ϒδ T - A summary on gamma delta T cells. PDF

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ϒδ T-cells What is ϒδ T-cells Gamma delta (ϒδ) T-cells, along with αβ T cells and B cells represent the three-lymphocyte lineage found in all vertebrate species. Most T-cells are αβ T cells with TCR composed of an α chain and a β chain. In contrast ϒδ T-cells TCR is made of a ϒ and δ chain. ϒδ T-cel...

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ϒδ T-cells What is ϒδ T-cells Gamma delta (ϒδ) T-cells, along with αβ T cells and B cells represent the three-lymphocyte lineage found in all vertebrate species. Most T-cells are αβ T cells with TCR composed of an α chain and a β chain. In contrast ϒδ T-cells TCR is made of a ϒ and δ chain. ϒδ T-cells are among the very first T-cells to develop in the thymus. Just like αβ T-cells, the TCR is made by somatic recombination of VDJ gene segments. There is more diversity in ϒδ gene segments compared to αβ gene segments. This creates a more diverse repertoire of ϒδ TCRs. Where are ϒδ T-cells found? Unlike αβ T cells, ϒδ T-cells in both human and mice constitute only a small proportion (1-5%) of lymphocytes circulating in the blood. The signature human ϒδ T-cell population residing in the blood are identified by their Vϒ9/Vδ2+ TCR. These largely response to phosphorylated metabolites such IPP. Due to metabolic dysregulation in cancer cells, IPP often accumulated and presented to ϒδ T-cells TCR. With the help of a drug group called bisphosphonates, which boost the accumulation of metabolites in cancer cells even further. This allows blood resident ϒδ T-cell to recognise and kill tumour cells. However, ϒδ T-cells are found more widely in epithelial rich tissues such as the skin, intestine and reproductive tract where they make up 50% of T-cells. These epithelial ϒδ T-cell subsets form a larger group of epithelial tissue-resident lymphocytes named intra-epithelial lymphocytes (IELs). The skin is composed of the epidermal upper layer and the dermis-bottom later. In the murine epidermis, ϒδ T-cell are known as dendritic epidermal ϒδ T-cell (DETCs) (Vϒ5/Vδ1+). Human epidermis contains αβ T-cells and ϒδ T-cells but these are

not equivalent to DETCs but have similar effector functions. In the murine dermis, the ϒδ T-cell re known as dermis ϒδ T-cell.

The gastrointestinal tract is made of a single cell layer of epithelial cells that is vigorously dividing. The majority of cells in the GI tract are enterocytes which facilitate the absorption of nutrients and water from the lumen. The intestinal epithelium and the underlying tissue are home to plethora of immune cells such as B-cells, ILCs, macrophages, dendritic cells, ϒδ T-cells and αβ Tcells. This is like the human intestine. Human tissue resident ϒδ T-cell are identified by a different set of TCR that differ from the blood derived ϒδ T-cell. These cells migrate and stay within the human epithelial tissues such as the skin, respiratory tract and gut mucosa, where they surveillance for any signs of stress such as infection, transformation or malignant cells. Upon encounter, they immediately kill them.

Mouse gamma delta T-cell development

ϒδ T-cell at different sites have different signature ϒδ chains produced by VDJ somatic recombination. ϒδ T-cell bearing specific Vϒ gene segments in TCR are exported from the thymus at defined periods of foetal and neonatal development. These then migrate to and populate different epithelial rich tissues. DETC ϒδ T-cell are the first to be developed at around embryonic day 14 and they are potent IFN-y T-cells. These thymocytes preferentially rearrange Vδ1 and Vϒ5 loci. Those that fail to do so, will attempt to rearrange the genes encoding Vϒ6 at around E16. Resulting Vϒ6+Vδ1+ form IEL compartments in the uterus, tongue and lungs. By contract to Vϒ5+Vδ1, Vϒ6+Vδ1+ are a critical source of IL-17 during infection and autoimmunity. Vϒ4 TCR ϒδ T-cell are developed at E18 and migrate and populate the blood, spleen and lymph nodes. The development of Vγ1+ TCR ϒδ T-cell take place after birth and some Vγ7+ intestinal intraepithelial lymphocytes might be thymic independent.

Regulations of the composition of local IEL compartments. https://ac.els-cdn.com/S0092867416310819/1-s2.0S0092867416310819-main.pdf?_tid=0ee6a9ca-d6b3-11e7803b00000aacb360&acdnat=1512145199_78e0dea1797639850a93 44f3c332a83e Epithelial cells provide signals that instruct the development and function of their local ϒδ T cell compartment. Skint1- a butyrophilin like gene, has shown to be essential for the development of DETCs. Skint is specifically expressed by TECs and karatinocytes in the dermis. During Vϒ5Vδ1 TCR rearrangement, these T cells are positively selected in the thymus by binding to Skint1. Signalling from Skint matures the ϒδ T-cell expressing Vϒ5Vδ1 TCR. Upon migration to the dermis,

DETCs must receive the same signalling from the residential site for survival. In Skint1 mutant mice, 90% of DETCs are ablated whilst other Tcells are unaffected. Unlike for DETCs, no critical epithelial determinant has been identified for other ϒδ T-cells. Skint gene is part of the Btnl family that include 6 rodent genes and 5 human genes. Several of these Btnl genes are found in immune regulation. Human BTN3A1 = facilitate peripheral blood ϒδ T-cell responses to endogenous metabolites such as phosphoantigens. How this is mediated is not known, we don’t know whether its mediated by direct TCR-BTN3A1 binding. The mouse gut is a major site of Btnl1, Btnl4, and Btnl6 expression. Btnl1 is expressed in small intestinal villus epithelial cells and it selectively promotes the maturation and expansion of Vϒ7+ T-cells, thereby shaping the IEL compartments of the gut. In Blnt1KO mice, no Vϒ7+ T-cells T- cells found in the small intestine. Similarly, Human Vϒ4+ T-cells are selectively regulated by gut epithelial cells expressing BTNL3 and BTNL8. This tells us that in both humans and mice, epithelia uses organ specific btnl genes to shape the local T cell compartments. Role of ϒδ T-cells https://ac.els-cdn.com/S1074761309003331/1-s2.0S1074761309003331-main.pdf?_tid=5173075c-d6b8-11e7b65a00000aacb361&acdnat=1512147459_5f1a81a3622e2c4be642f 7bad80520b1 https://f1000researchdata.s3.amazonaws.com/manuscripts/119 25/f59e08ef-caf6-4490-b525-53043b3477e1_11057__dieter_kabelitz.pdf?doi=10.12688/f1000research.11057.1 Recognition of target cells by ϒδ T-cells

ϒδ T-cells participate in stress surveillance, this small subset of T-cells are capable of recognising generic stress signals induced by infected or transformed cells. Compared to αβ, ϒδ T-cells recognise more generic sentinels of dysregulation and not specific antigens in the context of MHC. Mice models that have been genetically engineered to be deficient in ϒδ T-cells showed that ϒδ T-cells are involved in early control of infection. Loss of ϒδ T-cells result in increase in virus titres immediately post infection and increased mortality. Once ϒδ T-cells recognise alterations of self, they respond very quickly without needing priming by antigen presenting cell or clonal expansion in the lymphatic system. They can kill transformed or infected cells immediately as well as inducing the adaptive immune system. Antigen recognition by ϒδ T-cells does not require antigen processing and MHC-complex presentation of peptide epitopes. ϒδ T-cells can recognise molecular patterns of dysregulation in stressed, infected or transformed cells. These molecules may be self-encoded ligands that are upregulated during stress, inflammation or infection OR these ligands can be non-selfencoded. Upon recognition, the lymphoid stress-surveillance response’ is initiated. Lead to: - Limit the dissemination of infected and malignant cells - Sustain tissue integrity - Regulate and activate the downstream adaptive responses. Self-encoded ligands include MHC class I polypeptide related sequences such as MICA and MICB and the T10/T22, RAE-1 proteins (in mice). Reactivity to these antigens predominates amongst ϒδ T-cells that reside in epithelia.

Non-self -encoded ligands include Phospho-antigens such as microbial HMBPP (a metabolic intermediate specific to many prokaryotes and parasites such as malaria) and alkyamines that are found in bacteria, plants or animal cells. As well as bacterial and mammalian homologues of heat shock proteins HSP60/GroEL. Reactivity to these antigens is common amongst ϒδ T-cells that reside in the blood and lymphoid tissues. HMBPPs are intermediates of the non-mevalonate pathway of isoprenoid biosynthesis. It is an essential metabolite in pathogenic bacteria such as Mycobacterium tuberculosis and malaria, but absent from the human mevalonate pathway. Human Vϒ9Vδ2+ ϒδ T-cells in peripheral blood circulation are highly reactive to phosphoantigens such as HMBPPs. HMB-PP functions in this capacity by binding the cytosolic B30.2 domain of BTN3A1. This result in a conformation change in the BTN3A1 molecule. This leads to the presentation of pAg on the extracellular immunoglobulin V domain of BTN3A1.

The structure of microbial HMBPP is similar to the human pyrophosphate IPP found in the human mevalonate pathway. This can lead to cross reactivity. Transformed or virus infected cells tend to have metabolic dysregulation, therefore IPP tend to accumulate. Therefore, many solid tumours or

lymphoma cells are quiet sensitive to ϒδ T-cells mediated lysis. In a prostate cancer TRAMP mouse model, deficiency in ϒδ Tcells resulted in a more severe form of cancer.

Interestingly, the sensitivity of tumour cells to lysis by Vδ2Vγ9 γδ T cells can be pharmacologically manipulated. Nitrogen containing bisphosphonates (N-BPs) such as zoledronic acid are in clinical use to treat diseases associated with bone resorption. In addition to their anti-resorptive bone activity, N-BPs also interfere with the mevalonate metabolic pathway. N-BPs block an enzyme downstream of the synthesis of isopentenyl pyrophosphate (IPP), leading to increased accumulation of IPP and thereby to γδ T-cell activation. Therefore, pre-treatment of tumour cells with N-BP increases their susceptibility to γδ T-cell mediated lysis. In addition to the TCR, ϒδ T-cells express other cell surface receptors such as NK-type receptor NKG2D. It is expressed in all ϒδ T-cells and is a receptor for multiple stress inducible MHCclass I related molecules such as MICA, MICB, ULBP1-6 and RAE1 in mice. Normal cells do not express NKG2D ligands, but the cell surface expression is induced by cell stress, DNA, damage and cellular transformation. Upon ligand binding, NKG2D transmits cellular activation via PI3 kinase pathway. This result in TNF-α production and release of cytolytic granules. NKG2D ligands can be released from the surface of tumour cells via protease-mediated shedding or via exosome secretion, and soluble NKG2D ligands may block NKG2D receptor activation and thereby serve as a tumour immune escape mechanism Unlike αβ T cells, IELs are already resident in tissues to carry out tissue surveillance and maintain homeostasis. So, they don’t need to travel through the lymphatic system before being recruited to sites of infection.

Antigen presentation by ϒδ T-cells. Human blood Vγ9Vδ2 T cells express inflammatory chemokine receptors, which allows them to be immediately recruited to sites of infection. In here, they become activated and turn into ϒδ APC T-cells - for the induction of microbe or tumor specific CD4+ and CD8+ T-cell response. Unlike dendritic cells, the ϒδ T-cells must be activated first before they can take up antigens. Several TCR dependent activators of ϒδ T-cells include IPP, HMBPP. The activated ϒδ Tcells then express the lymph node homing receptor CCR7- this allows them to relocate to the draining lymph node in a similar manner to DCs. They also increase their expression of MHC-II, co-stimulatory molecules such as CD40, CD80, CD86. ϒδ T-cells can also readily take up and degrade exogenous soluble proteins for peptide loading on MHC-I – antigen cross presentation. Similar to DCs, the intracellular protein degradation and endosomal acidification were significantly delayed in ϒδ T-cells. This is a condition that favours antigen cross presentation. In

contrast to various human DC subsets, ϒδ T-cells are capable of efficiently translocating soluble exogenous antigens into the cytosol for processing via cytosolic proteasome. It’s been reported that ϒδ T-cells can cross present influenza antigens derived from virus infected cells. This emphasizes the rapid innate-like response of human Vγ9Vδ2 T cells to microbial antigens and to tumor cells, as well as the contribution of γδ

ϒδ T-cells in cancer immunotherapy ϒδ T-cells mediate killing of malignant cells in a number of ways IFN-ϒ  directly inhibit tumor growth, stimulate macrophages and block angiogenesis. Th2 like cytokines IL-4, IL-10 Control CD8+ T-cell expansion Recruit neutrophils and monocytes. Upregulate expression of Fas ligands (Fas-L) and TRAIL  enhance tumor killing activity in Fas or Trail receptor sensitive tumors. CD16- a receptor for Fc portion of IgG  CD16 can enhance the antibody-dependent cellular cytotoxicity (ADCC) in the presence of anti-tumor cell monoclonal antibodies.

Immunotherapy using activated ϒδ T-cells ex vivo. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352452/pdf/onc otarget-08-8900.pdf Targeting the immune system against tumors is one of the primary focus in cancer studies.

There has been suggestion of adoptive transfer of ex vivo activated and expanded ϒδ T-cells for immunotherapy. As a result, adoptive transfer of Vγ9Vδ2 T cells has already showed a strong lytic activity towards a spectrum of tumour cell lines such as hepatocellular carcinoma, breast cancer, lung carcinoma and colorectal cancer. One of the main obstacles is that only a small quantity of gamma-delta T-cells can be amplified. There need to be a reliable SOP in currently clinical trials to amplify loads. One of the most widely and effective method to amplify ϒδ Tcells is to use a phosphorylated antigen or anti- ϒδ T-cells TCR antibody. Emerging Pathogenic Roles of ϒδ T-cells in Cancer Progression. Despite the well-established concept of ϒδ T-cells as potent antitumor TILs, a study in 2007 on human breast cancer surprisingly revealed a potential protumor function. ϒδ T-cells cells isolated from breast cancer biopsies were reported to inhibit the function of several immune cell populations in vitro, and consequently suppress their antitumor responses. Consistent with these observations, the presence of ϒδ T-cells was shown to positively correlate with advanced tumour stages and inversely correlate with patient survival. In mice there are two distinct populations of ϒδ T-cells that have opposite roles in melanoma progression. Vϒ4+ ϒδ T-cells display antitumor properties by secreting IFN-y. Vϒ1+ ϒδ T-cells can suppress Vϒ4+ cells promoting tumour escape. This is because Vϒ1+ ϒδ T-cells can produce IL-17 rather than IFN-Y. IL-17 can:

 Recruit immunosuppressive myeloid populations  Inhibit antitumor responses  Enhance angiogenesis http://cancerres.aacrjournals.org/content/canres/75/5/798.full.p df...