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: Clinical and Experimental Immunology

Article Title: Interleukin (IL)‐39 [IL‐23p19/Epstein–Barr virus‐induced 3 (Ebi3)] induces differentiation/expansion of neutrophils in lupus‐prone mice

doi: 10.1111/cei.12840

Figure Lengend Snippet: Interleukin (IL)‐39 [IL‐23p19/Epstein–Barr virus‐induced 3 (Ebi3) expanded CD11b+ cells. (a,b) Splenocytes were separated from 8‐week‐old C57BL/6 mice and cultured in vitro for 3 days in the presence of 50 ng/ml Ebi3 and IL‐39. All live cells, including large granule cells, were gated on the basis of forward‐ and side‐scatter and analysed by fluorescence activated cell sorter (FACS). The percentages of CD11c+ and CD11b+ cells (a) and statistical analysis of the percentage (b) are shown; (c–e) 400 ng/mouse p19, Ebi3 and IL‐39 were injected intravenously (i.v.) into 8‐week‐old C57BL/6 mice (six mice per group). On day 7 after injection, live lymphocyte‐sized cells were gated on the basis of forward‐ and side‐scatter and analysed by FACS. The percentages of CD138, IL‐10 or GL7‐expressing B220+ B cells (c,e) and statistical analysis of the percentage (d) are shown; (f–i) 400 ng/mouse p19 and IL‐39 were injected i.v. into 8‐week‐old C57BL/6 mice (six mice per group). On day 7 after injection, live lymphocyte‐sized cells were gated on the basis of forward‐ and side‐scatter and analysed by FACS. The percentages of CD3+ and CD4+ T cells (f) and statistical analysis of the percentage (g), IL‐10, forkhead box protein 3 (FoxP3), IL‐4, interferon (IFN)‐γ, IL‐17A‐expressing CD4+ T cells (i) and statistical analysis of the percentage (h) are shown. (j) Splenocytes were separated from 8‐week‐old C57BL/6 mice and cultured in vitro for 3 days in the presence of 50 ng/ml Ebi3 or IL‐39. Live lymphocyte‐sized cells were gated on the basis of forward‐ and side‐scatter and analysed by FACS. The percentages of IL‐10, IFN‐γ, IL‐17A‐expressing CD4 + T cells are shown. Results represent at least three independent experiments. *P < 0·05 (two‐tailed Student's t‐test). Error bars, standard error of the mean.

Article Snippet: GL7 + B220 + cells were described as above and infected with p19, p28, p35, p40, Ebi3 (Santa Cruz Biotech, Santa Cruz, CA, USA; sc‐60028‐v, sc‐72185‐v, sc‐39639‐v, sc‐39641‐v, sc‐39411‐v, respectively)‐specific shRNA; 5 × 10 6 control, p19, p28, p35, p40, or Ebi3 ‐specific shRNA‐transfected GL7 + B220 + B cells per mouse were injected i.v. into 8‐week‐old female lupus‐prone MRL/lpr or CD19 cre mice.

Techniques: Virus, Cell Culture, In Vitro, Fluorescence, Injection, Expressing, Two Tailed Test

Journal: Clinical and Experimental Immunology

Article Title: Interleukin (IL)‐39 [IL‐23p19/Epstein–Barr virus‐induced 3 (Ebi3)] induces differentiation/expansion of neutrophils in lupus‐prone mice

doi: 10.1111/cei.12840

Figure Lengend Snippet: Interleukin (IL)‐39 expanded and/or induced neutrophils. (a–c) Splenocytes were separated from 8‐week‐old C57BL/6 mice, cultured for 3 days in the presence of medium, 50 ng/ml p19, Epstein–Barr virus‐induced 3 (Ebi3) or IL‐39 and analysed by fluorescence activated cell sorter (FACS). The percentages of Gr‐1+CD11b+ neutrophils (a), statistical analysis of the percentage (b) and cultured cell numbers (c) are shown; (d,e) 400 ng/mouse IL‐39 were injected intravenously (i.v.) into 8‐week‐old C57BL/6 mice (six mice per group). On day 7 after IL‐39 injection, splenocytes were analysed by FACS. The percentages of Gr‐1+CD11b+ neutrophils (d) and cultured cell numbers (e) are shown. (f,g) Lin– ScaI+ cells were sorted from 8‐week‐old C57BL/6 mice by microbeads and stimulated for 5 days with 1 ng/ml granulocyte–macrophage colony‐stimulating factor (GM‐CSF) in the presence of 50 ng/ml IL‐39. Cells were analysed by FACS and the percentages of Gr‐1+CD11b+ neutrophils (f) and cultured cell numbers (g) are shown. Results represent at least three independent experiments. *P < 0·05; **P < 0·01. One‐way analysis of variance (anova) plus Dunnett's multiple comparison test: compare all columns versus control column. Error bars, standard error of the mean.

Article Snippet: GL7 + B220 + cells were described as above and infected with p19, p28, p35, p40, Ebi3 (Santa Cruz Biotech, Santa Cruz, CA, USA; sc‐60028‐v, sc‐72185‐v, sc‐39639‐v, sc‐39641‐v, sc‐39411‐v, respectively)‐specific shRNA; 5 × 10 6 control, p19, p28, p35, p40, or Ebi3 ‐specific shRNA‐transfected GL7 + B220 + B cells per mouse were injected i.v. into 8‐week‐old female lupus‐prone MRL/lpr or CD19 cre mice.

Techniques: Cell Culture, Virus, Fluorescence, Injection, Comparison, Control

Journal: Clinical and Experimental Immunology

Article Title: Interleukin (IL)‐39 [IL‐23p19/Epstein–Barr virus‐induced 3 (Ebi3)] induces differentiation/expansion of neutrophils in lupus‐prone mice

doi: 10.1111/cei.12840

Figure Lengend Snippet: Interleukin (IL)‐39 activated signal transducer and activator of transcription‐3 (STAT‐3) pathways in neutrophils. (a) The expression of IL‐12Rβ1, IL‐12Rβ2, IL‐23R, gp130 or IL‐27R on the surface of neutrophils from the spleen of 8‐week‐old C57BL/6 mice was analysed by fluorescence activated cell sorter (FACS). Isotype antibody was used as the staining control. (b) GMean (mean fluorescence intensity) of IL‐12Rβ1, IL‐12Rβ2, IL‐23R, gp130 or IL‐27R staining from (a). (c,d) CD11b+Gr‐1+ neutrophils were sorted from the spleen of 8‐week‐old female C57BL/6 mice by FACS. The cells were cultured for 30 min in the presence of 50 ng/ml p19, Epstein–Barr virus‐induced 3 (Ebi3) and IL‐39 and analysed by FACS. The percentages (c) and the statistical analysis of the percentages (d) of phospho (p)‐STAT‐3‐expressing neutrophils are shown. (b,d) Data are shown as mean ± standard error of the mean (s.e.m.) (n = 3) from three independent experiments. *P < 0·05; **P < 0·01; ***P < 0·001. (b) Two tailed Student's t‐test; (d) one‐way analysis of variance (anova) plus Dunnett's multiple comparison test: compare all columns versus control column. Error bars, s.e.m.

Article Snippet: GL7 + B220 + cells were described as above and infected with p19, p28, p35, p40, Ebi3 (Santa Cruz Biotech, Santa Cruz, CA, USA; sc‐60028‐v, sc‐72185‐v, sc‐39639‐v, sc‐39641‐v, sc‐39411‐v, respectively)‐specific shRNA; 5 × 10 6 control, p19, p28, p35, p40, or Ebi3 ‐specific shRNA‐transfected GL7 + B220 + B cells per mouse were injected i.v. into 8‐week‐old female lupus‐prone MRL/lpr or CD19 cre mice.

Techniques: Expressing, Fluorescence, Staining, Control, Cell Culture, Virus, Two Tailed Test, Comparison

Journal: Clinical and Experimental Immunology

Article Title: Interleukin (IL)‐39 [IL‐23p19/Epstein–Barr virus‐induced 3 (Ebi3)] induces differentiation/expansion of neutrophils in lupus‐prone mice

doi: 10.1111/cei.12840

Figure Lengend Snippet: Interleukin (IL)‐39‐deficient GL7+ B cells could not induce neutrophils in lupus‐prone mice. (a,b) CD11b+Gr‐1+ neutrophils from spleen and peripheral blood mononuclear cells (PBMC) of 8‐month‐old female BALB/C, non‐lupus‐prone Murphy Roths large (MRL)+ and lupus‐prone MRL/lpr mice (six mice per group) were analysed by fluorescence activated cell sorter (FACS). The percentages of CD11b+Gr‐1+ neutrophils in the spleen and PBMC (a) and statistical analysis of the percentages in the spleen (b) are shown. (c,d) GL7+ B cells from 8‐month‐old female lupus‐prone MRL/lpr mice were sorted by FACS and infected with control shRNA or IL‐12 family subunits p28, p35 or p40, p19 or Epstein–Barr virus‐induced 3 (Ebi3)‐specific shRNA. On day 1 after infection, 5 × 106 control, p28, p35, p40, p19 and Ebi3‐specific shRNA‐infected GL7+B220+ B cells per mouse were injected intravenously (i.v.) into 8‐week‐old female lupus‐prone MRL/lpr mice (six mice per group). Age‐matched MRL/+ mice and control shRNA‐infected GL7+ B cells transferred group were used as non‐lupus‐prone mice and control shRNA, respectively. On day 14 after cell transfer, splenocytes are analysed by FACS. The percentages (c) and the absolute numbers (d) of CD11b+Gr‐1+ neutrophils per spleen are shown. We used two‐tailed Student's t‐test to analyse the difference between each of p28, p35 or p40, p19 or Ebi3‐specific shRNA‐infected GL7+ B cells transfer group and control shRNA‐infected GL7+ B cells transfer group. (b,d) Data are shown as mean ± standard error of the mean (s.e.m.) (n = 6) from one experiment representative of two other similar experiments. **P < 0·01; ***P < 0·001; ****P < 0·0001. One‐way analysis of variance (anova) plus Dunnett's multiple comparison test: compare all columns versus control column. Error bars, s.e.m.

Article Snippet: GL7 + B220 + cells were described as above and infected with p19, p28, p35, p40, Ebi3 (Santa Cruz Biotech, Santa Cruz, CA, USA; sc‐60028‐v, sc‐72185‐v, sc‐39639‐v, sc‐39641‐v, sc‐39411‐v, respectively)‐specific shRNA; 5 × 10 6 control, p19, p28, p35, p40, or Ebi3 ‐specific shRNA‐transfected GL7 + B220 + B cells per mouse were injected i.v. into 8‐week‐old female lupus‐prone MRL/lpr or CD19 cre mice.

Techniques: Fluorescence, Infection, Control, shRNA, Virus, Injection, Two Tailed Test, Comparison

Journal: Clinical and Experimental Immunology

Article Title: Interleukin (IL)‐39 [IL‐23p19/Epstein–Barr virus‐induced 3 (Ebi3)] induces differentiation/expansion of neutrophils in lupus‐prone mice

doi: 10.1111/cei.12840

Figure Lengend Snippet: Interleukin (IL)‐39‐deficient GL7+ B cells could not induce neutrophils in homozygous CD19cre mice. GL7+ B cells were sorted from 8‐month‐old female lupus‐prone Murphy Roths large (MRL)/lpr mice by fluorescence activated cell sorter (FACS) and infected with control shRNA or IL‐39 subunits p19 or Epstein–Barr virus‐induced 3 (Ebi3)‐specific shRNA. On day 1 after infection, 5 × 106 control, p19 and Ebi3‐specific shRNA‐infected GL7+B220+ B cells per mouse were injected intravenously (i.v.) into 8‐week‐old female CD19cre mice (six mice per group). On day 14 after cell transfer, splenocytes were analysed by FACS. The percentages (a) and absolute numbers (b) of CD11b+Gr‐1+ neutrophils per spleen are shown. Data are shown as mean ± standard error of the mean (s.e.m.) (n = 6) from one experiment representative of two other similar experiments. *P < 0·05; **P < 0·01. One‐way analysis of variance (anova) plus Dunnett's multiple comparison test: compare all columns versus control column. Error bars, s.e.m.

Article Snippet: GL7 + B220 + cells were described as above and infected with p19, p28, p35, p40, Ebi3 (Santa Cruz Biotech, Santa Cruz, CA, USA; sc‐60028‐v, sc‐72185‐v, sc‐39639‐v, sc‐39641‐v, sc‐39411‐v, respectively)‐specific shRNA; 5 × 10 6 control, p19, p28, p35, p40, or Ebi3 ‐specific shRNA‐transfected GL7 + B220 + B cells per mouse were injected i.v. into 8‐week‐old female lupus‐prone MRL/lpr or CD19 cre mice.

Techniques: Fluorescence, Infection, Control, shRNA, Virus, Injection, Comparison

Journal: Clinical and Experimental Immunology

Article Title: Interleukin (IL)‐39 [IL‐23p19/Epstein–Barr virus‐induced 3 (Ebi3)] induces differentiation/expansion of neutrophils in lupus‐prone mice

doi: 10.1111/cei.12840

Figure Lengend Snippet: Coordinate expression of B cell activating factor (BAFF) in neutrophils and interleukin (IL)‐39 in B cells. (a–c) BAFF‐expressing CD11b+Gr‐1+ neutrophils from 8‐month‐old female non‐lupus‐prone Murphy Roths large (MRL)+ and lupus‐prone MRL/lpr mice (six mice per group) were analysed by fluorescence activated cell sorter (FACS). The percentages (a), the statistical analysis of the percentages (b) and absolute numbers (c) of BAFF‐expressing CD11b+Gr‐1+ neutrophils per spleen are shown. (d) The cultured supernatant was collected from an in‐vitro IL‐39‐induced neutrophil differentiation cultured system (Fig. 2f,g). BAFF level was determined by sandwich enzyme‐linked immunosorbent assay (ELISA) assay. (e,f) B cells from 8‐week‐old C57BL/6 mice were sorted by B220 microbeads, cultured for 3 days with 50 ng/ml BAFF and analysed by FACS. The percentages of IL‐39 [p19 and Epstein–Barr virus‐induced 3 (Ebi3)]‐expressing B cells (e) and statistical analysis of the percentage (f) are shown. (g) B cells indicated in (Fig. 6e,f) were cultured for 3 days in the presence of a different concentration of Bcl6 inhibitor, and analysed by FACS. The percentages of IL‐39‐expressing B cells are shown. Results represent at least three independent experiments. *P < 0·05; **P < 0·01. (b,c,f) Two‐tailed Student's t‐test; (d) One‐way analysis of variance (anova) plus Dunnett's multiple comparison test: compare all columns versus control column. Error bars, standard error of the mean.

Article Snippet: GL7 + B220 + cells were described as above and infected with p19, p28, p35, p40, Ebi3 (Santa Cruz Biotech, Santa Cruz, CA, USA; sc‐60028‐v, sc‐72185‐v, sc‐39639‐v, sc‐39641‐v, sc‐39411‐v, respectively)‐specific shRNA; 5 × 10 6 control, p19, p28, p35, p40, or Ebi3 ‐specific shRNA‐transfected GL7 + B220 + B cells per mouse were injected i.v. into 8‐week‐old female lupus‐prone MRL/lpr or CD19 cre mice.

Techniques: Expressing, Fluorescence, Cell Culture, In Vitro, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Virus, Concentration Assay, Two Tailed Test, Comparison, Control

Journal: Scientific Reports

Article Title: Blocking protein phosphatase 2A signaling prevents endothelial-to-mesenchymal transition and renal fibrosis: a peptide-based drug therapy

doi: 10.1038/srep19821

Figure Lengend Snippet: ( a ) Representative immunohistochemical detection of PP2Ac in the kidney tissue of sham-operated and UUO mice (left). Co-localization of PP2Ac (green) with CD31(red) was visualized as yellow in the merged images from sham-operated and UUO-14d mice (n = 8). Scale bar: 20 μm. ( b,c ) Western blot analysis of PP2Ac expression in sham-operated and UUO-14d mice (n = 8) and in TGF-β1-treated HUVECs for the indicated periods with densitometry analysis (n ≥ 3). ( d ) HUVECs were incubated with or without TGF-β1 for 72 h and were harvested for immunoprecipitation analysis. Cell lysates were subjected to immunoprecipitation (IP) with an anti-PP2Ac anti-body, followed by immunoblotting (IB) with anti-3-nitrotyrosine (3-NT), anti-acetylation (acetyl), anti-phosphotyrosine (p-Tyr), and anti-methylation (unmethylated) antibodies, respectively. Densitometry analysis of 3-NT, acetyl, p-Tyr and methyl of PP2Ac in d. ( e ) Analysis of PP2A activity in immunoprecipitation of 3-NT-IP, Acetyl-IP, p-Tyr-IP and Unmethylated-IP, expressed as percentage of the value in control group, respectively (n ≥ 3). *P < 0.05 versus the basal condition. NS: no significance. Bars represent means ± sd.

Article Snippet: After transfer, the membranes were blocked and blotted routinely with antibodies against VE-cadherin (BD Biosciences), α-SMA (Abcam), collagen1 (Abcam), PP2Ac (Cell Signaling Technology), occludin (Invitrogen), P-threonine/P-serine (Santa Cruz), nitrotyrosine (Cayman), acetylated-lysine (Cell Signaling Technology), phosphotyrosine (PY-20,Biolgend), PP2A,C subunit, demethylated (Millipore), vimentin (Boster) and CD31 (Proteintech).

Techniques: Immunohistochemical staining, Western Blot, Expressing, Incubation, Immunoprecipitation, Methylation, Activity Assay, Control

Journal: Transplantation

Article Title: IL-35 Stabilizes Treg Phenotype to Protect Cardiac Allografts in Mice

doi: 10.1097/tp.0000000000004707

Figure Lengend Snippet: FIGURE 5. IL-35 directly promotes the activation and function of Treg and the secretion of IL-35 in a positive feedback mechanism in vitro. A, CD4+ Foxp3+ T cells were sorted and stimulated with anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) for 72 h, extracted RNA, and then qPCR analysis showed that only P35 and Ebi3 expressed in Treg. Lipopolysaccharide-activated DC as positive control; ND, not determined. B, In CD4+Foxp3+ T cells and CD4+Foxp3+Ebi3- T cells stimulated with or without anti-CD3 (5 μg/mL), anti-CD28 (2 μg/ mL), and PBS or IL-35 (200 ng/mL) for 72 h, the percentage of Foxp3+ and Zombie+ in Treg is shown by flow cytometry. The findings demonstrated that the IL-35 group had the highest expression of Foxp3+ cells and the lowest expression of Zombie+ cells in the 4 groups (n = 9/group, ***P < 0.001). C, In CD4+ Foxp3+ T cells stimulated with or without anti-CD3 (5 μg/mL), anti-CD28 (2 μg/mL), and PBS or IL-35 (200 ng/mL) or anti-IL-35 (200 ng/mL) for 72 h, the result found that Ki-67+, and PD-1+, CD44+ CD62L–, and P35+ EBI3+ expression in Treg were increased in the IL-35 group by flow cytometry (n = 3/group, *P < 0.05). D, In CD4+ Foxp3+ T cells stimulated with or without anti-CD3 (5 μg/mL), anti-CD28 (2 μg/mL), PBS or IL-35 (200 ng/mL) or anti-IL-35 (200 ng/mL) for 72 h, the supernatant was retained and tested by a cytometric bead array and enzyme-linked immunosorbent assay, and the data showed that although IL-35 expression increased dramatically (n = 12/group, *P < 0.05), IL-17A expression in the culture supernatant fell significantly (n = 4/group, **P < 0.01). DC, dendritic cell; IL, interleukin; PD-1, programmed cell death 1; qPCR, quantitative real-time polymerase chain reaction; Treg, regulatory T cell.

Article Snippet: IL12a–/– and Ebi3–/– mice on B6 background were purchased from Cyagen.

Techniques: Activation Assay, In Vitro, Positive Control, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Transplantation

Article Title: IL-35 Stabilizes Treg Phenotype to Protect Cardiac Allografts in Mice

doi: 10.1097/tp.0000000000004707

Figure Lengend Snippet: FIGURE 6. Through gp130/STAT1 signaling, IL-35 promotes Treg functions. A, Treg cultured with α-CD3 (5 μg/mL), α-CD28 (2 μg/mL), and IL-2 (100 U/mL) for 18 h, and IL-35 (200 ng/mL) or IFN-γ (100 ng/mL) for several time points before the test. In Treg, representative flow cytometry histograms expression of pSTAT1 was more activated by IL-35 than PBS at various time points, but neither pSTAT3 nor pSTAT4 was stimulated. B, When STAT1 inhibitor was introduced to the culture media, STAT1 phosphorylation was significantly reduced (n = 3/group, **P < 0.01). C, The results showed that adding a gp130 inhibitor significantly reduced the pGP130 (n = 3/group, *P < 0.05) and pSTAT1 (n = 3/group, **P < 0.01). D, In WT CD4+ Foxp3+ T cells stimulated with or without anti-CD3 (5 μg/mL), anti-CD28 (2 μg/mL), and PBS/IL-35 (200 ng/mL)/STAT1 inhibitor (1 µg/mL), the percentage of Foxp3+, P35+Ebi3+, and Ki-67+ in Treg were decreased in the inhibitor group by flow cytometry (n = 3/group, P > 0.05). E, In CD8+ T cells stimulated with or without anti-CD3 (1 μg/mL), anti-CD28 (2 μg/mL), and STAT1 inhibitor (1 µg/mL), the percentage of GZMB+, Ki-67+ CD8+ T, and CD44+CD62L– in CD8+ T cells was unchanged by flow cytometry (n = 4/group, P > 0.05). F, Treg were coculture with CD8+ T cells (ratio 1:8) with or without anti-CD3 (1 μg/mL), anti-CD28 (2 μg/mL), IL-35 (100 ng/mL), and STAT1 inhibitor (1 µg/mL) for 72 h, and the percentage of GZMB+ and CD44+ CD62L– in CD8+ T cells were increased in inhibitor group by flow cytometry (n = 3/group, *P < 0.05). IFN-γ, interferon gamma; IL, interleukin; pSTAT, phospho signal transducer and activator of transcription; Treg, regulatory T cell; WT, wild type.

Article Snippet: IL12a–/– and Ebi3–/– mice on B6 background were purchased from Cyagen.

Techniques: Cell Culture, Flow Cytometry, Expressing, Phospho-proteomics

Journal: Biomarker Research

Article Title: Single-cell and spatial transcriptomics reveal alterations in trophoblasts at invasion sites and disturbed myometrial immune microenvironment in placenta accreta spectrum disorders

doi: 10.1186/s40364-024-00598-6

Figure Lengend Snippet: Enhanced IL-35 and HLA-G signal in invasive parts of PAS. A Enrichment score of IL-35 response pathways of each spot in the ST data. Gradient from blue (low score) to red (high score). B Expression of HLA-G and EBI3 in trophoblast cells in the PAS and normal placenta groups (****, p < 0.0001). C t-SNE color-coded for the expression of EBI3 signal associated genes in scRNA-seq. D t-SNE color-coded for the expression of HLA-G signal associated genes in scRNA-seq. E Spatial distribution of HLA-G and EBI3 and their coupled receptors in the ST data of PAS-2. The magnified view of the dashed box area is displayed on the right side of the figure. Arrows indicate the factors and adjacent receptors. F Immunofluorescence staining images indicate the expression of HLA-G and EBI3 in the PAS and normal placenta samples. The fourth column shows an enlarged view of the area within the dashed box

Article Snippet: We used the following primary antibodies: α-SMA/ACTA2 (1:300; ab7817, abcam), CK-7/KRT7 (1:200; ab154334, abcam), PD-L1/CD274 (1:2500; PA5-20343, INVTROGEN), INO1(1:2000; ab211017, abcam), TIM-3/HAVCR2 (1:1000; ab241332, abcam), EBI3 (1:5000; NBP-76976, Novus Biologicals), HLA-G (1:7500; ab283278, abcam), and WNT4 (1:500; GTX101085, GeneTex).

Techniques: Expressing, Immunofluorescence, Staining