Summary of China Journal of Immunology 1999, Volume 15, Issue 10.
Author: He Wei
SETTING: Institute of Basic Medicine, China Academy of Medical Sciences, Department of Immunology, School of Basic Medicine, Peking Union Medical College, Beijing 100005.
T cells express two antigen receptors (TCR): TCR α β and TCRγδ. TCRαβ can specifically recognize the antigen peptides presented by MHC class or class II molecules on the surface of antigen presenting cells (APC), while TCRγδ mainly recognizes various antigens in MHC non-limiting way. In recent years, the types and ways of TCRγδ recognition antigen have been deeply studied, and this paper reviews its progress.
The diversity and distribution characteristics of 1 TCRγδ indicate the particularity of antigen recognition.
Similar to TCRαβ and immunoglobulin (Ig), TCRγδ gene consists of recombinant V, D, J and C regions. Although the V region diversity of γ and δ loci is not as good as α and β, the diversity of their binding regions makes the existence of TCRγδ even exceed the potential of TCRαβ diversity [1]. However, many γδT cell subsets only use a limited part of their receptor library, and some specific combinations of Vγ, Vδ and junction sequences lead to the monotonicity of TCRγδ structure [1]. There are three ways for mouse γδT cells to develop: the first group develops in fetal thymus, and the γδT cells produced in batches enter specific epithelial tissues respectively. These cells recombine a single γ/δ gene and have a single connecting sequence, showing a single specificity. V Δ 5 cells enter the skin, and V Δ 6 cells enter the reproductive tract epithelium and tongue. The second group developed in adult thymus, and expressed mostly Vγ 1 or Vγ4 or a small amount of Vγ2 or Vγ7, which had extensive linkage polymorphism, mainly distributed in peripheral blood and occasionally entered mucosal tissue. The third group is thymus-independent, mainly Vγ7 and Vγ 1, with large junction polymorphism, mainly distributed in small intestinal epithelium. Therefore, the antigen specificity of γδT cells ranges from single specificity to extremely variable [2]. The pre-determination of γδT cells in different distribution sites suggests that they may be a special group of T cells that recognize specific antigens, rather than the random distribution of TCRαβ cells. In humans, Vδ only uses one of the δ chains. In adult peripheral blood, more than 70% of Vδ expressed Vδ2, and the rest were Vδ 1. Vδ2 and VR9*** represent, Vδ 1 and vγ* * * represent [3].
Types and mechanism of antigen recognition of 2 γδT cells
2. 1 MHC molecules It has been reported that mouse and human γδT cells can recognize MHC class ⅰ and ⅱ molecules. Human peripheral blood γδT cells (Vδ9) can recognize MHCⅱⅱ molecules on the surface of allogeneic dendritic cells (DC)/ monocytes [4].
1987 Matis et al. established some MHC-restricted γδT cell lines by stimulating spleen cells of athymic mice with allogeneic APC in vitro [5]. They recognize non-self MHC molecules on allogeneic cells and present specific reactions, but their specificity is different from that of traditional αβT cells. For example, the γδT cell line LBK5 can recognize multiple allele products of MHCⅱ class II molecules I-E [6]. IEK is a class II MHC molecule in mice, which can bind various peptides and superantigens and stimulate the activation of αβT cells. Schild et al. found that when LBK5 recognized IEK, the peptide bound to IEK did not transmit specificity, and classical antigen therapy did not start [7]. The different ability of various cells to stimulate LBK5 can be attributed to the expression of MHC molecules on their surfaces, which has nothing to do with the source and type of cells and the factors affecting the loading of MHC peptides. The stimulation intensity of IEK protein bound to the plate to LBK5 is similar to that caused by cells expressing IEK. These results show that LBK5 directly recognizes IEK molecules.
There are also many reports that γδT cells can recognize non-classical MHC molecules. T 10 and T22 antigens recognized by G8 strain were isolated from the spleen of Balb/c nude mice [6,7]. Porcelli et al. isolated CD 1c restricted γδT cells from immunocompromised patients. Schild et al. conducted in-depth research on G8 line and found that T 10 has 94% homology with T22 [7]. Similar to LBK5, T 10/T22 was recognized by G8 clone without traditional antigen treatment. Similarly, the ability of different cells to activate γδT cells is also attributed to the expression of MHC on their surfaces, and the treatment process of class I/II antigens has no effect on them. For example, both mouse cell line RMA-S and human cell line T2 have the defect of loading peptides on MHC molecules, while both RMA-S and T2 transfected with T22 can activate G8 cells. It is very interesting that G8 can recognize T 10/T22 expressed in Drosophila cells, but Drosophila does not have an immune system similar to that of mammals, and lacks any factors necessary for antigen processing and presentation. The above results indicate that these so-called MHC-restricted γδT cell clones seem unable to recognize classical MHC through antigen processing and presentation. MHC molecules themselves are considered as antigens, and peptides loaded on these cells do not function as ligands. It is also reported that the γ δ cell clone TgI4.4 can recognize a herpes simplex T transmembrane glycoprotein GI [8]. The whole wild-type gI expressed on RMA-S cells deficient in antigen treatment can be recognized by TgI4.4 cells, and the soluble recombinant gI-Ig coated on the plate can also be recognized, which indicates that gI can be directly and completely recognized without antigen treatment and other molecular presentation. It seems that the recognition of protein antigen by γδT cells tends to be recognized directly without treatment and presentation. Specific MHC molecules are considered as antigens, not antigen presenting molecules.
2.2 Non-MHC molecules Obviously, compared with the huge sequence polymorphism of TCRγδ, there are still too few kinds of classical antigen recognition. A large number of literatures show that TCRγδ has a completely different antigen recognition pathway from TCRαβ. At present, it has been proved that two kinds of molecules are TCRγδ ligands: non-peptide small molecules containing phosphate groups and heat shock proteins.
2.2. 1 phosphorylated group Vγ9/δ2, the main subgroup of human γδT cells, can exist in a large number in mycobacterial infection sites and react with bacteria and parasites in vitro. It is found that the effective components in mycobacteria are non-peptide compounds with low molecular weight (1 ~ 3 KD), including carbohydrate skeleton and phosphoric acid. Constant et al. isolated four different water-soluble substances from Mycobacterium tuberculosis H37RV strain: TUBag 1-4. TUBag4 is thymidine 5'- triphosphate, and its γ-phosphate is replaced by a low molecular weight group with undetermined composition [9]. TUBag3 is similar to 4 in structure, but it is uridine instead of thymidine. 1 and 2 are non-nucleotide fragments of 3 and 4, with minimal activity. TUBag4 can stimulate the expansion of peripheral blood Vγ9/δ2 T cells and other specific γδT cells. These compounds exist in microorganisms and mammals. Because it is difficult to isolate natural antigens from the culture filtrate or extract of mycobacteria, Tanaka Y et al. first synthesized a series of single-base phosphate compounds, and found that some of them, especially monoethyl phosphate compounds, can simulate the reaction of Vγ9/δ2 T cells to mycobacteria [10]. Later, they reported the natural ligands of γδT cells: isopentene pyrophosphate IPP and related terpenoid (isopentenyl) pyrophosphate derivatives. However, replacing pyrophosphate group with phosphate group will greatly weaken its antigenicity. IPP and related terpenoid pyrophosphates are active precursors of lipophilic compounds such as vitamins, lipids and steroids. These terpenoid pyrophosphate intermediates exist in both bacterial and mammalian cells, and their recognition by human Vγ9/δ2 T cell subsets can partially explain their reactivity to a series of tumor cell lines. All the above studies used activated γδT cell lines, which did not contain APC and other cytokines. Most of the subsequent research results further show that the activation of γδT cells by phosphate groups requires T-T cell interaction, but the recognition itself does not require the expression of MHC class I/II molecules, CD 1, TAP 1/TAP2 or DMA/DMB. Although APC exists in some research systems, it is considered to be non-MHC restrictive, and its role may be related to providing cytokines needed for the growth of γδT cells. Carena et al.' s research further shows the special significance of MHC molecules on APC surface in γδT cells recognizing phosphate group ligands [1 1]. CD94(NKG2-A/B heterodimer) is a receptor expressed on the surface of most γδT cells, which can specifically bind MHC molecules. They found that when CD94 binds to MHC molecules, it can down-regulate the activation of γδT cells through phosphorylated ligands. When the ligand is at a low concentration, the inhibitory effect of CD94 is more obvious, thus increasing the activation threshold of γδT cells. Under physiological conditions, this mechanism is of great significance to prevent autoimmune reaction.
Another important issue has also been preliminarily clarified, that is, whether the diversity of TCRCDR3 affects the specificity of phosphorylated ligands in Vγ9/δ2 T cells. By selecting a group of random cell clones and testing different ligands, it was found that all clones showed the same form of cross-reactivity. It is impossible to select clones specific for a single ligand. In addition, T cell lines or clones showed the same form of cross-reactivity regardless of strong or weak stimuli [12]. Despite the cross reaction, these cells are highly specific in ligand structure. The number and position of phosphate groups and the type of carbon chain skeleton are very important for the activation of T cells. Therefore, Vγ9/δ2 T oligoclonal T cell subsets have extensive cross-reactivity and ligand specificity.
2.2.2 heat shock protein (hsp) 1990 or so, there are a lot of reports that γδT cells recognize HSP family members. The phenotype of γδT cell subsets in peripheral blood or umbilical cord blood that recognize hsp is mainly Vγ9/δ2, which is rich in connection polymorphism and was originally found to come from bacterial infection. Hsp family members recognized by human and mouse γδT cells are mainly hsp60 and HSP 65 [13]. Subsequently, it was found that some heat shock proteins with high expression on the surface of tumor cells can activate Vγ9/δ2 T cells, such as hsp60 on the surface of Daudi lymphoma and HSP 72 on the surface of lung cancer cells [14, 15]. Monoclonal antibodies against heat shock protein can at least partially inhibit this reaction. This reaction is positively correlated with the expression of heat shock protein on the surface of target cells. In some autoimmune diseases, the recognition of hsp on the surface of target cells by γδT cells has also been confirmed. For example, γδT cells can recognize hsp on the surface of oligodendrocytes in patients with multiple sclerosis, causing cell killing [16].
Hsp, as a highly conserved molecular chaperone protein, exists widely in prokaryotic and eukaryotic cells. Besides constitutive expression, high expression can also be induced under various stress conditions such as high temperature, hypoxia, radiation, infection and poisoning. Heat shock proteins play an indispensable role in protein folding, transfer and subunit assembly, and they also play a role in many immune reactions. They combine with a series of protein and peptides and participate in antigen presentation, so APC can process the combined peptides to form stable MHC molecule-peptide complexes. On the other hand, hsp may also act as an antigen presenting molecule by expressing on the cell surface [17], because the three-dimensional structure of its N-terminal peptide binding site is similar to that of MHC peptides. Under various stress conditions, the high expression of heat shock protein induces the activation of γδT cells. By producing cytokines and cytotoxic activity, γδT cells may play a role in rapidly eliminating stress factors and damaged cells and initiating subsequent immune responses.
Structural basis of antigen recognition of 3 γδT cells
To sum up, the recognition of antigen by γδT cells is not similar to that of αβT cells, but more similar to the direct recognition of antigen by Ig, and there is no MHC limitation. The comparative study on the molecular structures of TCRγδ and TCRαβ explains this difference to some extent. The secondary structures of TCRαβ and TCRγδ are similar to those of Ig. The three of them form a single Ig or TCR through the recombination of V, D and J, thus forming the specificity for antigen. X-ray diffraction results show that CDR3 ring of Ig and TCRαβ are the key structures for recognizing peptide segments, so it is speculated that similar regions of γ/δ chains also play a similar role. Rock et al. analyzed the CDR3 length of Ig and TCR receptor chains from mice to humans [18]. CDR3 of light chain of Ig is relatively short and its length is relatively fixed, while CDR3 of heavy chain is relatively long, and its length range varies greatly, which may suggest that Ig recognizes many antigens with different sizes in a large range from small molecules to large pathogens. The CDR3 length distribution range of TCRαβ is narrow, and the CDR3 length of α and β chains is similar, which may reflect the functional needs of α β chain, that is, contacting MHC and binding peptide segments at the same time. The γ chain CDR3 of TCRγδ is short, and its length range is small, while its δ chain CDR3 is long, and its variation range is large, so TCRγδ is closer to Ig than TCRαβ in CDR3 length.
In the mixed lymphocyte reaction, compared with the homologous reactive cloning of αβT cells, the cloning frequency of γδT cells recognizing the homologous MHC molecules is very low. Moreover, most cell clones have high cross-reactivity, which is extremely rare in the homologous reactive clones of αβT cells, suggesting that TCRγδ reacts to MHC similar to Ig's recognition of MHC.
4 Conclusions and questions
The diversity and complexity of antigen recognition of γδT cells make it difficult for people to summarize all the biological significance of γδT cells at present. However, the existing research results seem to have revealed the basic functional characteristics of γδT cells. The non-MHC restriction of γδT cells on antigen and the lack of antigen processing and presentation suggest that γδT cells can react faster than αβT cells when the body changes abnormally (such as stress). In addition, γδT cells can also respond to antigens that αβT cells can't recognize, and they are functionally complementary to the latter. In addition, the immune monitoring function of γδT cells is extensive, because its recognition ligands such as hsp and phosphate are ubiquitous in nature. After a long period of evolution, through the complex processing and accurate presentation of antigen peptides by APC, αβT cells have realized their immune response characteristics of high antigen specificity, strict MHC restrictions and detailed division of responsibilities (Th and CTL), enabling the immune system to efficiently, cooperatively and orderly eliminate foreign biomolecules or pathogens. On the other hand, γδT cells respond to stress events in vivo more widely, rapidly and directly, and their response means are more common, that is, γδT cells can play the dual functions of cytotoxicity and cytokine secretion at the same time; However, in some specific sites, such as epithelial cells, γδT cells that express specific and single TCR receptors seem to exist for local high-frequency emergencies. In a word, in the process of immune response, γδT cells may play the role of starting, coordinating and supplementing the function of αβT cells.
At present, the research on antigen recognition of γδT cells still needs to be deepened in many aspects. What are the basic structural requirements for γδT cells to recognize protein antigen? It is not clear in what way hsp plays a role in the activation of γδT cells. Do γδT cells directly recognize hsp molecules on the cell surface or recognize the peptides they present? In fact, the role of peptides carried by hsp has not been clearly and completely studied. What is the significance of TCR CDR3 diversity? Since hsp and phosphate metabolites are self-and non-self components at the same time, why are the self-reactive γδT cell clones not screened from the cell bank during development? In the immune response caused by these substances, does the specificity of γδT cells point to both exogenous components and autologous components? Only by thoroughly understanding these problems can people make a comprehensive and profound evaluation of the biological function of γδT cells, and its theoretical results can be used for the treatment of tumors and autoimmune diseases.
Since 1994, we have systematically studied its characteristics, distribution, subsets, selective acquisition of receptor molecules, functional characteristics and its participation in tumors and autoimmune diseases, in order to provide information for revealing the mystery of its function and promote the application of its theoretical achievements in disease prevention, diagnosis and treatment.
About the author: He Wei, male, 43 years old. Doctor of Medicine, professor, German doctoral supervisor, vice president of School of Basic Medicine of Peking Union Medical College, and deputy director of Institute of Basic Medicine of China Academy of Medical Sciences. Member of Basic Immunology Branch of Chinese Immunology Society, Director of Beijing Immunology Society, Editors of Foreign Medical Immunology, China Journal of Microbial Immunology and China Journal of Immunology. /kloc-0 returned to China in 1994, and * * presided over the National Key Basic Research and Development Project (973), 95 Key Research Project, National Natural Science Foundation, 863 Biotechnology High-tech Plan, Ministry of Health Fund, National Doctoral Program Fund, Education Commission Fund, Sino-US, Sino-Japanese, Sino-German Cooperative Research Project and China Academy of Medical Sciences 15 Project. Scientific research focuses on the role of γ δ T cells in anti-infection immunity, tumor immunity and autoimmunity, the cloning and expression of IL- 15 gene and the anti-tumor effect of IL- 15 transgenic tumor vaccine, the relationship between peroxidation and immunity in aging, gene regulation of thymic degeneration and immunological diagnosis of senile dementia. At present, * * * has published 35 papers (8 foreign papers) and 2 monographs, and won the second prize of provincial and ministerial scientific research.