Effects And Mechanism Of Tumors On Migration Of CD8~+T Cells To Secondary Lymphoid Organs

Background:CD8+T lymphocytes(T cells) play important roles in anti-infection and anti-tumor immune response in vivo. Antigen-specific cytotoxic T lymphocytes(CTLs) can exert the killing effect through the release of effector molecules, such as perforin and granzyme. CTLs in tumor patients suffer from activation barriers(e.g. immune tolerance) or CTLs after activation in the tumor microenvironment suffer from functional inhibition(e.g. T cells failure). However, recent research shows that CD8+T cells in tumor patients suffer from not only immune tolerance in terms of anti-cancer, but also from activation barriers in resistance against foreign pathogens.As is well-known, when antigen presenting cells(APCs) after swallowing or antigen processing are migrating to secondary lymphoid organs(SLOs), APCs bind with T cell receptor(TCR) through MHC-peptide complexes, and finally exert the killing effect by activating CTLs and migrating from the lymphatic vessels to peripheral tissues. The probability of random encounter between APCs and antigen-specific T cells in vivo is very small. Nevertheless, the presence of SLOs creates conditions for their rapid encounter and binding, which leads to the rapid production of abundant CTLs. Besides the low expressions of tumor antigens by tumor cells and MHC molecules, previous research about the mechanisms of T-cell immune tolerance or failure formation in tumor patients focuses on the regulatory effects of tumor microenvironment on APCs and CD4+T cells, and the direct inhibitory effects on CTLs. For example, myeloid-derived suppressor cells(MDSCs), tumor-associated macrophages(TAMs), T regulatory cells(Tregs), and tumor cells will induce immune tolerance of CD8+T cells or functional failure of CTLs through the expressions of immunosuppressive molecules(e.g. PD-L1) or secretion of relevant cytokines(e.g. TGF-β and IL-10). As stated earlier, SLOs(including lymph nodes and spleen) are the key place for reactivation of the effector cells, CTLs.Objective:However, there is no exploration into the activation process of tumor-bearing CTLs in SLOs. Research from this perspective will provide new clues for design of new anti-cancer active immunotherapy schemes and for enhancement of anti-infection ability in tumor patients.The aim of this study is to clarify the effects of tumor-bearing on immune response of CTLs in SLOs.Methods:We first randomly divided 6- to 8-week-old female C57BL/6J mice into 2 groups: a normal group infected by OVA-Listerial monocytogenes(LM), and a test group of Lewis lung cancer mice infected by OVA-LM. The response abilities of OVA-specific CTLs were detected. The above experiments were repeated with LCMV. Then CD8+T cells were screened out using magnetic beads, CFSE-marked, and retransfused via caudal-vein into the mice to detect the changes in their directional migration ability to SLOs. Quantitative real-time PCR(q RT-PCR) and enzyme-linked immunosorbent assay(ELISA) were used to detect the expressions of chemokines CCL21 and CCL19. Then to clarify the specific causes for the changes in SLOs, we used flow cytometry to detect the cell compositional changes in SLOs. Since all mice at late tumor-bearing stage suffered from anemia, we also detected the changes of blood hemoglobin, and used ELISA to detect the expression of blood erythropoietin(EPO). We further analyzed the effects of anemia and EPO hypersecretion during anemia on SLOs, and built an anemia model by treating mice with intraperitoneal injection of phenylhydrazine. CD8+T cells were screened out using magnetic beads, CFSE-marked, and retransfused via caudal-vein into the mice to detect the changes in their directional migration ability to SLOs. q RT-PCR and ELISA were used to detect the expressions of CCL21 and CCL19. Intraperitoneal injection of EPO alone was used to repeat the above experiments. Finally, an EPO antibody was used to block the action of EPO in tumor-bearing mice and then the changes of chemokines were observed. The main results and conclusions are listed below.Results:1. Changes of immune response levels of CD8+T cells in SLOs of tumor-bearing mice(1) After different tumor-bearingdurations, the mice were inoculated with OVA-containing LM. After 7 days, the immune response levels of CD8+T cells specific to OVA epitope in spleen were detected. Results show that after CD8+T cells in spleen of tumor-bearing mice were stimulated with OVA peptide in vitro, the intracellular IFN-γ and Granzym-B secretion capacities were significantly reduced with the prolonging of tumor-bearing days.(2) After different tumor-bearingdurations, the mice were inoculated with GP33-containing LCMV. After 8 days, the immune response levels of CD8+T cells specific to GP33 epitope in spleen were detected. Results show that after stimulation in vitro with GP33 peptide, the intracellular secretion of IFN-γ by CD8+T cells was significantly downregulated, and the ratio of specific GP33 epitope in CD8 cells was also significantly down-regulated.2. Directional migration ability of CD8+T cells to spleen in tumor-bearing mice, and expressions of chemokines in spleen.(1) The directional migration ability of CD8+T cells to SLOs was detected with spleen as example. Primary CD8+T cells were marked with CFSE, and a test group was built by caudal-vein transplantation of tumor for different periods, with a normal group as control. Then distributions of CFSE-marked cells in spleen were analyzed and compared between groups. Results show that in the test group, the count of CFSE-marked CD8+T cells migratingtospleen detected by flow cytometry was significantly reduced. Fluorescent observations show that the count of CFSE-marked CD8+T cells migrating to T cells zone is significantly smaller versus the control group.(2) Previous research shows that the directional migration to SLOs of CD8+T cells is regulated by the secretion of chemokines CCL21 and CCL19 by stromal cells. Then the expressions of splenic chemokines were detected by ELISA and q RT-RCR. Results show that the expressions of CCL21 and CCL19 were gradually downregulated with the prolonging of tumor-bearing time at both m RNA and protein levels. These results were confirmed by fluorescence tests performed later.(3) To clarify whether this phenomenon is common, we inoculated C57 mice with C57-derived malignant melanocytoma cells B16F10, and inoculated BALB/C mice with a BALB/C-derived colon cancer cell line CT26. Then we observed the directional migration ability of CD8+T cells to SLOs in tumor-bearing mice. Primary CD8+T cells were marked with CFSE, and a test group was built by caudal-vein transplantation of tumor for different periods, with a normal group as control. Then distributions of CFSE-marked cells in the spleen were analyzed and compared between groups. Fluorescent observations show that the count of CFSE-marked CD8+T cells migrating to the T-cell zone is significantly smaller versus the control group, and the secretions of CCL21 and CCL19 were also significantly reduced.3. The directional migration ability of CD8+T cells to spleen in tumor-bearing mice, and expressions of chemokines in spleen were investigated after knockout of MDSCs.(1) Previous research shows that the count of splenic MDSCs is significantly increased under tumor-bearing condition. To clarify the changes of MDSCs in the tumor-bearing group, we analyzed the changes of major cell components in the spleen using flow cytometry. Results show that the proportions of splenic CD4+T and CD8+T cells were significantly reduced. The proportions of CD45- cells and MDSCs were significantly increased along with the prolonging of tumor-bearing time, while the proportion of Treg cells did not change obviously.(2) Previous research shows that treatment of low-dose 5-FU can specifically knock out MDSCs. In this study, the proportion of splenic MDSCs is significantly reduced after such treatment. However, the treatment with 5-FU could neither recover the directional migration ability of T cells to the spleen, nor the expressions of CCL21 and CCL19 at m RNA or protein level.(3) Though knockout of MDSCs did not improve the directional migration ability of CD8+T cells to SLOs, we further tested CD45- cells with flow cytometry. Results show that the CD45- cells were dominated by CD45-TER119+ cells. Since cancer patients at late stage are usually accompanied with varying degrees of anemia, we speculate that the significant increase of CD45- TER119+ cells is attributed to anemia. Then we extracted heart blood from mice after different tumor-bearing periods and tested. Results show that the count of red blood cells(RBCs), the amount of hemoglobin, and the hematocrit of RBCs were reduced to different degrees along with the prolonging of tumor-bearing period.4. Effects of anemia on the directional migration ability of CD8+T cells to spleen in tumor-bearing mice, and on expressions of chemokines in spleen.(1) Since all mice at late stage suffered from varying degrees of anemia, we investigated the effects of anemia on the spleen. The mice were injected via caudal vein with 4×106 OVA-LM. Then the response ability of anti-OVA-specific CD8+T cells in the spleen was detected. Results show that the response abilities of anti-OVA-specific CD8+T cells in blood and in spleen were both significantly lower versus the control group. Then T cells of OT-1 mice were retransfused and the mice were injected via caudal vein with 4×106 OVA-LM. The response ability of anti-OT-1 splenic CD8+T cells was detected. Results show that after occurrence of anemia, the response level of the OT-1-derived CD8+T cells was significantly lower versus the control group, and the proliferation of CD8+T cells was significantly weakened. These results indicate that the immune response level of CD8+T cells in anemic mice was downregulated.(2) Adaptive immune response mainly occurs in SLOs. To clarify the cause for the decline of response level in CD8+T cells, flow cytometry was used to investigate the changes in spleen. Results show that the cells showing negative regulatory effects on immune response reportedly such as MDSCs and Treg did not show any change. Then CD8+T cells of normal mice were screened out using magnetic beads, marked by CFSE and retransfused via caudal vein into anemic mice. Results show that with the aggregation of anemia(at 3-5 days after modeling, the hemoglobin level was minimized and then gradually recovered), the proportion of CFSE-positive CD8+T cells in the spleen was gradually reduced. Moreover, fluorescence microscopy showed that the count of CFSE-positive cells in the T cell zone was significantly down-regulated in the spleen. This result indicates that the directional migration ability of CD8+T cells to SLOs was weakened.(3) The chemoattraction of T cells by SLOs was realized mainly through the secretion of CCL21 and CCL19. Then the expressions of CCL21 and CCL19 in the spleen following anemia were detected. Results show that both m RNA and protein levels were reduced with the aggravation of anemia. When anemia was gradually alleviated, the expressions were slowly recovered to normal levels(Fig. 3A, B, C). These results indicate that anemia could impact the immune response of T cells by affecting the chemokines in SLOs.(4) The changes of cell components in the spleen were tested. Results show that the proportions of CD4+T and CD8+T cells gradually decrease with the aggravation of anemia, while the proportion of CD45-negative cells increases. The proportions return to normal levels after alleviation of anemia(Fig. 11A). Specifically, the CD45-negative cells were dominated by TER119-positive cells(Fig. 11B), which is consistent with the early-stage tumor tests. These results indicate that the response changes of SLOs and CD8+T cells at late tumor-bearing stage were mainly induced by anemia.5. Effects of anemia-induced significant upregulation of EPO secretion on the directional migration ability of CD8+T cells to spleen, and on expressions of chemokines in spleen.(1) Preliminary tests show that anemia occurring at late tumor-bearing stage induced changes of directional migration ability of CD8+T cells to SLOs, and down-regulated the expressions of CCL21 and CCL19. As is well-known, EPO level significantly increases with the occurrence of anemia. We further validated the expressions of EPO following anemia. Results show that EPO expression gradually increased with the prolonging of tumor-bearing time, and also increased with the presence of pure anemia. Intraperitoneal injection of EPO alone induced the significant increase in the count of CD45- cells, which were dominated by TER119+ cells. After intraperitoneal injection of 4IU/g/d recombinant human EPO, the migrating-to-spleen ability of CD8+T cells was significantly reduced since the 3rd day. This result is fully consistent with the changes of SLOs at late tumor-bearing stage.(2) We further detected the expressions of chemokines after injection of EPO. Results show that the secreted amounts of CCL21 and CCL19 in the spleen gradually decreased with the increase of dosage, and with a single injection, the amounts decreased most obviously at day 2 and recovered at day 4. The downregulation was more obvious after a longer continuous injection. These results were confirmed by fluorescence tests. Such consistence indicates that the effects of anemia on SLOs may be realized through the high secretion of EPO.(3) To further validate the effects of EPO on tumor-bearing SLOs, we provided caudal-vein injection of 250 ug of EPO antibody every two days to each tumor-bearing mouse since the 14 th day, and killed the mice at day 22. Results show that tumor-bearing did not down-regulate the expressions of chemokines in the spleen, or reduce the homing-to-SLOs ability of CD8+T cells. These results further validate that the changes of SLOs under tumor-bearing state were attributed to the increased expression level of EPO induced by anemia.6. Effects of tumor-bearing on the directional migration ability of CD8+T cells to SLOs, and on expressions of chemokines in SLO.(1) Lymph nodes are another major part of SLOs. Then we aim to clarify whether anemia under tumor-bearing state would exert the same effect on lymph nodes as on the spleen. First, we built an anemia model and detected the expressions of lymph-inoculated chemokines with the occurrence of anemia. Results show that expressions of neither CCL21 nor CCL19 were significantly different at m RNA or protein level. Then magnetic beads were used to screen out CFSE-positive CD8+T cells, which were retransfused via caudal vein into mice. The directional migration ability to lymph nodes was tested. No significant difference was found. Then the mice were inoculated with Lewis lung cancer cells. The changes of chemokines under tumor-bearing state were observed. The results were not significantly different versus the pure anemia condition. The changes of cell components in lymph nodes were also tested. No obvious change was found in the CD45-negative cells.7. Effects of tumor-bearing T cells on secretion of splenic chemokines(1) We aim to clarify whether tumor-bearing T cells are involved in regulation of chemokine expressions in SLOs. First, we detected the changes of chemokines in the spleen in BALB/C-derived nude mice and tumor-bearing BALB/C mice. Results show that expressions of CCL21 or CCL19 were not significantly different the two types of mice. As reported, T cells are involved in maintaining the chemokine expressions in SLOs. Our results also indicate that because of absence of T cells, the chemokine expressions in the tumor-free mice were also significantly down-regulated. We aim to exclude the possibility that the insignificance of chemokine secretion levels between the two was induced by the too low initial expression level of chemokines in nude mice. Then in mice with knockout of CD4 or CD8 alone, we detected the chemokine expressions between knockout mice and non-knockout mice under tumor-bearing state. Results show that in tumor-bearing mice, the knockout of CD4 or CD8 did not induce significant difference in the expressions of CCL21 or CCL19. As reported, TNF-a receptor is highly expressed in mesenchymal cells, and TNF-a expression level under tumor-bearing state is significantly elevated. We then test the expressions of chemokines in tumor-bearing mice before and after knockout of TNF-a. Results show that in tumor-bearing mice, the knockout of TNF-a did not induce significant difference in the expressions of either chemokine. This result indicates that T cells are not involved in the regulation of chemokines under the tumor-bearing state.Conclusions:The above analyses indicate that with the prolonging of tumor-bearing time, the directional migration ability to SLOs of CD8+T cells was weakened, and the expressions of CCL21 and CCL19 in the spleen were both down-regulated. These changes were caused mainly by the anemia-induced significant enhancement of EPO secretion. However, these changes were not observed in lymph nodes, which are another important part of SLOs. In conclusion, this work helps to further clarify the effects of microenvironment "remodeling" in SLOs on the anti-cancer immune response as well as the molecular mechanism. Italso helps to probe into the specific causes for the decline of overall immune functions in tumor patients in clinic, and provides a new clue for design of tumor immunotherapy.