Research On Alginate Enhancing The Antitumor Immunity Memory Effects Of CD8~+T Cells

Alginate,an FDA-approved natural biomaterial usually used as therapeutic adjuvant,drug carrier and biological scaffold,reportedly assists the immune system to activate cytotoxic T cells in antitumor assays.In this study,by incubating sorted cytotoxic CD8~+T cells with alginate,we found this material promote the secretion of cytokines IFN-?and IL-2 by CD8~+T cells and facilitated the antitumor cytotoxic activities of T cells.Alginate incubation significantly promoted memory properties of CD8~+T cells and elevated the proportion of CD62L~+CD44~+central memory T cell(TCM),improved the long-term antitumor memory effects of CD8~+T cells in vivo.Mechanistically,alginate increased the CD8~+T CM cells by increasing intracellular glutathione generation and reducing reactive oxide species in CD8~+T cells.Further,we found that guluronic acid,a constituent component(G unit)of alginate,was responsible for inducing glutathione and promoting TCM.Collectively,we reported new biological activities of alginate on scavenging reactive oxide species and regulating the function of cytotoxic T cell,which suggests that alginate and guluronic acid may be used for improving cytotoxic T cell functions in immunotherapy.Part I Research on enhancing the antitumor effects of CD8~+ T cells by alginateObjective:To investigate the effects of alginate on the activity of CD8~+ T cells and to detect the influences of alginate on the anti-tumor effect in vivo and in vitro of cytotoxic T cells through incubating CD8~+ T cells in RPMI1640 medium mixed with 1% alginate.Methods: ? Spleen,Lymph nodes and peripheral blood were obtained and digested into a single cell suspension from B16-10 melanoma bearing mice.CD8~+ T cells were sorted from the mixed suspension of splenocytes and lymphocytes using the BD FACS Aria II cell sorter.? We had measured the endotoxin of 1%(w/v)premixed alginate solution by Chromogenic LAL Endpoint Assay Kit in this study.? The sorted CD8~+ T cells were cultured in RPMI1640 medium(Control)and RPMI1640 medium mixed with 1%(w/v)alginate for 48 h,respectively.The dead cells were determined by 0.4% trypan blue dye exclusion using a microscope.Apoptosis measurement was performed according to the instruction of Annexin V-APC(PI)apoptosis kit.? The levels of cytokine secretion of CD8~+ T cells were detected by real-time q PCR,flow cytometry and ELISA.? The cytotoxicity effect of CD8~+ T cells which cultured in RPMI1640 medium mixed with 1%(w/v)alginate for 48 h on B16-F10 melanoma cells was evaluated by MTS assay.? The in vivo antitumor effects of CD8~+ T cells which cultured in RPMI1640 medium mixed with 1%(w/v)alginate were verified by adoptively treating B16-10 melanoma bearing C57BL/6 mice and nude mice.Results: ? The LPS concentration of 1%(w/v)premixed alginate solution was 0.139 EU/m L.? There were no significant differences on apoptosis and death rates of CD8~+ T cells between the two groups were identified.? The results of real-time q PCR,flow cytometry and ELISA showed that IL-2 and IFN-? secretion from CD8~+ T cells incubated with alginate were elevated.? The test of the cytotoxic effect showed that the CD8~+ T cells incubated with RPMI1640 medium mixed with 1%(w/v)alginate for 48 h had stronger killing ability than normally cultured CD8~+ T cells.? Compared to the untreated control and the conventional CD8~+ T cells treatment,alginate-incubated CD8~+ T cells treatment displayed more potent antitumor activity in melanoma bearing mice.Meanwhile,the alginate-incubated CD8~+ T cells also significantly prolonged survival time of tumor bearing mice.Conclusions: ? Alginate promoted the secretion of cytokines IFN-? and IL-2 by CD8~+ T cells.? Alginate enhanced the antitumor effect of CD8~+ T cells in vivo and in vitro.Part II Research on alginate promoting the proportion of memory CD8~+ T cells and enhancing the anti-tumor immune memory effect of CD8~+ T cellsObjective:To investigate the effect of alginate on CD8~+ T cell subsets and anti-tumor ability by detecting the expression levels of memory phenotypes and stemness-related genes in alginate-incubated CD8~+ T cells.Methods: ? CD8~+ T cells were sorted from the mixed suspension of splenocytes and lymphocytes of B16-10 melanoma bearing mice using the BD FACS Aria II cell sorter.?Using T cell pre-treatment model of C57BL/6 mice investigate the long term anti-tumor memory effects of alginate-incubated CD8~+ T cells in vivo.? The expression levels of lymph node homing molecules,stemness-related genes and transcription factors regulating CD8~+ memory T cell differentiation in CD8~+ T cells were detected by real-time q PCR and flow cytometry.? Flow cytometry was used to detect the memory subsets of CD8~+ T cells cultured in RPMI1640 medium mixed with 1%(w/v)alginate.? The viscosities of alginate solution and glucan solution were measured with an ubbelohde capillary viscometer.Results: ? The pre-inoculation of alginate-incubated CD8~+ T cells but not conventionally cultured CD8~+ T cells effectively suppressed tumor generation and promoted survival.? Alginate incubation induced CD8~+ T cells to express higher levels of memory markers(CD44,CD62 L,CCR7 and CD122),stemness associated genes(Ctnnb1,c-Kit,Runx2 and ABCB1)and the transcription factor Runx2.? Flow cytometry showed that alginate could promote the proportion of CD44+CD62L+CD8~+ T(TCM)cells,and the proportion increased gradually with alginate concentration increasing,the difference was significant.? The alginate-contained medium has higher viscosity than regular medium.The possibility of high viscosity of alginate solution increased TCM proportion in cultured T cells was excluded by showing that glucan with the similar viscosity.Conclusions: ? Alginate increases the proportion of CD8~+ TCM and may improve the long-term antitumor memory effects of CD8~+ T cells in vivo.? Alginate incubation increased the expression levels of memory markers,stemness associated genes and the transcription factor Runx2.? Alginate could promote the proportion of CD8~+ TCM cells.? The viscosity of alginate could not increased CD8~+ TCM proportion.Part III Research on the underlying mechanism of alginate enhancing antitumor memory effects of CD8~+ T cellsObjective:To further explore the mechanism of alginate promoting the proportion of memory CD8~+ T cells,and to reveal the internal mechanism of alginate enhancing the anti-tumor immune memory of CD8~+ T cells.Methods: ? Real-time q PCR was used to measure the antioxidants levels of human Jurkat T cells,mouse CD8~+ T cells and CD3+ T cells cultured in RPMI1640 medium mixed with 1%(w/v)alginate.? ROS levels of human Jurkat T cells,mouse CD8~+ T cells and CD3+ T cells were measured using DCFH-DA and analyzed by flow cytometric.? The contents of GSH in human Jurkat T cells,mouse CD8~+ T cells and CD3+ T cells were determined by reduced GSH assay kit.Results: ? Real-time q PCR results showed that the antioxidant genes levels of GCLC,GCLM,GST,CAT and SOD2 were increased significantly when human Jurkat T cells,mouse CD8~+ T cells and CD3+ T cells were cultured in RPMI1640 medium mixed with 1%(w/v)alginate.The results also showed that ROS was decreased and GSH content was increased.? BSO inhibited the ability of alginate increasing the expression of CD62 L,CCR7 gene and the proportion of CD8~+ TCM cells.? By further examined the effects of mannuronic acid and guluronic acid on CD8~+ T cells,Guluronic acid(G units)but not mannuronic acid(M units)significantly elevated the proportion of CD8~+ TCM cells.Meanwhile,the results showed the guluronic acid upregulated m RNA levels of the antioxidant genes GST,CAT and SOD2 in CD8~+ T cells.Conclusions: ? Alginate elevated the generation of intracellular GSH and antioxidant,reducing ROS in CD8~+ T cells,thus promoting the proportion of CD8~+ TCM,leading to enhanced in vivo antitumor effects.? The inhibition of GSH production decreased the ability of alginate promoting CD8~+ TCM.? By further examined the effects of alginate constituents on CD8~+ T cells,Guluronic acid(G unit)was found to increase the antioxidant gene expressions,decrease ROS levels.Therefore,G unit was the substance within alginate that was capable of promoting abundance of TCM.