Identification of a small molecule Tim-3 inhibitor to potentiate T cell-mediated antitumor immunotherapy in preclinical models
Data files
Nov 07, 2023 version files 9.14 MB
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ML-T720201210fpkm.anno.zip
9.13 MB
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README.md
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Abstract
T cell immunoglobulin and mucin-containing molecule 3 (Tim-3), expressed in dysfunctional and exhausted T cells, has been widely acknowledged as a promising immune checkpoint target for tumor immunotherapy. Here, using a strategy combining virtual and functional screening, we identified a compound named ML-T7 that targets the FG-CC’ cleft of Tim-3, a highly conserved binding site of phosphatidylserine (PtdSer) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). ML-T7 enhanced the survival and antitumor activity of primary CD8+ cytotoxic T lymphocytes (CTLs) and human chimeric antigen receptor (CAR) T cells and reduced their exhaustion in vitro and in vivo. In addition, ML-T7 promoted NK cells’ killing activity and DC antigen-presenting capacity, consistent with the reported activity of Tim-3. Notably, ML-T7 strengthened DCs’ functions through both Tim-3 and Tim-4, consistent with the hypothesis that Tim-4 contains a similar FG-CC’ loop. Intraperitoneal dosing of ML-T7 showed comparable tumor inhibitory effects to Tim-3 blocking antibody. ML-T7 reduced syngeneic tumor progression in both wildtype and Tim-3 humanized mice and alleviated the immunosuppressive microenvironment. Furthermore, combined ML-T7 and anti-PD-1 therapy had greater therapeutic efficacy than monotherapy in mice, supporting further development of ML-T7 for tumor immunotherapy. Our study demonstrates a potential small molecule for selectively blocking Tim-3 and warrants further study.
https://doi.org/10.5061/dryad.9ghx3ffpr
Methods and results: As mentioned in the paper, splenocytes from OT-Ι mice were stimulated with 10 nM OVA257-264 peptide in RPMI 1640 medium containing 10% FBS, 50 U/mL penicillin-streptomycin, 50 μM 2-Mercaptoethanol, 20 U/mL human recombinant IL-2 and 10 μM ML-T7/DMSO for 3 days, followed by 3 days culture in the presence of ML-T7/DMSO to get mature CTLs unless otherwise indicated.Total RNA was isolated from DMSO or ML-T7 treated CD8+ CTLs using TRIzol reagent (Invitrogen). RNA-Seq analysis was performed with an Illumina HiSeq 2500 or BGISEQ-500 by BGI (Shenzhen, Guangdong, China). Data quality was assessed with fastqc software and aligned to the mouse genome (mm10 build) using TopHat (with Bowtie2), and data was curated and visualized using the BGI Interactive analysis platform.RNAseq detected 1271 differentially expressed transcripts (1.5-fold or more) in ML-T7-treated CD8+ CTLs. Specifically, ML-T7 treatment upregulated several effector T cell-associated genes, including Lck, Zap70, Erk1, Il-2 and Cd69, while downregulated several exhausted T cell-associated genes, including Havcr2, Tigit and Ctla4. Gene set enrichment analysis (GSEA) showed that TCR signaling pathway and IL2-STAT5 pathway were significantly enriched in ML-T7-treated CTLs.
Description of the data and file structure
File list:
ML-T720201210fpkm.anno
File description:
The following text describes files named “ML-T720201210fpkm.anno”.This csv data file contains differential gene expression patterns and gene signatures.
| File name | File category | File type | Experiment or Analysis | Related to Figure(s) | Description |
ML-T720201210fpkm.anno | inh7-vs-MOCK.csv | fpkm list | RNA sequencing of DMSO(MOCK)-treated cells and ML-T7(inh7)-treated cells | Fig2 and FigS3 | Differential expression results table for ML-T7(inh7)-treated cells vs DMSO(MOCK)-treated cells
**Plain text (.csv) files can be interpreted as follows:**
Stand-alone plain csv files, containing a csv file of differential expression results, for all genes. These tables can be used to search for results of genes not discussed in the text. Tables contain the following information (in columns) for each gene (in rows):
ML-T720201210fpkm.anno.csv result file description:
- name = ID of the gene
- MOCK_1,2,3 = DMSO1,2,3-Control group in the article
- inh7_1,2,3 = ML-T71,2,3-Experimental group in the article
- GeneName = Name of the gene
- Biotype = gene type
- Positon = coordinates of genes
- NR:Seq-id = optimal comparison results between genes and NR database
- NR:Score = the score of gene comparison with NR database
- NR:Evalue = The Evalue value of genes compared with the NR database
- NR: Description = Function description of the gene in the NR database
- NT:Seq-id = optimal comparison results between genes and NT database
- NT:Score = comparison score between gene and NT database
- NT:Evalue = The Evalue value of genes compared with NT database
- NT: Description = Description: Function description of the gene in the NT database
- Uniprot:UniprotKB-AC = optimal comparison results between genes and Uniprot database
- Uniprot:Score = the score of genes compared with Uniprot database
- Uniprot: Evalue = The Evalue value of genes compared with Uniprot database
- Uniprot: Description = Function description of the gene in niprot database
- COG:gene =gene name in the COG database compare
- COG:Score = the score compared with the COG database
- COG:Eval = comparison with the COG database Evalue value
- COG:num = gene ID in the COG database compared
- Pfam:pfam_ID = gene ID of the protein family Pfam compared
- Pfam:pfam_Name = the gene name of the protein family Pfam compared
- Pfam:pfam_Description = Functional description of the protein family Pfam compared
- GO:biological_process = Annotated GO Term describing the biological process
- GO:cellular_component = Annotated GO Term describing cell components
- GO:molecular_function = the commented GO Term describing the molecular function
- KEGG:KO = the ID in KEGG commented to
- KEGG:Description = Function description in KEGG
Sharing and access information
No licenses or restrictions are placed on the data.
Splenocytes from OT-Ι mice were stimulated with 10 nM OVA257-264 peptide in RPMI 1640 medium containing 10% FBS, 50 U/mL penicillin-streptomycin, 50 μM 2-Mercaptoethanol, 20 U/mL human recombinant IL-2 and 10 μM ML-T7/DMSO for 3 days, followed by 3 days culture in the presence of ML-T7/DMSO to get mature CTLs unless otherwise indicated.Total RNA was isolated from DMSO or ML-T7 treated CD8+ CTLs using TRIzol reagent (Invitrogen). RNA-Seq analysis was performed with an Illumina HiSeq 2500 or BGISEQ-500 by BGI (Shenzhen, Guangdong, China). Data quality was assessed with fastqc software and aligned to the mouse genome (mm10 build) using TopHat (with Bowtie2), and data was curated and visualized using the BGI Interactive analysis platform.RNAseq detected 1271 differentially expressed transcripts (1.5-fold or more) in ML-T7-treated CD8+ CTLs. Specifically, ML-T7 treatment upregulated several effector T cell-associated genes, including Lck, Zap70, Erk1, Il-2 and Cd69, while downregulated several exhausted T cell-associated genes, including Havcr2, Tigit and Ctla4. Gene set enrichment analysis (GSEA) showed that TCR signaling pathway and IL2-STAT5 pathway were significantly enriched in ML-T7-treated CTLs.