The Roles And Underlying Mechanisms Of Eosinophils In Memory CD8~+T Cell Generation During Bacterial Infection

The rapid and efficient response of memory T cells after the second encounter with the pathogen constitutes a hallmark trait of adaptive immunity.The T cell response to acute infection can typically be divided into three phases—priming and expansion,resolution and contraction,and memory.T cell subsets in different stage of infection can vary in the phenotype,function,location,and long-term fate in terms of the ability to populate the memory T cell pool.Over the past decades,the signalling pathways and transcriptional programmes that regulate the formation of heterogeneous populations of effector and memory CD8~+T cells have been well characterized.However,the mechanisms for deciding the fate of different T cell subsets,especially for the generation and maintainence of memory CD8~+T cells,need to be further elucidated.Eosinophils have been traditionally perceived as the terminally differentiated cytotoxic effector cells.Recent studies about the molecular pathways that control the development,trafficking and degranulation of eosinophils have improved our understanding of the functions of eosinophils in immune response and inflammation.It’s well known that eosinophils are extensively involved in the infection and immunity.However,there is no report about the possible roles of eosinophils in the regulation of CD8~+T cellmemory generationin adaptive immunity,which has great significance therefore worth the further investigation.In this study,we infected wild-type and GATA-1 micewith OVA-expressing Listeria monocytogenes(LM-OVA)to prepare the experimental mouse model for primary and secondary immune response.It’s well accepted that GATA-1 mice exhibit eosinophil deficiency.We found that the proportion and numbers of memory CD8~+Tcells in spleen from eosinophil-deficient mice were significantly lower than those in wild-type mice 60 days after LM infection.Moreover,after secondary immunization on day 60 with the same LM injection,the proportion of splenicIFN-γ+CD8~+T cells and the concentration of circulating IFN-γof eosinophil-deficient mice were significantly decreased than that of wild-type mice.These results suggest that eosinophil deficiency leads to impaired formation of memory CD8~+T cells and weakened secondary immune response in mice in response to bacterial infection.By dynamic analysis of antigen-specific CD8~+T cells on day 8 and 14 after primary infection of LM,we found that the proportion and numbers of antigen-specific CD8~+T cells in eosinophil-deficient mice were also lower than that in wild-type mice.In the early stage of infection(D0-D3),the numbers of CD8~+T cells were decreased in eosinophil-deficient mice,while the numbers of CD8~+T cells and eosinophils both were increased in wild-type mice.Therefore,we speculated that eosinophils could inhibit the apoptosis of CD8~+T cells in the early stage of infection.In vitro experiments showed that LM infection induced high expression of Annexin-V and PD1 in CD8~+Tcells,indicating more apoptosis of CD8~+T cells.Meanwhile co-culture of eosinophils with CD8~+T cells decreased the LM infection-induced expression of Annexin-V and PDI,indicating co-culture of eosinophils could inhibit apoptosis of CD8~+T cells.iranswell experiment proved that eosinophils could inhibit LM infection-induced apoptosis of CD8~+T cells without cell-cell interaction.Instead,the supernatant of eosinophils after infection inhibitedLM infection-induced apoptosis of CD8~+T cells.We then separated theeosinophils-derived supenatantby ultracentrifuge and found that only the upper layer with molecular weight greater than 10kD could efficiently inhibit LM infection-induced apoptosis of CD8~+T cells.These results suggested that the soluble molecules such as cytokines secreted by eosinophils stimulated by LM infection may be responsible for the inhibition of LM-induced apoptosis of CD8~+T cells.Nextly,we performed RNA-seq of eosinophils with or without LM infection and discovered the high expression level of IL-4 in eosinophils after LM infection.ELISA results showed that theprotein level of IL-4 was also significantly increased.When CD8~+T cells were pretreated with exogenous recombinant IL-4,LM infection-induced apoptosis of CD8~+T cells was inhibited with the reduction of Annexin-V and PD1 expression.While using anti-IL-4 antibody to neutralize IL-4 in the supernatant,the antiapoptotic effect of the supernatant was weakened.Besides,immunofluresence results showed the obvious co-localization of eosinophils and IL-4 in the early stage of LM infection.These results suggested that IL-4 derived from eosinophils mediates the anti-apoptotic effect of eosinophils on CD8~+T cells after LM infection.We went further to examine what’s the key signaling pathways of T cells in mediating the anti-apoptotic effect,and found that LM infection activated TLR4-MD2 and induced T cell apoptosis through LLO.Using the specific inhibitor to block the downstream pathways P38/MAPK,NF-kappaB and JNK/AP-1 respectively,we found that P3 8/MAPK inhibited LM infection-inducedCD8~+T cell apoptosis,while NF-kappaBhad no effect on apoptosis,and JNK/AP-1 promoted the apoptosis.Moreover,exogenous IL-4 reduced the phosphorylation of JNK in CD8~+T cells and thus inhibited apoptosis of CD8~+T cells.In summary,our results demonstrated that eosinophils can secrete IL-4 to inhibit LM infection-induced apoptosis of CD8~+T cells at the early stage of LM infection,thus ensuring the effective adaptive immune response and the formation of T cell memory in response to bacterial infection.