Tumor Cell-released Autophagosomes (TRAP) Upregulate Inhibitory Receptors On T Cell And Its Molecular Mechanism And Function

In the process of chronic infection or cancer,T cells are exposed to persistent antigen stimulation or inflammatory signals for a long time,resulting in the gradual loss of effector functions of T cells in a graded manner,commonly known as T cell exhaustion.The high expression and co-expression of inhibitory receptors such as PD-1,CTLA-4,LAG-3,Tim-3 and BTLA on T cells is one of the main characteristics of exhausted T cells.In our previous study,secretory autophagosomes have been successfully recruited from the culture supernatant of a variety of mouse/human tumor cells and from the hydrothorax/ascites of tumor patients by differential centrifugation,and named as tumor cell-released autophagosome?TRAP?.It was further found that TRAPs could induce B cells to differentiate IL-10⁺Bregs with immunosuppressive function in tumor microenvironment;induce macrophages to polarize to M2-like,and highly express PD-L1;promote neutrophil apoptosis by triggering neutrophils to produce ROS and inducing caspase-3 activation,also induce CD4⁺T cells to secrete IL-6 to inhibit T cell function and promote tumor growth and metastasis.However,it is not clear whether TRAPs is involved in inducing the expression of inhibitory receptors on T cells and its mechanism.Objectives:To investigate whether TRAPs could induce inhibitory receptors expressing on T cells and its molecular mechanism,and to provide a new idea for reversing exhausted T cell to enhance tumor immunotherapy.Methods:1.Investigation of the expression of inhibitory receptors on T cells induced by TRAPs1)TRAPs were extracted in tumor cell culture supernatant by differential centrifugation,and then were identified the purity of TRAPs by Western blot and flow cytometry,and the particle size of TRAPs by NTA.2)Isolated mouse spleen cells were co-cultured in the presence of?-CD3/CD28 mAbs,and then cultured with TRAPs for 72 hours in vitro.The expression of PD-1,CTLA-4,LAG-3,Tim-3 and BTLA on CD4⁺T cells and CD8⁺T cells was detected by flow cytometry.Then,under the above culture system,different concentrations of TRAPs were added to induce culture in vitro for 72 hours,and the expression of LAG-3 on CD4⁺T cells and CD8⁺T cells was detected by flow cytometry.3)The mouse spleen cells were induced and co-cultured with TRAPs in the presence of?-CD3/CD28 mAbs for 72 hours in vitro.The co-expression of PD-1 with CTLA-4,LAG-3,Tim-3 and BTLA on CD4⁺T cells and CD8⁺T cells was detected by flow cytometry.4)The mouse spleen cells were induced and co-cultured with different concentrations of TRAPs in the presence of?-CD3/CD28 mAbs for 72 hours in vitro.The expression of IFN-?in CD4⁺T cells and CD8⁺T cells was detected by flow cytometry.2.Investigation of the molecular mechanism of high expression of LAG-3 on T cells induced by TRAPs1)The spleen cells of WT,TLR2^-/-,TLR4^-/-and MyD88^-/-mice were induced and cocultured respectively with TRAPs in the presence of?-CD3/CD28 mAbs for 72 hours in vitro.The expression of LAG-3 on CD4⁺T cells and CD8⁺T cells was detected by flow cytometry.2)The spleen cells of WT mice were induced and co-cultured with TRAPs and anti-IL-6 neutralizing antibody,or isolating IL-6^-/-mice and co-cultured with TRAPs in the presence of?-CD3/CD28 mAbs for 72 hours in vitro.The expression of LAG-3 on CD4⁺T cells and CD8⁺T cells was detected by flow cytometry.3)The mouse spleen cells were pretreated with different concentrations of STAT3 inhibitors?Stattic?,JAK2/STAT3 inhibitors?AG490?and JAK1/JAK2/STAT3 inhibitors?CYT387?for 1 hours,and then were induced and co-cultured with TRAPs in the presence of?-CD3/CD28 mAbs for 72 hours in vitro.The expression of LAG-3 on CD4⁺T cells and CD8⁺T cells was detected by flow cytometry.3.Study on the function of high expression of LAG-3 on T cells induced by TRAPs1)The mouse spleen cells were induced and co-cultured with TRAPs in the presence of?-CD3/CD28 mAbs for 72 hours in vitro.The cells were collected and washed once with PBS,and named the obtained normal cultured splenocytes SP_CM and TRAPs-induced and high-expressed LAG-3 splenocytes SP_TRAP.Then,the two groups of spleen cells were re-added to the 24-well plate,and the BMDM was added to mixed culture to the?CD3/CD28 antibody culture system or isolated culture by Transwell chamber which the spleen cells induced by TRAPs were planted in the low chamber,and the BMDM was planted in the up chamber for 24 hours.The expression of IFN-?in CD4⁺T cells and CD8⁺T cells was detected by flow cytometry.2)The mouse spleen cells were induced and co-cultured with TRAPs in the presence of?-CD3/CD28 mAbs for 72 hours in vitro.The TRAPs-induced and high-expressed LAG-3 splenocytes(SP_TRAP)were collected and washed once with PBS,and then re-added to the 24-well plate.In the presence of?-CD3/CD28 mAbs,SP_TRAP was pretreated with?LAG-3 and?PD-L1 mAbs separately or combinedly,and then BMDM was added to mixed culture for 24 hours.The expression of IFN-?in CD4⁺T cells and CD8⁺T cells was detected by flow cytometry.Results:1.Investigation of the expression of inhibitory receptors on T cells induced by TRAPs1)Secretory autophagosomes can be extracted Hepa1-6 tumor cells culture supernatant with a purity greater than 90%by differential centrifugation,and the particle size is mainly between 200-400?nm?.2)In TRAPs-treated mouse spleen cells,the expressions of PD-1,CTLA-4,LAG-3,Tim-3 and BTLA on CD4⁺T cells and CD8⁺T cells were up-regulated,and the up- regulation of LAG-3 was most significant.And with the increase of the concentration of TRAPs,the expression level of LAG-3 on the surface also gradually increased.3)In the mouse spleen cells treated with TRAPs,the expression levels of PD-1⁺ CTLA-4⁺,PD-1⁺LAG-3⁺,PD-1⁺Tim-3⁺,PD-1⁺BTLA⁺CD4⁺T cells and CD8⁺T cells increased significantly.4)The amount of IFN-?secreted by CD4⁺T cells and CD8⁺T cells decreased significantly in the mouse spleen cells treated with TRAPs,and the expression of IFN-?decreased with the increase of TRAPs concentration.2.Investigation of the molecular mechanism of high expression of LAG-3 on T cells induced by TRAPs1)The expressions of LAG-3 on CD4⁺T cells and CD8⁺T cells of WT and TLR4^-/- mice treated with TRAPs were significantly increased.However,the expressions of LAG-3 on CD4⁺T cells and CD8⁺T cells in TLR2^-/-and MyD88^-/-mice did not change significantly.2)After adding anti-IL-6 neutralizing antibody,the expression of LAG-3 on CD4⁺T cells and CD8⁺T cells in the spleen cells of WT mice treated with TRAPs was significantly decreased,and was similar to that of LAG-3 without TRAPs treated.The expression of LAG-3 on CD4⁺T cells and CD8⁺T cells of IL-6^-/-mice treated with TRAPs did not change significantly.3)STAT3 inhibitor Stattic,JAK2/STAT3 inhibitor AG490 and JAK1/JAK2/STAT3 inhibitor CYT387 could significantly inhibit the expression of LAG-3 on T cells induced by TRAPs.3.Study on the function of high expression of LAG-3 on T cells induced by RAPs1)In the group of normal cultured spleen cells(SP_CM),the co-culture of BMDM had o effect on the secretion of IFN-?by CD4⁺T cells and CD8⁺T cells,but in the roup of the spleen cells induced by TRAPs(SP_TRAP),the secretion of IFN-?by CD4⁺T cells and CD8⁺T cells decreased significantly after co-culture with BMDM.In Transwell chamber,it was found that the amount of IFN-?secreted by CD4⁺T ells and CD8⁺T cells recovered significantly after isolation and culture of BMDM nd SP_TRAP,which was similar to the level of IFN-?secreted by T cells in the group f SP_TRAP cells without co-culturing with BMDM.2)The expression of IFN-?in CD4⁺T cells and CD8⁺T cells decreased significantly in BMDM cell group,but the expression level of IFN-?in CD4⁺T cells and CD8⁺ T cells did not change significantly when?LAG3 mAbs was used alone.When ?PD-L1 mAbs was blocked alone,it could restore the inhibition of BMDM on the secretion of IFN-?by CD4⁺T cells and CD8⁺T cells.When?LAG-3 and?PD-L1 mAbs were blocked combined,the expression of IFN-?in CD4⁺T cells and CD8⁺ T cells was significantly increased,and was higher than the expression level of IFN-?when?PD-L1 mAbs was blocked alone.Conclusions:1.TRAPs induce the upregulation and co-expression of multiple inhibitory receptors on CD4⁺T cells and CD8⁺T cells,and inhibit the secretion of IFN-?by CD4⁺T cells and CD8⁺T cells.2.TRAPs promote the secretion of IL-6 by the spleen cells mainly through TLR2-MyD88 signaling pathway,and then IL-6 plays a role mainly through JAK/STAT3 signaling pathway to promote the expression of LAG-3 on CD4⁺T cells and CD8⁺T cells.3.Blocking LAG-3 alone has no effect on the recovery of T cell function.When combined with PD-1/PD-L1,it can further enhance the function of T cells.