Discovery Of βNMN And 3MOBA As Promoters Of T Cells Proliferation,Differentiation,and Function

Background:The birth of adoptive cell therapy(ACT)such as CAR-T and Treg therapies has undoubtedly brought good news to patients with cancer and autoimmune diseases,respectively.Two significant obstacles for a broad and effective use of ACT are 1)generation of a large number of functional CAR-T or Treg cells in vitro and 2)prevention of the transferred cells from becoming dysfunctional in vivo by immunosuppressive tumor microenvironment(TME)and continuous antigen exposure.Persistent stimulation necessary for generating enough T cells for ACT in vitro induces T cell exhaustion due to the elevated and sustained expression of inhibitory receptors such as PD-1 and CTLA-4.In order to overcome these challenges,we looked for small molecules that promote T cell proliferation and suppress expression of the inhibitory receptors.We isolated two such molecules,3MOBA andβΝMN,from culture medium of the human colorectal carcinoma cell line HCT-8.When human T cells are activated in the presence of these molecules,they proliferate much faster and express a significantly less amount of PD-1 and CTLA-4.As these chemicals are cheap and natural metabolites,they can be easily applied to generation of T cells for ACT without cumbersome genetic manipulations.Objective:1.To discover small molecules that promote T cell proliferation and differentiation but inhibit T cell exhaustion2.To characterize their effects on T cell proliferation,differentiation,effector function,and expression of the inhibitory receptorsMethod:1.Screen conditioned medium(CM)from various cell lines to search for a cell line that secrets factors promoting T cell proliferation: The HCT-8,HCA-7,Caco-2and MEF conditioned medium were collected differently and applied to the T cells culture with 50% bulk concentration.After 2-3 days,the proliferation of T cells was detected by flow cytometry according to Cell Trace Violet fluorescent dyes,so as to screen cell lines that promote T cells proliferation.2.Select a cell line,HCT-8,that secrets factors promoting T cell proliferation:HCT-8,HCA-7,Caco-2 are epithelial cell lines,and their conditioned medium promoted T cells proliferation.Hence we selected HCT-8 CM for following study.3.Test whether the factors are small molecules by biochemical techniques: since we aim to look for small molecules that promote T cells proliferation,thus we separate small molecules from HCT-8CM by 15 ml protein concentrator with the cutoff molecular weight of 3KD and gel chromatography.We confirmed that the HCT-8 CM(<3KD)promoted in vitro proliferation of T cells,and we named promoting factors as LNY2.4.Perform metabolomics to identify secreted metabolites: In order to identify LNY2,we performed metabolomics analysis for HCT-8 CM(<3 KD).There were 27 metabolites enriched in HCT-8 CM and 17 metabolites metabolites uniquely secreted by HCT-8.According to the literature,we purchased some of the metabolic drugs and applied them to T cells proliferation and Tregs differentiation assays.We observed the effects of these drugs on the activation,proliferation,and differentiation of T/Treg cells.5.Identify the factors promoting T cell proliferation among the secreted metabolites:On the basis of the characteristics that LNY2 can promote the proliferation,activation and differentiation of T cells in a long-term culture in vitro,we finally identified 3MOBA and βNMN are the factors that promote the proliferation of T cells.6.Characterize the effects of the discovered molecules(3MOBA and βNMN)on T cell proliferation,differentiation,effector function,and expression of the inhibitory receptors: We first optimized the concentration of 3MOBA andβNMN and found that the combined use of the two drugs is better than that of single use.We investigated if 3MOBA and βNMN make any difference on the expression of CTLA-4 and PD-1 in human T cells.We also performed Seahorse analysis to see their effect on the respiratory activity of T cells.Results:1.We discovered 3MOBA and βNMN secreted by HCT-8 cells as promoters of T cells proliferation.2.3MOBA and βNMN promoted in vitro expansion of total human T cells while suppressing expression of the inhibitory receptors PD-1 and CTLA-4significantly.3.βNMN and 3MOBA promoted T Cells respiratory activity,suggested that they promoted energy metabolism in T cells while inhibiting excessive activation of T cells(decreasing PD-1 and CTLA-4 expression).4.3MOBA and βNMN also significantly promoted in vitro expansion and differentiation as well as the cytotoxic function of human Treg cells.Conclusion:3MOBA and βNMN promote T cell proliferation,differentiation,and functions,and therefore can be easily applied to expansion of T cells for ACT to obtain more T cells with the superior functionality.