Journal: eBioMedicine

Article Title: Tryptophan metabolism promotes immune evasion in human pancreatic β cells

doi: 10.1016/j.ebiom.2023.104740

Figure Lengend Snippet: Induction of PD-L1 by TRP is AHR-independent. (A and B) Time course in ECN90 cells of FICZ (200 nM) and TRP (10 mM) induction of CYP1A1 (A) and PD-L1 (B) measured by RT-qPCR. (C) Dose response effect of TRP on PD-L1 expression measured by RT-qPCR in ECN90 cells (12 h treatment). (D and E) Effects of AHR inhibitors StemRegenin (1 μM) and CH 223191 (1 μM) on CYP1A1 (D) and PD-L1 (E) measured by RT-qPCR in ECN90 cells treated for 12 h with TRP. (F) Representative flow cytometry plots of HLA-A2 and PD-L1 expression in ECN90 cells treated with IFN− γ (25 ng/ml) or TRP (10 mM) for 48 h. Gate is on propidium iodide-negative live cells. (G) Mean fluorescence intensity (MFI) of PD-L1. (H) HLA-ABC expression measured by RT-qPCR in ECN90 treated 12 h with TRP (10 mM) or IFN− γ (25 ng/ml). (I) MFI of HLA-A2. (N = 3–6) ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005. Data were statistically analyzed using a parametric Mann–Whitney test.

Article Snippet: Membranes were immunoblotted with the following antibodies: PD-L1 (1:1000; 28-8, ABCAM, RRID: AB_268878), Phospho-Tyr701 STAT1 (1:1000; Cell Signaling Technology, RRID: AB_10950970), STAT1 (1:1000; Cell Signaling Technology, RRID: AB_2240087), Phospho-Ser473 AKT (1:1000; Cell Signaling Technology, RRID: AB_2315049), Pd-l1 (1:1000; BioTechne, RRID: AB_354540), β-ACTIN (1:2000; Sigma, RRID: AB_476744), α-TUBULIN (1:2000; Sigma, RRID: AB_477593).

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Fluorescence, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Tryptophan metabolism promotes immune evasion in human pancreatic β cells

doi: 10.1016/j.ebiom.2023.104740

Figure Lengend Snippet: TRP is the only AA that robustly induces PD-L1 in ECN90 β cells. (A) ECN90 cells were treated with or without amino acids (10 mM/each) for 12 h and analyzed for PD-L1 expression by RT-qPCR. (B) PD-L1 RT-qPCR expression in ECN90 cells depleted of GLN for 48 h and then cultured for 12 h with TRP (10 mM). (C) ECN90 cells were treated with multiples stressors and PD-L1 was analyzed by RT-qPCR. (D) ECN90 cells were depleted of TRP for 48 h and then treated with IFN− γ (25 ng/ml) for 12 h. PD-L1 was analyzed by RT-qPCR. (N = 3–6) ∗ P < 0.05; ∗∗∗ P < 0.005. Data were statistically analyzed using a parametric Mann–Whitney test.

Article Snippet: Membranes were immunoblotted with the following antibodies: PD-L1 (1:1000; 28-8, ABCAM, RRID: AB_268878), Phospho-Tyr701 STAT1 (1:1000; Cell Signaling Technology, RRID: AB_10950970), STAT1 (1:1000; Cell Signaling Technology, RRID: AB_2240087), Phospho-Ser473 AKT (1:1000; Cell Signaling Technology, RRID: AB_2315049), Pd-l1 (1:1000; BioTechne, RRID: AB_354540), β-ACTIN (1:2000; Sigma, RRID: AB_476744), α-TUBULIN (1:2000; Sigma, RRID: AB_477593).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Tryptophan metabolism promotes immune evasion in human pancreatic β cells

doi: 10.1016/j.ebiom.2023.104740

Figure Lengend Snippet: TRP-derived amines induce PD-L1 expression. (A) Schematic of TRP catabolic pathways. (B–F) ECN90 cells were treated with TRP or its catabolites from (B) the kynurenine (KYN 200 μM, KA 200 μM), (C) serotonin (5-HTP 1 mM, 5-HT 1 mM), (D) indole (I3PA 1 mM, I3A 1 mM) pathways, (E and F) TRY (5 mM) and ISO (10 μM) catabolites. PD-L1 was analyzed by RT-qPCR. (N = 3) ∗∗ P < 0.01; ∗∗∗ P < 0.005. Data were statistically analyzed using a parametric Mann–Whitney test.

Article Snippet: Membranes were immunoblotted with the following antibodies: PD-L1 (1:1000; 28-8, ABCAM, RRID: AB_268878), Phospho-Tyr701 STAT1 (1:1000; Cell Signaling Technology, RRID: AB_10950970), STAT1 (1:1000; Cell Signaling Technology, RRID: AB_2240087), Phospho-Ser473 AKT (1:1000; Cell Signaling Technology, RRID: AB_2315049), Pd-l1 (1:1000; BioTechne, RRID: AB_354540), β-ACTIN (1:2000; Sigma, RRID: AB_476744), α-TUBULIN (1:2000; Sigma, RRID: AB_477593).

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Tryptophan metabolism promotes immune evasion in human pancreatic β cells

doi: 10.1016/j.ebiom.2023.104740

Figure Lengend Snippet: Tryptophan modulates β-cell responses to IFN-γ. ECN90 cells were treated with TRP (10 mM), IFN− γ (25 ng/ml) or both. (A) PD-L1 expression was measured by RT-qPCR (12 h treatment); (B and C) PD-L1 was analyzed and quantified by Western blot (48 h treatment); (D–F) flow cytometry plots and mean fluorescence intensity (MFI) and frequency of PD-L1 (G and H) and HLA-A2 (I and J) (48 h treatment). (G–I) HLA-I (ABC) , and CXCL10 were measured by RT-qPCR (12 h treatment). (N = 9) ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005. Data were statistically analyzed using a two-tailed unpaired student’s t test.

Article Snippet: Membranes were immunoblotted with the following antibodies: PD-L1 (1:1000; 28-8, ABCAM, RRID: AB_268878), Phospho-Tyr701 STAT1 (1:1000; Cell Signaling Technology, RRID: AB_10950970), STAT1 (1:1000; Cell Signaling Technology, RRID: AB_2240087), Phospho-Ser473 AKT (1:1000; Cell Signaling Technology, RRID: AB_2315049), Pd-l1 (1:1000; BioTechne, RRID: AB_354540), β-ACTIN (1:2000; Sigma, RRID: AB_476744), α-TUBULIN (1:2000; Sigma, RRID: AB_477593).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: eBioMedicine

Article Title: Tryptophan metabolism promotes immune evasion in human pancreatic β cells

doi: 10.1016/j.ebiom.2023.104740

Figure Lengend Snippet: TRP effects on mouse islets. Islets were prepared from 12-week-old C57BL/6 male mice and treated with TRP (10 mM), IFN− γ (250 pg/ml) or both. (A) Pd-l1 gene expression was measured by RT-qPCR (12 h treatment). (B and C) PD-L1 was analyzed and quantified by Western blot (48 h treatment); (D–H) Flow cytometry plots of Pd-l1 and MHC Class I and mean fluorescence intensity (MFI) and Frequency on β cells from dispersed islet cells treated for 48 h as above. Gate on β cells was defined as CD71 + CD49f + among live Lin − EpCam + CD24 low cells. (I and J) h2(DLB1Q1) and Cxcl10 were measured by RT-qPCR (12 h treatment). (N = 3–6) ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005. Data were statistically analyzed using a two-tailed unpaired student’s t test.

Article Snippet: Membranes were immunoblotted with the following antibodies: PD-L1 (1:1000; 28-8, ABCAM, RRID: AB_268878), Phospho-Tyr701 STAT1 (1:1000; Cell Signaling Technology, RRID: AB_10950970), STAT1 (1:1000; Cell Signaling Technology, RRID: AB_2240087), Phospho-Ser473 AKT (1:1000; Cell Signaling Technology, RRID: AB_2315049), Pd-l1 (1:1000; BioTechne, RRID: AB_354540), β-ACTIN (1:2000; Sigma, RRID: AB_476744), α-TUBULIN (1:2000; Sigma, RRID: AB_477593).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: eBioMedicine

Article Title: Tryptophan metabolism promotes immune evasion in human pancreatic β cells

doi: 10.1016/j.ebiom.2023.104740

Figure Lengend Snippet: Effects of TRP on human islets. Human islets were treated with TRP (10 mM), IFN− γ (25 ng/ml) or both. (A) PD-L1 expression was measured by RT-qPCR (12 h treatment); ( B) HLA-ABC and (C) CXCL10 were measured by RT-qPCR (12 h treatment). (D – H ) Flow cytometry plots of PD-L1 and MHC Class I and mean fluorescence intensity (MFI) and frequency on dispersed islet cells treated for 48 h. Gate on endocrine islet cells was defined as GP2 − among live Lin − EpCam + CD24 low cells. (N = 3–4) ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005; ∗∗∗∗∗ P < 0.005. Data were statistically analyzed using a parametric Mann–Whitney test.

Article Snippet: Membranes were immunoblotted with the following antibodies: PD-L1 (1:1000; 28-8, ABCAM, RRID: AB_268878), Phospho-Tyr701 STAT1 (1:1000; Cell Signaling Technology, RRID: AB_10950970), STAT1 (1:1000; Cell Signaling Technology, RRID: AB_2240087), Phospho-Ser473 AKT (1:1000; Cell Signaling Technology, RRID: AB_2315049), Pd-l1 (1:1000; BioTechne, RRID: AB_354540), β-ACTIN (1:2000; Sigma, RRID: AB_476744), α-TUBULIN (1:2000; Sigma, RRID: AB_477593).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Fluorescence, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Tryptophan metabolism promotes immune evasion in human pancreatic β cells

doi: 10.1016/j.ebiom.2023.104740

Figure Lengend Snippet: PD-L1 induction by TRP is AKT-dependent. (A and B) ECN90 cells were treated for 12 h with TRP (10 mM) with or without cycloheximide (50 μg/ml) or rapamycin (50 nM). PD-L1 was measured by RT-qPCR. (C) Western blot of pSTAT1, STAT1 and pAKT and (D) pAKT/TUBULIN quantification in ECN90 cells treated with TRP, IFN-γ or both for 12 h. (E and F) PD-L1 was measured by RT-qPCR in ECN90 cells treated for 12 h with TRP (10 mM), with or without (E) the Akt inhibitor 10-DEBC (30 μM) or (F) the AKT activator PS48 (30 μM) or IGF-LR3 (100 ng/ml). (G) Representative flow cytometry plots of HLA-A2 and PD-L1 expression on ECN90 cells treated with TRP (10 mM) or IGF-LR3 (100 ng/ml) for 48 h. Gate is on propidium iodide-negative live cells (H and I) Mean fluorescence intensity (MFI) of PD-L1 and HLA-A2. (N = 3–6) ∗∗ P < 0.01; ∗∗∗ P < 0.005. Data were statistically analyzed using a two-tailed unpaired student’s t test.

Article Snippet: Membranes were immunoblotted with the following antibodies: PD-L1 (1:1000; 28-8, ABCAM, RRID: AB_268878), Phospho-Tyr701 STAT1 (1:1000; Cell Signaling Technology, RRID: AB_10950970), STAT1 (1:1000; Cell Signaling Technology, RRID: AB_2240087), Phospho-Ser473 AKT (1:1000; Cell Signaling Technology, RRID: AB_2315049), Pd-l1 (1:1000; BioTechne, RRID: AB_354540), β-ACTIN (1:2000; Sigma, RRID: AB_476744), α-TUBULIN (1:2000; Sigma, RRID: AB_477593).

Techniques: Quantitative RT-PCR, Western Blot, Flow Cytometry, Expressing, Fluorescence, Two Tailed Test

Journal: eBioMedicine

Article Title: Tryptophan metabolism promotes immune evasion in human pancreatic β cells

doi: 10.1016/j.ebiom.2023.104740

Figure Lengend Snippet: TRP-treated ECN90 β cells downregulate activation and PD-1 expression of TCR-transduced Jurkat T cells. (A) The experimental design involved lentiviral transduction of Jurkat T cells with a TCR recognizing an HLA-A2-restricted peptide. These cells expressed three fluorescent reporters controlled by AP-1, NF-AT, and NF-kB promoters. (B) Representative flow cytometry plots of HLA-A2 and PD-L1 expression on live ECN90 cells treated for 48 h with TRP (10 mM), IFN- γ (25 ng/ml) or both. These cells were subsequently used for T-cell co-culture experiments. (C–J) TCR-transduced Jurkat T cells were cultured for 6 h with ECN90 β cells left unpulsed (for incubation with anti-PPI TCR-transduced T cells) or preliminarily pulsed or not with viral peptide (0.1 μM for 2 h; for incubation with anti-viral TCR-transduced T cells). (C–F) Flow cytometry analysis of the percentage of positive cells expressing the indicated markers (AP-1, NF-AT, NF-kB or PD-1) in non-transduced or anti-PPI 15–24 peptide TCR transduced Jurkat T cells. (N = 15). (G–J) Percentage of positive cells expressing the indicated markers in an HLA-A3-restricted anti-viral peptide CVB1 1356–1364 TCR transduced Jurkat T cells cultured with ECN90 cells that were pulsed or not with the viral peptide. (N = 10) ∗∗ P < 0.01; ∗∗∗ P < 0.005. Data were statistically analyzed using a two-tailed unpaired student’s t test.

Article Snippet: Membranes were immunoblotted with the following antibodies: PD-L1 (1:1000; 28-8, ABCAM, RRID: AB_268878), Phospho-Tyr701 STAT1 (1:1000; Cell Signaling Technology, RRID: AB_10950970), STAT1 (1:1000; Cell Signaling Technology, RRID: AB_2240087), Phospho-Ser473 AKT (1:1000; Cell Signaling Technology, RRID: AB_2315049), Pd-l1 (1:1000; BioTechne, RRID: AB_354540), β-ACTIN (1:2000; Sigma, RRID: AB_476744), α-TUBULIN (1:2000; Sigma, RRID: AB_477593).

Techniques: Activation Assay, Expressing, Transduction, Flow Cytometry, Co-Culture Assay, Cell Culture, Incubation, Two Tailed Test