The Development Of Two IL-15 Complexes In Pichia Pastoris The Function And Mechanism Of LSP1 In Macrophage Polarization

Part Ⅰ:Development of two novel IL-15 complexes in the Pichia pastorisInterleukin 15(IL-15)is a pleiotropic cytokine that stimulates the differentiation and proliferation of NK cells,T cells and B cells,enhances the cytotoxic activity of NK cells and CD8+ T cells,and induces the generation and maintenance of memory CD8+T cells.Multiple studies have shown the therapeutic potential of IL-15 in anti-tumor immunotherapy.However,there are several limitations in applying IL-15 in therapy,such as the short half-life in vivo and the toxicity and side effects caused by multiple effects of native IL-15.In recent years,based on the findings that the IL-15/IL-15Rαcomplex has a high affinity for IL-15Rβ/γc,variety of recombinant IL-15 superagonists have been developed through mutation and/or configuration modification,that can efficiently and selectively induce IL-15R(β/γc expressing NK cells and CD8+T cells,including RLI,the IL-15/IL-15Rα-IgG1-Fc complex,and ALT-803,which shown promising anti-tumor effects.In this study,we designed two different IL-15/SuIL-15Rα-IgG4 Fc complexes,which were formed by the noncovalent assembly of IL-15 with monomeric or dimeric IL-15 Rα sushi domain-IgG4 Fc fusion protein and designated IL-15/SuIL-15Rα-mFc and IL-15/SuIL-15Rα-dFc,respectively.The two complexes were expressed in Pichia pastoris,a low cost and stable recombinant protein expression system.Compared with recombinant human IL-15(rhIL-15),the activity of the two complexes to stimulate CTLL-2 cell proliferation decreased by 50%,but the half-life of the two complexes increased from 0.7 hours(rhIL-15)to 9.16 hours(IL-15/SuIL-15Rα-mFc)and 14.26 hours(IL-15/SuIL-15Rα-dFc),respectively.Under the condition of using the same molar amount of IL-15,the two complexes significantly induced the proliferation of NK cells,NKT cells and memory CD8+ T cells in the spleen and peripheral blood of mice,and the proliferation and activation of NK cells and CD8+T cells in PBMCs.These findings indicate that compared with the natural IL-15,the two IL-15/SuIL15Rα-IgG4 Fc complexes exhibit potent activity in selectively inducing the proliferation and activation of NK cells and CD8+T cells,as well as longer half-life in vivo,which have great potential for clinical application.In addition,the Pichia pastoris expression system used in this study can express the active IL-15/SuIL-15Rα-IgG4 Fc complexes efficiently and stably.Compared withPart Ⅱ:The function and mechanism of LSP1 in macrophage polarizationLeukocyte-specific protein 1(LSP1)is an intracellular F-actin bundling protein,which is mainly expressed in hematopoietic cells,such as T and B lymphocytes,neutrophils and macrophages,and also expressed in endothelial cells.LSP1 plays an important role in regulating leukocyte chemotaxis and transendothelial cell migration,and has been proved to be involved in the regulation of various immune cell functions,such as T cell maturation and survival,and B cell apoptosis.Moreover,LSP1 is associated with the pathogenesis of various cancers,including lymphoma,breast cancer,and glioma,and may serve as a diagnostic marker for a variety of diseases.Recent studies have shown that high LSP1 expression is accompanied by increased infiltration of M2 macrophages,neutrophils,and regulatory T cells in the tumor microenvironment of glioblastoma,but its function and specific mechanism remains elusive.In this study,we investigated the function of LSP1 in macrophages by comparing bone-marrow derived macrophages(BMDMs)from wild-type(WT)mice and LSP1 deficient mice induced in vitro.Our study showed that LSP1 inhibited the mRNA expression of pro-inflammatory cytokines Tnf and Il1b in BMDMs and promoted the mRNA expression of 116 in response to LPS stimulation.Since macrophages exhibit different functional phenotypes in response to different environmental signals,we then examined the effect of LSP1 on the polarization of macrophages,and found that LSP1 could inhibit the polarization of macrophages toward M1-like phenotype.Further study revealed that LSP1 interacted with IKKα and inhibited its phosphorylation,thereby inhibiting the activation of the NF-κB signaling pathway.These results suggest that LSP1 affects the polarization of macrophages,possibly by regulating the activation of NF-κB signaling pathway via interacting with IKKα.Our study reports for the first time that LSP1 can participate in the regulation of the NF-κB signaling pathway by interacting with IKKα,thereby regulating the polarization of macrophages.The results of this study may contribute to a more comprehensive understanding of the role of LSP1 in the immune system.the mammalian cell expression system,the Pichia pastoris expression system can greatly reduce the production cost and production cycle,which has significant potential and advantages in the development of engineering transformation applications.