Journal: Journal for Immunotherapy of Cancer

Article Title: Phenotypic and functional testing of circulating regulatory T cells in advanced melanoma patients treated with neoadjuvant ipilimumab

doi: 10.1186/s40425-016-0141-1

Figure Lengend Snippet: Association between the change in peripheral blood Treg frequency and Treg suppressive function (change in % Treg proliferation after the treatment) across different Treg:Th ratios. The change (week 6 level – baseline level) in circulating Treg was measured phenotypically as CD4 + /CD25 hi /Foxp3 + (Diff CD4 + CD29 hi Foxp3 + , left column), CD4 + /CD25 hi /CD39 (Diff CD4 + CD25 + CD39 + , middle column), or the percent of circulating Treg (Diff %Treg in CD4 + Cells, right column). The percent suppressive activity, calculated as the week 6 % Treg inhibition – baseline % Treg function, at 1:1 (Diff 1:1, top row), 1:2 (Diff 1:2, middle row), or 1:5 (Diff 1:5, bottom row) was plotted against the frequency of Treg across all patients with valid test results. There was no statistically significant association (p values on each part). Fewer than 18 data points were generated due to missing values on some patients

Article Snippet: The Miltenyi Biotec Treg isolation (CD4 + CD25 + CD127 dim/- ) kit was used to purify Treg according to manufacturer’s instructions.

Techniques: Activity Assay, Inhibition, Generated

Journal: Journal for Immunotherapy of Cancer

Article Title: Phenotypic and functional testing of circulating regulatory T cells in advanced melanoma patients treated with neoadjuvant ipilimumab

doi: 10.1186/s40425-016-0141-1

Figure Lengend Snippet: Association between circulating Treg frequency and Treg function (% Treg function, pre- and post-treatment). Baseline phenotypic (baseline circulating Treg cells) and suppressive function at each ratio (baseline %Treg function; top row), and post treatment measures (bottom row) were tested across patients for correlations. There was no significant association (p values on each part). Fewer than 18 data points were generated due to missing values on some patients

Article Snippet: The Miltenyi Biotec Treg isolation (CD4 + CD25 + CD127 dim/- ) kit was used to purify Treg according to manufacturer’s instructions.

Techniques: Generated

Journal: Journal for Immunotherapy of Cancer

Article Title: Phenotypic and functional testing of circulating regulatory T cells in advanced melanoma patients treated with neoadjuvant ipilimumab

doi: 10.1186/s40425-016-0141-1

Figure Lengend Snippet: Association between reduced progression free survival with increase in circulating Treg. The patients were divided into two groups: those with % inhibition increased and those with % inhibition decreased. We defined the group by taking a majority of the three ratios. That is, if the results of at least 2 of the 3 ratios indicated a higher % inhibition at week 6 than at baseline, the patient was included in the WK6 > BL group, and if the results of at least 2 of the 3 ratios indicated a lower % inhibition at week 6 than at baseline, then the patient was included in the WK6 < BL group. Suppressive activity over time is shown in a Kaplan-Meier plot. Increase in Treg suppressive function was significantly associated with a decrease in PFS ( p = 0.02381)

Article Snippet: The Miltenyi Biotec Treg isolation (CD4 + CD25 + CD127 dim/- ) kit was used to purify Treg according to manufacturer’s instructions.

Techniques: Inhibition, Activity Assay

Journal: Clinical and Experimental Immunology

Article Title: The influence of bone marrow-and synovium-derived mesenchymal stromal cells from osteoarthritis patients on regulatory T cells in co-culture

doi: 10.1111/cei.12122

Figure Lengend Snippet: (a) CD4+CD25+CD127− Treg percentages of total CD4+ T cells at d0. Magnetic separation of CD4+CD25+CD127− cells was performed with each circle representing a single culture. Percentages varied from less than 20% to over 70%. (b) Gating strategy for regulatory T-cells (CD127−/FoxP3+).

Article Snippet: PBMC were then separated into a mixture of CD4 + CD25 – and CD4 + CD25 + CD127 – cells using magnetic separation (CD4 + CD25 + CD127 dim/– regulatory T cell Isolation Kit II, LS and LD columns, MidiMACSTM separator, all from Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques:

Journal: BMC Bioinformatics

Article Title: The development of a comparison approach for Illumina bead chips unravels unexpected challenges applying newest generation microarrays

doi: 10.1186/1471-2105-10-186

Figure Lengend Snippet: Technical replication on subsequent array versions . (A) Experimental analysis for the T reg data set: FACS analysis and sorting windows of CD4 + CD127 low CD25 + T reg cells and CD4 + CD127 + CD25 - T conv cells ( left ). Expression of FOXP3 in the respective T cell subsets was assessed by flow cytometry ( middle ) and quantitative RT-PCR ( right ). Boxplots were used to compare the dynamic range of signal intensities on the arrays for (B) the T reg data set and (C) the whole blood data set. Only signals for the 8299 identical oligonucleotides were used. Technical replicates were checked both by principle component analysis based on the 100 most variable genes for (D) the T reg data set and (E) the whole blood data set as well as hierarchical cluster analysis (see Additional file ).

Article Snippet: CD4 + CD127 low CD25 + (T reg ) and CD4 + CD127 + CD25 - (T conv ) T cells were stained with CD4, CD25 and CD127 mAb (all from BD Pharmingen) and sorted on a FACSDiva cell sorter.

Techniques: Expressing, Flow Cytometry, Quantitative RT-PCR

Journal: Oncoimmunology

Article Title: Zoledronic acid inhibits NFAT and IL-2 signaling pathways in regulatory T cells and diminishes their suppressive function in patients with metastatic cancer

doi: 10.1080/2162402X.2017.1338238

Figure Lengend Snippet: PBMC from cancer patients (n = 10) were stained and analyzed by flow cytometry for; A) Treg (CD3+CD4+CD127−CD25+FOXP3+ of lymphocytes), B) NK cell (CD3−CD56+ of lymphocytes), γδ T cell (CD3+γδ+), and Conventional T cell (CD3+FOXP3−) frequencies before and after ZA treatment. Statistical analyses were performed on pooled data using Student's t test.

Article Snippet: Alternatively, Treg were sorted on a BD FACSJazz CD4 + CD127 − CD25 high (>95%) after CD4 enrichment (Miltenyi Biotech).

Techniques: Staining, Flow Cytometry

Journal: Oncoimmunology

Article Title: Zoledronic acid inhibits NFAT and IL-2 signaling pathways in regulatory T cells and diminishes their suppressive function in patients with metastatic cancer

doi: 10.1080/2162402X.2017.1338238

Figure Lengend Snippet: A) Enriched Treg from healthy blood donors (n = 6) were stimulated with IL-2 in the presence or absence of ZA for 3 days and analyzed for the expression of CD25 by flow cytometry. A representative histogram and accumulated data mean ± SEM are shown and statistical analysis was performed by Wilcoxon test. Each symbol represents a distinct healthy individual. B) Enriched Treg from healthy blood donors (n = 3) were stimulated with and without IL-2 in the presence or absence of ZA for 15, 30, and 90 minutes and analyzed for the expression of phosphorylated STAT5 by flow cytometry. Representative histograms and accumulated data mean ± SEM are shown and statistical analysis was performed by Two-way ANOVA. **p = 0.004, *p = 0.01. C) Enriched or sorted Treg (n = 6) were overnight stimulated with IL-2 and OKT3 in the presence or absence of ZA and stained for total TGFβ. A representative histogram and accumulated data are shown and statistical analyses were performed by Wilcoxon test.

Article Snippet: Alternatively, Treg were sorted on a BD FACSJazz CD4 + CD127 − CD25 high (>95%) after CD4 enrichment (Miltenyi Biotech).

Techniques: Expressing, Flow Cytometry, Staining

Journal: Oncoimmunology

Article Title: Zoledronic acid inhibits NFAT and IL-2 signaling pathways in regulatory T cells and diminishes their suppressive function in patients with metastatic cancer

doi: 10.1080/2162402X.2017.1338238

Figure Lengend Snippet: A) PBMC from cancer patients (n = 6) were stained and analyzed by flow cytometry for conventional (CD3+FOXP3−) T cell and NK cell proliferation (Ki67-positive cells) pre and one week post ZA administration. B) Correlation between percent circulating Treg and NK and T cell proliferation (Ki67). Statistical analyses were performed on pooled data using A) Wilcoxon test, and B) Pearson's correlation. PBMC from cancer patients (n = 10) were stained and analyzed by flow cytometry for the expression of C) FOXP3 and D) CD25. Representative histograms and pooled data are shown and Statistical analyses were performed using Student's t test.

Article Snippet: Alternatively, Treg were sorted on a BD FACSJazz CD4 + CD127 − CD25 high (>95%) after CD4 enrichment (Miltenyi Biotech).

Techniques: Staining, Flow Cytometry, Expressing

Journal: Allergy

Article Title: Butyrate as a bioactive human milk protective component against food allergy

doi: 10.1111/all.14625

Figure Lengend Snippet: Effects of HM butyrate in basal condition on gut barrier and on cytokine response. Four‐week‐old female C3H/HeJ mice were divided into two groups: (1) control mice (n = 4), receiving water as vehicle; (2) treated mice (n = 4), receiving oral butyrate once daily (30 mg/kg of body weight). After 14 d all mice were killed, and intestine, in particular ileum, jejunum, and colon, spleen, and mesenteric lymph nodes (MLN) were removed. A‐E, Oral butyrate treatment elicited a significant reduction in gut permeability, measured by plasma FITC‐dextran quantification, and increase in IL‐22, occludin, ZO‐1, and Muc2 expression. F‐L, Oral butyrate treatment induced a significant reduction in IL‐4, IL‐5, and IL‐13 and a significant increase in IFN‐γ and IL‐10 expression in MLN and spleen. M‐N, CD4+/CD25+/FoxP3+ cell numbers were increased after butyrate treatment in mouse spleen MLN and colon. Data are representative of at least 2 independent experiments, reported as median with range, and analyzed using the unpaired t test. * P < .05

Article Snippet: Specifically, the staining and enumeration of CD4+CD25+CD127 dim/ⁿeg Tregs were performed by using the Treg Detection Kit (CD4/CD25/CD127) (Miltenyi Biotec), according to the manufacturer's instructions.

Techniques: Permeability, Expressing

Journal: Allergy

Article Title: Butyrate as a bioactive human milk protective component against food allergy

doi: 10.1111/all.14625

Figure Lengend Snippet: Evaluation of direct immunoregulatory action of HM butyrate on human peripheral blood mononuclear cells (PBMCs): Th2/Th1 and IL‐10 response and evaluation of FoxP3 expression. A, PBMCs from children with IgE‐mediated FA were stimulated with butyrate HM median concentration (0.75 mM) or with BLG, OVA, and PN (100 μL/well) in the presence or in the absence of butyrate HM median concentration for 24 h. BLG, OVA, and PN induced a significant increase in IL‐4 (A), IL‐5, and IL‐13 (C) production, but the presence of 0.75 mM butyrate significantly reduced the release of these Th2 cytokines. Butyrate alone or in the presence of specific allergen stimulated, at the same dose, IL‐10 (D) and IFN‐γ (E) production and FoxP3 expression (F), through a demethylation of respective gene (G, H, I) in CD4+ T cells purified from stimulated PBMCs from children with IgE‐mediated FA. (L) Butyrate treatment significantly reduced HDAC activity in a dose‐dependent manner in PBMCs. Data represent the median with range of 2 independent experiments, each performed in triplicate. Data were analyzed using the unpaired t test. BLG, β‐lactoglobulin; NT, untreated cells; OVA, ovalbumin; PN, peanut extracts. * P < .05; ** P < .01

Article Snippet: Specifically, the staining and enumeration of CD4+CD25+CD127 dim/ⁿeg Tregs were performed by using the Treg Detection Kit (CD4/CD25/CD127) (Miltenyi Biotec), according to the manufacturer's instructions.

Techniques: Expressing, Concentration Assay, Purification, Activity Assay

Journal: Allergy

Article Title: Butyrate as a bioactive human milk protective component against food allergy

doi: 10.1111/all.14625

Figure Lengend Snippet: Evaluation of direct immunoregulatory action of HM butyrate on human peripheral blood mononuclear cells (PBMCs): modulation of mono‐dendritic cell expression. PBMCs from children with IgE‐mediated FA were stimulated with butyrate HM median concentration (0.75 mM) or with specific allergen (BLG, OVA, PN) (100 μL/well of each allergen) in the presence or in the absence of butyrate for 48 h (A‐B). CD45hi CD14+ monocytes were stained with flow cytometric markers CD206+ or CD86+ to discern the CD206 M2 type from CD86 M1 type of total monocytes. A representative dot plot panel (left panel) is presented. The graph on the right reports the mean ± SD of ratios of M1 vs M2 peripheral monocytes (CD86/CD206 ratio) from different FA patients (ANOVA; *** P < .001 vs CTRL) (B). Purified CD14+ monocytes from IgE‐mediated FA were cultured with regular medium containing GM‐CSF (50 ng/mL) and IL‐4 (1000 U/mL), with or without butyrate HM median concentration (0.75 mM). After 7 d, cells were collected, stained with the reported antibodies, and analyzed by flow cytometry (C‐E). Representative example of dot plots of iDCs generated in the presence or in the absence of butyrate (C). The bars graph reports mean and standard deviation of % of CD14‐CD1a+ from independent experiments using different IgE‐mediated FA (ANOVA; *** P < .001 vs CTRL). (D). Immature DCs were collected and exposed to LPS‐activating stimulus (1 μg/mL). After 24 h, cells were stained for CD86 maturation marker. Bar graphs report the percentage of positive cells for the indicated markers in gated viable CD14‐CD1a+ cells (ANOVA; *** P < .001 vs CTRL) (E). Phenotypical characterization of iDCs generated in the presence or in the absence of 0.75 mM of butyrate for 7 d in healthy subjects (left panel) and in IgE‐mediated FA (right panel). Bar graph reports mean and standard deviation of % of CD11b+ CD103+ from independent experiments using IgE‐mediated FA (ANOVA; *** P < .001 vs CTRL). (F‐G). Butyrate‐DC had no effect on regulatory cell differentiation. Naïve CD4+ T cells from IgE‐mediated FA were cultured with immature or mature DCs generated in the presence of butyrate at 1:10 DC:T ratio. After being cultured for 5 d, cells were collected. CD4+CD25+CD127 dim/ⁿeg Tregs were identified and enumerated with a flow cytometer (H). The bar graphs report the percentage of positive cells for the indicated markers

Article Snippet: Specifically, the staining and enumeration of CD4+CD25+CD127 dim/ⁿeg Tregs were performed by using the Treg Detection Kit (CD4/CD25/CD127) (Miltenyi Biotec), according to the manufacturer's instructions.

Techniques: Expressing, Concentration Assay, Staining, Purification, Cell Culture, Flow Cytometry, Generated, Standard Deviation, Marker, Cell Differentiation

Journal: Allergy

Article Title: Butyrate as a bioactive human milk protective component against food allergy

doi: 10.1111/all.14625

Figure Lengend Snippet: Evaluation of direct immunoregulatory action of HM butyrate on human peripheral blood mononuclear cells (PBMCs): effect on CD4+ T‐cell subsets. Butyrate directly affects the Treg generation. Naïve CD4+ T cells were collected from healthy controls and from IgE‐mediated FA children and were maintained in culture with or without 0.75 mM of butyrate. After 5 d, cells were collected. CD4+CD25+CD127 dim/neg Tregs were identified and enumerated with a flow cytometer. The bar graphs on the right report the percentage of positive cells for the indicated markers (ANOVA; * P < .05 vs CTRL) (A‐B). Butyrate‐DC had an effect on TCD4+ cell differentiation. Naïve CD4+ T cells from IgE‐mediated FA patients and healthy controls were cultured with DCs generated in the presence of butyrate at 1:10 DC:T ratio. After 5 d, cells were collected and stained with the indicated markers prior to FACS analysis. CD45RA and CCR7 markers allow the definition of most peculiar subsets among CD4+ T‐cell compartment including effector memory (TEM; CD45RA−CCR7), effector memory RA (TEMRA; CD45RA+CCR7−), naïve (TN; CD45RA+CCR7+), and central memory (TCM; CD45RA‐CCR7+) T cells (C‐E). Representative example of dot plots of TCD4+ cells from control and IgE‐mediated FA subjects after autologous T:DC co‐culture (D‐E). The bar graphs report the percentage of positive cells for the indicated markers in CD4+ gated viable cells (ANOVA; * P < .05, ** P < .01, *** P < .001 vs CTRL)

Article Snippet: Specifically, the staining and enumeration of CD4+CD25+CD127 dim/ⁿeg Tregs were performed by using the Treg Detection Kit (CD4/CD25/CD127) (Miltenyi Biotec), according to the manufacturer's instructions.

Techniques: Flow Cytometry, Cell Differentiation, Cell Culture, Generated, Staining, Co-Culture Assay