mediating protection from infection, including for SARS-CoV-2. (1,5,6) In addition, studies in cancer (8,17) and transplant (14,15) suggest that the γδ T-cell repertoire can provide prognostic information and insights into treatment outcomes.

    The immunoSEQ Assay provides a fast, easy, quantitative, and high-throughput approach to monitor the γδ T-cell repertoire across healthy and disease states or in response to treatment. The immunoSEQ Assay has already revealed valuable insights into features of the γδ T-cell repertoire associated with disease remission in TNBC and immune reconstitution following transplant. Still, there is much more to understand if we are to translate these insights into immune-based biomarkers or new T-cell-based therapies.

    If you’re ready to use the immunoSEQ Assay in your research or have more questions, visit our immunoSEQ Assay products page or get in touch with our product team. We’re here to offer you end-to-end support from sample collection to data analysis to help propel your research forward.

    You can easily explore your immunoSEQ Data in the immunoSEQ Analyzer, our powerful and easy-to-use analytics platform. View our series of tutorials to see how simple data analysis is in the immunoSEQ Analyzer.

    For Research Use Only. Not for use in diagnostic procedures.

    References

    1. Fichtner AS, Ravens S, Prinz I. Human γδ TCR repertoires in health and disease. Cells 2020;9(4):E800.

    2. Vantourout P, Hayday A. Six-of-the-best: unique contributions of γδ T cells to immunology. Nat Rev Immunol. 2013;13(2):88–100.

    3. Fahl SP, Coffey F, Wiest DL. Origins of γδ T cell effector subsets: a riddle wrapped in an enigma. J Immunol Baltim Md 1950. 2014;193(9):4289–94.

    4. Castro CD, Boughter CT, Broughton AE, Ramesh A, Adams EJ. Diversity in recognition and function of human γδ T cells. Immunol Rev. 2020;298(1):134–52.

    5. De Libero G, Mori L. The T-cell response to lipid antigens of Mycobacterium tuberculosis. Front Immunol. 2014;5:219.

    6. Lei L, Qian H, Yang X, Zhang X, Zhang D, Dai T, et al. The phenotypic changes of γδ T cells in COVID-19 patients. J Cell Mol Med. 2020;24(19):11603–6.

    7. Sullivan LC, Shaw EM, Stankovic S, Snell GI, Brooks AG, Westall GP. The complex existence of γδ T cells following transplantation: the good, the bad and the simply confusing. Clin Transl Immunol. 2019;8(9):e1078.

    8. Wu Y, Kyle-Cezar F, Woolf RT, Naceur-Lombardelli C, Owen J, Biswas D, et al. An innate-like Vδ1+ γδ T cell compartment in the human breast is associated with remission in triple-negative breast cancer. Sci Transl Med. 2019;11(513):eaax9364.

    9. Born WK, Kemal Aydintug M, O’Brien RL. Diversity of γδ T-cell antigens. Cell Mol Immunol. 2013;10(1):13–20.

    10. Sewell AK. Why must T cells be cross-reactive? Nat Rev Immunol. 2012;12(9):669–77.

    11. Poccia F, Agrati C, Castilletti C, Bordi L, Gioia C, Horejsh D, et al. Anti-severe acute respiratory syndrome coronavirus immune responses: the role played by V gamma 9V delta 2 T cells. J Infect Dis. 2006;193(9):1244–9.

    12. Rijkers G, Vervenne T, van der Pol P. More bricks in the wall against SARS-CoV-2 infection: involvement of γ9δ2 T cells. Cell Mol Immunol. 2020;17(7):771–2.

    13. Ogongo P, Steyn AJ, Karim F, Dullabh KJ, Awala I, Madansein R, et al. Differential skewing of donor-unrestricted and γδ T cell repertoires in tuberculosis-infected human lungs. J Clin Invest. 2020;130(1):214–30.

    14. Arruda LCM, Gaballa A, Uhlin M. Graft γδ TCR sequencing identifies public clonotypes associated with hematopoietic stem cell transplantation efficacy in acute myeloid leukemia patients and unravels cytomegalovirus impact on repertoire distribution. J Immunol Baltim Md 1950. 2019;202(6):1859–70.

    15. Gaballa A, Arruda LCM, Rådestad E, Uhlin M. CD8+γδ T cells are more frequent in CMV seropositive bone marrow grafts and display phenotype of an adaptive immune response. Shamsi T, editor. Stem Cells Int. 2019;2019:6348060.

    16. Gaballa A, Alagrafi F, Uhlin M, Stikvoort A. Revisiting the role of γδ T cells in anti-CMV immune response after transplantation. Viruses 2021;13(6):1031.

    17. Daniels J, Doukas PG, Escala MEM, Ringbloom KG, Shih DJH, Yang J, et al. Cellular origins and genetic landscape of cutaneous gamma delta T cell lymphomas. Nat Commun. 2020;11(1):1806.

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