rorγt inhibitors  (Vitae Pharmaceuticals)

Journal: The Journal of Clinical Investigation

Article Title: Selective disruption of ROR γ t-CBF β interaction by IMU-935 prevents ROR γ t-dependent Th17 autoimmunity but not thymocyte development

doi: 10.1172/JCI185942

Figure Lengend Snippet: ( A and B ) Flow cytometric analysis of CD4 + and CD8 + thymocytes ex vivo developed for 3 days from sorted ROR γ t –/– CD4 – CD8 – or WT CD4 – CD8 – in the presence of different concentrations of IMU-935 ( A ) ( n = 2–4/group, from 4 experiments) or RORγt modulators ( B ) (1 μM; n = 4/group, from 4 experiments). Percentages of CD4 + plus CD4 + CD8 + T cells among live Thy1.2 + thymocytes are shown. ( C and D ) Percentages of live cells among CD4 + CD8 + thymocytes relative to the vehicle control different times after culture in the presence of indicated concentrations of IMU-935 ( C ) or RORγt modulators ( D ) (1 μM; n = 3/group, from 1 experiment). Thymocytes from ROR γ t –/– mice were used as a control. ( E ) Picture and cellularity of the thymus from mice ( n = 8–9/group; n = 8 for vehicle and IMU-935 groups, and n = 9 for Cpd-1 and cintirorgon groups) treated with vehicle or indicated RORγt inhibitors or cintirorgon (100 mg/kg orally, twice daily) for 28 days. ( F ) H&E staining (scale bar: 500 μm) of the thymus from mice treated as described in E . M, medullar; C, cortex. Bottom: The ratio of the medullary/cortical region. ( G ) Flow cytometric analysis of CD4 and CD8 in the thymocytes from mice treated as described in E . ( H ) TUNEL staining (top; scale bar: 500 μm) and percentage of TUNEL-positive apoptotic cells in thymus obtained from mouse treated as described in E . Bottom right: The qRT-PCR analysis of Bcl2l1 mRNA. ( I ) Number of alive, human CD4 + CD8 + thymocytes cultured in the presence of indicated RORγt inhibitors (1 μM) for 24 hours (thymocytes collected from 1 donor, n = 3 replicates/group). Data were assessed by 1-way ANOVA with Dunnett’s post hoc test ( A – H ) or 2-tailed Student’s t test ( I ). * P < 0.05; ** P < 0.01.

Article Snippet: Indeed, an RORγt inhibitor developed by Novartis affected thymocyte development similarly to RORγt –/– mice, including accelerated thymocyte apoptosis and thymic lymphoma ( ).

Techniques: Ex Vivo, Control, Staining, TUNEL Assay, Quantitative RT-PCR, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: Selective disruption of ROR γ t-CBF β interaction by IMU-935 prevents ROR γ t-dependent Th17 autoimmunity but not thymocyte development

doi: 10.1172/JCI185942

Figure Lengend Snippet: ( A ) Venn diagram displaying the number of DEGs (1.5-fold, up or down; P < 0.05) identified by RNA-Seq assays in WT versus ROR γ t –/– thymocytes and in WT thymocytes treated with IMU-935 (1 μM) or not. ( B and C ) Volcano plots displaying the global gene expression. The horizontal dotted line marks P = 0.05, the vertical dotted lines mark fold change of ±1.5. ( D ) Heatmap showing antiapoptotic, apoptotic, and cell cycle gene expression in ROR γ t –/– or IMU-935–nontreated (WT-vehicle control) or treated (WT–IMU-935) thymocytes. Gene expression was normalized by total counts of each gene. ( E ) Heatmap depicting changes in the activity of upstream regulators predicted by IPA. Activation z score indicates increased (orange; z > 0) or decreased (blue; z < 0) activity. All z score values are labeled within the corresponding cell. Genes shown are known to regulate apoptosis ( Mycn , E2f1 , Esr1 ), survival ( Nfkbia , TP53 ), cell cycle ( Gli1 , Cdkn2a ), oxidative stress ( Nfe2l2 ), and thymocyte development ( Ctnnb1 , Lef1 ). ( F ) Heatmap showing the activity of the pathways determined by IPA. Pathway activity was obtained by comparing WT and ROR γ t –/– thymocytes (left column) and IMU-935–nontreated versus -treated WT thymocytes (right column). N/A indicates pathway status is not predictable due to the small number of genes affected by IMU-935 treatment in thymocyte RNA-Seq. ( G ) Genome-wide RORγt-DNA binding signal intensity determined by ChIP-Seq assays near the TSS in ROR γ t –/– or WT (RORγt + ) or WT–IMU-935 thymocytes. ( H ) RORγt binding peaks at the Bcl2l1 locus. The results are the representative of overlays from 2 separate experiments. Veh, vehicle.

Article Snippet: Indeed, an RORγt inhibitor developed by Novartis affected thymocyte development similarly to RORγt –/– mice, including accelerated thymocyte apoptosis and thymic lymphoma ( ).

Techniques: RNA Sequencing, Gene Expression, Control, Activity Assay, Activation Assay, Labeling, Genome Wide, Binding Assay, ChIP-sequencing

Journal: The Journal of Clinical Investigation

Article Title: Selective disruption of ROR γ t-CBF β interaction by IMU-935 prevents ROR γ t-dependent Th17 autoimmunity but not thymocyte development

doi: 10.1172/JCI185942

Figure Lengend Snippet: ( A ) Changes in Th17 differentiation after knockout of indicated genes, relative to nontargeting group (NonT; 100%) in Cas9-expressing CD4 + T cells ( n = 3–10/group from 3 pooled independent experiments). ( B ) Relative luciferase activity from a promoterless control (pGL3) or RORγt reporter (RORBE) in HEK293T cells transfected with indicated expression plasmids for 24 hours. ( C ) A visualization of the computer-predicted interaction between RORγt and CBFβ (top left) or RUNX1 and CBFβ (top right). Dashed circle highlights the interaction interface between indicated proteins. The cyan fragment indicates the RORγt amino acids 403–413 critical for binding to CBFβ. The black box in the bottom panel indicates the amino acids with >40% contact frequency with CBFβ; the white box indicates <40% contact frequency. ( D and E ) IP analysis of the RORγt-CBFβ interaction in ROR γ t –/– CD4 + T cells expressing GFP (EV) or with Flag-RORγt polarized in Th17 conditions ( D ) in ROR γ t –/– or WT thymocytes ( E ) and treated with indicated concentrations of IMU-935. IP with anti–Flag-RORγt ( D ) or anti-RORγt ( E ) antibody and immunoblot with anti-CBFβ antibody. Input was analyzed by Western blot (bottom 2 panels). Right: The relative intensity of immunoprecipitated CBFβ band. ( F ) Flow cytometric analysis of RORγt (top), RUNX1 (middle), and CBFβ (bottom) levels in differentiated Th17 cells, peripheral naive CD4 + T cells, and different subsets of thymocytes. ( G and H ) IP analysis of the RORγt-CBFβ interaction in control (NonT) or Runx1 -deleted Th17 cells (sg Runx1 ) ( G ) or thymocytes expressing EV or RUNX1 ( H ) treated with indicated concentrations of IMU-935, similar to what is described in D and E . Data represent 2 ( F ), 3 ( H ), or 4 ( B , D , E , and G ) independent experiments. Data were assessed by 1-way ANOVA with Dunnett’s ( A , D , and E ), Tukey’s ( B ) post hoc test, or 2-tailed Student’s t test ( G and H ). * P < 0.05; ** P < 0.01.

Article Snippet: Indeed, an RORγt inhibitor developed by Novartis affected thymocyte development similarly to RORγt –/– mice, including accelerated thymocyte apoptosis and thymic lymphoma ( ).

Techniques: Knock-Out, Expressing, Luciferase, Activity Assay, Control, Transfection, Binding Assay, Western Blot, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: Selective disruption of ROR γ t-CBF β interaction by IMU-935 prevents ROR γ t-dependent Th17 autoimmunity but not thymocyte development

doi: 10.1172/JCI185942

Figure Lengend Snippet: ( A – C ) IP analysis of the RORγt-CBFβ interaction in HEK293T cells with indicated expression plasmids for WT ( A ), deletion mutation ( B ), or alanine scanning mutation ( C ) RORγt. ΔDBD, DNA binding domain-deleted RORγt; ΔHinge, hinge-deleted RORγt; ΔLBD, Lihand-binding domain-deleted RORγt. ( D ) The percentage of Th17 differentiation activity supported by the RORγt with the indicated point mutation expressed in ROR γ t –/– CD4 + T cells relative to those expressing WT RORγt (defined as 100%). ROR γ t –/– CD4 + T cells expressing GFP alone (EV) or with WT RORγt or RORγt with the indicated single amino acid mutated to alanine were polarized under Th17 conditions. ( E ) Visualization of computer-predicted interaction between RORγt and CBFβ. The cyan fragment indicates RORγt amino acids 403–413. R407, L410, and E412 of RORγt critical for binding to CBFβ are indicated. Red dashed lines indicate the ionic lock between R407 and E336. ( F ) IP analysis of CBFγ interaction with RORβt or R407A/L410A DM. ( G and H ) Relative luciferase activity from HEK293T cells transfected with pGL3 control or RORγt reporter (RORBE) together with plasmids expressing WT or RORγt with indicated mutation ( G ) with or without CBFβ ( H ). ( I ) The percentage of Th17 differentiation activity supported by the RORγt with indicated mutation expressed in ROR γ t –/– CD4 + T cells relative to those expressing WT RORγt (defined as 100%). ( J ) Flow cytometric analysis of CD4 + and CD8 + thymocytes ex vivo developed for 3 days from sorted ROR γ t –/– CD4 – CD8 – transduced with WT or RORγt with indicated mutation. Right: The summary of the percentages of CD4 + plus CD4 + CD8 + T cells among live Thy1.2 + cells. Data represent 3 ( D and G ) or 4 ( H – J ) independent experiments; representative Western blots are shown from 3 independent experiments ( A – C , and F ). Data were analyzed by 1-way ANOVA with Dunnett’s ( D ) or Tukey’s ( G – J ) post hoc test. * P < 0.05; ** P < 0.01. WCL, whole-cell lysate.

Article Snippet: Indeed, an RORγt inhibitor developed by Novartis affected thymocyte development similarly to RORγt –/– mice, including accelerated thymocyte apoptosis and thymic lymphoma ( ).

Techniques: Expressing, Mutagenesis, Binding Assay, Activity Assay, Luciferase, Transfection, Control, Ex Vivo, Transduction, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Selective disruption of ROR γ t-CBF β interaction by IMU-935 prevents ROR γ t-dependent Th17 autoimmunity but not thymocyte development

doi: 10.1172/JCI185942

Figure Lengend Snippet: ( A ) IP analysis of the RORγt-CBFβ interaction in Th17 cells (left) or thymocytes (right) from indicated mice. Cell lysates from differentiated Th17 cells and thymocytes were subjected to IP with anti-RORγt antibody and immunoblotting with anti-CBFβ or (top) and anti-RORγt (bottom) antibody. Input CBFβ and RORγt were analyzed by Western blot (bottom 2 panels). Representative Western blots are shown from 3 independent experiments. ( B ) Flow cytometric analysis and percentage of IL-17A + cells among CD4 + T cells from indicated mice polarized under Th17 conditions for 60 hours ( n = 4 mice/group, from 1 experiment). ( C ) Clinical score of EAE among indicated mice ( n = 6/group) different days after induction of disease by immunization with MOG 35–55 . ( D ) Flow cytometric analysis (left 2 panels), percentages (third panel), and numbers (right) of CD4 + T cells infiltrated into the CNS of EAE-induced mice shown in C . ( E ) Flow cytometric analysis (left 2 panels), percentages (third panel), and numbers (right) of CNS-infiltrated CD4 + T cells producing IL-17A in EAE-induced mice shown in C . ( F ) Flow cytometric analysis of CD4 + and CD8 + thymocytes ex vivo developed for 3 days from sorted WT or ROR γ t DM/DM CD4 – CD8 – cells. Right: Summary of the percentage of CD4 + plus CD4 + CD8 + T cells among live Thy1.2 + cells ( n = 3 mice/group, from 1 experiment). ( G ) Percentage of live cells among thymocytes from indicated mice and cultured for different times in vitro ( n = 4 mice/group, from 1 experiment). Data were analyzed by 2-tailed Student’s t test ( B – G ). * P < 0.05; ** P < 0.01.

Article Snippet: Indeed, an RORγt inhibitor developed by Novartis affected thymocyte development similarly to RORγt –/– mice, including accelerated thymocyte apoptosis and thymic lymphoma ( ).

Techniques: Western Blot, Ex Vivo, Cell Culture, In Vitro