th17 polarization  (Galectin Therapeutics)

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 1. The short isoform of LKB1 is predominantly expressed in Th17 cells CD4 T cells isolated from were differ entiated under Th17, iTreg, or non- polarizing (NP) conditions for 7 days prior to harvesting. We utilized flow cytometry to assess a) the percent of LKB1 positive cells within each cell subset and b) LKB1 protein expression, as measured by median fluorescence intensity (MFI). c) We visualized LKB1S expression by immunoblotting and quantified LKB1S expression relative to NP. Vinculin is shown as a loading control. We amplified the d) short and e) long splice variants of Stk11 using qRT-PCR, as well as f) the ratio of Stk11S to Stk11L transcript expression. Stk11 transcripts were quantified using the 2-ΔΔCt method, normalized to ActB (β-actin), and are expressed relative to NP. Protein band intensity was quanti fied using ImageJ. Data are the mean of three independent experiments. * p < 0.05; * * p < 0.01; * ** p < 0.001; unpaired, two-tailed Student’s t-test.

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

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

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 2. HnRNPLL associates with Stk11S in Th17 cells and requires PKCθ for its expression CD4 T cells isolated from WT mice were differentiated under Th17, iTreg, or non-polarizing (NP) conditions for 7 days prior to harvesting. a) We assessed hnRNPLL expression by immunoblotting and b) Hnrnpll levels by qRT-PCR. Vinculin is shown as a loading control. c) For RNA-IP, hnRNPLL was immunoprecipitated from whole cell lysate with RNase inhibitor to preserve RNA content. RNA was extracted from the lysate after immunoprecipitation, and we used qRT-PCR to amplify Stk11S transcripts. Hnrnpll transcripts were quantified using the 2-ΔΔCt method, normalized to ActB (β-actin), and are expressed relative to the WT NP sample. Stk11S is expressed as bound units (BU) relative to Stk11S bound to hnRNPLL immunoprecipitated from NP samples. Protein band intensity was quantified using ImageJ. Data are the mean ± S.E.M of three independent experiments. * p < 0.05; * * p < 0.01; * ** p < 0.001; * ** * p < 0.0001; two-way ANOVA with Bonferroni correction applied.

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Expressing, Isolation, Western Blot, Quantitative RT-PCR, Control, Immunoprecipitation

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 3. PKCθ regulates Stk11 splice variant and LKB1 isoform expression upstream of hnRNPLL CD4 T cells isolated from WT or PKCθ-/- mice were differentiated under Th17, iTreg, or non-polarizing (NP) conditions for 7 days prior to harvesting. We used qRT-PCR to quantify a) Prkcq and b) Hnrnpll transcript levels in WT Th17 and iTreg cells, as well as the levels of c) Stk11S and Stk11L. Relative gene expression was determined using the 2-ΔΔCT method. For Il17f and Prkcq, For Prckq and Hnrnpll, results are presented as fold expression of the gene of interest normalized to the housekeeping gene, ActB (β-actin), for each sample, and expressed relative to the For Stk11 splice variants, the results are presented as percent of the variant expressed relative to total Stk11 (common) for each sample. d) We used immunoblotting to assess differences in LKB1 isoform in differentiated CD4 T cells from WT and PKCθ-/- mice. Vinculin is shown as a loading control. Protein band intensity was quantified using ImageJ. Data are the mean ± S.E.M of three independent experiments. Statistical analyses shown in gray refer to comparisons between conditions for cells derived from PKCθ-/- mice. * p < 0.05; * * p < 0.01; * ** * p < 0.0001; two-way ANOVA with Bonferroni correction applied.

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Variant Assay, Expressing, Isolation, Quantitative RT-PCR, Gene Expression, Western Blot, Control, Derivative Assay

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 4. HnRNPLL regulates Stk11S expression CD4 T cells isolated from WT or PKCθ-/- mice were differentiated under Th17, iTreg, or non-polarizing (NP) conditions for 7 days prior to har vesting. On day 3 of the differentiation process, Th17 polarized cells were transfected with 50 μM of Hnrnpll siRNA (siLL) or with a scrambled control (siSCR). At the end of the differentiation period, we used qRT-PCR to quantify a) Stk11S and b) Stk11L. c) LKB1 isoform expression was assessed by immuno blotting. Vinculin is shown as a loading control. We also quantified d) Il17f and e) Prkcq by qRT-PCR. Relative gene expression was determined using the 2-

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Expressing, Isolation, Transfection, Control, Quantitative RT-PCR, Gene Expression

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 5. IL-6 signaling acts upstream of PKCθ-regulated Stk11 splicing CD4 T cells isolated from WT or PKCθ-/- mice were differentiated under Th17, iTreg, or non-polarizing (NP) conditions for 7 days prior to harvesting. iTregs were dosed with 20 ng/ml of IL-6 on day 5 of the differentiation process, then har vested 48 h later. We used qRT-PCR to quantify a) Prkcq, b) Hnrnpll, and d) Rorc and relative gene expression was determined using the 2-ΔΔCt method. The results are presented as fold expression of the gene of interest normalized to the housekeeping gene, ActB (β-actin), for each sample, and is expressed relative to the WT NP sample. c) Stk11S is expressed as a percent of the total Stk11 (common) for each sample. e) In vitro differentiated iTregs from WT or PKCθ-/- mice were labeled with Red650 and mixed with Ultra Green labeled responder cells (CD4 T cells) at three different ratios. Percent suppres sion was determined as described in Materials & Methods. Data are the mean ± S.E.M of three independent experi ments. Statistical analyses shown in gray refer to comparisons between conditions for cells derived from PKCθ-/-

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Isolation, Quantitative RT-PCR, Gene Expression, Expressing, In Vitro, Labeling, Derivative Assay

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 6. Stk11 splicing proceeds through a Stat3-mediated pathway CD4 T cells isolated from WT or PKCθ-/- mice were differentiated under Th17, iTreg, or non- polarizing (NP) conditions for 7 days prior to harvesting. iTregs were dosed with 20 ng/ml of IL-6 on day 5 of the differentiation process. For some cultures, iTregs were also treated with 10 μM of the specific STAT3, Stattic, on days 5 and 6 of polarization. All cells were harvested on day 7. We used qRT-PCR to quantify a) Prkcq and b) Hnrnpll using the 2-ΔΔCt method. The results are presented as fold expression of the gene of interest normalized to the housekeeping gene, ActB (β-actin), for each sample, and is expressed relative to levels in WT NP sample. We used flow cytometry to assess the percentage of c) IL-17-expressing or e) Foxp3-expressing cells generated for each treatment condition and the median fluorescence intensity (MFI) of d) IL-17 or f) Foxp3 expressed by each population. Data are the mean ± S.E.M of three independent experiments. Statistical analyses shown in gray refer to comparisons between conditions for cells derived from PKCθ-/- mice. Statistical analyses represented by symbols refer to comparisons between cells derived from WT and PKCθ-/- mice, subjected to identical treatments. $ or * p < 0.05; & or * * p < 0.01; * ** p < 0.001; # or * ** * p < 0.0001; two-way ANOVA with Bonferroni correction applied.

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Flow Cytometry, Generated, Fluorescence, Derivative Assay

Journal: Molecular immunology

Article Title: LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

doi: 10.1016/j.molimm.2023.03.020

Figure Lengend Snippet: Fig. 7. Stk11s expression is sufficient to induce Th17 programming CD4 T cells were isolated from WT or PKCθ-/- mice. Prior to differentiation, cells to be differ entiated into Th17 cells were transduced with empty vector (Th17 +EV) or with Stk11L (Th17 +Stk11L), while cells to be differentiated into iTreg cells were transduced with empty vector (iTreg+EV) or with Stk11S (iTreg+Stk11S). At the end of the 7-day polarization process a) LKB1 expression was visualized by immunoblotting. Vinculin as a loading control. We quantified b) Foxp3, c) Rorc, and d) Il17f by qRT-PCR. Relative gene expression was determined using the 2-ΔΔCt method. The results are presented as the fold expression of the gene of interest normalized to the housekeeping gene, ActB (β-actin), for each sample and is expressed relative to the WT NP sample. Protein band intensity was quantified using ImageJ. Data are the mean ± S.E.M of three independent experiments. Statistical analyses shown in gray refer to comparisons between conditions for cells derived from PKCθ-/- mice. Statistical analyses represented by symbols refer to comparisons between cells derived from WT and PKCθ-/- mice, subjected to identical treatments. $ or * p < 0.05; & or * * p < 0.01; * ** p < 0.001; # or * ** * p < 0.0001; two-way ANOVA with Bonferroni correction applied.

Article Snippet: For some experiments, CD4 T cells were polarized under iTreg or Th17 polarizing conditions and on days 5 and 6 of the differentiation process were treated with 10 μM of the specific Stat3 inhibitor, Stattic (Santa Cruz Biotechnology, Dallas, TX), according to the manufacturer’s D. Mohan et al. Molecular Immunology 157 (2023) 129–141 recommendations.

Techniques: Expressing, Isolation, Transduction, Plasmid Preparation, Western Blot, Control, Quantitative RT-PCR, Gene Expression, Derivative Assay