Journal: Veterinary Research

Article Title: CD27 expression discriminates porcine T helper cells with functionally distinct properties

doi: 10.1186/1297-9716-44-18

Figure Lengend Snippet: Expression of CCR7, CD62L and CX3CR1 on CD27-defined T-helper cell subsets. ( A ) CCR7 expression of T helper cells from blood (PBMCs; left panel), spleen (middle panel) and mediastinal lymph node (Med. LN; right panel) was investigated by five-colour FCM including a live/dead discrimination dye. CD4 + T helper cells (upper left contour plots; black gates) were gated and analysed for CD8α (y-axis) and CD27 (x-axis) expression (lower left contour plots). Gates were set on CD8α - CD27 + (blue gates), CD8α + CD27 + (green gates) and CD8α + CD27 - (red gates) cell subsets for analyses of CCR7 expression indicated by coloured histograms. Numbers show percentage of gated cell subsets. See main text for description of arrow heads and numbers. Representative data from one individual out of six is shown. Per sample at least 1 × 10 5 lymphocytes were acquired. ( B ) CD62L and CX3CR1 mRNA expression of total PBMCs and CD27-sorted T-helper cell subsets ex vivo was examined by use of quantitative RT-PCR. The graphs show normalized 2^ΔΔCt values obtained from cells of three animals (symbols) and geometric means (coloured bars) relative to IS. Significant difference is indicated (* p < 0.05).

Article Snippet: The following primary antibodies were used: CD3-eFluor450 (mIgG1, clone PPT3; custom conjugated to eFluor450 by eBioscience, San Diego, CA, USA), CD4 (mIgG2b, clone 74-12-4), CD8α-PE (mIgG2a, clone 76-2-11; BD Biosciences, San Jose, CA, USA), CD27-Alexa647 (mIgG1, clone b30c7), CD45RC-PerCP-Cy5.5 (mIgG1, clone 3a56), SLA-DR-Qdot605 (mIgG2a, clone MSA3, custom conjugated to Qdot605 by Life Technologies, Carlsbad, CA, USA) and rat anti-human CD197-PE-Cy7 (CCR7) (rat IgG2a, clone 3D12; BD Biosciences).

Techniques: Expressing, Ex Vivo, Quantitative RT-PCR

Journal: Arthritis Research & Therapy

Article Title: Altered circulating CCR6 + and CXCR3 + T cell subsets are associated with poor renal prognosis in MPO-ANCA-associated vasculitis

doi: 10.1186/s13075-021-02576-x

Figure Lengend Snippet: Surface expression of CCR4, CCR6, and CXCR3 on human T cell functional subset. A , B Representative plots and statistical data of CD4 + T cell and CD8 + T cell subsets. CD4 + T cell subsets and CD8 + T cells subsets from the peripheral blood were identified according to the expression of CCR7 and CD45RO. The percentage of T naïve (CCR7 + CD45RO − ), T CM (CCR7 + CD45RO + ), T EM (CCR7 - CD45RO + ), and T EMRA (CCR7 − CD45RO − ) in the peripheral blood of HC and active MPO-AAV patients is shown (n MPO-AAV=33, HC=20). C , D Percentages of CCR6 + T cells, CCR4 + T cells CXCR3 + T cells in the T EM (CCR7 − CD45RO + ), and T EMRA (CCR7 − CD45RO − ) subpopulation were shown

Article Snippet: Freshly collected PBMCs were stained immediately using the following fluorochrome-conjugated anti-human antibodies: BV510-conjugated anti-CD3, BB515-conjugated anti-CD4, APC-Cyanine7 (APC-Cy7)-conjugated anti-CD8, PerCP/Cyanine5.5-conjugated anti-CD45RO, PE/Cyanine7 (PE-Cy7)-conjugated anti-CCR7, PE-conjugated anti-CCR6, Brilliant Violet 421-conjugated anti-CCR4, and APC-conjugated anti-CXCR3 (BD Biosciences).

Techniques: Expressing, Functional Assay

Journal: Arthritis Research & Therapy

Article Title: Altered circulating CCR6 + and CXCR3 + T cell subsets are associated with poor renal prognosis in MPO-ANCA-associated vasculitis

doi: 10.1186/s13075-021-02576-x

Figure Lengend Snippet: The distribution of CD4 + effector memory T cell subsets in HC and active MPO-AAV patients. A Representative plots of CD4 + effector memory T cell subsets. Gating strategy for the identification of T EM 1, T EM 2, T EM 17, and T EM 17.1 according to the expression of chemokine receptors. The CD4 + T EM cell subset (CD4 + CCR7 − CD45RO + ) was gated for T EM 1 cells (CD4 + CD45RO + CCR7 − CCR6 − CXCR3 + CCR4 − ), T EM 2 cells (CD4 + CD45RO + CCR7 − CCR6 − CXCR3 − CCR4 + ), T EM 17 cells (CD4 + CD45RO + CCR7 − CCR6 + CXCR3 − CCR4 + ), and T EM 17.1 cell (CD4 + CD45RO + CCR7 − CCR6 + CXCR3 + CCR4 − ) subsets. B The summary data of the percentages of CD4 + T EM cell subsets in the blood of patients with active MPO-AAV patients and HC (n MPO-AAV=33 HC=20). C Serum CCL20 level in active MPO-AAV patients and HC was evaluated by ELISA (n MPO-AAV=33 HC=20)

Article Snippet: Freshly collected PBMCs were stained immediately using the following fluorochrome-conjugated anti-human antibodies: BV510-conjugated anti-CD3, BB515-conjugated anti-CD4, APC-Cyanine7 (APC-Cy7)-conjugated anti-CD8, PerCP/Cyanine5.5-conjugated anti-CD45RO, PE/Cyanine7 (PE-Cy7)-conjugated anti-CCR7, PE-conjugated anti-CCR6, Brilliant Violet 421-conjugated anti-CCR4, and APC-conjugated anti-CXCR3 (BD Biosciences).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Metabolism

Article Title: Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4 + T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals

doi: 10.1016/j.cmet.2017.01.008

Figure Lengend Snippet: HFD-Induced Metabolic Stress Promotes the Differentiation of Primed CD4 + T Cells to a CXCR3 + -LFA1 + Effector Memory-like Phenotype in Mice (A–F) Cell surface staining of CCR7 (A), CD62L (B), CXCR3 (C), LFA1 (D), CD25 (E), and CD44 (F) in in vivo-primed CD4 + T cells isolated from pooled lymph nodes of the HFD or CD Marilyn female donor mice used in A–1D (absolute numbers). (G) Ex vivo chemotaxis (3 hr, 6 hr, and 24 hr time points) of in vivo-primed CD4 + T cells isolated from pooled lymph nodes of the HFD or CD Marilyn female donor mice used in A–1D in response to CXCL10 (300 ng/mL), CCL19/21 (200 ng/mL of each chemokine), or media only. (H and I) Intracellular staining of IFNγ (H) and IL4 (I) in in vivo-primed CD4 + T cells isolated from mesenteric lymph nodes of HFD or CD mice. (A–F) n = 3–6 independent donors. (G) n = 3 independent donors (each donor was tested in duplicate). (H and I) n = 5 independent mice. The values denote mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.001.

Article Snippet: In particular, the panel used consisted of the subsequent cellular surface markers: CD3 (Pacific Blue-labeled, clone HIT3a, BD Pharmingen), CD4 (APC-Cy7, SK3, BD Biosciences), CCR5/CD195 (PE, 2D7, BD Pharmingen), CXCR3/CD183 (FITC, 49801, R&D Systems), CCR7/CD197 (PE-Cy7, 3D12, BD Pharmingen), HLA-DR (Quantum Red, HK14, Sigma-Aldrich), CD45RO (APC, UCHL1, Caltag), and CD45RA (ECD, 2H4, IOTest, Beckman Coulter).

Techniques: Staining, In Vivo, Isolation, Ex Vivo, Chemotaxis Assay

Journal: Cell Metabolism

Article Title: Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4 + T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals

doi: 10.1016/j.cmet.2017.01.008

Figure Lengend Snippet: Antigen Presentation by DC from Metabolically Stressed Donors Does Not Affect T Cell Differentiation to a Pro-inflammatory Effector Memory-like Phenotype (A) Dot plots and quantification of in vitro proliferation of CFSE-labeled CD4 + T cells isolated from pooled lymph nodes of HFD or CD Marilyn female mice incubated for 3 days with CD11c + DC isolated from the spleen of HFD or CD C57BL/6 male mice. The undivided and fourth division populations are quantified by dilution of the CFSE-label. (B) Cell surface staining of CD44, CCR7, CD62L, and CXCR3 in the undivided population of CFSE-labeled CD4 + T cells shown in (A). (C) Cell surface staining of CD40, CD80, and CD86 in CD11c + DC used in (A). (A and B) n = 3 independent mice (each mouse was tested in triplicate). (C) n = 3 independent mice. The values denote mean ± SEM. ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: In particular, the panel used consisted of the subsequent cellular surface markers: CD3 (Pacific Blue-labeled, clone HIT3a, BD Pharmingen), CD4 (APC-Cy7, SK3, BD Biosciences), CCR5/CD195 (PE, 2D7, BD Pharmingen), CXCR3/CD183 (FITC, 49801, R&D Systems), CCR7/CD197 (PE-Cy7, 3D12, BD Pharmingen), HLA-DR (Quantum Red, HK14, Sigma-Aldrich), CD45RO (APC, UCHL1, Caltag), and CD45RA (ECD, 2H4, IOTest, Beckman Coulter).

Techniques: Metabolic Labelling, Cell Differentiation, In Vitro, Labeling, Isolation, Incubation, Staining

Journal: Cell Metabolism

Article Title: Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4 + T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals

doi: 10.1016/j.cmet.2017.01.008

Figure Lengend Snippet: Direct Exposure of CD4 + T Cells to Saturated FA Causes Enhanced Activation of a PI3K p110δ-Akt-Dependent Pathway upon Priming (A) Levels and densitometric quantification of p-Akt (S473), Akt, p-S6 (S235/236), and β-actin protein expression in in vivo-primed CD4 + T cells isolated from mesenteric lymph nodes of CD and HFD mice i.p. injected with the PI3K inhibitor IC87114 or left untreated. (B) Levels and densitometric quantification of p-Akt (S473) and Akt protein expression in CD4 + T cells purified from the peripheral blood of lean and obese human subjects. (C) Membrane fluidity index in in vivo-primed CD4 + T cells isolated from mesenteric lymph nodes of CD and HFD mice, calculated as the ratio of oleic acid (C18:1) to linoleic acid (C18:2). (D) Cell surface staining of CTxB in in vitro activated (Act) CD4 + T cells isolated from the lymph nodes of CD and HFD mice. The quantification of aggregation of CTxB is measured by bright detail intensity of the signal. (E–H) Cell surface staining of CD44 with CCR7 (E), CD62L (F), CXCR3 (G), and LFA1 (H) in in vivo-primed CD4 + T cells isolated from mesenteric lymph nodes of PED and PCD mice. (I and J) Protein levels and densitometric quantification of p-Akt (S473), Akt, p-S6 (S235/236), and S6 protein expression (I), and gene expression of CCR7 and CD62L (J) in CD4 + T cells isolated from pooled lymph nodes of mice and cultured overnight with IL-7 (1 ng/mL) in the presence or absence of palmitic, linoleic, or stearic acid (200 μM), followed by activation with plate bound anti-CD3 (0.5 μg/mL) and anti-CD28 (2.5 μg/mL) for the indicated time points. (K and L) Levels of CCR7 and CD62L gene expression (K) and CCR7 protein (L) in CD4 + T cells isolated from pooled lymph nodes of mice, then activated with plate bound anti-CD3 (0.5 μg/mL) and anti-CD28 (2.5 μg/mL) for the indicated time points in the presence or absence of the Akt activator SC79 (500 nM). (A and B) Each lane shows data from independent mouse/human samples. (C–H) n = 3–6 independent mice. (I) Representative data of n = 3 independent mice. (J–L) 3–6 pooled mice. The values denote mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.001.

Article Snippet: In particular, the panel used consisted of the subsequent cellular surface markers: CD3 (Pacific Blue-labeled, clone HIT3a, BD Pharmingen), CD4 (APC-Cy7, SK3, BD Biosciences), CCR5/CD195 (PE, 2D7, BD Pharmingen), CXCR3/CD183 (FITC, 49801, R&D Systems), CCR7/CD197 (PE-Cy7, 3D12, BD Pharmingen), HLA-DR (Quantum Red, HK14, Sigma-Aldrich), CD45RO (APC, UCHL1, Caltag), and CD45RA (ECD, 2H4, IOTest, Beckman Coulter).

Techniques: Activation Assay, Expressing, In Vivo, Isolation, Injection, Purification, Staining, In Vitro, Cell Culture

Journal: Cell Metabolism

Article Title: Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4 + T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals

doi: 10.1016/j.cmet.2017.01.008

Figure Lengend Snippet: The PI3K, Akt Pathway Is Key for the Differentiation of CXCR3 + Effector Memory T Cells (A–D) Cell surface staining of CD44 with CCR7 (A), CD62L (B), CXCR3 (C), and LFA1 (D) in in vivo-primed CD4 + T cells isolated from mesenteric lymph nodes of HFD P110δ D910A or WT C57BL/6 mice. (E) Levels and densitometric quantification of p-Akt (S473), Akt, p-S6 (S235/236), and S6 protein expression in in vivo-primed CD4 + T cells from (A)–(D). (F) Ex vivo chemotaxis (3 hr, 6 hr, and 24 hr time points) of in vivo-primed CD4 + T cells isolated from mesenteric lymph nodes of HFD or CD OT-II P110δ D910A mice in response to CXCL10 (300 ng/mL), CCL19/21 (200 ng/mL of each chemokine), or media only. (G–J) Cell surface staining of CD44 with CCR7 (G), CD62L (H), CXCR3 (I), and LFA1 (J) in in vivo-primed CD4 + T cells isolated from mesenteric lymph nodes of CD and HFD mice i.p. injected with the PI3K inhibitor IC87114, the Akt activator SC79, or left untreated. (A–E and G–J) n = 5 independent mice. (F) n = 4 independent mice (each mouse was tested in duplicate). The values denote mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; and ∗∗∗ p < 0.001.

Article Snippet: In particular, the panel used consisted of the subsequent cellular surface markers: CD3 (Pacific Blue-labeled, clone HIT3a, BD Pharmingen), CD4 (APC-Cy7, SK3, BD Biosciences), CCR5/CD195 (PE, 2D7, BD Pharmingen), CXCR3/CD183 (FITC, 49801, R&D Systems), CCR7/CD197 (PE-Cy7, 3D12, BD Pharmingen), HLA-DR (Quantum Red, HK14, Sigma-Aldrich), CD45RO (APC, UCHL1, Caltag), and CD45RA (ECD, 2H4, IOTest, Beckman Coulter).

Techniques: Staining, In Vivo, Isolation, Expressing, Ex Vivo, Chemotaxis Assay, Injection

Journal: Cell Metabolism

Article Title: Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4 + T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals

doi: 10.1016/j.cmet.2017.01.008

Figure Lengend Snippet: Inhibition of FAO Prevents Enhanced Effector Memory Differentiation in Saturated Fatty Acid-Enriched Diet (A) OCR of naive CD4 + T cells isolated from pooled lymph nodes of mice and cultured overnight with IL-7 (1 ng/mL) in the presence or absence of palmitic or stearic acid (200 μM). (B–E) Cell surface staining of CD44 with CCR7 (B), CD62L (C), CXCR3 (D), and LFA1 (E) in in vivo-primed CD4 + T cells isolated from mesenteric lymph nodes of PED or PCD mice i.p. injected with etomoxir or left untreated. (A) Six pooled mice tested in 6–10 replicates per treatment. (B–E) n = 5 independent mice. The values denote mean ± SEM. ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: In particular, the panel used consisted of the subsequent cellular surface markers: CD3 (Pacific Blue-labeled, clone HIT3a, BD Pharmingen), CD4 (APC-Cy7, SK3, BD Biosciences), CCR5/CD195 (PE, 2D7, BD Pharmingen), CXCR3/CD183 (FITC, 49801, R&D Systems), CCR7/CD197 (PE-Cy7, 3D12, BD Pharmingen), HLA-DR (Quantum Red, HK14, Sigma-Aldrich), CD45RO (APC, UCHL1, Caltag), and CD45RA (ECD, 2H4, IOTest, Beckman Coulter).

Techniques: Inhibition, Isolation, Cell Culture, Staining, In Vivo, Injection