Journal: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: (A) Cartoon depicting IFNL bound to each IFNLR1 variant and complexed with IL10RB with proximal kinases JAK1 and TYK2 engaged. Note the truncation in the Box1 and Box2 JAK1 binding motifs within IFNLR1 variant 2. (B) Alignment of the JAK1 binding motifs for IFNLR1 variant 1 (Q8IU57; top sequence) and IFNLR1 variant 2 (Q8IU57-2; bottom sequence). Box 1 (blue highlight) and Box 2 (yellow highlight) domains are indicated and reveal the 29 amino acid truncation within the cytoplasmic domain of IFNLR1 variant 2 that eliminates the C-terminal residues of Box 1 and all but two residues of Box 2. Created in BioRender. Novotny, L. (2025) https://BioRender.com/24aqcgc .

Article Snippet: Variant-expressing HEK293T cells and WT iHeps treated +/-dox (100ng/ml) and IFNLR1 -KO iHeps treated + dox (100ng/ml dox) for 22h were incubated in medium +/-0.01-1.0μM upadacitinib (a JAK1 inhibitor, Tocris) or 0.01-1.0μM deucravacitinib (a TYK2 inhibitor, TargetMol) for 2h prior to stimulation +/- IFNL3 (100ng/ml, 24h) in the continued presence of inhibitor.

Techniques: Variant Assay, Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: Western blot analysis of whole cell lysates from ( A ) WT iHeps and ( B ) IFNLR1 -KO iHeps +/-dox-induced for 24h then treated +/-IFNL3 or IFNA2 for 15min. GAPDH served as an indicator of equivalent protein loading per lane. The ratio of phosphorylated to total protein for ( C ) dox-uninduced WT iHeps and ( D ) dox-induced IFNLR1 -KO iHeps +/-IFNL3 is shown as a percentage, based on integrated band intensity determined in ImageJ.

Article Snippet: Variant-expressing HEK293T cells and WT iHeps treated +/-dox (100ng/ml) and IFNLR1 -KO iHeps treated + dox (100ng/ml dox) for 22h were incubated in medium +/-0.01-1.0μM upadacitinib (a JAK1 inhibitor, Tocris) or 0.01-1.0μM deucravacitinib (a TYK2 inhibitor, TargetMol) for 2h prior to stimulation +/- IFNL3 (100ng/ml, 24h) in the continued presence of inhibitor.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: ( A ) Model depicting the mechanisms of distinct signaling outcomes imparted by ternary complexes composed of IFNL3, IL10RB and either IFNLR1 variant 1 or 2. Greater arrow width indicates higher association and/or phosphorylation (JAK1, STAT1, STAT2), greater internalization of receptor complexes, or higher induction of gene expression. IFNLR1 variant 1 containing heterodimers are depicted to have greater stability of JAK1 and/or pJAK1 binding and to be more prone to internalization than IFNLR1 variant 2 containing heterodimers. This correlates with variant 1 mediating more efficient phosphorylation of STAT1 and STAT2 and supporting higher expression of antiviral ISGs and de novo expression of proinflammatory ISGs compared to variant 2. ( B ) Model depicting assemblage of multimeric clusters containing multiple heterodimers of IFNL3-bound IFNLR1 variants in complex with IL10RB. In this model, proximity of JAK1 molecules bound to the cytoplasmic domains of IFNLR1 variants 1 and 2 could participate in TYK2-independent transphosphorylation. The relative abundance of IFNLR1 variants within multimeric complexes would influence the nature of receptor internalization, STAT phosphorylation, and downstream gene expression. The TYK2 dependence of signaling differs for antiviral vs. proinflammatory ISG expression and has a complex relationship with the relative abundance of IFNLR1 variants. Soluble variant 3, not studied in these experiments, is included in the model for consideration. Created in BioRender. Novotny, L. (2025) https://BioRender.com/24aqcgc .

Article Snippet: Variant-expressing HEK293T cells and WT iHeps treated +/-dox (100ng/ml) and IFNLR1 -KO iHeps treated + dox (100ng/ml dox) for 22h were incubated in medium +/-0.01-1.0μM upadacitinib (a JAK1 inhibitor, Tocris) or 0.01-1.0μM deucravacitinib (a TYK2 inhibitor, TargetMol) for 2h prior to stimulation +/- IFNL3 (100ng/ml, 24h) in the continued presence of inhibitor.

Techniques: Variant Assay, Phospho-proteomics, Gene Expression, Binding Assay, Expressing

Journal: bioRxiv

Article Title: Pan-cancer tumor classification by a holistic tumor microenvironment atlas

doi: 10.64898/2025.12.27.696641

Figure Lengend Snippet: a, GO enrichment analysis on genes differentially expressed in CXCL9 + and IFIT1 + TAMs. Red and blue represent pathways enriched in CXCL9 + and IFIT1 + TAMs, respectively. P values are adjusted by BH in the hypergeometric test. b, Dot plot showing the expression of representative signature genes of CXCL9 + and IFIT1 + TAMs. The dot size represents the proportion of expressing cells. The color indicates the average level of gene expression. c, Quantification of the mRNA expression of CXCL9 + macrophage-specific gene ( CXCL9 ) and IFIT1 + macrophage-specific genes ( IFIT1 ) in monocyte-derived macrophages, following a 24h stimulation with IFNA1, IFNA13, IFNA14, IFNB1, IFNG or control. P values are calculated using the two-sided t-test, where * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. d, ATAC-seq tracks showing the chromatin accessibility in the CXCL9 and IFIT1 loci for macrophage subsets in TNBC tumors. e, Scatter plots showing the spatial distribution pattern of CD68 + CD163 + CXCL9 + cells and CD68 + CD163 + IFIT1 + cells in representative Xenium samples. Each dot represents an individual cell, and color represents the cell identity. The right bar plots showing the proportion of IFIT1 + or CXCL9 + macrophages across Xenium samples. f, Scatter plots showing effect sizes of the cancer cell state comparisons between c68-CXCL9 high versus c68-CXCL9 low and c69-IFIT1 high versus c69-IFIT1 low tumors. Left: recurrent cancer gene programs (Barkley et al.); right: HALLMARK pathways (MsigDB). Dots represent cancer cell states, and colors represent statistical significance categories. Effect sizes are calculated as Hedge’s g values derived from Student’s t-test, and P values are adjusted by the BH-method. Dots with a BH-adjusted P value < 0.05 and an absolute effect size >0.2 are highlighted.

Article Snippet: Recombinant proteins IFNA1 (Novoprotein, CC75), IFNA13 (MedChemExpress, HY-P72796), IFNA14 (MedChemExpress, HY-P72243), IFNB1 (PeproTech, 300-02BC), and IFNG (PeproTech, 300-02) were then added to the medium at a final concentration of 20 ng/mL.

Techniques: Expressing, Gene Expression, Derivative Assay, Control