il 4  (R&D Systems)

Journal: Exploration

Article Title: Specifically Breaking Through the Injured Blood‐Brain Barrier With Tannic Acid‐Based Nanomedicine for Ischemic Stroke Ischemia Reperfusion Treatment

doi: 10.1002/EXP.20240388

Figure Lengend Snippet: TPM reverses neuroinflammation induced by ISIR. (A) Mechanism of TPM inhibiting the activation of the microglial cGAS‐STING pathway. (B) Schematic illustration of the HT22 cells and BV2 cells coincubation experimental process. (C) Immunofluorescence staining of dsDNA of BV2 cells in different treatment groups. (D) Immunohistochemical staining of STING in the cerebral infarction area of each treatment group. Scale bar: 20 µm. (E) Quantitative analysis of STING immunohistochemical staining. (F) WB of inflammation‐related proteins in cerebral infarct area, including cGAS, STING, IRF3, P‐IRF3, P65, and P‐P65. (G–K) Determination of the levels of pro‐inflammatory factors (IL‐1β, IL‐6, and TNF‐α) and anti‐inflammatory factors (IL‐4 and IL‐10) in the cerebral infarction area of each treatment group. (L–O) Quantitative analysis of inflammation‐related proteins in BV2 cells, including cGAS (l), STING (m), P‐IRF3/ IRF3 (N), P‐P65/ P65 (O). Data were expressed as mean ± SE. ( n = 3, ns, p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Rat IL‐4 (Interleukin 4) ELISA kit , Elabscience , E‐EL‐R0014.

Techniques: Activation Assay, Immunofluorescence, Staining, Immunohistochemical staining

Journal: Blood

Article Title: Deficiency of T follicular helper cell Tet3 DNA demethylation inhibits pathogenic IgG2c class switching and chronic GVHD

doi: 10.1182/blood.2024025036

Figure Lengend Snippet: Tet3 cKO T cells do not reduce the GC response or total IgG deposition in cGVHD. (A) Flow cytometry analysis of mouse splenocytes taken at 49 days after transplant. TFHs are the CXCR5 + , PD1 + percentage of FOXP3 − , CD4 + live splenocytes. TFRs are the CXCR5 + , PD1 + percentage of FOXP3 + , CD4 + live splenocytes. The TFR/TFH ratio, as a more biologically representative measure, is also shown. GCBs are the GL7 + , Fas hi percentage of the CD19 + live splenocytes. The results shown are pooled from 3 experimental replicates with BM only (n = 20), cGVHD (n = 21), Tet3 fl/fl ;CD4-Cre – (n = 11), and Tet3 fl/fl ;CD4-Cre + (n = 22). The results show reduced TFH frequency, an improved TFR/TFH ratio, and no change in the GCB frequency with Tet3 fl/fl ;CD4-Cre + T-cell donors. Gating is shown in supplemental Figure 7B-C. (B) Representative images of cryopreserved spleen sections stained to show GCs from mice at 49 days after transplant. Sections are stained with B220 (blue), PNA rhodamine (red), and CD4 FITC (green). (C) Quantification of the average size and frequency of GCs observed in the spleen sections. In Tet3 fl/fl ;CD4-Cre + recipients, the GC area is reduced, whereas frequency is unchanged. A representative experiment is shown. (D) Representative images of cryopreserved lung sections from mice 49 days after transplant stained with anti-IgG FITC (green) and DAPI (blue). (E) Quantification of the total IgG deposited in lung tissue sections. The results show no reduction in the total IgG deposition in Tet3 fl/fl ;CD4-Cre + T-cell transplanted mice. A representative experiment is shown. Images are at 200× magnification. For all panels, the statistics shown are the results of the 1-way analysis of variance with Holm-Sidak correction for multiple comparisons. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: Purified rat IgG1 IL-4 neutralizing antibody (clone 11B11 [BSF-1]; American Type Culture Collection) 53 was given intraperitoneally from day 28 to day 49 thrice weekly at 1.5 mg per dose for 3 doses and then at 0.5 mg per dose.

Techniques: Flow Cytometry, Staining

Journal: Blood

Article Title: Deficiency of T follicular helper cell Tet3 DNA demethylation inhibits pathogenic IgG2c class switching and chronic GVHD

doi: 10.1182/blood.2024025036

Figure Lengend Snippet: Tet3 cKO T-cell transplanted mice have IgG1 type antibody deposited in lungs and show reduced expression of GATA3 in TFHs. (A) Representative images of lung sections stained with DAPI (blue), Cy3 nonisotype specific anti-Ig (red), and a type-specific FITC conjugated anti-immunoglobulin (green). From left to right these are anti-IgM, anti-IgG1, anti-IgG2c, and anti-IgG3. Images are at 200× magnification. The results from a representative experiment are shown. (B) Quantification by group as percentage of the total positive immunoglobulin area that is double positive for the type specific immunoglobulin. The results show that Tet3 fl/fl ;CD4-Cre + T-cell transplanted mice have IgG1 type antibody deposition, whereas all other conditions show IgG2c type antibody deposition. For all panels, the statistics shown are the results of 1-way analysis of variance with Holm-Sidak correction for multiple comparisons. ∗∗ P < .01; ∗∗∗∗ P < .0001.

Article Snippet: Purified rat IgG1 IL-4 neutralizing antibody (clone 11B11 [BSF-1]; American Type Culture Collection) 53 was given intraperitoneally from day 28 to day 49 thrice weekly at 1.5 mg per dose for 3 doses and then at 0.5 mg per dose.

Techniques: Expressing, Staining

Journal: Blood

Article Title: Deficiency of T follicular helper cell Tet3 DNA demethylation inhibits pathogenic IgG2c class switching and chronic GVHD

doi: 10.1182/blood.2024025036

Figure Lengend Snippet: Treatment with IL-4 neutralizing antibody reversed both pulmonary improvements and IgG1 class switching in Tet3 cKO T-cell transplanted mice. (A-C) Results of cGVHD/BO transplant of BM only (n = 6), cGVHD (n = 8), Tet3 fl/fl ;CD4-Cre – (n = 7), or Tet3 fl/fl ;CD4-Cre + (n = 8) T cells with and without the additional treatment of IL-4 neutralizing antibody (aIL-4) [ Cre – +aIL-4 n = 6; Cre + +aIL-4 n = 5]. In treated groups, IL-4 neutralizing antibody was delivered interperitoneally thrice weekly from day 28 to day 49 after transplant. (A) Pulmonary findings on day 49 after transplant. Pulmonary function was significantly improved in those with Tet3 fl/fl ;CD4-Cre + T-cell transplant, and this improvement was lost with IL-4 neutralizing antibody treatment. Elastance (middle panel), for which the results were not significant, had a P value of .08 for the comparison between Tet3 fl/fl ;CD4-Cre + T-cell recipients and their aIL-4 treated counterparts. (B) Flow cytometric analysis of splenic GCBs conducted 49 days after transplant. GCBs are gated as CD19 + , GL7 + , Fas hi cells. Panels show the frequency of each immunoglobulin type positive GCB as a percentage of the total GCBs, with the right-most panel showing the ratio of the two. (C) Flow cytometric analysis of lung infiltrating plasma cells. Plasma cells were gated as CD138 + , CD19 − , B220 − lymphocytes. Analysis of the immunoglobulin typing was performed as in panel B. For all panels, statistics shown are the results of 1-way analysis of variance with Holm-Sidak correction for multiple comparisons. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: Purified rat IgG1 IL-4 neutralizing antibody (clone 11B11 [BSF-1]; American Type Culture Collection) 53 was given intraperitoneally from day 28 to day 49 thrice weekly at 1.5 mg per dose for 3 doses and then at 0.5 mg per dose.

Techniques: Comparison, Clinical Proteomics

Journal: Blood

Article Title: Deficiency of T follicular helper cell Tet3 DNA demethylation inhibits pathogenic IgG2c class switching and chronic GVHD

doi: 10.1182/blood.2024025036

Figure Lengend Snippet: Increased IL-4 and GATA3 expression in TFHs from recipients of Tet3 fl/fl ;CD4-Cre + T cells. (A) The results of an in vitro coculture assay. Sorted B cells were cocultured with TFHs for 6 days. After culture, cells were stained for TFH markers as defined in Figure 2 , and the percentage of GATA3 + and IL-4 + TFHs was determined. There was a significant increase in the expression of GATA3 and IL-4 in the TFHs that lacked Tet3 . (B-C) Flow cytometric analysis of the splenocytes taken from mice transplanted and treated as described in Figure 5 . Representative sample histograms are shown in panel B, whereas quantifications are shown in panel C. With or without aIL-4 treatment, Tet3 fl/fl ;CD4-Cre + T-cell recipients showed increased expression of GATA3 and IL-4 in their splenic TFHs. For panels A-C, the statistics shown are from 1-way analyses of variance with Holm-Sidak correction for multiple comparisons. (D) Tet3 knockdown in human TFH skews B-cell antibody production from inflammatory to immunosuppressive isotypes. Naïve human CD4 + T cells were purified and expanded under TFH-skewing conditions. On day 5, T-cell cultures were electroporated in the presence of control or Tet3 siRNA. Expanded CXCR5 + cells were purified and combined in a 1:1 ratio with autologous memory B cells in the presence of SEB for 5 additional days of culture. The results of relative mean fluorescence intensity (MFI) differences in markers are shown. The left 2 panels show the relative expression of IL-4 and GATA in cultured TFH s with either the control or TET3 siRNA. For these samples, cells were intercellularly stained for their respective markers. The right 2 panels show the relative expression of inflammatory IgG1 and inhibitory IgG4 on GCBs from coculture. Expression of IgG1 was reduced, whereas expression of IgG4 was increased with the Tet3 -specific siRNA vs control siRNA. For panel D, the statistics shown are the results of unpaired t tests with ROUTS outlier test. ∗ P < .05; ∗∗ P < .01; ∗∗∗∗ P < .0001.

Article Snippet: Purified rat IgG1 IL-4 neutralizing antibody (clone 11B11 [BSF-1]; American Type Culture Collection) 53 was given intraperitoneally from day 28 to day 49 thrice weekly at 1.5 mg per dose for 3 doses and then at 0.5 mg per dose.

Techniques: Expressing, In Vitro, Co-culture Assay, Staining, Knockdown, Purification, Control, Fluorescence, Cell Culture