| Diffuse large B-cell lymphoma(DLBCL)is the most common subtype of non-Hodgkin's lymphomas with rapid progression.Approximately 30%of patients fail to respond to first-line treatment or present relapsed DLBCL.For these patients,current treatment options are limited and have poor efficacy.Growing evidence supports that host T cell immunity could be utilized to eliminate refractory/relapsed B cell lymphomas.CXCR5+CD8+ T cell is a T cell subgroup discovered recently.This subgroup was found presenting high cytotoxicity in chronic virus infection.CXCR5+CD8+ T cells coexpress CXCR5 with CD 19+ tumor cells.Compared with CXCR5-CD8+ T cells,CXCR5+CD8+ T cells may show stronger interaction with B cells.This study explored the cytotoxicity of CXCR5+CD8+ T cells in DLBCL.Among healthy people,the proportion of CXCR5+CD8+ T cells in T cells is generally below 1%.However,this proportion among DLBCL patients varies and is higher than the healthy control(0.11-0.51%vs 0.18-3.25%,p=0.0031).The proportion of CXCR5+CD8+ T cells in CD8+T cells is higher too(0.38?1.72%vs 0.62?14.38%,p=0.0044).In the cytotoxicity experiment,CD3/CD38 coactivated CD8+ T cells were taken as the effector cells,the autologous CD109+ tumor cells were taken as the target cells,when the effector/target(E:T)ratio was 16:1,no difference of cytotoxicity was found between the CXCR5+CD8+ T cells group and the control group.However,when the E:T ratio was 4:1 or 1:1,the cytotoxicity of the CXCR5+CD8+ T cells group was significantly higher than the control group.We separated the CD8+ T cells and tumor cells by a membrane with pores of 3.0 ?m in diameter,which allowed the transfer of soluble protein molecules but not whole cells.When the CD8+ T cells and tumor cells were separated,we noticed a large drop in tumor cell killing in all E:T ratios.No difference in CXCR5+CD8+ T cell-mediated and CXCR5-CD8+ T cell-mediated cytotoxicity was observed at any E:T ratio.Following cytotoxicity assay,CXCR5+CD8+ and CXCR5-CD8+ T cells were harvested for mRNA analyses and the supernatant in the bottom tumor cell-containing chamber was harvested for ELISA of granzyme and perforin proteins.We found that in all 6 DLBCL patients examined,the CXCR5+CD8+ T cells expressed elevated levels of granzyme A,granzyme B,and perforin mRNA than CXCR5-CD8+ T cells from the same patient.However,in the corresponding supernatant from wells containing the tumor cells,no significant differences in granzyme B and perforin protein levels were observed between CXCR5+CD8+ T cells and CXCR5-CD8+ T cells.The CD107a expression by stimulated CXCR5+CD8+ T cells and CXCR5-CD8+T cells was examined by flow cytometry.The results showed no significant difference in the level of degranulation.DLBCL were known to secrete high level of interleukin 10,which could potentially change the inflammatory status of cancer-reactive immunity.Therefore,we blocked the IL-10/IL-1OR pathway,and found that the expressions of granzymeA,granzyme B,and perforin by CXCR5+CD8+ T cells were significantly elevated,and the viability in corresponding tumor cells was lower.In summary,the percentage of CXCR5+CD8+ T cells in DLBCL patients is upregulated.The cytotoxicity of CXCR5+CD8+ T cells is stronger than CXCR5-CD8+T cells.Low E:T ratio allowed the CXCR5+CD8+ T cells to present higher cytotoxicity.It might be due to the possibility that CXCR5+CD8+ T cells were able to mediate longer and more stable interactions with tumor cells,thus resulting in more efficient delivery of cytotoxic molecules.Compared with CXCR5-CD8+ T cells,CXCR5+CD8+ T cells expressed more granzyme and perforin,but no significant difference in the level of degranulation was found.When the IL-10/1 OR pathway was blocked,the expression of granzyme and perforin of CXCR5+CD8+ T cells would be significantly higher.In conclusion,CXCR5+CD8+ T cells are potential candidates of CD8+ T cell-based immunotherapies,could mediate elimination of autologous tumor cells in DLBCL patients,but are also susceptible to IL-10 mediated suppression. |
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