Journal: PLoS ONE

Article Title: A Convenient Model of Severe, High Incidence Autoimmune Gastritis Caused by Polyclonal Effector T Cells and without Perturbation of Regulatory T Cells

doi: 10.1371/journal.pone.0027153

Figure Lengend Snippet: (A) The number of cells in the paragastric and inguinal lymph nodes from recipient mice 8 weeks after transfer of either wildtype or H/Kα −/− CD4 + T cells. (B) The percentage of effector/memory T cells in the donor population with the phenotype CD44 hi CD62L lo in CD90.2 + CD4 + T cells. Data pooled from four independent experiments. Each circle represents the data from one mouse. Mann-Whitney U test was used; bars, mean, **, P<0.01 and ***, P<0.001.

Article Snippet: Anti-CD3-FITC (145-2C11), anti-CD90.1-FITC (HIS51), anti-CD90.2-FITC (30H-12), anti-CD62L-PE (MEL-14), anti-CD4 PerCP (RM4-5), and anti-CD8-APC (53-6.7) were purchased from BD Pharmingen; anti-CD44-APC (IM-7) was purchased from eBioscience.

Techniques: MANN-WHITNEY

Journal: PLoS ONE

Article Title: A Convenient Model of Severe, High Incidence Autoimmune Gastritis Caused by Polyclonal Effector T Cells and without Perturbation of Regulatory T Cells

doi: 10.1371/journal.pone.0027153

Figure Lengend Snippet: Cells were harvested from (A, B) paragastric lymph node, inguinal lymph node and (A, C) stomach tissue for intracellular staining of Foxp3. The numbers indicate the percentages of Foxp3 + Treg cells in donor CD90.2 + CD4 + T cells in paragastric and inguinal lymph nodes and Foxp3 + Treg cells in total CD4 + T cells in stomach tissue. Data pooled from three independent experiments. In (B) and (C), each circle represents the data from one mouse. Mann-Whitney U test was used; bars, mean, and **, P<0.01.

Article Snippet: Anti-CD3-FITC (145-2C11), anti-CD90.1-FITC (HIS51), anti-CD90.2-FITC (30H-12), anti-CD62L-PE (MEL-14), anti-CD4 PerCP (RM4-5), and anti-CD8-APC (53-6.7) were purchased from BD Pharmingen; anti-CD44-APC (IM-7) was purchased from eBioscience.

Techniques: Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: IL-21 restricts T follicular regulatory T cell proliferation through Bcl-6 mediated inhibition of responsiveness to IL-2

doi: 10.1038/ncomms14647

Figure Lengend Snippet: Mixed BM chimeras were generated by reconstituting lethally irradiated 8-week-old CD45.1 recipients with 5 × 10 6 each of CD45.2 BM cells from either Il21r + / + Thy1.1 + and Il21r −/− Thy1.2 + or control chimeras consisting of BM from Il21r + / + Thy1.1 + and Il21r + / + Thy1.2 mice. Eight weeks after BM transfer, mice were immunized intravenously with 2 × 10 8 SRBC and the CD4 + T-cell populations in the spleen were assessed on day 7. ( a ) Control chimeras showing relative percentages of Thy1.1 and Thy1.2 CD4 + T cells within the total CD45.2 CD4 + T cell population. ( b ) Relative percentages of Thy1.1 and Thy1.2 Foxp3 + Treg cells and ( c ) Thy1.1 and Thy1.2 Foxp3 + CXCR5 + PD-1 + Tfr cells within the donor CD45.2 populations, respectively. ( d ) Chimerism was assessed by tail bleed at 7 weeks. ( e ) Relative percentages of Thy1.2 Il21r −/− CD4 + T cells within the total CD45.2 CD4 + T-cell population (shown in e – g ) and Thy1.2 CXCR5 + PD-1 + Foxp3 − Tfh cells. ( e ) Relative percentages of Thy1.2 Foxp3 + Treg cells. ( f ) Relative percentages of Thy1.2 Foxp3 + Treg cells and ( g ) relative percentages Thy1.2 Foxp3 + CXCR5 + PD-1 + CD4 + Tfr cells within the total CD45.2 Tfr population. Percentages of ( h ) Tfh cells as a percentage of CD4 + T cells. ( i ) Treg cells as a percentage of CD4 + T cells. ( j ) Tfr cells as a percentage of CXCR5 + PD-1 + CD4 + T follicular cells and ( k ) the ratio of Tfr:Tfh in individual mice within the Thy1.1 Il21r +/+ and Thy1.2 Il21r −/− CD4 + donor populations in chimeras. Data are shown as individual mice n =5 from two separate experiments with similar results. Statistical analyses were performed by paired Student's t -test, P -values >0.05 were deemed not significant. Localization of Thy1.1 (WT) Il21r + / + and Thy1.2 Il21r −/− Foxp3 + cells within GCs of the spleen from mixed BM chimeras: GCs were identified as IgD lo . ( l ) Tfr cells within the GCs were identified with Foxp3 (red), Thy1.1 (blue) and Thy1.2 (green). Individual antibody immunostains are shown in grey scale. Scale bars, 100 μm. ( m ) Quantification of the percentage of Tfr cells counted from 80 immunostained GCs in sections of spleen as in k . Data are shown as mean ±s.d., n =5 mice.

Article Snippet: Antibodies against surface molecules purchased from BD Bioscience were anti-CD4-PE-Cy7 (RM4–5, dilution 1/300), anti-CD25-APC & BV605 (PC61, dilution 1/100), anti-CD45.1-PE-Cy7 (A20, dilution 1/200), anti-CD45.2-APC-Cy7 (104, dilution 1/200), anti-CD90.1-eF450 (HIS51, dilution 1/200), anti-CD90.2-BV605 (53-2.1, dilution 1/200), anti-CXCR4-PE (2B11/CXCR4, dilution 1/50), anti-CXCR5-biotin (2G8, dilution 1/100), anti-GL7-PE (GL7, dilution 1/300), anti-FAS-FITC (Jo2, dilution 1/200), anti-IgG1-biotin (A85-1, dilution 1/100) and anti-Ki67-A647 (B56, dilution 1/100).

Techniques: Generated, Irradiation

Journal: Nature Communications

Article Title: IL-21 restricts T follicular regulatory T cell proliferation through Bcl-6 mediated inhibition of responsiveness to IL-2

doi: 10.1038/ncomms14647

Figure Lengend Snippet: A direct comparison of CD25 expression on Thy1.1 Il21r +/+ cells and Thy1.2 Il21r −/− Treg populations from mixed BM chimeras described in . ( a ) Representative FACS dot plot showing CD25 and Foxp3 expression in CD4 + T cells. Relative percentages of Thy1.1 Il21r +/+ cells and Thy1.2 Il21r −/− cells in individual mice showing Foxp3 + CD4 + T cells that either ( b ) lack CD25 expression or ( c ) express CD25. ( d ) MFI of CD25 on Thy1.1 Il21r +/+ cells and Thy1.2 Il21r −/− Foxp3 + CD4 + T cells. ( e ) Representative FACS dot plot showing CD25 and Foxp3 expression in CXCR5 + PD-1 + CD4 + T cells. Relative percentages of Thy1.1 Il21r +/+ cells and Thy1.2 Il21r −/− Foxp3 + Tfr cells that either ( f ) lack CD25 expression or ( g ) express CD25. ( h ) MFI of CD25 on Thy1.1 Il21r +/+ cells and Thy1.2 Il21r −/− Foxp3 + Tfr cells. IL-2:IL-2mAb complex expansion of Treg cells in Il21r −/− and WT C57BL/6 mice: ( i ) Quantification of Foxp3 + CD25 + Treg cells as a proportion of CD4 + TCRβ + T cells in IL2:IL2 monoclonal antibody-injected mice and ( j ) fold increase of Treg cells in response to IL2:IL2 monoclonal antibody treatment relative to SRBC immunized alone. ( k ) Quantification of Foxp3 + Tfr cells as a proportion of CXCR5 + PD-1 + CD4 + TCRβ + T cells and ( l ) fold increase of Tfr cells on IL2:IL2 monoclonal antibody treatment. Data are shown as individual mice with means ±s.d., from two replicate experiments, P- values were determined by Student's t -test, P -values >0.05 were deemed not significant (NS).

Article Snippet: Antibodies against surface molecules purchased from BD Bioscience were anti-CD4-PE-Cy7 (RM4–5, dilution 1/300), anti-CD25-APC & BV605 (PC61, dilution 1/100), anti-CD45.1-PE-Cy7 (A20, dilution 1/200), anti-CD45.2-APC-Cy7 (104, dilution 1/200), anti-CD90.1-eF450 (HIS51, dilution 1/200), anti-CD90.2-BV605 (53-2.1, dilution 1/200), anti-CXCR4-PE (2B11/CXCR4, dilution 1/50), anti-CXCR5-biotin (2G8, dilution 1/100), anti-GL7-PE (GL7, dilution 1/300), anti-FAS-FITC (Jo2, dilution 1/200), anti-IgG1-biotin (A85-1, dilution 1/100) and anti-Ki67-A647 (B56, dilution 1/100).

Techniques: Expressing, Injection

Journal: bioRxiv

Article Title: Macrophages orchestrate the expansion of a pro-angiogenic perivascular niche during cancer progression

doi: 10.1101/2020.10.30.361907

Figure Lengend Snippet: ( A ) Representative images of frozen sections of MMTV-PyMT tumors stained with DAPI (nuclei; blue) and antibodies against F4/80 (magenta) and αSMA (red); functional vasculature was labeled in vivo using i.v. dextran-FITC (green). Scale bar represents 100μm. ( B ) Representative images of frozen sections from human invasive ductal mammary carcinoma (left) and DCIS (right) stained with DAPI (nuclei; blue) and antibodies against CD31 (green), CD68 (magenta) and αSMA (red), images representative of 5 patients. Scale bar represents 100μm (left panel) and 50μm (right panel). ( C ) Representative flow cytometry gating strategy for live (7AAD - ) CD45 - cells and CD31 + endothelial cells and CD90 + CAFs (left) and the abundance of CAFs at different tumor volumes (right), n=6 mice per condition. ( D ) Identification of CAF subsets by unsupervised clustering from multiparametric flow cytometry data using the FlowSOM algorithm. UMAP and unsupervised clustering was performed using the markers shown in the heatmap (right). UMAP plot shows individual cells colored by their unsupervised clustering assignment (left). Heatmap displays the relative marker expression of each marker among the two subsets (right), n=4 mice. ( E ) Representative gating strategy for flow cytometry sorting the predicted subsets of CAFs by unsupervised clustering analysis. ( F ) Bar plots depicting normalized gene expression values for the indicated genes in the two bulk RNA-sequenced CAF subsets (across n=5 mice), showing that the SMA + CAF population expresses pericyte markers ( Acta2, Des, Pdgfb and Cspg4 ) in MMTV-PyMT tumors. ( G ) Abundance of the respective CAF populations during distinct stages of tumor progression, n=6 mice per stage. (MG; mammary gland). ( H ) Schematic for experimental approach and dosing Edu into MMTV-PyMT mice to assess in vivo proliferation (left), proportion EdU + cells within each CAF subset (right). ( I ) Established tumors in Kaede MMTV-PyMT mice were photoconverted to kaede red and then at 72h post photoconversion tumors were analyzed (schematic left) for their respective kaede red/green proportion using flow cytometry for evidence of peripheral recruitment (Kaede green cells). A representative unconverted tumor is shown for comparison (right top). ( J ) Representative images of frozen sections of MMTV-PyMT tumors stained with antibodies against F4/80 (green), αSMA (magenta) and the proliferation marker Ki67 (red). White arrows show αSMA + Ki67 + cells in contact with F4/80 + TAMs. ( K ) Schematic for experimental approach and dosing strategy to acutely deplete Lyve-1 + pvTAM with clodronate-filled liposomes. ( L ) Abundance of TAM CD206 + TAM populations following cntrl- or clodronate-filled liposome treatment (n=6 mice cntrl-lip and n=5 mice clod-lip). ( M ) Abundance of CD45 - cell populations (cohorts of n=6 mice). ( N ) Proportion of EdU + cells within each CD45 - cell subset, (cohorts of n=6 mice). Bar charts represent mean, error bars represent s.d. and the dots show individual data points from individual tumors and mice. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The following antibodies against the indicated antigen were purchased from Thermo Fisher Scientific and were used at 1 μg/ml unless stated otherwise: CD3ε APC and PE (145-2C11), CD4 FITC (RM4-5), CD8β eFluor®450 (H35-17.2), CD11b APC-eFluor ® 780 (M1/70), CD11b BV510 (M1/70), CD11c APC (N418), CD16/32 (2.4G2; Tonbo Biosciences), CD19 APC (6D5; Biolegend ® ), CD29 APC (eBioHMb1-1), CD31 eFluor ® 450 and PE (390), CD34 FITC and APC (RAM34), CD45 APC-eFluor ® 780, FITC and PerCP-Cy5.5 (30-F11), CD90.2 eFluor ® 450 (53-2.1), CD90.1 eFluor ® 450 (HIS51), CD90.1 BV510 (OX-7), CD103 PE (2E7), CD206 APC (FAB2535A; Bio-Techne), F4/80 PE (BM8; Biolegend ® ), F4/80 BV421 (BM8; Biolegend ® ), FAP (10 μg/ml, AF3715, Bio-Techne), Ly6C PE and eFluor ® 450 (HK1.4), Ly6G FITC (1A8; Biolegend ® ), Lyve-1 AlexaFluor ® 488 (ALY7), MHCII PE and FITC (M5/114.15.2), NK1.1 APC (PK136), PDGFRα PerCP-Cy5.5 (APA5), Ly6A/E AlexaFluor ® 700 (D7).

Techniques: Staining, Functional Assay, Labeling, In Vivo, Flow Cytometry, Marker, Expressing, Comparison, Liposomes