Journal: bioRxiv

Article Title: Enhancer hubs govern chromatin topology and Th17 identity

doi: 10.64898/2026.04.02.715458

Figure Lengend Snippet: a) Genome browser plot of the Il17a / Il17f locus (70kb window) integrating 500bp resolution region capture Micro-C (RCMC; ICE balanced, normalized by observed/expected), with 3D contacts annotated by dashed line and Il17a-5 enhancer contacts indicated by blue triangles; ATAC-STARR-seq pooled input DNA library coverage track containing DNA fragments from Th0 Th1 Th2 Th17 and Treg ATAC-seq (grey); ATAC-STARR-seq activity score (Log2 fold change CPM) from Th0 (blue), Th1 (orange), Th2 (red), Th17 (yellow) and Treg (green) RNA versus Input DNA; Effect sizes for gRNA in CRISPRi for Il17a and Il17f (grey = tested; red = FDR < 0.05). OCRs are labeled with direction (+/-) and distance (in Kbp) relative to nearest gene. b) Scatter plot comparing sgRNA effect sizes (Log2 fold change high vs low bin) for CRISPRi screens using Il17a and Il17f reporters (green = only Il17f, red = only Il17a, blue = both, grey = non-significant; FDR < 0.05). c) Distribution of elementwise sgRNA effect sizes grouped by top functional OCRs in both Il17a (left) and Il17f (right) CRISPRi screens (lines = tested gRNA per element, blue = FDR < 0.05). Density plot (top) shows distribution of effect sizes for all gRNA. d) Flow cytometry analysis summarizing frequency of IL-17a+ cells or e) geometric MFI of Il17f (HCR-FlowFish) expression from in vitro derived Th17 cells following CRISPRi-mediated perturbation with candidate gRNAs. f) Representative stacked histograms to show distribution of in vitro derived Th17 cell Il17a and Il17f signal (red) relative to non-transduced (grey) following CRISPRi-mediated repression with top candidate single gRNA. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-hoc test versus NTC and sandwich standard error ( d ) or one-sample t-tests with Benjamini-Hochberg correction (e) . Data are shown as mean ± s.e.m. for gRNA-transduced (Thy1.1 + ) relative to non-transduced (Thy1.1-) cell signal; *** p<0.001; ** p<0.0001; * p<0.05.

Article Snippet: Cells were pre-hybridized in probe hybridization buffer containing 30% formamide and 10% dextran sulfate, then incubated overnight at 37°C with Il17a and Il17f HCR probe sets (Molecular Instruments) at a final concentration of 4 nM.

Techniques: Activity Assay, Labeling, Functional Assay, Flow Cytometry, Expressing, In Vitro, Derivative Assay

Journal: bioRxiv

Article Title: Enhancer hubs govern chromatin topology and Th17 identity

doi: 10.64898/2026.04.02.715458

Figure Lengend Snippet: a) Schematic of the CRISPR-based screening workflow for identifying regulatory elements involved in Th17 differentiation. Naive CD4+ T cells were activated in vitro under Th0 conditions for 24h, followed by transduction with gRNAs targeting open chromatin regions. Cells were then polarized under Th17 conditions for 3 days and prepared for FACS using one of three readouts: i) eGFP expression (i.e Il17a), ii) fixed intracellular staining (i.e RORγt, BATF), or iii) hybridized chain reaction fluorescence in situ hybridization (i.e Il17a Il17f). Cells were finally sorted into high or low expression bins where gRNA abundance was compared. b) Scatter plot comparing sgRNA effect sizes (Log2 fold change hi/lo) between Th17 differentiation noncoding CRISPRi screens with Il17a-eGFP and RORγt readouts (blue = sgRNA significant in both; padj < 0.05). c) Distribution of element-wise effect sizes for gRNA (vertical lines) targeting OCRs in the Il17a- and RORγt-CRISPRi screens (lines = element-targeting gRNA, blue = padj < 0.05). d) Volcano plots depicting sgRNA effect sizes (Log2 fold change) comparing high/low bins for Il17a-eGFP (left) and RORγt(right), with top gRNA labelled (red = padj < 0.05). e ) Mean fluorescence intensity (MFI) of Il17a-eGFP (left) or RORyt (right) from in vitro derived Th17 cells following CRISPRi-mediated repression with individual candidate gRNAs, shown relative to non-targeting control (NTC). Box plots summarize n=5 per targeting gRNA, n=3 for Th0, n=3 for NTC. Statistical analysis was performed using one-way ANOVA with Dunnett’s test versus the NTC and sandwich standard error. Data are shown as mean ± s.e.m. relative to the NTC; * p <0.01; ** p < 0.001

Article Snippet: Cells were pre-hybridized in probe hybridization buffer containing 30% formamide and 10% dextran sulfate, then incubated overnight at 37°C with Il17a and Il17f HCR probe sets (Molecular Instruments) at a final concentration of 4 nM.

Techniques: CRISPR, In Vitro, Transduction, Expressing, Staining, Fluorescence, In Situ Hybridization, Derivative Assay, Control

Journal: bioRxiv

Article Title: Enhancer hubs govern chromatin topology and Th17 identity

doi: 10.64898/2026.04.02.715458

Figure Lengend Snippet: STARR-seq signal (Log2 FC) at all ATAC-STARR-seq tested OCRs within Batf , Rorc(t), and Il17a/f loci categorized by CRISPR-screen result (untested = no CRISPR gRNA coverage). b) Waterfall plot of Th17 ATAC-STARR-seq signal (Log2 fold change RNA/DNA) for OCRs with at least 1 significant gRNA in Il17a-CRISPRi (blue circle) or Il17f-CRISPRi (orange circle), c) RORγt-CRISPRi (blue circle) and RORγt-CRISPRa (red circle) or d) Batf-CRISPRi (blue circle) and Batf-CRISPRa (red circle)

Article Snippet: Cells were pre-hybridized in probe hybridization buffer containing 30% formamide and 10% dextran sulfate, then incubated overnight at 37°C with Il17a and Il17f HCR probe sets (Molecular Instruments) at a final concentration of 4 nM.

Techniques: CRISPR

Journal: Molecular Medicine

Article Title: IL17F + naïve and IFNγ + memory CD8 T cells drive hepatic dysfunction in the cecal ligation and puncture model of sepsis

doi: 10.1186/s10020-025-01411-2

Figure Lengend Snippet: Effects of Immune Education, CLP, and Specific Cytokine Blockade on T cell Cytokine Production. A-C Immune Educated or Control IFNγ-eYFP reporter mice on a C57Bl/6 background were sacrificed prior to or 24 h. following CLP. IFNγ + T cells were assessed. Percent IFNγ + CD4 (Left) and CD8 (Right) T cells in the ( A ) spleen and ( B ) liver. C Histograms of IFNγ + (Red) and negative (Gray) splenic CD4 T cells in Educated mice following CLP. D-E Immune Educated or Control IL17A EGFP /IL17F mCherry reporter mice on a C57Bl/6 background were sacrificed prior to or 24 h. following CLP. IL17A + and IL17F + T cells were assessed. Percent IL17A + CD4 (Left), IL17A + CD8 (Middle Left), IL17F + CD4 (Middle Right) and IL17F + CD8 (Right) T cells in the ( D ) inguinal lymph node (ILN) and ( E ) liver. F Histograms of IL17A + (Orange) vs IL17F + (Green) ILN CD4 T cells in Educated mice following CLP. A-F * = p < 0.05 for comparisons between two groups, † = p < 0.05 for interaction effect, # = p < 0.05 for CLP effect, ^ = p < 0.05 for Education effect. N = 3–4/group. G Hepatic leukocytes were isolated from Educated or control C57Bl/6 mice prior to or 24 h. following CLP alone or with specific cytokine blockade and intracellular cytokine production was assessed following in vitro PMA/Ionomycin stimulation. Percent IFNγ + (Left) and IL17A + (Right) CD8 T cells. Analyzed by 2-way ANOVA. * = p < 0.05 for comparisons between two groups, # = p < 0.05 for specific cytokine effect, p values for interactions, effect of education or effect of blockade displayed. N = 3–5/group. Gating: FSC/SSC, Singlets, Live, CD90/CD4 or CD90/CD8, eYFP, eGFP, mCherry, IFNγ, IL17A, or TNF

Article Snippet: CD4 −/− (B6.129S2-CD4 tm1Mak /J), CD8 −/− (B6.129S2-CD8a tm1Mak /J), IFNγ −/− (B6.129S7-Ifng tm1Ts /J IFNγ), IFNγ-eYFP reporter mice (B6.129S4-Ifng tm3.1Lky /J), and IL17A-eGFP/IL17F-mCherry reporter mice. (C57Bl/6-Il17a tm1Bcgen Il17f em1Litt /J IL17A EGFP /IL17F mCherry ), all on a C57Bl/6 genetic backgound, were obtained from the Jackson Laboratory and bred and maintained in our barrier SPF animal facility.

Techniques: Control, Isolation, In Vitro

Journal: Molecular Medicine

Article Title: IL17F + naïve and IFNγ + memory CD8 T cells drive hepatic dysfunction in the cecal ligation and puncture model of sepsis

doi: 10.1186/s10020-025-01411-2

Figure Lengend Snippet: Effects of Immune Education, CLP, and Specific Cytokine Blockade on T cell Cytokine Production. A-C Immune Educated or Control IFNγ-eYFP reporter mice on a C57Bl/6 background were sacrificed prior to or 24 h. following CLP. IFNγ + T cells were assessed. Percent IFNγ + CD4 (Left) and CD8 (Right) T cells in the ( A ) spleen and ( B ) liver. C Histograms of IFNγ + (Red) and negative (Gray) splenic CD4 T cells in Educated mice following CLP. D-E Immune Educated or Control IL17A EGFP /IL17F mCherry reporter mice on a C57Bl/6 background were sacrificed prior to or 24 h. following CLP. IL17A + and IL17F + T cells were assessed. Percent IL17A + CD4 (Left), IL17A + CD8 (Middle Left), IL17F + CD4 (Middle Right) and IL17F + CD8 (Right) T cells in the ( D ) inguinal lymph node (ILN) and ( E ) liver. F Histograms of IL17A + (Orange) vs IL17F + (Green) ILN CD4 T cells in Educated mice following CLP. A-F * = p < 0.05 for comparisons between two groups, † = p < 0.05 for interaction effect, # = p < 0.05 for CLP effect, ^ = p < 0.05 for Education effect. N = 3–4/group. G Hepatic leukocytes were isolated from Educated or control C57Bl/6 mice prior to or 24 h. following CLP alone or with specific cytokine blockade and intracellular cytokine production was assessed following in vitro PMA/Ionomycin stimulation. Percent IFNγ + (Left) and IL17A + (Right) CD8 T cells. Analyzed by 2-way ANOVA. * = p < 0.05 for comparisons between two groups, # = p < 0.05 for specific cytokine effect, p values for interactions, effect of education or effect of blockade displayed. N = 3–5/group. Gating: FSC/SSC, Singlets, Live, CD90/CD4 or CD90/CD8, eYFP, eGFP, mCherry, IFNγ, IL17A, or TNF

Article Snippet: IFNγ (XMG1.2, dose: 0.5 mg), IL12p40 (C17.8, dose: 1 mg), TNF (XT3.11, 1 mg), IL17A (17F3, dose: 0.5 mg) and IL17F (MM17F8F5.1A9, dose: 0.5 mg) blocking antibodies were all obtained from BioXCell (Lebanon, NH), diluted from stock concentration in sterile PBS and administered through intraperitoneal injection at in vivo blocking doses as previously described in the literature. (Jordan et al. ; Taylor et al. ; Naik et al. ; Lemaire et al. ).

Techniques: Control, Isolation, In Vitro

Journal: Molecular Medicine

Article Title: IL17F + naïve and IFNγ + memory CD8 T cells drive hepatic dysfunction in the cecal ligation and puncture model of sepsis

doi: 10.1186/s10020-025-01411-2

Figure Lengend Snippet: Effects of Immune Education, CLP, and Specific Cytokine Blockade on Innate and T Cell Immune Responses. Hepatic leukocytes were isolated from Educated or control C57Bl/6 mice prior to or 24 h. following CLP alone or with specific cytokine blockade; innate immune cell numbers and cytokine production were assessed. Total number of hepatic neutrophils ( A ) or MoDCs ( B ), and percent TNF + ( C ), IL1β + ( D ) or IL12p40 + MoDCs ( E ) and total number of hepatic CD8 T cells ( F ) in control (Blue) or Educated (Red) mice at baseline, following CLP or following CLP with IFNγ, IL12p40, TNF, or IL17A/F combined blockade. CLP with IL17A or IL17F blockade shown to the right. Analyzed by 2-way ANOVA. IL17A and IL17F analyzed separately. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 for comparisons between two groups, # = p < 0.05 for cytokine effect compared to CLP. N = 3–12/group. Cytokine production analyzed in a subset of samples. Gating: Neutrophils = FSC/SSC, Singlets, Live Cells, CD11b + /CD11c − , Ly6G + /Ly6C + , MoDCs = FSC/SSC, Singlets, Live, CD11b + /CD11c − , Ly6G − , MHCII + , Macrophages = FSC/SSC, Singlets, Live, CD11b + /CD11c − , Ly6G − , MHCII − , Ly6C Hi /CD64 + , CD8 + T cells = FSC/SSC, Singlets, Live, CD90 + /CD8 +

Article Snippet: IFNγ (XMG1.2, dose: 0.5 mg), IL12p40 (C17.8, dose: 1 mg), TNF (XT3.11, 1 mg), IL17A (17F3, dose: 0.5 mg) and IL17F (MM17F8F5.1A9, dose: 0.5 mg) blocking antibodies were all obtained from BioXCell (Lebanon, NH), diluted from stock concentration in sterile PBS and administered through intraperitoneal injection at in vivo blocking doses as previously described in the literature. (Jordan et al. ; Taylor et al. ; Naik et al. ; Lemaire et al. ).

Techniques: Isolation, Control

Journal: Molecular Medicine

Article Title: IL17F + naïve and IFNγ + memory CD8 T cells drive hepatic dysfunction in the cecal ligation and puncture model of sepsis

doi: 10.1186/s10020-025-01411-2

Figure Lengend Snippet: Effects of Immune Education, CLP, and Specific Cytokine Blockade on CLP-Induced Hepatic Dysfunction. Immune Educated (Red) or Control (Blue) C57Bl/6 mice were sacrificed prior to or 24 h. following CLP alone or with cytokine blockade. A Hepatic tissue was assessed by RT-PCR for log 10 transformed relative transcription levels of SCLO1a1, SLC10, and ABCC2. B Serum Alanine aminotransferase (ALT). C Log 10 transformed peritoneal bacterial colony forming units. D Probability of Survival in the days following CLP with blockade as noted. Data as mean ± SD. Analyzed by 2-way ANOVA. * = p < 0.05 for comparisons between two groups. P Int = Significant Interaction between Blockade and Education, P Block = Significant Effect of Treatment (CLP + antibody blockade), P Edu = Significant Effect of Control vs Immune Education. For A , primary 2-way ANOVA comparison between Baseline, CLP, CLP + αIFNγ, CLP + αIL12p40, CLP + αTNF, and CLP + αIL17A/F. IL17A and IL17F blockade compared to each other separately from other comparisons. Survival analyzed by Log-rank Test. N = 4–22/group

Article Snippet: IFNγ (XMG1.2, dose: 0.5 mg), IL12p40 (C17.8, dose: 1 mg), TNF (XT3.11, 1 mg), IL17A (17F3, dose: 0.5 mg) and IL17F (MM17F8F5.1A9, dose: 0.5 mg) blocking antibodies were all obtained from BioXCell (Lebanon, NH), diluted from stock concentration in sterile PBS and administered through intraperitoneal injection at in vivo blocking doses as previously described in the literature. (Jordan et al. ; Taylor et al. ; Naik et al. ; Lemaire et al. ).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Blocking Assay, Comparison

Journal: Molecular Medicine

Article Title: IL17F + naïve and IFNγ + memory CD8 T cells drive hepatic dysfunction in the cecal ligation and puncture model of sepsis

doi: 10.1186/s10020-025-01411-2

Figure Lengend Snippet: Effects of CLP on Pet Shop and SPF J:ARC(S) Swiss Outbred Mice. Pet Store or Control J:ARC(S) Swiss Outbred mice were sacrificed 24 h. following CLP. A Serum levels of IFNγ, IL12p40, and IL17. B Total number of hepatic neutrophils, MoDCs, or cDCs. C RT-PCR log 10 transformed relative transcription levels of SCLO1a1 from hepatic tissue, serum alanine aminotransferase (ALT), and log 10 transformed peritoneal bacterial colony forming units. Analyzed by 2-way ANOVA. P Int = Significant Interaction between Blockade and Pet Store/Laboratory, P Block = Significant Effect of Treatment (CLP + antibody blockade), P group = Significant Effect of Pet Store/Laboratory Strain. 2-way ANOVA comparison between Baseline, CLP, CLP + αIFNγ, and CLP + αIL17F. * = p < 0.05 for comparisons between two groups. N = 4–7/group. Gating: Neutrophils = FSC/SSC, Singlets, Live, CD11b + /CD11c − , Ly6G + /Ly6C + , MoDCs = FSC/SSC, Singlets, Live, CD11b + /CD11c − , Ly6G − , MHCII + , cDCs = FSC/SSC, Singlets, Live, CD11c + , MHCII +

Article Snippet: IFNγ (XMG1.2, dose: 0.5 mg), IL12p40 (C17.8, dose: 1 mg), TNF (XT3.11, 1 mg), IL17A (17F3, dose: 0.5 mg) and IL17F (MM17F8F5.1A9, dose: 0.5 mg) blocking antibodies were all obtained from BioXCell (Lebanon, NH), diluted from stock concentration in sterile PBS and administered through intraperitoneal injection at in vivo blocking doses as previously described in the literature. (Jordan et al. ; Taylor et al. ; Naik et al. ; Lemaire et al. ).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Blocking Assay, Comparison