Journal: Science Advances

Article Title: Cathepsin W restrains peripheral regulatory T cells for mucosal immune quiescence

doi: 10.1126/sciadv.adf3924

Figure Lengend Snippet: ( A and B ) OTII Rag2 −/− and OTII Rag2 −/– Ctsw −/− CD45.2 + CD4 + T cells were transferred into congenic CD45.1 WT recipient mice, followed by administration of ovalbumin (OVA) in the drinking water for 5 days. (A) Representative flow cytometry analysis and (B) quantification of CD45.2 + Foxp3 + pT reg cells from the spleen (Spl) and lamina properia (LP) in the recipient mice. ( C to G ) CD4 + CD45RB hi CD25 − T cells were intraperitoneally transferred into Rag2 −/− mice for colitis induction. Mice were euthanized and analyzed 10 weeks after colitis induction. (C) Body weight of Rag2 −/− mice received WT or Ctsw −/− T cells during colitis. (D) Hematoxylin and eosin staining of colon samples from the different groups as in (C). Scale bars, 50 μm. (E) Colon lengths (left) and histological score (right) from the different groups as in (C). (F to G) (F) Representative flow cytometry and (G) quantification of IL-17 and interferon-γ (IFN-γ) production, Foxp3 expression by CD4 + T cells isolated from LP from indicated groups as in (C). Data are representative of three independent experiments (A to G). ** P < 0.01 [(B, E, and G) Student’s t test; (C) two-way ANOVA with Tukey’s multiple comparisons test; error bars represent SD].

Article Snippet: The CD25 and its mutation were amplified from mouse CD25 cDNA clone (Sino Biological).

Techniques: Flow Cytometry, Staining, Expressing, Isolation

Journal: Science Advances

Article Title: Cathepsin W restrains peripheral regulatory T cells for mucosal immune quiescence

doi: 10.1126/sciadv.adf3924

Figure Lengend Snippet: ( A and B ) Proliferation of CellTrace Far Red–labeled WT naïve CD4 + T cells in the presence of anti-CD3, irradiated CD4 + T cell–depleted splenocytes, and green fluorescent protein (GFP) + (Foxp3 + ) pT reg cells from Foxp3 GFP and Foxp3 GFP Ctsw −/− mice. Data shown are determined by (A) flow cytometry and (B) quantification. T eff , effector T cells. ( C ) Body weight of Rag2 −/− mice transferred with WT CD4 + CD45RB hi CD25 − T cells with or without WT or Ctsw −/− Foxp3 + pT reg cells. ( D ) Hematoxylin and eosin staining of colon samples from the different groups as in (C). Scale bars, 50 μm. ( E ) Colon lengths (left) and histological score (right) from the different groups as in (C). Data are representative of three independent experiments (A to D) or are pooled from two independent experiments (E). * P < 0.05 [(B and C) two-way ANOVA with Tukey’s multiple comparisons test; (E) one-way ANOVA with Tukey’s multiple comparisons test; error bars represent SD].

Article Snippet: The CD25 and its mutation were amplified from mouse CD25 cDNA clone (Sino Biological).

Techniques: Labeling, Irradiation, Flow Cytometry, Staining

Journal: Science Advances

Article Title: Cathepsin W restrains peripheral regulatory T cells for mucosal immune quiescence

doi: 10.1126/sciadv.adf3924

Figure Lengend Snippet: ( A ) Volcano plot comparing the P value versus fold change for reads of mRNA sequencing (mRNA-seq) data from in vitro–differentiated T reg cells from WT versus Ctsw −/− mice. The genes of significant change are highlighted in red (transcripts up-regulated in Ctsw −/− pT reg cells) and in blue (transcripts down-regulated in Ctsw −/− pT reg cells), respectively. ( B ) Quantitative polymerase chain reaction (qPCR) analysis of indicated genes from WT and Ctsw −/− pT reg cells. ( C ) Gene set enrichment analysis of up-regulated signaling pathway in Ctsw −/− pT reg cells. ( D ) Immunoblot analysis of p-STAT5, STAT5, and β-actin in WT and Ctsw −/− in vitro–differentiated pT reg cells with IL-2 stimulation. ( E and F ) (E) Representative flow cytometry analysis and (F) quantification of CD25, CD122, and CD132 expression on WT and Ctsw −/− in vitro–differentiated Foxp3 + pT reg cells. ( G and H ) (G) Representative flow cytometry analysis and (H) quantification of CD25, CD122, and CD132 on Foxp3 + T reg cells from the spleen and LP of Foxp3 cre Ctsw fl/fl and control mice. Data are representative of two independent experiments (A to H). ** P < 0.01 and *** P < 0.001 [(B, F, and H) Student’s t test; error bars represent SD].

Article Snippet: The CD25 and its mutation were amplified from mouse CD25 cDNA clone (Sino Biological).

Techniques: Sequencing, In Vitro, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Expressing

Journal: Science Advances

Article Title: Cathepsin W restrains peripheral regulatory T cells for mucosal immune quiescence

doi: 10.1126/sciadv.adf3924

Figure Lengend Snippet: ( A ) Co-immunoprecipitation (IP) of CTSW and CD25 from extracts of cotransfected 293 T cells. HEK293 T cells were transfected with mouse V5-tagged active form and inactive form of CTSW and Flag-tagged CD25. Anti-V5 immunoprecipitates (IP) and total lysates were immunoblotted with anti-V5 and anti-Flag antibodies. ( B ) Naïve CD4 + T cells retroviral transduced with V5-tagged CTSW in the presence of transforming growth factor–β (TGF-β). Confocal and stimulated emission depletion (STED) imaging of CD25 and V5 on in vitro–differentiated pT reg cells were shown. Heat imaging was generated on the basis of STED microscopy (bottom row). ( C ) CD25 and V5 signals in (C) were reconstructed to three-dimensional illustration. ( D ) Quantification of colocalization CD25 and V5 on the cell membrane and cytoplasm as exemplified in (C). ( E ) Amino acid alignment illustrating conservation in CTSW cleave motif across different species. ( F ) HEK293 T cells were cotransfected with mouse V5-tagged CTSW and Flag-tagged CD25. Total lysates were immunoblotted with anti-CD25 and anti-V5 antibodies (], glycosylated CD25; *, nonglycosylated CD25; #, cleaved CD25). ( G ) HEK293 T cells were cotransfected with indicated forms of V5-tagged CTSW and Flag-tagged CD25. Total lysates were immunoblotted with anti-CD25 and anti-V5 antibodies (], glycosylated CD25; *, nonglycosylated CD25; #, cleaved CD25). ( H and I ) Naïve CD25-deficient CD4 + T cells retroviral transduced with CTSW and CD25 or CD25RR in the presence of TGF-β. (H) Representative flow cytometry analysis and (I) quantification of Foxp3 expression. Data are representative of two independent experiments (A to C and E to I) or are pooled from two independent experiments (D). ** P < 0.01 and *** P < 0.001 [(D) Student’s t test; (I) two-way ANOVA with Tukey’s multiple comparisons test; error bars represent SD].

Article Snippet: The CD25 and its mutation were amplified from mouse CD25 cDNA clone (Sino Biological).

Techniques: Immunoprecipitation, Transfection, Transduction, Imaging, In Vitro, Generated, Microscopy, Flow Cytometry, Expressing

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Elevated bone marrow sympathetic drive precedes systemic inflammation in angiotensin II hypertension

doi: 10.1152/ajpheart.00510.2018

Figure Lengend Snippet: Antibodies used for flow cytometry and brain immunohistochemistry analysis in rat

Article Snippet: 9 , Mouse anti-rat CD25:RPE , FC , Bio-Rad , mca273pe.

Techniques: Flow Cytometry, Immunohistochemistry