Journal: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: (A, E) Pretreatment with IL-35 neutralizing antibodies (anti-P35 or anti-Ebi3) aggravated LPS- and CLP-induced ARDS. Lungs from each experimental group were processed for histological examination after H&E staining. Compared with the LPS + IgG group, thickened alveolar wall, alveolar hemorrhage and collapse, inflammatory cells in filtration were more severe and pretreated with IL-35 neutralizing antibodies prior to LPS challenge. (B, D) Lung injury scores were estimated by the method of Mikawa, which is from the following four indicators of lung injury score: alveolar congestion; bleeding; gap or vascular wall neutrophil infiltration or aggregation; alveolar septal thickening or transparent membrane formation. 0 marks: no or very slight damage, 1 marks: mild injury, 2 marks: moderate injury, 3 marks: severe injury, 4 marks: very severe damage, the cumulative increase in the number of lesions of the total score is the pathological score of the ARDS. Pretreatment with IL-35 neutralizing antibodies lungs injury were more severe than LPS + IgG group. **p < 0.01, ***p < 0.001, by the two-way ANOVA followed by LSD multiple comparisons test, compared with the LPS + IgG group.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Staining, Filtration, Membrane

Journal: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: Interleukin 35 (IL-35) blockade upregulated the production of proinflammatory cytokines and downregulated anti-inflammatory cytokines during ARDS models. Cytokine and chemokine concentrations in BALF and blood specimens from five mice treated with or without anti–Ebi3 or anti–P35 blocking antibodies were determined by mice cytokine/chemokine magnetic bead panel kit assays 24 h after onset of ARDS. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by the one-way ANOVA followed by LSD multiple comparisons test, compared with mice treated with isotypical IgG control. ARDS featured increased CXCL-1, TNF-α, IL-6, and IL-17A and reduced IFN-γ, IL-10, IL-2, and IL-13 after neutralizing antibody treatment (anti-IL-35 Ebi3 or anti-IL-35 P35) in BALF and serum.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Blocking Assay, Control

Journal: bioRxiv

Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

doi: 10.64898/2026.02.23.707416

Figure Lengend Snippet: ( A ) The frequency of IL-35-expressing (i.e. IL-12p35 + EBI3 + ) BMDCs, either uninfected (UI) or infected with LDPm for indicated time points, was determined by flow cytometry. In this and other flow cytometry figures, numbers in each quadrant indicate the percentage of cells in the respective quadrant (representative of n = 3 experiments; left). Right: summary of three experiments. ( B ) The frequency of IL-35 expressing BMDCs infected with LDAm for indicated time points was analyzed by flow cytometry as described in (A) and is presented graphically (data pooled from three experiments). ( C ) EBI3 and IL12A mRNA expression in uninfected BMDCs and BMDCs infected with LDPm for 12 or 24 h was assessed by RT-qPCR. Results were normalized to ACTB mRNA (encoding β-actin) expression and are presented as fold change relative to uninfected BMDCs ( n = 9 replicates per group). ( D ) Confocal microscopic analysis of the colocalization (merge; yellow) of IL-12p35 (green) and EBI3 (red) in uninfected and LDPm-infected (48 h) BMDCs; nuclei were stained with Hoechst (blue) (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: IL-12p35/EBI3 colocalization quantified by Pearson’s and Manders’ Coefficients. ( E ) The association between IL-12p35 and EBI3 in uninfected BMDCs or BMDCs infected with LDPm for 48 h was assessed by immunoprecipitation (IP) followed by immunoblotting (IB); β-actin serves as a loading control (representative of n = 3 experiments). WCL, whole-cell lysate (no IP); IgG, immunoglobulin G (IP control). ( F ) Interaction between EBI3 and IL-12p35 in BMDCs infected with LDPm for 48 h, assessed by FRET (representative of n = 3 experiments; left). Scale bar, 10 μm. Right: FRET efficiency. ( G ) IL-35 production by uninfected and LDPm-infected (48 h) BMDCs measured by ELISA (combined data from three experiments, each with n = 3 replicates). ( H ) HuMoDCs were infected with LDPm for indicated times, and the frequency of IL-35-expressing DCs was analyzed by flow cytometry as in (A) (representative plots from n = 3 experiments; left). Right: pooled data from three independent experiments. ( I ) Frequency of IL-35-expressing DCs, T cells, and other cells (i.e., non-DC, non-T cells; CD11c - CD3 - cells) in the spleen of LD-infected mice at indicated days postinfection, analyzed by flow cytometry [representative plots (left) and pooled data (right); n = 18 mice per time point]. The gating strategy is shown in Fig. EV1A. The levels of the IL-35 subunits EBI3 and IL-12p35 in these cell populations is shown in Fig. EV1B. Each symbol represents data from one experiment [A (right panel), B and H (right panel)], replicate (C and G), field [D (right panel)], cell [F (right panel)], or mouse [I (right panel)]. Horizontal bars (B, G, and right panels of A, D, F and H) indicate means and error bars (C, D and F ) represent SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

Techniques: Expressing, Infection, Flow Cytometry, Quantitative RT-PCR, Staining, Immunoprecipitation, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

doi: 10.64898/2026.02.23.707416

Figure Lengend Snippet: ( A ) Top: putative STAT sites in the EBI3 and IL12A promoters. Bottom: ChIP-qPCR analysis of STAT3 recruitment to the indicated regions of EBI3 and IL12A promoters in BMDCs at 0.5 h after LDPm infection ( n = 6 replicates). Results are presented as fold enrichment relative to uninfected BMDCs. ( B ) Left: Details of EBI3 and IL12A promoter-specific oligonucleotides containing wild-type or mutated STAT sites (mutated bases in italics) used for the DNA pull-down assay. Right: DNA pull-down analysis using streptavidin (SA)-conjugated Dynabeads, followed by immunoblotting to assess the binding of STAT3 [present in the nuclear lysates of LDPm-infected (0.5 h) BMDCs] to the biotin (Btn)-labeled oligonucleotides shown in the left panel (representative of n = 3 experiments). ( C ) Immunoblot analysis confirming STAT3 silencing by siRNA; β-actin serves as a loading control (representative of n = 3 experiments). Ctrl siRNA, control siRNA. ( D ) IL-35 expression in uninfected and LDPm-infected BMDCs (48 h infection) transfected with the indicated siRNAs, analyzed by flow cytometry [representative data (left) and compiled data (right) from n = 3 experiments]. ( E ) Effect of TIM-3 blockade using an anti-TIM-3 antibody on IL-35 production by BMDCs infected with LDPm for 48 h, analyzed by flow cytometry [representative (left) and compiled (right) data from n = 3 experiments]. Uninfected BMDCs without any antibody treatment (no Ab) serve as controls. Each symbol represents data from one replicate (A) or one experiment (right panels of D and E). Horizontal bars (right panels of D and E) denote means; error bars (A) indicate SD. *** P < 0.001; ns, not significant.

Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

Techniques: ChIP-qPCR, Infection, Pull Down Assay, Western Blot, Binding Assay, Labeling, Control, Expressing, Transfection, Flow Cytometry

Journal: bioRxiv

Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

doi: 10.64898/2026.02.23.707416

Figure Lengend Snippet: ( A ) Schematic of the adoptive transfer protocol for anti-IL-35 antibody-transfected DCs: BALB/c BMDCs (1 x 10 6 ), either untransfected or transfected with an isotype control (Ctrl) or a neutralizing anti-IL-35 antibody (transfection efficiency shown in Fig. EV5B), were adoptively transferred intravenously into LD-infected BALB/c mice on the indicated days postinfection (shown by arrows). In some experiments, no DC was transferred into LD-infected or uninfected mice. At day 60 postinfection, spleen and liver of these mice were collected for subsequent analyses (see panels B to E). ( B and C ) Spleen and liver weights (B) and parasite burdens (C; expressed as LDU) are shown (combined data from two experiments; n = 3 mice per group in each experiment). ( D and E) Frequencies of IFNγ- or IL-10-producing CD4 + and CD8 + T cells (D) and IL-35-expressing total T cells (IL-12p35 + EBI3 + CD3 + cells; E) in the spleen were analyzed by flow cytometry. Numbers above the outlined regions (D) or within quadrants (E) indicate the percentage of cells in the respective region or quadrant (representative of n = 6; left). Right: combined data from two separate experiments ( n = 3 mice per group in each experiment). Gating strategies are shown in Fig. EV6, A and B. In (B), (C), and the right panels of (D) and (E), each symbol represents the data from one mouse, and horizontal bars indicate mean values. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

Techniques: Adoptive Transfer Assay, Transfection, Control, Infection, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: IL-35 produced by dendritic cells via TIM-3-STAT3 signaling contributes to the development of visceral leishmaniasis

doi: 10.64898/2026.02.23.707416

Figure Lengend Snippet: LD activates STAT3 in DCs via the TIM-3 receptor and its downstream signaling mediator Btk (as shown in our previous report ( Mishra et al ., 2023 )). Activated STAT3 then directly promotes IL-35 production in DCs by binding to the IL12A and EBI3 promoters ( IL12A and EBI3 encode the IL-35 subunits IL-12p35 and EBI3, respectively). DC-derived IL-35, in turn, inhibits the activation and maturation of bystander DCs by suppressing the NF-κB signaling pathway (inner green shaded box), promotes IL-35 expression in T cells, reduces T cell proliferation, and drives pathogenic type-2 T cell responses. Collectively, these events (summarized in the blue-outlined box) impair anti-leishmanial immunity and exacerbate disease pathogenesis. Notably, pharmacological blockade of STAT3 activation by WP1066 reduces IL-35 production by DCs, suppresses disease-promoting type-2 T cell responses, enhances host-protective type-1 T cell responses, and ultimately lowers parasite burden in vivo .

Article Snippet: The following antibodies were used for flow cytometry: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A) (both from R&D Systems); eFluor 660-anti-mouse IL-12p35 (50-7352-82), PerCP-anti-mouse/human IL-12p35 (MA5-23622) and Alexa Fluor 594-anti-mouse IgG (A-11020; all from Thermo Fisher Scientific); PE-anti-human EBI3 (360903), FITC-anti-mouse CD40 (124608), FITC-anti-mouse CD86 (105006), FITC-anti-mouse CD80 (104706), FITC-anti-mouse CD11c (117306), PE-anti-mouse CD8α (100708), FITC-anti-mouse CD8α (100706), PE/Cyanine7-anti-mouse CD3 (100220), PerCP/Cyanine5.5-anti-mouse CD4 (100434), Alexa Fluor 647-anti-mouse IDO1 (654003), APC-anti-mouse IL-10 (505010), FITC-anti-mouse IFNγ (505806) and isotype control antibodies such as FITC-rat IgG2a,κ (400505) and FITC-armenian hamster IgG (400905; all from Biolegend, CA, USA).

Techniques: Binding Assay, Derivative Assay, Activation Assay, Expressing, In Vivo