| Background:CAR-T cell therapy has shown great promise in the advancement of individualized cancer immunotherapy.This technology is based on a combination of the specific interaction of an antigen and a corresponding antibody and cytotoxic T-lymphocyte(CTL)activity against target cells.However,there are still some formidable challenges for broader application of the CAR-engineered T cells,including the low lentiviral transfection efficacy of T cells,suppression of CAR-T cell function,the lack persistence of CAR-T cells after infusion,and immune escape of the tumor cells by selective antigen low-expression.Lentivirus vectors are capable of stably infecting the dividing or non-dividing cells by integrating into the host genome.Moreover,these vectors are nontoxic to the human body which enables transduced T cells to obtain long term stable gene expression.A limitation of lentivirus-mediated T-cell transfection is the low transfection efficacy.To date,how to increase the lentiviral transfection efficiency of T cells is still challenging.Considering the limited persistence of CAR-T cells in patients,one promising approach to enhance the efficacy of CAR-T cells is to increase the percentages of less differentiated T cell phenotypes.Despite a clear advantage of the less-differentiated populations,the majority of ACT trials utilize unfractionated T cell subsets,due to the lack of appropriate methods for generation of less differentiated CAR-T cells.Thus,it is of great importance to optimize the production methods to obtain CAR-T cells with advanced quality and potentials for antitumor immunotherapy.CARs have been empirically designed to emulate the antigen-binding properties of monoclonal antibodies(m Abs)and the signaling properties of the T cell antigen receptor(TCR).They typically feature an extracellular,m Ab-derived,single-chain variable fragment(sc Fv)that targets a given tumor-associated antigen(TAA)and is linked to a spacer and transmembrane domain as well as intracellular signaling modules.This modular and one-dimensional architecture affords much flexibility in clinical applications,meanwhile,it limits the extent to which CARs reproduce the complexities of the signaling responses that have coevolved with TCRs.T cells in their natural state respond to the presence of even a single nominal peptide–major histocompatibility complex(p MHC)on the surface of antigen-presenting cells.However,despite a strong affinity for antigen-antibody recognition,more target antigen molecules are required to effectively activate CAR-T cells,compared to the T cell receptor(TCR)stimulation.On the other hand,experimental studies showed more than20%of patients suffered from cancer relapses after CD19-CAR-T cell therapy,and more than 50%of patients who had attained complete remission subsequently relapsed in a recent CD22-CAR clinical trial,due to the emergence of tumors that express antigen at low or undetectable levels.Thus,how to optimize the structure of CAR-T according to the natural TCR signaling pathway to improve the recognition and killing ability of T cells to tumor cells expressing low target antigen is conducive to the development of more effective CAR-T therapy.The common?Cfamily have been widely utilized for the CAR-T cell preparation,because these cytokines play pivotal roles in fueling T cells to thrive,combat tumors and drive long-lived memory against tumor metastasis or relapse.Because the primary signal transfection and transcriptional activators activated by IL-21 are different from these activated by other?C family cytokines,IL-21 has its own unique roles in anti-tumor immunity.The purpose of this study is to explore the effects of adding IL-21 to the CAR-T cells in the CAR-T preparation,so as to provide a certain experimental basis for optimizing the CAR-T cell preparation.At least three tyrosine kinases are known to be involved in the early phosphorylation of T cell activation.Lymphocyte-specific tyrosine kinase(LCK)is mainly located in the cytoplasm and on the cell membrane of T cells.Genetic studies have demonstrated that LCK plays an important role in the T cell activation,and deletion or mutations of LCK can lead to T cell aplasia.During immunological synapse formation,a ligand binding to the receptor causes alterations in the location and configuration of the membrane receptor,then crosslinking takes place in the membrane receptor.Subsequently,the cytoplasmic tails of CD3,CD4 or CD8 molecules aggregate together,and the LCK attached to the intracellular regions of CD4 or CD8 molecules undergoes phosphorylation,which in turn mediates immunoreceptor tyrosine-based activation motif(ITAM)phosphorylation of CD3 molecules.The region involved in binding of CD4 or CD8 molecules with LCK contains similar negatively charged chain molecules with two cysteines.We explored the functional role of attachment of the negatively charged chain molecules to the intracellular region of CAR in CAR-T antitumor immunity.Our study optimized the CAR-T preparation and CARs structure,to provide new insights into the optimization of CAR-T cell antitumor therapy.Methods:(1)HER-2-CAR plasmid,28CAR,137CAR,137LCK0,137LCK1 and 137LCK2 and the other related plasmids were constructed by molecular cloning and verified by sequencing and restriction enzyme digestion.(2)8 cytokine cocktails were designed(not contain IL-21:IL-2?IL-7?IL-15 and IL-7+IL-15;contain IL-21:IL-2+IL-21?IL-7+IL-21?IL-15+IL-21 and IL-7+IL-15+IL-21).The number of T cells cultured with the different cytokine conditions was calculated with an automatic cell counter.Flow cytometry was used to detect the transfection efficiency of T cells and phenotype of CAR-T cells cultured with different cytokine combinations.(3)The expression of T cell subtypes and inhibitory molecules of several TR1419-CAR-T cells were detected by flow cytometry seven days after T cell transfection.Then mean fluorescence intensities(MFI)of CAR expression were compared.(4)Detection of IFN-?expression of T cells at the early stage of T cell activation.Flow cytometry and enzyme-linked immunosorbent assay were used to detect IFN-?expression of T cells cultured with different cytokine combinations at different time periods.(5)The cytotoxic activities detection of CAR-T cells cultured with different cytokine combinations.target antigen positive tumor cells were selected as target cells based on the antigen expression of tumor cells,which was detected by flow cytometry.The ability of CAR-T cells to kill tumor target cells was measured with Calcein AM release-based cytotoxic cell assay,IFN-?and granzyme B secretion in cell culture supernatants were detected by ELISA.(6)TR1419-CAR-T cells were co-incubated with tumor cells for 7 days to detect the proliferation,subtype differentiation and inhibitory molecular expression of several CAR-T cells.Results:Part I)(1)we successfully prepared HER-2 CAR plasmid and established a lentivirus transfection protocol,CAR-T cells can effectively be prepared.(2)The addition of IL-21 efficiently increased the transfection efficiency of T cells during the CAR-T cell preparation.There was no significant difference in the transfection efficiency of T cells cultured with the 4 cytokine conditions without adding IL-21,and the frequency of CAR expression of each condition ranged from 15%to 30%.Meanwhile,no difference in transfection efficiency of T cells was found between the 4 cytokine conditions with adding IL-21,and the frequency of CAR expression of each condition ranged from 25%to 40%.T cells cultured with the addition of IL-21 had a higher transfection efficiency compared to the corresponding T cells cultured without IL-21.(3)IL-21regulated the secretion of IFN-?at the early stage of T cell activation.We observed that IL-2+IL-21,IL-7+IL-21 and IL-15+IL-21 exhibited higher IFN-?expression of T cells than the corresponding IL-2,IL-7 and IL-15cytokine conditions during the first 6h of T cell activation.However,the 4cytokine conditions with the addition of IL-21 exhibited lower IFN-?expression of T cells than the 4 corresponding cytokine conditions without the addition of IL-21 24-48h after T cell activation.Lastly,we found that the transfection efficiency of CD8+T cells showed significant negative correlation with its IFN-?expression.(4)IL-21 improved T cells proliferation capacity.The addition of IL-21 to IL-7,IL-15 or IL-7+IL-15combo markedly enhanced T cell expansion.For IL-7 condition,despite the slight increase of T cell numbers induced by the addition of IL-21,IL-7+IL-21 exhibited significantly lower T cell proliferation than the other cytokine combinations.(5)IL-21 increased the proportion of Tn in CAR-T cells.The subtype of CAR-T cells cultured with the 8 cytokine combinations was dominated by Tn,accounting for 50%-80%.The IL-7+IL-21 led to the highest percentage of Tn cells,followed by IL-15+IL-21.IL-2 caused the generation of the lowest percentage of Tn cell,followed by IL-2+IL-21.Lastly,our data showed that CAR-T cells cultured with the addition of IL-21 had a higher proportion of T cells that exhibited a Tn phenotype as compared to the corresponding CAR-T cells cultured without IL-21.(6)The tumor-specific killing ability of CAR-T cells was enhanced by IL-21.All CAR-T cells cultured with different cytokines conditions were effective in killing antigen-expressing tumor cells,accompanied by the secretion of IFN-?and granzyme B.However,IL-21 enhanced the CAR-T cell antitumor activity and secretion of effector cytokines.As for the HER-2-negative tumor cells,all CAR-T cells had no significant killing effect and secretion of effector cytokines,compared with the mock T cells.Part ii)(1)we completed the construction of the relatived CAR plasmids targeting TRAIL-R1.The sequencing results of the plasmids matched the original sequence.Jurkat cells and T cells were successfully transfected to express the corresponding CAR gene.(2)137CAR,137LCK0,137LCK1 and137LCK2 plasmids were transfected into Jurkat cells.Then CD69 expression of several CAR-T cells was detected after stimulation with different concentrations of target antigen.137LCK2-Jurkat cells expressed higher levels of CD69 than the other CAR-T cells in response to antigen stimulation with low concentrations.Therefore,137LCK2 was used in all subsequent experiments.(3)28CAR,137CAR and 137LCK2 plasmids were transfected into T cells.The transfection rates of the T cells in several groups were 30%-40%,and the mean fluorescence intensity in several CAR-T cells was not significantly different.In addition,all CAR-T cells in several groups expressed comparable amounts of inhibitory molecule PD-1,LAG-3 and Tim-3.Moreover,CD4+CAR-T cells expressed higher levels of PD-1 than CD8+CAR-T cells,while CD8+CAR-T cells expressed higher levels of LAG-3 and Tim-3.Then we analyzed the proportion of differentiated subtypes in CAR-T cells.Tn constituted the majority of CAR-T cells(about 50%),and the proportion of Tn in 28CAR group was slightly higher than that in the other groups.(4)The several CAR-T groups were stimulated with different concentrations of target antigen.The CD137 expression of the several CAR-T cells decreased with the reduction of the target antigen concentration,and 137LCK2-T cells had the highest level of CD137 expression in several CAR-T cells with multiple concentrations antigen stimulation,while the other CAR-T cells showed no significant difference in CD137 expression.HS578-T and SW480 were selected as the target cells according to the surface antigen expression levels.All CAR-T cells were effective in killing antigen-expressing tumor cells,and there was no significant difference in killing HS578-T and SW480 among the three CAR-T cells.(5)The several CAR-T cells were cultured with the target antigen stimulation for 7 days,and the proportion of Tn was dominant in all the CAR-T groups.The CD4+CAR-T cells were dominated by the proportion of TCM,while the CD8+CAR-T cells were dominated by the proportion of TN.In addition,there was no significant difference in the proportion of TEM and TE among the CAR-T groups,but more TN differentiated into TCM in 137LCK2-T cells,compared to the other CAR-T groups.On the other hand,the proliferation rates of both CD4+137LCK2-T cells and CD8+137LCK2-T cells were significantly higher than that of the other groups,and 137LCK2-T cells also showed lower LAG-3 expression.Conclusion:On the one hand,we found that IL-21 improved lentivirus transfection efficiency of T cells,and regulated the secretion of IFN-?at the early stage of T cell activation.IL-21-mediated regulation of IFN-?expression provided potential explanation for the increased transfection efficiency of T cells.We confirmed that IL-21 enhanced the enrichment and expansion of less differentiated CAR-T cells,and augmented the CAR-T cell cytotoxicity.On the other hand,the intracellular segments of CD4/CD8 molecules were designed and cloned into CAR lentiviral vectors by linking a specific sequence within the intracellular region of the CAR plasmids.The sensitivity of CAR-T cells to target antigen was improved by using this optimization method.Meanwhile,CAR-T cell proliferation in response to target antigen stimulation was enhanced,and the proportion of Tcm in the CAR-T cells was improved.Further,this optimization reduced the expression level of LAG-3 in CAR-T cells.Our study provides the basis for the optimization of CAR-T cell antitumor therapy by optimizing the CAR-T preparation and CARs structure. |
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