Preliminary Verification Of Liver As An Extrathymic Development Organ For γδ T Cells

ObjectiveThe liver is not only an important metabolic organ but also a crucial immune organ. The liver is rich in innate immune cells, whose unique cellular compositionexerts a great influence on the formation of its functional features. Many studies show that adult liver contains hemopoietic stem cells, which can grow into various kinds of immune cells for a long time after transfusion to the body. Therefore, adult liver is also thought to a hematopoietic organ. Especially when bone marrow hematopoietic function is damaged or inhibited, the liver may become a extramedullary and compensatory hematopoietic organ. But the effect and mechanism still needs further research and confirmed. For now, few people take part in fields that whether liver specific hematopoietic function will affect the component and development of immune cells in the liver microenvironment.A feature of the liver immune system is abundent in innate immune cells, such as NKT, NK, γδ T cells and so on. Recently, studies reveal that a group of NK cells resident in liver, which are quite different from classic NK cells in phenotypes, distribution, function and development, very probably develop from hematopoietic cells in liver. Other researches also show that γδ T cells can develop independent of thymus, such as in small intestine. As the proportion of y8 T cells in liver is much richer than spleen, lymph node and peripheral blood, is it possible that the liver couble be the extrathymic developmental organ for γδ T cells and has further influence on the formation of y8 T cells in liver? In order to discuss those questions, the report studies four aspects based on that liver is richer in y8 T cells than other immune organs. First of all, we tested the content of T progenitor cells and observe the dynamic change of their content. Besides, in terms of functions, we detected whether T progenitor cells had the potency to develop into γδ T cells. Moreover, we examined whether liver contained γδ T precursor cells and further observed whether they had some different features. Last but not least, we carried out a preliminary study on the features of y8 T cells in liver through the distribution, phenotypes as well as their traits of immune responses when infected with Salmonella Typhimurium.Methods1 Mononuclear cells were separated from the liver, small intestine, skin, thymus, spleen and lymph node, and flow cytometry was used to analyse the proportion of γδ T cells inlymphocyte.2 With the stimulation of PMA and ionomycin in vitro, flow cytometry was applied to examine the dynamic curves of γδ17 and γδ T1 subsets in liver, thymus and spleen.3 Flow cytometry was used to determine the content of T precursor cells DN1-DN4 in various tissues and the time phase changes of DN1-DN2 cells in liver.4 Flow cytometry was performed to test the phenotype of liver γδ T precursor cells, the expression of transcription factors and the secretion of cytokines.5 The ability for T precursor cells developing into γδ T cells was observed through the OP9-DL1 system cells in vitro and adoptive transfer experiments in vivo6 After infection with Salmonella typhimurium by gavage, the activation and the cytokine secretions of liver γδ cells was analyzed by flow cytometry.Results1 The proportion of γδ T cells in liver was significantly higher than thymus, spleen and lymph nodes. The proportion of y8 T17 cells in thymus gradually decreased with the increase of ageing, while in spleen and liver the ratio gradually increased. The ratio of γδ T1 cells in thymus and liver had no obvious links with age, while in spleen gradually decreased with the increase of ageing.2 Compared with wild-type mice, athymic nude mice had no difference in the ratio of γδ T cells in intestinal IEL, but γδ T cells in liver significantly decreased and were almost non-existent in spleen.γδ5 T cells in nude mice liver had normal cytokine secretion capacity.3 Athymic nude mice and wild-type mouse liver were rich in DN1 and DN2 T precursor cells. DN2 cells gradually appeared in liver from postnatal two weeks, and the percentage of DN2 cells peaked at fourth weeks. Compared with thymus DN2 cells, liver DN2 cells had higher expression of CD 117, lower expression of CD90 and CD25.4 Adult mouse liver contained CD24hiCD73+γδ T precursor cells, the proportion of which gradually increased with ageing. CD24h’CD73+γδ T precursor cells in adult mouse liver had a strong ability to secrete cytokines, higher expression of CD44, transcription factor T-bet and RORyt, lower expression of CD27.5 Co-incubated with OP9-DL1 cells, liver-derived DN1 cells could develop into γδ cells, the proportion of which was higher than thymus-derived DN1 or DN2 cells. Adoptive transfer experiments showed that liver-derived DN2 cells could develop into γδ precursor cells and mature γδ cells.6 The phenotype of γδ cells in liver was characterized by low expression of CD27 and high expression of CD44. In adult mice, γδ T cells in liver was rich in y8 T17 cells, while γδ T cells in intestine contained more γδ T1 cells, and there is no specific distribution of subpopulations in spleen or thymus.7 On the third day after infection, the IL-17a secretion by γδ cells in liver was increased significantly. On the fifth day after infection, the γδ cells’ expression of CD69 in liver was rapidly increased and the secretion of IFN-y was also increased obviously.Conclusions1 Apart from the small intestine and skin, the liver contains relatively abundant γδ T cells.2 The liver contains y8 T precursor cells, which have unique phenotypic and functional characteristics.3 Liver T precursor cells DN1 or DN2 cells have the ability to develop into γδ T cells. The liver may be one of extrathymic developmental organs for γδ T cells.4 y8 T cells in liver are unique in phenotype, distribution of subsets and functional features.