Analysis Of Tissue Location And Biological Characteristics Of Differentiated CD8~+T Cells In Mice Infected And Re-infected By Lethal Plasmodium Yoelii 17XL

Objective:Malaria is a global public health problem that endangers human health.Its pathogenic mechanism is complex and it is difficult to establish long-term immune protection.T cell mediated cellular immunity plays an essential role in the anti-malarial immune response.Compared with the liver stage,CD8⁺T cells play both immune protection and immunopathology roles in the erythrocyte stage of plasmodium.Our previous study found that lethal Plasmodium yoelii 17XL infection was harmful to the liver,and large amounts of accumulation of plasmodium-derived hemozoin can cause liver tissue damaged.However,P.y17XL infected survival mice showed abundant recruitment of CD8⁺T cells in the lymph nodes,spleen,and liver.Hepatic T cell mediated cellular immune response plays an important role in immune protection against P.y17XL re-infection in artesunate-cured mice.At present,the migration and tissue localization of the CD8⁺T cells of erythrocyte stage in the primary immune response and immune memory stage have not been fully understood,and the formation mechanism and function of CD8⁺memory T cells remain to be further explored.In this study,the characteristics of CD8⁺effector T cells,memory T cells and exhausted T cells subsets were analyzed by establishing a mouse model of P.y17XL resistance to primary infection and re-infection.The differences of chemokines between lymph nodes,spleen and liver after P.y17XL infection were compared.Combining with the characteristics of chemokine receptors expressed by CD8⁺T cells,the roles of chemokine receptors CXCR3 and CXCR6 in the functional effect of CD8⁺T cells and the formation of memory cells were expounded.The chemotactic characteristics of CD8⁺T cells of erythrocyte stage were verified by blocking lymphocyte migration experiment with FTY720.The The way to work and proliferate characteristics of CD8⁺memory T cells in vivo were determined by adoptive lymph node transfer test.The purpose of this study is to provide a new target for malaria immune defense and a new idea for the development of malaria in erythrocyte stage vaccine.Methods:1.Mouse model:1)P.y 17XL-1st infection mice model:C57BL/6 mice and BALB/c mice were infected via intraperitoneal injection of 1×10⁶parasitized red blood cells（p RBCs）.2)P.y 17XL-2nd infection mice model:After a month,resistant C57BL/6 mice were infected via intraperitoneal injection of 1×10⁶p RBCs to establish resistant mice model.3)For FTY720 trial,C57BL/6 mice that infected by P.y17XL were randomly divided into two groups.One of groups were treated with oral administration of FTY720（0.1mg/kg）once from day 0 post infection（p.i.）to day 5 p.i.and control group were given equal volumes of sterile water.4)For C57BL/6 adoptive tranfer experiments,C57BL/6 mice were intravenous injected with5×10⁶ highly purified CD8⁺T cells that selected from lymph node of resistant C57BL/6 mice infected P.y 17XL for twice,then intraperitoneal injected of 1×10⁶p RBCs.5)For CD45.1⁺-C57BL/6 adoptive tranfer experiments,CD45.1⁺-C57BL/6recipient mice were intravenous injected with 5×10⁶lymphnode cells from CD45.2⁺-C57BL/6 resistant mice and CD45.2⁺-C57BL/6 normal mice and then infected with 1×10⁶P.y 17XL plasmodium.2.The thin blood smear of tail vein was prepared from the d3 p.i.and the parasitemia,survival rate were observed dynamically.Paraffin sections were prepared from spleen and liver tissues on d5 p.i.,and histopathological changes of spleen and liver were detected by HE staining.3.Lymphocyte suspension of lymph nodes,spleen and liver were prepared by described methods.We used flow cytometry to detect the number of central memory(T_CM,CD44⁺CD62L⁺),effector T cells(T_EFF,CD44⁺CD62L^-)and exhausted T(T_EX,PD-1⁺Tim-3⁺)of CD8⁺T cell subpopulation.The expression of surface chemokine receptors CXCR3,CXCR6 and CX3CR1 were detected.Intracellular effector cytokines IFN-γand Granzyme B（GB）levels;Intracellular TCF-1 level of CD8⁺memory T cells;The response characteristics of CD45.1⁺and CD45.2⁺cell subsets in adoptive cell transfer experiments were demonstrated.The differences of spleen,lymph node and liver were analyzed within and between groups.4.Spleen and liver tissue of BALB/c and C57BL/6 mice infected by P.y17XL,Spleen and liver tissue of C57BL/6 mice re-infected by P.y17XL,G-Series arrays was prepared to detect the expression level of chemokines in mouse tissues.Results:1.Compared with C57BL/6 mice,the erythrocyte infection rate of BALB/c mice increased rapidly during day5～day8（P<0.0001）and reached the peak on day8（79.57±3.823%）.The parasitemia of C57BL/6 mice reached the peak on day14（48.19±3.192%）and then decreased.All BALB/c mice were dead on day9 and survival rate was lower（0%vs 78.5%,P<0.0001）.The liver and spleen of BALB/c mice looked more swollen than those of C57BL/6 mice.A hematoxylin-eosin staining using day 5 p.i.-organ samples immersed with fixed in 4%paraformaldehyde in advance.We observed that the extensive remodelling of the mouse spleen during malaria infection of BALB/c mice,charateristically consisting of the number and size of white pulps from BALB/c mice lower and smaller,the edge more indistinct than that in C57BL/6 mice,presence of haemozoin granules shown in illustration and a diffuse hypercellularity in the splenic red pulp in BALB/c.In contrast,in the C57BL/6 mice,the red and white pulps of spleen are clearly demarcated and the number of white pulps is abundant.Moreover,liver tissue of mice in both groups was damaged,with extensive swelling,loose arrangement and uneven size of liver cells,and a small amount of inflammatory infiltration and hemozoin infiltration.These results indicate that the spleen and liver were involved with primary P.y17XL infection,and C57BL/6 mice are more resistant to infection than BALB/c mice.2.To determine the difference in response to P.y17XL primary infection between the BALB/c and C57BL/6 mice,we analyzed differences in CD8⁺T cell differentiated subsets in the spleen,lymph node,and liver.Flow cytometry results showed that compared with BALB/c mice,number of CD8⁺T cells in spleen,liver and lymph node in C57BL/6 mice obviously increased（P<0.05）.The percentage and total number of CD8⁺effector T cell(T_EFF,CD44⁺CD62L^-)and CD8⁺central memory T cell(T_CM,CD44⁺CD62L⁺)in lymphocytes of spleen and lymph node of C57BL/6 mice was significantly increased（P<0.05）,and the percentage of T_EFFsubset of liver was also increased（P<0.05）.Compared with C57BL/6 mice,the percentage of CD8⁺exhausted T cells(T_EX,PD-1⁺Tim-3⁺)in the liver and spleen of BALB/c mice was significantly higher（P<0.0001,P<0.01）.The total number of CD8⁺T_EX cells in spleen of BALB/c mice was increased（P<0.001）.It suggested that P.y17XL primary infection induced CD8⁺T cells proliferated and differentiated to T_EFF and T_CM cells in C57BL/6 mice.In contrast,BALB/c mice showed large CD8⁺T_EX cells in the spleen and liver,which may be closely related to the rapid rise in parasitemia.3.We used flow cytometry to detect splenic and liver CD8⁺T cells secreting expressing IFN-γand GB on day 5 p.i.of BALB/c and C57BL/6 mice.Experiment data showed that the total number of CD8⁺GB⁺T cells（P<0.01,P<0.05）and CD8⁺IFN-γ⁺T subsets（P<0.05,P<0.05）in liver and spleen of C57BL/6 mice was increased compared with BALB/c mice,and the CTL response in liver and spleen of C57BL/6 mice was enhanced.In addition,the median fluorescence intensity of IFN-γand GB in C57BL/6 and BALB/c mice showed that the CTL effect in liver was significantly enhanced compared with that in spleen,and the secretion of IFN-γand GB was significantly increased,suggesting that CTL effect function was more dominant in liver than spleen.These data showed that liver was an important site for the primary immune response of P.y17XL,and liver CD8⁺T cells played an important role in the clearance of plasmodium.4.The expression levels of CXCR3,CXCR6 and CX3CR1 on CD8⁺T cells in the liver,spleen and lymph nodes of BALB/c and C57BL/6 mice infected by P.y17XL were detected on day 5 p.i..Flow cytometry results showed that,in compared with BALB/c mice,CD8⁺CD44⁺CXCR3⁺（P<0.01,P<0.05）,CD8⁺CD44⁺CXCR6⁺（P<0.0001,P<0.01）and CD8⁺CD44⁺CX3CR1⁺（P<0.001,P<0.05）chemokine receptor levels were obviously increased in lymph nodes of C57BL/6 mice.We founded that CXCR3,CXCR6 and CX3CR1 chemokine receptors were also greatly expressed by CD8⁺T cells in liver and spleen of C57BL/6 mice,and the percentage of lymphocytes were significantly higher than that of BALB/c mice（P<0.05）.These results suggested that the high levels of chemokine receptors on activated CD8⁺T cells may mediate the migration and recruitment of CD8⁺T cells between secondary lymphatic organs and non-lymphoid tissues.5.In order to explore the role of chemokine receptors in mediating the chemotaxis and function of CD8⁺T cells,the lymphocyte migration blocker FTY720was used to observe P.y 17XL infected C57BL/6 mice.The parasitemia of experimental group（FTY720 group）had been rising from day4～7 after administration and was greatly higher than the control group(P_d4<0.05,P_d5<0.01,P_d6<0.01,P_d7<0.05).Mice died from day 5 after P.y17XL infection,with survival rate of 20%in experimental group（P<0.05）.We used flow cytometry to detect the number of CD8⁺T cells and their surface CXCR3 chemokine receptor expression levels after FTY720 treatment.The results suggested that compared with the control group,the percentage of CD8⁺T cells in lymph nodes（P<0.05）and the total number of CD8⁺T cells in liver and spleen（P<0.05,P<0.05）were significantly decreased in the experimental group,and the CXCR3 expression level of CD8⁺T cells in lymph nodes,liver and spleen was significantly decreased（P<0.05）.These results indicate that CD8⁺T cells mediate migration through CXCR3 and play a key role in the function of the anti-malarial immune response of CTL.6.As control group（P.y 17XL,C57BL/6-1st）,resistant C57BL/6 mice on day30 were attacked by P.y 17XL again（C57BL/6-2nd）.Using a C57BL/6 re-infection model,we analyzed the characteristics of memory CD8⁺T cell subsets.Compared with the control group,the experimental group showed completely resistance to P.y 17XL re-infection.During the observation period,no Plasmodium parasitized RBC（p RBC）was observed under the microscope,and the survival rate of mice reached 100%.In compared with the C57BL/6-1st mice,our data showed that there were a large number of T_CM cell subsets in the spleen of the experimental group（P<0.01）,and CXCR3⁺CXCR6⁺T_CM cells in the spleen and lymph nodes were significantly increased（P<0.01,P<0.05）.CD8⁺T cells in spleen and liver of C57BL/6-2nd mice showed high expression of IFN-γ（P<0.05,P<0.001）,while no increase in GB expression（Not supplied）.These results indicate that memory CD8⁺T cells exert the immune memory effect against P.y 17XL reinfection by secreting IFN-γin long-term.The antibody array was used to identify 25 cytokines.Compared with control group,the expressions of Eotaxin and MIP-2 were up-regulated in spleen of experimental group.7.In order to verify the mode of action and characteristics of memory CD8⁺T cells in vivo,P.y 17XL resistant mouse lymphocytes were analyzed by adoptive transfer method.CD8⁺T cells in lymph nodes of re-infected resistant mice were positive selected by magnetic beads for adoptive cell transfer.The functional characteristics of CD8⁺T cell subsets were analyzed by flow cytometry on day 5 p.i..Flow cytometry results showed that,in spleen and liver,the number of stem-like CD8⁺PD-1⁺TCF1⁺T cells（P<0.05,P<0.01）,CD8⁺GB⁺T cells（P<0.05,P<0.05）and CD8⁺IFN-γ⁺T cells（P<0.001,P<0.05）were obviously increased from experimental mice in compared with the control group.These results suggest that CD8⁺memory T cells in adoptive transferred cells proliferated in the recipient mice,and thus mediate the CTL immune response against P.y 17XL infection.To further clarify the T cell chemotactic pathway in lymph nodes,a mouse model of CD45.1⁺-C57BL/6 adoptive cell transfer was established.The proportion of CD8⁺CD45.2⁺cells in lymph node of the experimental group increased significantly（2.92%～7.32%）.As the control group,the proportion of CD8⁺CD45.2⁺cell subsets did not increase significantly（2.40%～3.67%）.It indicated that with the development of P.y 17XL infection,CD8⁺CD45.2⁺cells appeared to proliferate in experimental group.On day3,CD8⁺CD45.2⁺CD44⁺CD62L⁺T_CM（35.9%）in experimental group was higher than that in control group（8.82%）.On day8,in compared with control group（13.4%）,the CD8⁺CD45.2⁺CD44⁺CD62L^-T_EFF（73.9%）in experimental group was in higher levels.Our data indicate that memory CD8⁺T cells from donor mice proliferated and differentiate to effector memory subset and exert CTL effect in vivo.Conclusion:1.CD8⁺T cells secrete toxic granules GB and IFN-γto mediate primary cellular immune response against P.y17XL infection.2.CXCR3 mediates the chemotaxis of CD8⁺T cells from lymph nodes to spleen and liver.3.Activated memory CD8⁺T cells secrete IFN-γto mediate the immune memory effect of spleen and liver against P.y17XL re-infection.4.Chemotaxis of memory CD8⁺T cells provide important protection for anti malaria immunity.