cd5 kd mice  (Miltenyi Biotec)

Journal: Frontiers in Immunology

Article Title: CD5 Controls Gut Immunity by Shaping the Cytokine Profile of Intestinal T Cells

doi: 10.3389/fimmu.2022.906499

Figure Lengend Snippet: Loss of CD5 in NOD mice causes wasting disease (A) Doxycycline (dox)-inducible CD5 KD shRNA constructs were validated by luciferase reporter assay, using a dual-luciferase vector that incorporates the CD5 cDNA (n=2-3 biological replicates). Data are representative of 4 experiments. (B) Relative CD5 expression in blood CD4 + or CD8 + T cells from CD5 KD mice (n=3) compared to WT NOD mice after dox-feeding followed by dox-removal from the drinking water. (C) Spontaneous diabetes frequency in cohorts of dox-treated CD5 KD (KD, n = 32) and WT (n = 44) NOD mice, P-values were calculated using the Gehan-Breslow-Wilcoxon test and statistical significance (P < 0.05) is indicated. (D) Representative picture of 5 months-old dox-treated CD5 KD and WT NOD mice. (E) Weight curves of CD5 KD and WT NOD mice with or without dox-treatment (n=17-21). *P < 0.05, two-tailed unpaired t-test.

Article Snippet: For T cell transfer colitis, CD4 + T cells were pre-sorted from spleens of NOD WT or CD5 KD mice using CD4 microbeads (Miltenyi Biotec).

Techniques: shRNA, Construct, Luciferase, Reporter Assay, Plasmid Preparation, Expressing, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: CD5 Controls Gut Immunity by Shaping the Cytokine Profile of Intestinal T Cells

doi: 10.3389/fimmu.2022.906499

Figure Lengend Snippet: CD5 KD causes GALT hyperplasia and histological anomalies in the gut (A, B) Representative images of H&E stained histological sections of the intestine (duodenum, jejunum, ileum and colon) (A) or Peyer’s Patches (B) of dox-treated CD5 KD and WT NOD mice at 2 months (2m) or 5m of age. Scale bars are 100μm (A) or 500μm (b, upper panel). In b, the total cell number in Peyer’s Patches of 2m old dox-treated CD5 KD and WT NOD mice (n=7-8) is shown. *P < 0.05, two-tailed unpaired t-test. (C) Representative images of mesenteric lymph nodes (mLN) of dox-treated CD5 KD and WT NOD mice at 2m or 5m of age. The right panel shows total cell numbers in mLN of 2m old animals (n=28), **P < 0.01, two-tailed unpaired t-test.

Article Snippet: For T cell transfer colitis, CD4 + T cells were pre-sorted from spleens of NOD WT or CD5 KD mice using CD4 microbeads (Miltenyi Biotec).

Techniques: Staining, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: CD5 Controls Gut Immunity by Shaping the Cytokine Profile of Intestinal T Cells

doi: 10.3389/fimmu.2022.906499

Figure Lengend Snippet: CD5 deficiency compromises gut barrier integrity (A–C) WT (black) or CD5 KD (red) NOD mice were treated with doxycycline for 2 months. (A) Number of epithelial cells (EpCam + ) in the ileum of CD5 KD dox and their littermate controls (n = 8 per group). This data represent cells analyzed in 60 seconds at the same flow rate by flow cytometry. (B) Relative mRNA levels for Muc2, Muc4, Cldn4 and Cldn4 in ileum epithelial cells of CD5 KD dox and their littermate controls (n = 5 per group). Each symbol represents an individual mouse, lines indicate the mean). All statistical analyses were performed by two-tailed Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For T cell transfer colitis, CD4 + T cells were pre-sorted from spleens of NOD WT or CD5 KD mice using CD4 microbeads (Miltenyi Biotec).

Techniques: Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CD5 Controls Gut Immunity by Shaping the Cytokine Profile of Intestinal T Cells

doi: 10.3389/fimmu.2022.906499

Figure Lengend Snippet: Disease in CD5 KD NOD mice is driven by modified T cell function (A, B) Antibody-mediated T cell depletion in dox-treated CD5 KD or NOD WT mice. Relative percentages of CD4 + (A) or CD8 + (B) T cells in the blood (left panels) and weight curves (right panels) of dox-treated CD5 KD and WT NOD mice with or without anti-CD4 antibody (A) or anti-CD8 antibody (B) treatment (arrows) (n=5-12). *P < 0.05 (two-tailed unpaired t-test). (C, D) Weight curves for bone marrow (BM) chimeric mice, shown as percent of initial weight. In c, NOD Rag KO mice (n=5-7) were reconstituted with CD5 KD BM and either treated or not with doxycycline. In D, NOD Rag KO mice (n=11-12) were reconstituted with a mixture of BM from WT or CD5 KD T-cell deficient (TCR KO) and B-cell deficient (IgM KO) mice, and all BM transplant recipients received dox-treatment. *P < 0.05 (two-tailed unpaired t-test).

Article Snippet: For T cell transfer colitis, CD4 + T cells were pre-sorted from spleens of NOD WT or CD5 KD mice using CD4 microbeads (Miltenyi Biotec).

Techniques: Modification, Cell Function Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: CD5 Controls Gut Immunity by Shaping the Cytokine Profile of Intestinal T Cells

doi: 10.3389/fimmu.2022.906499

Figure Lengend Snippet: The frequency of peripherally-induced Tregs is decreased by CD5 KD, but loss of pTregs alone does not cause wasting disease (A–C) Lymphocytes from the colonic lamina propria (LP, a), mesenteric lymph nodes (mLN, b) and spleen (C) of dox-treated CD5 KD and WT mice were analyzed by flow cytometry. pTregs were characterized as RORγt + Nrp-1 lo cells gated within total CD4 + Foxp3 + cells in the colonic lamina propria (A) and as Foxp3 + Nrp-1 lo cells within total CD4 + cells in mLN and spleen (B , C) . All samples were gated on live CD4 + CD8 - lymphocytes. Representative FACS plots are shown on the left, cell frequency data (mean ± SEM) on the right. All mice were 7 weeks old, n=26-27 for spleen, n=26 for mLN and n=10-11 for colonic LP. *P < 0.05 (two-tailed unpaired t-test). (D) CD5 KD and WT NOD mice were treated with dox for 4 weeks then kept without dox-treatment for another 4 or 8 weeks, as indicated. pTreg frequencies in the colonic lamina propria, mesenteric lymph nodes (mLN) and spleen were analyzed by flow cytometry. P-values were calculated by two-tailed unpaired t-test. ns: P > 0.05. (E) CD5 KD and WT NOD mice were treated with dox either throughout the experiment or fed with normal water (without dox) from 3m (left panel; n=5-10 male mice) or 2m of age (right panel; n=15-28 female mice). Weight curves are shown (mean ± SEM), *P < 0.05, **P < 0.01 (two-tailed unpaired t-test). (F) pTreg frequencies in the colonic LP, mLN and spleen of CNS1 KO and WT NOD mice, analyzed by flow cytometry as described above. *P < 0.05 (two-tailed unpaired t-test). (G) Weight curves (mean ± SEM) for CNS1 KO or WT NOD male (left panel, n=19-26) and female (right panel, n=16-29) mice. ns - not significant.

Article Snippet: For T cell transfer colitis, CD4 + T cells were pre-sorted from spleens of NOD WT or CD5 KD mice using CD4 microbeads (Miltenyi Biotec).

Techniques: Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: CD5 Controls Gut Immunity by Shaping the Cytokine Profile of Intestinal T Cells

doi: 10.3389/fimmu.2022.906499

Figure Lengend Snippet: CD5 KD exacerbates colitis by modifying the function of effector T cells (A) DSS-induced colitis in dox-treated CD5 KD and WT NOD mice. Representative H&E stained colon and small intestine histology, weight curves depicting percent of initial weight, pathology scores and representative organ pictures are shown; n=6-8 mice per group. * P < 0.05 (two-tailed unpaired t-test). (B) DSS-induced colitis in thymectomized (lower panels) dox-treated CD5 KD and WT NOD mice. Controls include non-thymectomized mice (upper panels). Representative H&E stained colon histology, weight curves depicting percent of initial weight, pathology scores and representative organ pictures are shown; n=5-8 mice per group. (C) CD4 + CD45RB hi T cells were FACS-sorted from spleens of CD5 KD and WT NOD mice and injected intravenously into NOD Rag KO mice which were then treated with dox. Representative H&E stained colon histology, weight curves depicting percent of initial weight and colitis scores are shown; n=5-9 mice per group. *P < 0.05 (two-tailed unpaired t-test). **P < 0.01; ***P < 0.001 (two-tailed unpaired t-test). All scale bars are 100μm.

Article Snippet: For T cell transfer colitis, CD4 + T cells were pre-sorted from spleens of NOD WT or CD5 KD mice using CD4 microbeads (Miltenyi Biotec).

Techniques: Staining, Two Tailed Test, Injection

Journal: Frontiers in Immunology

Article Title: CD5 Controls Gut Immunity by Shaping the Cytokine Profile of Intestinal T Cells

doi: 10.3389/fimmu.2022.906499

Figure Lengend Snippet: Loss of CD5 increases the propensity of T cells to secrete IL-17A. (A) Frequency of IL-17A + and IL-22 + CD4 + T cell and serum concentrations of IL-17A and IL-22 in CD5 KD dox (red) and WT (black) mice treated with doxycycline for 2 months (n = 5 mice per group). (B) IL-17A and IL-22 concentration in the culture medium of cells (mLN or spleen) after overnight IL-23 stimulation (100ng/mL) (n = 5 mice per group). (C) IL-17A and IL-22 mRNA levels in WT and CD5KD CD4 + T cells after adoptive transfer into gender-matched NOD. scid mice treated with doxycycline (paired samples from 11 mice). (D, E) IL-17A and IL-22 mRNA levels (D) and cytokine concentration in the culture medium from CD4 + T cells from WT dox and CD5 KD dox mice after in vitro Th17 and Th22 differentiation. (n = 6-12 technical replicates per group, starting material was pooled from 2-3 mice). (F) CD5 surface expression (MFI: mean fluorescence intensity) on CD4 + T cells from CD5 KD dox (red), CD5KO (blue) and WT littermates analyzed by flow cytometry after 2 months of doxycycline (CD5 KD) or tamoxifen (CD5 KO) treatment. (n = 4-6 mice per group). (G) IL-17A and IL-22 concentration in the culture medium of WT and CD5KO CD4 + T cells after in vitro Th17 and Th22 differentiation. (n = 12 technical replicates per group, starting material was pooled from 2-3 mice). Data were compared by two-tailed Mann–Whitney test except in (C) where data were compared by two-tailed Wilcoxon matched-pairs test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns - not significant.

Article Snippet: For T cell transfer colitis, CD4 + T cells were pre-sorted from spleens of NOD WT or CD5 KD mice using CD4 microbeads (Miltenyi Biotec).

Techniques: Concentration Assay, Adoptive Transfer Assay, In Vitro, Expressing, Fluorescence, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CD5 Controls Gut Immunity by Shaping the Cytokine Profile of Intestinal T Cells

doi: 10.3389/fimmu.2022.906499

Figure Lengend Snippet: CD5 deficiency increases Stat3 phosphorylation. (A–C) Stat3 phosphorylation (Tyr705) levels in CD5 KD dox , CD5 KO and WT CD4 + T cells after in vitro Th17 and Th22 differentiation (A, B) and in WT and CD5 KD CD4 + T cells after adoptive transfer into gender-matched NOD. scid mice treated with doxycycline (C) . (D) Act-1 mRNA levels in WT dox and CD5 KD dox CD4 + T cells after in vitro Th17 differentiation. (n = 6 replicates per group). Data were compared by two-tailed Mann–Whitney test except in (C) where data were compared by two-tailed Wilcoxon matched-pairs test. *P < 0.05, **P < 0.01, ****P < 0.0001.

Article Snippet: For T cell transfer colitis, CD4 + T cells were pre-sorted from spleens of NOD WT or CD5 KD mice using CD4 microbeads (Miltenyi Biotec).

Techniques: In Vitro, Adoptive Transfer Assay, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CD5 Controls Gut Immunity by Shaping the Cytokine Profile of Intestinal T Cells

doi: 10.3389/fimmu.2022.906499

Figure Lengend Snippet:

Article Snippet: For T cell transfer colitis, CD4 + T cells were pre-sorted from spleens of NOD WT or CD5 KD mice using CD4 microbeads (Miltenyi Biotec).

Techniques: