Journal: eLife

Article Title: Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist

doi: 10.7554/eLife.65777

Figure Lengend Snippet:

Article Snippet: For quantitative polymerase chain reaction (qPCR), Tregs and CD8+ T cells were stimulated for 4 hr with 200 nM cytokine prior to RNA isolation using RNeasy kit (QIAGEN). cDNA was generated using the iScript reverse transcriptase kit. qPCR was performed using PowerTrack SYBR Green (ThermoFisher) and analyzed on StepOnePlus Real-Time PCR System (Applied Biosystems).

Techniques: Expressing, Recombinant, Modification, Sequencing, Cell Isolation, Selection, Reverse Transcription, SYBR Green Assay, Flow Cytometry, Staining, Activation Assay, Software

Journal: JCI Insight

Article Title: The XCL1/XCR1 axis is upregulated in type 1 diabetes and aggravates its pathogenesis

doi: 10.1172/jci.insight.178743

Figure Lengend Snippet: ( A ) Volcano blots of the gene expression profile of chemokine ligands and their receptors of laser-dissected islets from RIP-GP mice at day 1, 3, 7, 10, 14, and 28 after the LCMV infection in comparison with uninfected mice. XCL1 is highlighted in red and XCR1 in blue. ( B ) Quantification of the data obtained by gene array (curves) and reverse transcription PCR (RT-PCR) (bars) of laser-dissected islets for the expression of XCL1 and XCR1. Data are shown as mean ± SD. ( C ) Duplex RNAScope in situ hybridization XCL1 (red) and XCR1 (blue) of pancreas tissue sections from RIP-GP mice at days 0, 7, 10, 14, 28 after infection ( n = 3 mice per time point). Representative images are displayed per time. Original magnification, 63× (oil). Scale bar: 20 μm. ( D ) Duplex RNAScope in situ hybridization for XCL1 (red) and XCR1 (blue) of pancreas tissue sections from NOD mice obtained at different age and disease state ( n = 2–3). Mice were considered diabetic with nonfasting BG levels of > 300 mg/dL. Original magnification, 40×. Scale bars: 25 μm. ( E ) Quantification of XCL1 (left) and XCR1 (right) expression in the islets of RIP-GP mice at day 0, 7, 10, 14, and 28 after infection ( n = 3 per time point). Data are shown as mean ± SEM, and significant P values (2-way ANOVA) are indicated. ( F ) Quantification of XCL1 (left) and XCR1 (right) expression in the islets of NOD mice at different time points ( n = 2–3 per time point). Data are shown as mean ± SEM.

Article Snippet: The human pancreas sections have been tested for RNA integrity sufficient for the RNAScope duplex detection method (ACD/Bio-Techne) using positive control probe sets provided by the supplier.

Techniques: Gene Expression, Infection, Comparison, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Expressing, RNAscope, In Situ Hybridization

Journal: JCI Insight

Article Title: The XCL1/XCR1 axis is upregulated in type 1 diabetes and aggravates its pathogenesis

doi: 10.1172/jci.insight.178743

Figure Lengend Snippet: Human pancreas slides were obtained through the HPAP program. Groups of individuals were divided as follows: nondiabetic (ND), nondiabetic with known T1D familiarity (T1D familiarity), autoantibody positive (Aab + ), and patients with diabetes (T1D). ( A ) Representative pictures of duplex RNAScope in situ hybridization for XCL1 (red) and XCR1 (blue). In the left column, the original picture with a magnified example of a positive cell (square). On the right, XCR1-expressing cells are highlighted with a light blue dot, and XCL1-expressing cells are highlighted with a red dot. Islets are indicated with black lines. Original magnification, 63×. Scale bars: 20 μm. ( B ) Number of XCR1-expressing cells per islet microenvironment. Number of islets per section analyzed was 6–63. Each islet is represented by 1 dot. Data are shown as the mean ± SEM number of XCR1-expressing cell in one individual. ( C ) Mean ± SEM of XCR1-expressing cells per islet microenvironment in the groups of ND, T1D familiarity, Aab + , and T1D. Number of islets per section analyzed was 6–63. The mean for every individual is represented by dots.

Article Snippet: The human pancreas sections have been tested for RNA integrity sufficient for the RNAScope duplex detection method (ACD/Bio-Techne) using positive control probe sets provided by the supplier.

Techniques: RNAscope, In Situ Hybridization, Expressing

Journal: JCI Insight

Article Title: The XCL1/XCR1 axis is upregulated in type 1 diabetes and aggravates its pathogenesis

doi: 10.1172/jci.insight.178743

Figure Lengend Snippet: ( A ) Duplex RNAScope in situ hybridization for XCL1 (red) and XCR1 (blue) of pancreas tissue sections obtained from RIP-GP × XCL1 –/– mice at different times after infection, demonstrating that XCL1-deficient mice do not express XCL1. Original magnification, 63× (oil). Scale bars: 20 μm. ( B ) IHC staining for CD103 of quick-frozen pancreas sections at different times after LCMV infection, comparing RIP-GP with RIP-GP × XCL1 –/– mice. Representative pictures are shown. Original magnification, 40×. Scale bars: 25 μm. ( C ) Immunofluorescence double-staining for CD11c (green) and CD103 (red) of quick-frozen pancreas sections obtained at different times after LCMV infection, comparing RIP-GP with RIP-GP × XCL1 –/– mice. Nuclei are stained with DAPI (blue). White arrows indicate the double-positive cells (cDC1). Representative pictures are shown. Original magnification, 40×. Scale bars: 20 μm. ( D ) Quantification of the CD103 staining in RIP-GP (light gray) and RIP-GP × XCL1 –/– (dark gray) mice shown in B , expressed as a percentage of positive CD103 cell area per islet area. Numbers of mice per group are indicated. Data are shown as mean ± SEM. ( E ) Quantification of the CD103/CD11c double-staining in RIP-GP (light gray) and RIP-GP × XCL1 –/– (dark gray) mice shown in C , expressed as a percentage of CD103/CD11c–double-positive cell area per CD11c + cell area. Numbers of mice per group are indicated. Data are shown as mean ± SEM, and significant P values (Mann-Whitney t test) are indicated.

Article Snippet: The human pancreas sections have been tested for RNA integrity sufficient for the RNAScope duplex detection method (ACD/Bio-Techne) using positive control probe sets provided by the supplier.

Techniques: RNAscope, In Situ Hybridization, Infection, Immunohistochemistry, Immunofluorescence, Double Staining, Staining, MANN-WHITNEY