ebi3  (Jackson Laboratory)

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

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 confocal microscopy: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A; both from R&D Systems).

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 confocal microscopy: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A; both from R&D Systems).

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 confocal microscopy: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A; both from R&D Systems).

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 confocal microscopy: PE-anti-mouse EBI3 (IC18341P) and APC-anti-human/mouse IL-12p35 (IC2191A; both from R&D Systems).

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

Journal: Advanced Science

Article Title: Tumor‐Derived Interleukin 35 Promotes Fibrosis in the Tumor Microenvironment of Pancreatic Cancer by Activating Pancreatic Stellate Cells

doi: 10.1002/advs.202509074

Figure Lengend Snippet: Tumor‐derived IL‐35 promotes fibrosis in PDAC. A) Representative images of IHC staining for IL‐35 and Masson's trichrome staining for collagen fibers (collagen stained blue). B) Distribution of the percentage area occupied by blue‐stained collagen in 109 PDAC slices with different levels of IL‐35 expression. Data were analyzed using unpaired t‐test. C) Kaplan‐Meier analysis of the overall survival of 109 PDAC patients according to different Masson staining scoring. Collagen fiber area exceeding 50% was categorized as high (n = 67), Collagen fiber area of 50% or less was categorized as low (n = 42).D) Left, representative images of Masson trichrome staining in KPC, IL12A −/− KPC, and EBI3 −/− KPC mice (tumor diameter was ≈8 mm). Right, the percentage of collagen fiber area to the total pancreas in indicated mice was shown. Data were analyzed using unpaired t‐test. E) Kaplan‐Meier analysis comparing the survival of indicate group, the observation endpoint is set at 160 days postnatally (n=8). F) 1x10 5 indicated KPC cells were orthotopically injected into the pancreas of 5‐week‐old SCID mice (n = 6) to induce tumor formation. After 30 days, harvest the tumors. Representative images of Masson trichrome staining and the percentage of collagen fiber area to the total pancreas in the indicated group are shown. Data were analyzed using unpaired t‐test. G) Kaplan‐Meier analysis comparing the survival of indicate group (n = 8). Shown are mean ± SD; * p <.05. ** p <.01. *** p <.001. Scale bars,100 µm.

Article Snippet: IL‐12A flox/flox and EBI3 flox/flox mice were purchased from the Shanghai Model Organisms Center, PR China.

Techniques: Derivative Assay, Immunohistochemistry, Staining, Expressing, Injection

Journal: Advanced Science

Article Title: Tumor‐Derived Interleukin 35 Promotes Fibrosis in the Tumor Microenvironment of Pancreatic Cancer by Activating Pancreatic Stellate Cells

doi: 10.1002/advs.202509074

Figure Lengend Snippet: Molecular mechanism of IL‐35 transcriptional regulation of IGFBP2 and Tsp‐1. A) Western blotting was performed on EBI3, IL‐12A, IGFBP2, and Tsp‐1 in the indicated cell lines. B) Genome‐wide mRNA sequencing was conducted to compare the expression profiles between PANC‐1 cells with or without IL‐35 overexpression. The genes significantly altered by IL‐35 upregulation (|log2FC > 2|) are presented in a volcano plot. C) Representative images of IHC staining targeted on IL‐12A, EBI3, IGFBP2, and Tsp‐1 in PDAC tissues of KPC, IL‐12A −/− KPC and EBI3 −/− KPC mice. D) ELISA was used to measure the levels of IGFBP2 and Tsp‐1 in the peripheral blood of 4‐month‐old KPC, IL‐12A −/− KPC, and EBI3 −/− KPC mice. E) Representative images of IHC staining targeted on IL‐12A, EBI3, IGFBP2, and Tsp‐1 in human PDAC tissues, and statistical analysis of IHC results of IGFBP2/Tsp‐1 and IL‐35 expression in 109 human PDAC surgical samples in the table. Data were analyzed using Spearman correlation analysis. F) PANC‐1 cells were pretreated with or without anti‐GP130 antibody (100 ng mL −1 ) and/or anti‐IL12RB2 antibody (2 µg mL −1 ) for 30 min, and then, rhIL‐35 (100 ng mL −1 ) was added to culture for another 16 h. The cells were subjected to western blotting assays. G) Human IGFBP2 and THBS1 promoter, and two of the identified STAT binding motifs were investigated. CHIP analysis of PANC‐1 cells pretreated with or without rhIL‐35 (100 ng mL −1 ) for 90 min. Chromatin was immunoprecipitated with anti‐STAT1 or anti‐STAT4 antibodies and then subjected to RT‐PCR analysis. Then, CHIP‐reCHIP analysis of PANC‐1 cells prepared as in above, chromatin was first immunoprecipitated with anti‐STAT1 or anti‐STAT4 antibody and then immunoprecipitated again with anti‐STAT4 (S1→S4) or anti‐STAT1 (S4→S1). H) Left, Schematic of the binding motifs mutation in IGFBP2 and THBS1 promoter. Right, Dual luciferase activity of reporter plasmids with the wild‐type or mutation of IGFBP2/THBS1 fused to the luciferase gene following IL‐35 co‐transfection in HEK293T cells. Data were analyzed using unpaired t‐test. Experiments above were repeated three times independently. Shown are mean ± SD; * p <.05. ** p <.01. *** p <.001. **** p <.0001. ns no significance. Scale bars,100 µm.

Article Snippet: IL‐12A flox/flox and EBI3 flox/flox mice were purchased from the Shanghai Model Organisms Center, PR China.

Techniques: Western Blot, Genome Wide, Sequencing, Expressing, Over Expression, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Binding Assay, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Luciferase, Activity Assay, Cotransfection

Journal: Science Advances

Article Title: Ligand-receptor interactions induce and mediate regulatory functions of BATF3 + B cells

doi: 10.1126/sciadv.adx9917

Figure Lengend Snippet: ( A ) Expression distribution of IgV(D)J gene transcripts in purified B cells after stimulation. ( B ) FACS analysis of Ca 2+ flux in pregenerated B.LPS and B.LC cells in response to BCR-cross-linking anti-IgM. ( C and D ) FACS of (C, left) CSR to IgG1 and (D) plasma cell differentiation in the presence of IL-4, and (C, right) ELISA of different Ig isotypes secreted by stimulated B cells. ( E ) CSR to IgG2a in CD45.2 + B.CD154, B.C21, or B.LPS cells as target cells (Cell R ) in the presence of CD45.1 + B.LC21 as Cell L and/or IFN-γ, as indicated, for 96 hours. When B.Nil cells were used as Cell R , no B cell activation or CSR occurred. ( F ) Pregenerated CD45.2 + B.LA21 cells of different genotypes, as indicated, were used as Cell L to coculture with preactivated CD45.1 + B.C21 and B.LPS cells as Cell R for 72 hours before FACS analysis of CSR to IgG2a in Cell R . Representative of three independent experiments. ( G ) Volcano plot of DEGs induced by IL-27 in B.CD154 cells. ( H to J ) RNAscope analysis of VV WR -specific transcripts (pink signals) in the lung from (H) mb1 cre Il27p28 fl/fl , (I) μMT: Ebi3 −/− , and (J) μMT: Il27ra −/− mice and their respective “wild-type” control mice 10 days after VV WR infection and quantification of the transcripts (four areas in three different mice in each group), ELISA of virus-specific IgG2a, IgG1, or IgG in the circulation, FACS analysis of IgG2a + and IgG1 + B cells and plasma cells, and/or VV WR titers in different organs. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, one-way analysis of variance (ANOVA) multiple comparisons Tukey test in (E) and unpaired two-tailed t test in (C), (D), and (H) to (J). TPM, tags per million. PFU, plaque forming unit.

Article Snippet: C57BL/6J (C57, CD45.2 + , stock #000664) as well as Batf3 −/− (#013755), CD45.1 + ( Ptprc a , #002014), Cxcl10 −/− (#006087), Ebi3 −/− (#008691), Cxcr3 −/− (#005796), Ifngr1 −/− (#003288), Il10 IRES-Gfp (#008379), Il27ra −/− (#018078), and μMT ( Igh μ MT/ μ MT , #002288) mice, all on the C57 background, were originally from the Jackson Laboratory (JAX).

Techniques: Expressing, Purification, Clinical Proteomics, Cell Differentiation, Enzyme-linked Immunosorbent Assay, Activation Assay, RNAscope, Control, Infection, Virus, Two Tailed Test

Journal: Science Advances

Article Title: Ligand-receptor interactions induce and mediate regulatory functions of BATF3 + B cells

doi: 10.1126/sciadv.adx9917

Figure Lengend Snippet: ( A ) Flow imaging analysis of B220, IL-27p28, and GFP (indicating Il10 transcription) expression in B cells (left) and FACS analysis of proportions of IL-27p28 + and GFP + B cells (right) in Il10 IRES-Gfp mice immunized with NP-CGG/alum/LPS for 14 days. Also depicted were “secretory vesicle-like structures” on the plasma membrane in bright field. ( B ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of transcript levels of cytokine genes (top; data were normalized to Gapdh expression) and ELISA of IL-27 (bottom) in the spleen of C57 and μMT mice 7 days after NP-CGG/alum/LPS immunization. ( C ) ELISA of IL-27 in the spleen from mb1 cre Il27p28 fl/fl and “wild-type” mb1 cre Il27p28 +/fl littermates 14 days after NP-CGG/alum/LPS immunization (first panel), qRT-PCR analysis of cytokine gene transcript levels of (second panel), and CSR-related gene expression (third panel), as indicated, as well as FACS analysis of GL-7 + , IgG2a + , and plasma cells (fourth to sixth panels) and ELISA of NP 32 -binding IgG2a (last panel). ( D ) ELISA of IL-27 in the spleen in μMT: Ebi3 −/− mice and their μMT: Ebi3 +/+ counterparts immunized with NP-CGG/alum/LPS for 14 days (first panel) and in the circulation at different time points (second panel), FACS analysis of IgG2a + and GL-7 + B cells as well as plasma cells in the spleen (third to fifth panels), and ELISA of total (NP 32 -binding) specific IgG2a and high-affinity (NP 4 -binding) IgG1 and IgG2b (sixth to eighth panels) in these mice 14 days after immunization. ( E ) ELISA of NP-specific IgG and FACS analysis of B and T cell differentiation in the spleen in single and double KO mixed bone chimera mice, as indicated, 14 days after NP-CGG/LPS/alum immunization. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, unpaired two-tailed t test in (B) to (D) and one-way ANOVA multiple comparisons Tukey test in (E).

Article Snippet: C57BL/6J (C57, CD45.2 + , stock #000664) as well as Batf3 −/− (#013755), CD45.1 + ( Ptprc a , #002014), Cxcl10 −/− (#006087), Ebi3 −/− (#008691), Cxcr3 −/− (#005796), Ifngr1 −/− (#003288), Il10 IRES-Gfp (#008379), Il27ra −/− (#018078), and μMT ( Igh μ MT/ μ MT , #002288) mice, all on the C57 background, were originally from the Jackson Laboratory (JAX).

Techniques: Imaging, Expressing, Clinical Proteomics, Membrane, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Gene Expression, Binding Assay, Cell Differentiation, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Pro-Resolving Macrophage-Induced IL-35 + but Not TGF-β1 + Regulatory B Cell Activation Requires the PD-L1/PD-1 Pathway

doi: 10.3390/ijms26115332

Figure Lengend Snippet: M2 macrophages promoted the expression of IL-35 and TGF-β1 in B cells. B cells were co-cultured with macrophages for 48 h, followed by flow cytometry and immunofluorescence staining detection. ( A ) A schematic of B cells co-cultured with macrophages. ( B ) IL-35 (Ebi3 + IL-12a + ) expression in B cells detected by flow cytometry. ( C ) Statistical data of IL-35 expression in B cells ( n = 12). ( D ) IL-35 expression in B cells detected by immunofluorescence staining (The white arrow points to IL-35 + Breg; The enlarged image on the right shows the portion of the red box). ( E ) Statistical data of IL-35 expression in B cells ( n = 7). ( F ) TGF-β1 expression in B cells detected by flow cytometry. ( G ) Statistical data of TGF-β1 expression in B cells ( n = 12). ( H ) IL-10 expression in B cells detected by flow cytometry. ( I ) Statistical data of IL-10 expression in B cells. Statistical significance was analyzed by one-way ANOVA followed by Tukey’s test and Dunnett’s t -test. NS: no significant difference. ** p < 0.01; **** p < 0.0001.

Article Snippet: Cells were stained with rat anti-mouse EBi3 (1:150, cat: 210-501-B66, Rockland, ThermoFisher Scientific, Waltham, MA, USA) and rabbit anti-mouse IL-12a (1:100, cat: BS-0767R, Bioss, ThermoFisher Scientific, Waltham, MA, USA) overnight at 4 °C, and the Alexa flour 488-labeled goat anti-rabbit IgG (1:500) and Alexa flour 594-labeled goat anti-rat IgG (1:500) secondary antibodies were used to recognize the primary antibody.

Techniques: Expressing, Cell Culture, Flow Cytometry, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Pro-Resolving Macrophage-Induced IL-35 + but Not TGF-β1 + Regulatory B Cell Activation Requires the PD-L1/PD-1 Pathway

doi: 10.3390/ijms26115332

Figure Lengend Snippet: M2 macrophage-induced IL-35 and TGF-β1 expression in B cells requires direct cell–cell contact. B cells were co-cultured with M2 macrophages with or without a trans-well insert, followed by flow cytometry and immunofluorescence staining. ( A ) A schematic of B cells co-cultured with macrophages with a trans-well insert. ( B ) A typical picture of IL-35 expression in B cells detected by immunofluorescence staining (The white arrow points to IL-35 + Breg; The enlarged image on the right shows the portion of the red box). ( C ) Statistical data of IL-35 expression in B cells detected by immunofluorescence staining ( n = 7). ( D ) IL-35 (Ebi3 + IL-12a + ) expression in B cells detected by flow cytometry. ( E ) Statistical data of IL-35 expression in B cells detected by flow cytometry ( n = 6). ( F ) TGF-β1 expression in B cells detected by flow cytometry. ( G ) Statistical data of TGF-β1 expression in B cells ( n = 6). Statistical significance was analyzed using a one-way ANOVA followed by Tukey’s test and Dunnett’s t -test. NS: no significant difference. ** p < 0.01; **** p < 0.0001.

Article Snippet: Cells were stained with rat anti-mouse EBi3 (1:150, cat: 210-501-B66, Rockland, ThermoFisher Scientific, Waltham, MA, USA) and rabbit anti-mouse IL-12a (1:100, cat: BS-0767R, Bioss, ThermoFisher Scientific, Waltham, MA, USA) overnight at 4 °C, and the Alexa flour 488-labeled goat anti-rabbit IgG (1:500) and Alexa flour 594-labeled goat anti-rat IgG (1:500) secondary antibodies were used to recognize the primary antibody.

Techniques: Expressing, Cell Culture, Flow Cytometry, Immunofluorescence, Staining