Project description:Tumor antigen recognition and subsequent cytotoxic T lymphocyte (CTL) tumor infiltration are essential but alone are insufficient for an antitumor response. T cells nutrient uptake and metabolic reprogramming may dictate T cell differentiation and effector function to control CTL function in the tumor microenvironment. Slc7a5 is a system L transporter of large neutral amino acids and regulates T cell receptor (TCR)-mediated T cell activation during viral infection. Using tumor-specific CTL and antigen (Ag)-expressing tumor cell co-culture models, we determined that tumor-specific CTLs respond to tumor by increasing tryptophan uptake while responding to antigen alone without tumor cells by decreasing tryptophan uptake. ATAC-Seq analysis of the Ag-activated and tumor-activating CTLs revealed the transcriptional activation of a large set of transporters, including Slc7a5, a transporter for tryptophan. Slc7a5-specific inhibitor significantly decreased tryptophan uptake in the CTL-tumor co-culture while only tumor-specific CTLs and tumor are present in the microenvironment, indicating an essential role of Slc7a5 in transporting tryptophan in tumor-activated CTLs. We then generated mice with Slc7a5 deficiency only in T cells (Slc7a5.TKO). Transplanted breast tumor grew in a similar rate in WT and Slc7a5.TKO mice. However, Slc7a5.TKO mice formed significant more lung metastases than WTR mice. Further, transplanted melanoma tumor grew significantly faster in Slc7a5.TKO mice than in WT mice. Slc7a5.TKO mice show no significant difference in T cells as compared to WT mice. However, both CD4+ and CD8+ T cells diminished in tumors in the tumor-bearing mice, indicating an essential role of the Slc7a5-tryptophan metabolic pathway in T cell activation and expansion in the tumor microenvironment. scRNA-seq analysis determined that, unlike in the virus-infected host where MOTRC1 pathway is diminished, Slc7a5 deficiency leads to decreased AhR signaling and FasL expression in tumor-infiltrating T cells. Accordingly, FasL blockade increased lung metastases in breast tumor-bearing mice and increased tumor growth in melanoma tumor-bearing mice, indicating that Slc7a5 regulates the tryptophan-AhR metabolic pathway to regulate FasL expression in T cells to control tumor-infiltrating CTL effector function. Analysis of scRNA-seq dataset of human breast cancer patients revealed that FasL expression in T cells is correlated with SLC7A5 expression and patient response to immune checkpoint inhibitor immunotherapy. Furthermore, FasL is positively correlated with patient survival length in pooled datasets of 33 human cancers, and in human patients with breast cancer and melanoma in particular. Our findings determine that Slc7a5 controls tryptophan uptake in tumor-infiltrating CTLs to regulate the tryptophan-AhR metabolic pathway to regulate FasL expression to control CTL anti-tumor immune response.

Project description:BACKGROUND & AIMS- More frequent interaction of bacteria with the colonic epithelium is associated with ulcerative colitis (UC). The identities of all proteins which promote bacterial clearance in colonic epithelial cells are unknown. Previously, we discovered that dCAP-D3 (Chromosome Associated Protein-D3), regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS- Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing CAP-D3 shRNA. CAP-D3 levels in colonic epithelial cells from healthy and UC patient tissues were analyzed by immunoblot. RNA-sequencing identified bacterially-induced CAP-D3 target genes. The role of CAP-D3 target genes in bacterial clearance was analyzed by gentamycin protection assays, immunofluorescent staining, and by using pharmacologic inhibitors. RESULTS- CAP-D3 expression was reduced in colonic epithelial cells from UC patients with active disease. Reduction of CAP-D3 expression inhibited autophagy and decreased intracellular bacterial clearance. The components of the heterodimeric SLC7A5/SLC3A2 amino acid transporter were identified as CAP-D3 target genes; their levels increased in infected, CAP-D3 deficient cell lines and in cells from UC patients. In HT-29 cells, this resulted in earlier SLC7A5 recruitment to Salmonella-containing vacuoles, increased mTOR activity, and enhanced bacterial survival. Inhibition of SLC7A5/SLC3A2 or mTOR activity rescued the bacterial clearance defect in CAP-D3 deficient cells. CONCLUSIONS- CAP-D3 attenuates amino acid transporter transcription to promote bacterial autophagy in colon epithelial cells. CAP-D3 protein levels are decreased in patients with active UC, suggesting that CAP-D3 is a potential therapeutic target to restore mucosal homeostasis in UC patients. Three RNA samples from 3 independent experiments including timepoints taken at 0, 0.5 and 7 hours post-infection were analyzed on a bioanalyzer for quality; one of the 0.5 hour post-infection samples was excluded at this time due to poor RNA purity. Directional, cDNA libraries made from cellular mRNAs were generated from the other 8 samples and sequenced (paired-end sequencing of 100 bp reads) in the Genomics Core at the University of Chicago on an Illumina HiSeq2000.

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	ATX_P1_1proz.raw	Raw
	ATX_P1_1proz_rep1.raw	Raw
	ATX_P1_1proz_rep2.raw	Raw
	ATX_P2_1proz.raw	Raw
	ATX_P2_1proz_rep1.raw	Raw

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TXNIP mediates LAT1/SLC7A5 endocytosis to limit amino acid uptake in cells entering quiescence

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TXNIP mediates LAT1/SLC7A5 endocytosis to limit amino acid uptake in cells entering quiescence.

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