easysep human naïve cd4 + t cell isolation kit ii  (STEMCELL Technologies Inc)

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Naive CD4+ and CD8+ T-cells, were separately isolated by negative selection from PBMCs, and rested overnight prior to compound exposure. (A) Cells were treated with saturating concentrations of IL-7 (10 nM), MDK1472 (1 μM), and MDK-703 (1 μM) for up to 2 hours and scored for pSTAT5 accumulation by ELISA. (B) The dose response for each compound was measured in human naive CD4+ and CD8+ cells, utilizing an exposure time of 15 min (T max ).

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Isolation, Selection, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: Frozen PBMCs from 5 healthy donors were rested overnight and treated with 100 nM MDK-703 or 1nM IL-7, or no added compound, and cultured for 30 days in the presence of compounds. On days 3, 7, 16, and 30, cell aliquots were taken and analyzed by flow cytometry for (A) Ki-67 expression and (B) absolute numbers of CD8 + , CD4 + , Treg, and NK cells. Data are shown as mean ± SEM. Flow cytometry gating data shown in .

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Cell Culture, Flow Cytometry, Expressing

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: NSG mice (n = 10 per treatment) engrafted with human CD34+ cells from two donors (5 mice/donor) were dosed once intravenously with 1 mg/kg Fc (gray bars) or MDK-703 (blue bars), and peripheral blood (B-D) and spleen (E-G) were collected and analyzed at the indicated times by flow cytometry. (A) Diagram of the experimental plan. (B) Frequencies of Ki-67+ in CD3+, CD4+, CD8+, Treg, and NK cell populations in peripheral blood on day 7. (C) Absolute cell numbers of CD3+, CD4+, CD8+, Treg, and NK cell populations in peripheral blood on day 12. (D) CD8+ T memory subpopulations in peripheral blood on day 12. (E) Absolute cell numbers of CD3+, CD4+, CD8+, Treg, and NK cell populations in the spleen on day 12. (F) CD8+ T memory subpopulations in the spleen on day 12. (G) TCF1 expression in CD8+ T memory subpopulations in the spleen on day 12. Population gates were drawn based on FMO controls. Statistical analysis was done using Student’s T-Test. *p<0.05, **p<0.005, and ***p<0.0005. Detailed gating information is provided in .

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Flow Cytometry, Expressing

Journal: PLOS ONE

Article Title: A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy

doi: 10.1371/journal.pone.0286834

Figure Lengend Snippet: (A) PK of MDK-703: Animals (n = 3) were administered a single dose of 1 mg/kg MDK-703 via IV, SC, or IM. The serum concentration of MDK-703 at the indicated time points was determined by sandwich ELISA. (B and C) PD effect of MDK-703: Three animals were dosed once subcutaneously with 0.3 mg/kg, and blood samples were collected at the indicated time points for absolute lymphocyte counts (B) and immune profiling of CD8, CD4, Treg, and NK cells by flow cytometry (C). Data show mean ±SEM.

Article Snippet: For preparation of lymphocyte subsets, naïve CD4+ T-cells were isolated via two-stage negative selection with EasySep™ Human Naïve CD4+ T-Cell Isolation Kit II (Stemcell Technologies Cat# 17555); and CD8+ T-cells were isolated by negative selection from PBMCs with Stem Cell Technology Easy Sep CD8+ kit, or obtained freshly prepared from Stanford Blood Center.

Techniques: Concentration Assay, Sandwich ELISA, Flow Cytometry

Journal: bioRxiv

Article Title: The guanine nucleotide exchange factor Rin-like acts as a gatekeeper for T follicular helper cell differentiation via regulating CD28 signaling

doi: 10.1101/2022.06.23.497284

Figure Lengend Snippet: RNA was isolated from sorted WT and Rinl-KO naïve CD4 + T cells (CD62L + CD44 − ) and subjected to RNA sequencing. (A) Volcano plot depicts the comparison of global gene expression profiles between WT and Rinl-KO naïve CD4 + T cells. The y-axis indicates adjusted p-values (-log10), the x-axis shows the log2 fold change. (B) Diagram shows top dysregulated upstream regulators identified by Ingenuity Pathway Analysis (Qiagen Inc). Regulators are plotted on y-axis and are predicted to be up-(positive z-score, red) or downregulated (negative z-score, blue) in Rinl-KO naïve CD4 + T cells. (C) Summary of CD28 internalization kinetic in naïve and effector CD4 + T cells. Internalization was calculated as percentage (%) of CD28 expression from time point 0 (T=0). (D) Summary of phosphorylation of signaling molecules 2 minutes after αCD3ε or αCD3ε+αCD28 stimulation of CD4 + T cells. (E) Representative histograms of Erk1/2 (pT202/pY204) of WT and Rinl-KO CD4 + T cells activated with immobilized αCD3+αCD28 for 48 hours are shown. Summary is depicted below. (F) Representative histograms of proliferation after activation of naïve CD4 + T cells with low (0,75 μg/mL) and high (3 μg/mL) αCD28 for 3 days. Summary of data is shown alongside. (G) Summary of IL-2 measured from supernatants of cells cultured as in (F). (H) Expression of Cxcr5 was determined by RT-qPCR in naïve CD4 + T cells stimulated as described in (F). (I) Experimental setup. (J) Representative contour plots of Tfh of WT and Rinl-KO mice treated with isotype control or αCD28 (Clone 37.15) one day before immunization. (K) Summary of (J) . Data show a summary of 6-7 (C) , 3-4 (D) , 5 (E) 6 (F , G) , 4 (H) and 4-7 (J , K) mice per group analyzed in 3-4 (C , D, F, G, H) , 5 (E) or 2 (J , K) independent experiments. Data were statistically analyzed using two-way ANOVA using Tukey multiple comparison’s test (C) , paired two-tailed (E) or unpaired two-tailed t-tests (F , K) . For the comparison of the relative geoMFI values between WT (set as 1 for each phospho protein) and Rinl-KO, a one-sample t-test was performed (D) . *p < 0.05, **p < 0.01.

Article Snippet: Naïve human CD4 + T cells were isolated using the EasySep™ Human Naïve CD4 + T Cell Isolation Kit II (Stem Cell Technologies) according to the manufacturers’ instructions.

Techniques: Isolation, RNA Sequencing Assay, Expressing, Activation Assay, Cell Culture, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: The guanine nucleotide exchange factor Rin-like acts as a gatekeeper for T follicular helper cell differentiation via regulating CD28 signaling

doi: 10.1101/2022.06.23.497284

Figure Lengend Snippet: (A) Representative contour plots showing human PD-1 + CXCR5 + Tfh cells. Rinl was knocked-out in human CD4 + T cells and control and Rinl-KO cells were subsequently cultured under Tfh-skewing conditions for 5 days. (B) Summary of data shown in (A). (C) Rinl, Cxcr5 and Bcl6 expression in T cells retrieved from synovial biopsies of different patient cohorts ( Zhang et al , 2019 ). OA, Osteoarthritis; RA, Rheumatoid arthritis; TPM, transcripts per million. Data show a summary of 6 donors analyzed in 2 independent experiments (A , B) . Data were statistically analyzed using paired two-tailed t-tests (B) or 1-way ANOVA analysis followed by Tukey’s multiple-comparisons test (C) . *p < 0.05, ** p<0.01, ***p<0.001.

Article Snippet: Naïve human CD4 + T cells were isolated using the EasySep™ Human Naïve CD4 + T Cell Isolation Kit II (Stem Cell Technologies) according to the manufacturers’ instructions.

Techniques: Cell Culture, Expressing, Two Tailed Test

Journal: medRxiv

Article Title: Aberrant naive CD4+ T Cell differentiation in systemic juvenile idiopathic arthritis is committed to B cell help

doi: 10.1101/2022.02.01.22270100

Figure Lengend Snippet: SJIA naïve CD4+ T helper (Th) cell differentiation towards Th1 results in low IFNγ and Eomesodermin expression. A , Naive Th cells were isolated from pediatric healthy controls (HC, n=7), active disease (AD, n=5), and inactive disease (ID, n=8) sJIA patients (see ) and cultured under T0 (anti-CD3/anti-CD28) and different Th1 and Th17 polarizing conditions. IL-17A and IL-18 were included in Th1 conditions to somewhat mimic a sJIA-cytokine milieu. Following six days of culture and super-stimulation with phorbol 12- myristate 13-acetate/ionomycin (4h), cells were analyzed by flow cytometry. B , Heatmap of z-scores indicating T-bet, RORγt, IL-17A, IFNγ, and Eomesodermin (Eomes) expression (geometric mean fluorescence intensity (MFI) normalized to unstimulated samples) in cells arising from the indicated culture conditions and following super-stimulation as described. C - E , IFNγ (left panels), Eomes (center panels), and T-bet (right panels) expression (MFI), as well as ( F - H) correlation of IFNγ with Eomes (left panels) or T-bet (right panels) expression (MFI) in cells as in B is shown. B - H , Each data point represents a value derived from an individual patient or HC. C – E , Lines indicate median values, data were analyzed by Kruskal-Wallis test followed by Dunn’s post hoc test; * = P < 0.05, ** = P <0.01. F - H, Simple linear regression (OLS) and F-test; r s (r-squared value) and P -values are indicated.

Article Snippet: Naïve Th cells were isolated from PBMCs by immunomagnetic negative selection using the EasySep TM Human Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies Inc., Cologne, Germany) according to the manufacturer’s instruction.

Techniques: Cell Differentiation, Expressing, Isolation, Cell Culture, Flow Cytometry, Fluorescence, Derivative Assay

Journal: medRxiv

Article Title: Aberrant naive CD4+ T Cell differentiation in systemic juvenile idiopathic arthritis is committed to B cell help

doi: 10.1101/2022.02.01.22270100

Figure Lengend Snippet: SJIA naïve CD4+ T helper (Th) cell differentiation is not skewed toward a T h 17 fate or high IL-17A expression. A , Naive Th cells were isolated from pediatric healthy controls (HC, n=7), active disease (AD, n=5), and inactive disease (ID, n=8) sJIA patients (see ) and cultured under T0 (anti-CD3/anti-CD28) and different Th17 polarizing conditions. Following six days of culture and super-stimulation with phorbol 12-myristate 13- acetate/ionomycin (4h), cells were analyzed by flow cytometry. B , Heatmap of z-scores indicating T-bet, RORγt, IL-17A, IFNγ, and Eomesodermin (Eomes) expression (geometric mean fluorescence intensity (MFI) normalized to unstimulated samples) in cells arising from the indicated culture conditions and following super-stimulation as described. C , IL-17A expression (MFI) in cells as in B is shown. B and C , Each data point represents a value derived from an individual patient or HC. C , Lines in scattered dot plots indicate median values, data were analyzed by Kruskal-Wallis test followed by Dunn’s post hoc test.

Article Snippet: Naïve Th cells were isolated from PBMCs by immunomagnetic negative selection using the EasySep TM Human Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies Inc., Cologne, Germany) according to the manufacturer’s instruction.

Techniques: Cell Differentiation, Expressing, Isolation, Cell Culture, Flow Cytometry, Fluorescence, Derivative Assay

Journal: medRxiv

Article Title: Aberrant naive CD4+ T Cell differentiation in systemic juvenile idiopathic arthritis is committed to B cell help

doi: 10.1101/2022.02.01.22270100

Figure Lengend Snippet: SJIA naïve CD4+ T helper (Th) cell differentiation towards Th1 results in low IFNγ and Eomesodermin expression. Naive Th cells were isolated from pediatric healthy controls (HC, n=7), active disease (AD, n=5), and inactive disease (ID, n=8) sJIA patients (see ) and cultured under T0 (anti-CD3/anti-CD28) and different Th1 and Th17 polarizing conditions. Following six days of culture and super-stimulation with phorbol 12- myristate 13-acetate/ionomycin (4h), cells were analyzed by flow cytometry (see ). IFNγ, Eomes, and T-bet expression (geometric mean fluorescence intensity (MFI) normalized to unstimulated samples), as well as correlation of IFNγ with Eomes or T-bet expression (MFI) in cells cultured under indicated conditions. Each data point represents a value derived from an individual patient or HC. Lines in scatter plots indicate median values, data were analyzed by Kruskal-Wallis test followed by Dunn’s post hoc test; * = P < 0.05. Correlation data were analyzed by simple linear regression (OLS) and F-test; r s (r-squared value) and P -values are indicated.

Article Snippet: Naïve Th cells were isolated from PBMCs by immunomagnetic negative selection using the EasySep TM Human Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies Inc., Cologne, Germany) according to the manufacturer’s instruction.

Techniques: Cell Differentiation, Expressing, Isolation, Cell Culture, Flow Cytometry, Fluorescence, Derivative Assay

Journal: medRxiv

Article Title: Aberrant naive CD4+ T Cell differentiation in systemic juvenile idiopathic arthritis is committed to B cell help

doi: 10.1101/2022.02.01.22270100

Figure Lengend Snippet: SJIA naïve CD4+ T helper (Th) cell differentiation towards Th1 is hallmarked by IL-21 release in negative association with IFNγ and Eomesodermin expression. A , Naive Th cells were isolated from pediatric healthy controls (HC, n=7), active disease (AD, n=5), and inactive disease (ID, n=8) sJIA patients (see ) and cultured under T0 (anti-CD3/anti-CD28) and different Th1 and Th17 polarizing conditions. IL-17A and IL-18 were included to mimic a sJIA-cytokine milieu. On day six of culture, cytokine release (IFNγ, IL-17A, IL-21, IL-22) into culture supernatants following phorbol 12-myristate 13-acetate/ionomycin super-stimulation was analyzed by multiplexed bead array assay. B, Heatmaps of IFNγ (left panel) and IL-21 (right panel) release (pg/ml) by naive CD4+ cells following culture under the indicated T0, Th1, and Th17 polarizing conditions and super-stimulation as described. C - E , IFNγ (left panels) and IL-21 (middle panels) release and IFNγ−IL-21 correlation (right panels) of naive CD4+ cells cultured under the indicated Th0 and Th1 polarizing conditions and following super-stimulation. Data is shown in scatter dot plots of the measured cytokine concentrations in pg/ml. The triangular data point refers to a patient who developed MAS subsequent to the analysis. F , Correlations of IL-21 release (pg/ml) with intracellular IFNγ (left panel), Eomesodermin (Eomes) (middle panel) and T-bet (right panel) expression (geometric mean fluorescence intensities (MFIs) normalized to unstimulated samples) by cells cultured under the indicated Th1 polarizing condition and super-stimulated. G , Correlation of IL-21 release (pg/ml) and intracellular IL-17A (left panel) and RORγt (right panel) expression (MFI) by naïve CD4+ Th cells upon culture under the indicated Th17 polarizing condition and following super-stimulation. B - G , Each data point presents a value derived from an individual patient or HC. C – E, Lines indicate median values. Data in left and middle panels were analyzed by Kruskal-Wallis followed by Dunn’s post hoc test; * = P < 0.05. C - G , All correlation data were analyzed for simple linear regression (OLS) and F-test; r s (r-squared value) and P -values are indicated.

Article Snippet: Naïve Th cells were isolated from PBMCs by immunomagnetic negative selection using the EasySep TM Human Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies Inc., Cologne, Germany) according to the manufacturer’s instruction.

Techniques: Cell Differentiation, Expressing, Isolation, Cell Culture, Fluorescence, Derivative Assay

Journal: medRxiv

Article Title: Aberrant naive CD4+ T Cell differentiation in systemic juvenile idiopathic arthritis is committed to B cell help

doi: 10.1101/2022.02.01.22270100

Figure Lengend Snippet: Developing CD4+ T helper type 1 (T h 1) and type 17 (T h 17) cells in systemic juvenile idiopathic arthritis (sJIA) do not release elevated levels of IL-17A or IL-22. A - C , Naive Th cells were isolated from pediatric healthy controls (HC, n=7), active disease (AD, n=5), and inactive disease (ID, n=8) sJIA patients (see ) and cultured under T0 (anti-CD3/anti-CD28) and different Th1 and Th17 polarizing conditions as indicated. On day six of culture, cytokine release (IFNγ, IL-17A, IL-21, IL-22) into culture supernatants following phorbol 12-myristate 13-acetate/ionomycin super-stimulation was analyzed by multiplexed bead array assay. A , Heatmaps of IL-17A (left panel) and IL-22 (right panel) release (pg/ml) by naive CD4+ cells following culture under the indicated T0, Th1, and Th17 polarizing conditions and super-stimulation as described. B , IFNγ release following Th0 polarization and super-stimulation with one patient developing MAS subsequent to the analyses excluded from the data set. C , IL-21 (left panels) and IL-17A (right panels) release by naive CD4+ cells cultured under the indicated Th17 polarizing conditions and following super-stimulation. Data is shown in scatter dot plots of the measured cytokine concentrations in pg/ml. A - C , Each data point presents a value derived from an individual patient or HC. B and C, Lines indicate median values. Data were analyzed by Kruskal-Wallis followed by Dunn’s post hoc test; * = P < 0.05.

Article Snippet: Naïve Th cells were isolated from PBMCs by immunomagnetic negative selection using the EasySep TM Human Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies Inc., Cologne, Germany) according to the manufacturer’s instruction.

Techniques: Isolation, Cell Culture, Derivative Assay

Journal: medRxiv

Article Title: Aberrant naive CD4+ T Cell differentiation in systemic juvenile idiopathic arthritis is committed to B cell help

doi: 10.1101/2022.02.01.22270100

Figure Lengend Snippet: IL-21 restricts IFNγ release by developing CD4+ T helper type 1 (Th1) cells derived from naïve adult healthy donor CD4+ T cells. Naïve Th cells were isolated from adult healthy donors (HD, n=18) and cultured under Th1 polarizing conditions in the absence or presence of recombinant human IL-21. IL-18 was included to somewhat mimic the sJIA-cytokine environment. On day six of culture, IFNγ release into culture supernatants following phorbol 12-myristate 13-acetate/ionomycin super-stimulation was analyzed by ELISA. Scatter dot plots show IFNγ release in pg/ml by naive HC CD4+ cells cultured under the indicated Th1 polarizing conditions (left: n=11, right: n=18) in the absence (wo) and presence (+IL-21) of IL-21 and following super-stimulation as described. Each data point presents a value derived from an individual HD and data were analyzed by Wilcoxon signed-rank test; ** = P < 0.01, **** = P <0.0001.

Article Snippet: Naïve Th cells were isolated from PBMCs by immunomagnetic negative selection using the EasySep TM Human Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies Inc., Cologne, Germany) according to the manufacturer’s instruction.

Techniques: Derivative Assay, Isolation, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: medRxiv

Article Title: Aberrant naive CD4+ T Cell differentiation in systemic juvenile idiopathic arthritis is committed to B cell help

doi: 10.1101/2022.02.01.22270100

Figure Lengend Snippet: Early CD4+ T helper (Th) cell differentiation in systemic juvenile idiopathic arthritis (sJIA) is shifted toward a (PD-1+ICOS+CXCR5-) peripheral T helper (Tph) cell phenotype. A , Naïve Th cells were isolated from pediatric healthy controls (HC, n=5), active disease (AD, n=3), and inactive disease (ID, n=4) sJIA patients (see ) were cultured under T0, Th1, and T follicular helper (Tfh) / peripheral T helper (Tf/ph) polarizing conditions. Following six days of culture and super-stimulation with phorbol 12-myristate 13- acetate/ionomycin (4h), cells were analyzed by flow cytometry. B , Heatmap of z-scores indicating ICOS, PD-1, IL-21, CXCR5, and IFNγ expression (geometric mean fluorescence intensity (MFI) normalized to unstimulated samples) by naïve CD4+ T cells arising from the indicated Th0, Th1, and Tf/ph polarizing conditions and following super-stimulation. C , Multiple correlation analyses of IFNγ, IL-21, PD-1, ICOS, and CXCR5 expression (MFI)as shown in B . Spearman’s rank correlation coefficients are indicated. D , IFNγ, IL-21, PD-1, and ICOS expression (MFI, left and middle columns) and correlation of IL-21 and IFNγ or PD-1 expression (MFI, right columns) by naïve CD4+ T cells cultured under the indicated Th0 (left panel) and Tf/ph polarizing (right panel) conditions and following super-stimulation. B - D , Each data point presents a value derived from an individual patient or HC. D , Lines indicate median values. Data in left and middle columns were analyzed by Kruskal-Wallis followed by Dunn’s post hoc test; * = P < 0.05. Correlation data were analyzed by simple linear regression (OLS) and F-test; r s (r-squared value) and P -values are indicated.

Article Snippet: Naïve Th cells were isolated from PBMCs by immunomagnetic negative selection using the EasySep TM Human Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies Inc., Cologne, Germany) according to the manufacturer’s instruction.

Techniques: Cell Differentiation, Isolation, Cell Culture, Flow Cytometry, Expressing, Fluorescence, Derivative Assay

Journal: medRxiv

Article Title: Aberrant naive CD4+ T Cell differentiation in systemic juvenile idiopathic arthritis is committed to B cell help

doi: 10.1101/2022.02.01.22270100

Figure Lengend Snippet: Early CD4+ T helper (Th) cell differentiation in systemic juvenile idiopathic arthritis (sJIA) is shifted toward a (PD-1+ICOS+CXCR5-) peripheral T helper (Tph) cell phenotype. Naïve Th cells were isolated from pediatric healthy controls (HC, n=5), active disease (AD, n=3), and inactive disease (ID, n=4) sJIA patients (see ) were cultured under T0, Th1, and T follicular helper (Tfh) / peripheral T helper (Tf/ph) polarizing conditions. Following six days of culture and super-stimulation with phorbol 12-myristate 13- acetate/ionomycin (4h), cells were analyzed by flow cytometry. IFNγ, IL-21, PD-1, and ICOS expression (MFI, left and middle columns) and correlation of IL-21 and IFNγ or PD-1 expression (MFI, right columns) by naïve CD4+ T cells cultured under the indicated conditions and following super-stimulation. Each data point presents a value derived from an individual patient or HC. Lines in scatter dot plots indicate median values. Data in left and middle columns were analyzed by Kruskal-Wallis followed by Dunn’s post hoc test; * = P < 0.05. Correlation data were analyzed by simple linear regression (OLS) and F-test; r s (r-squared value) and P -values are indicated.

Article Snippet: Naïve Th cells were isolated from PBMCs by immunomagnetic negative selection using the EasySep TM Human Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies Inc., Cologne, Germany) according to the manufacturer’s instruction.

Techniques: Cell Differentiation, Isolation, Cell Culture, Flow Cytometry, Expressing, Derivative Assay

Journal: medRxiv

Article Title: Aberrant naive CD4+ T Cell differentiation in systemic juvenile idiopathic arthritis is committed to B cell help

doi: 10.1101/2022.02.01.22270100

Figure Lengend Snippet: Developing CD4+ T helper type 1 (Th1) cells in systemic juvenile idiopathic arthritis (sJIA) release elevated levels of IL-21 and CXCL13. Supernatants of naïve Th cells isolated from pediatric healthy controls (HC, n=4), active disease (AD, n=4), and inactive disease (ID, n=5) sJIA patients (see ) cultured under indicated Th1 and T follicular helper (Tfh) / peripheral T helper (Tf/ph) polarizing conditions (6d) and super-stimulation with phorbol 12-myristate 13-acetate/ionomycin (4h) were analyzed by multiplexed bead array assay for IL-21 and CXCL13 expression. Data is shown in scatter dot plots of the measured cytokine expression in pg/ml. Each data point presents a value derived from an individual patient or HC. Lines indicate median values and data were analyzed by Kruskal-Wallis test followed by Dunn’s post hoc test; * = P < 0.05, ** = P <0.01.

Article Snippet: Naïve Th cells were isolated from PBMCs by immunomagnetic negative selection using the EasySep TM Human Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies Inc., Cologne, Germany) according to the manufacturer’s instruction.

Techniques: Isolation, Cell Culture, Expressing, Derivative Assay

Journal: medRxiv

Article Title: Aberrant naive CD4+ T Cell differentiation in systemic juvenile idiopathic arthritis is committed to B cell help

doi: 10.1101/2022.02.01.22270100

Figure Lengend Snippet: Circulating CD4+ T helper (cTh) cells in systemic juvenile idiopathic arthritis (sJIA) reveal a T follicular helper (Tfh) / peripheral T helper (Tph) cell signature. A , IL-21 (left), IL-6 (middle), and IL-23 (right) serum levels in pediatric healthy controls (HC, n=10), active disease (AD, n=27), and inactive disease (ID, n=9) sJIA patients (see ). Data is shown in violin plots of the measured cytokine concentrations in pg/ml. B, Flow cytometry analyses of IFNγ and IL-21 in indicated cTh cell subsets in peripheral blood mononuclear cells (PBMCs) isolated from pediatric HCs (n=5), AD (n=3), and ID (n=4) sJIA patients (see ) following super-stimulation with phorbol 12-myristate 13-acetate/ionomycin (4h). C , Correlation of IL-21 with CXCR5 (left panel) and PD-1 expression (geometric mean fluorescence intensities (MFIs) normalized to unstimulated samples, right panels) in cTh cells. D , Heatmap of unsupervised clustering using correlation distance and ward.D linkage of indicated gene expression in whole blood RNA sequencing data (GSE112057) from HC (n=12) as well as sJIA (n=26), polyarticular (n=46) (polyJIA) and oligoarticular JIA (oligoJIA) patients (n=43). E and F , Expression of SJIA-hallmark genes ( E , IL1B , IL1R1 , IL1RN , S100A12 ) as well as ( F ) reported T cell associated genes IFNG , TNFSF13B ) (T effector memory), BCL6 and FOS (Tfh) as in D depicted in violin plots as the relative difference in expression of the respective genes. B - D , Each data point presents a value derived from an individual patient or HC. A , B , E , and F , Lines indicate median values. Data were analyzed by Kruskal-Wallis followed by Dunn’s post hoc test; * = P <0.05, ** = P <0.01, *** = P <0.001, **** = P <0.0001. C , Simple linear regression (OLS) and F-test; r s (r-squared value) and P -values are indicated.

Article Snippet: Naïve Th cells were isolated from PBMCs by immunomagnetic negative selection using the EasySep TM Human Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies Inc., Cologne, Germany) according to the manufacturer’s instruction.

Techniques: Flow Cytometry, Isolation, Expressing, Fluorescence, RNA Sequencing Assay, Derivative Assay

Journal: medRxiv

Article Title: Aberrant naive CD4+ T Cell differentiation in systemic juvenile idiopathic arthritis is committed to B cell help

doi: 10.1101/2022.02.01.22270100

Figure Lengend Snippet: T follicular helper / peripheral T helper cells (Tf/ph) derived from sJIA naïve CD4+ T helper (Th) cells can drive enhanced B cellular plasma blast generation . A, Naïve Th cells were isolated from pediatric healthy controls (HC, n=4), active disease (AD, n=2), and inactive disease (ID, n=3) sJIA patients (see ) and cultured under T0, Th1, and Tf/ph polarizing conditions. Following six days of culture polarized Th cells were co-cultured with allogenic HC memory B cells isolated from a single healthy donor and the generation of plasma blasts was assessed by flow cytometry following another six days of culture. B-D , Representative flow cytometry pseudo-color ( B, C ) or histogram plots (D) to monitor plasma blast (CD19+IgD-CD27+CD38+CD138-) generation from co-cultured memory B cells. E , Frequency of plasma blasts in co-cultures with Th cells polarized under the indicated Th0, Th1, and Tf/ph polarizing conditions. F and G , Relative change in the frequency of plasma blasts in co-cultures with Th cells cultured under the indicated Th1 (F) and Tf/ph (G) polarizing conditions, normalized to the Th0. E-G , Each data point presents a value derived from an individual patient or HC. F and G, Lines indicate median values. Data were analyzed by Mann-Whitney U test; * = P <0.05.

Article Snippet: Naïve Th cells were isolated from PBMCs by immunomagnetic negative selection using the EasySep TM Human Naïve CD4+ T Cell Isolation Kit II (STEMCELL Technologies Inc., Cologne, Germany) according to the manufacturer’s instruction.

Techniques: Derivative Assay, Isolation, Cell Culture, Flow Cytometry, MANN-WHITNEY