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mLN‐iFRCs possess superior de novo Treg‐inducing capacity. (A) FRCs were ex vivo isolated from skin‐draining pLNs and gut‐draining mLNs and immortalized via lentiviral transduction. After doxycycline‐induced expansion, pLN‐ and mLN‐iFRCs were phenotyped by flow cytometry. Representative histograms depict expression of gp38 among CD45 − CD24 − CD31 − iFRCs originating from mLN and pLN. Histogram is from a single experiment representative of three independent experiments with one mLN‐ and one pLN‐iFRCs per experiment. (B, C) CTV‐labeled naïve CD4 + T cells from Foxp3 hCD2 xRag2 ‐/‐ xDO11.10 mice were added to iFRCs (B) or iFRC‐derived SNs (C) in presence of IL‐2 and anti‐CD3/CD28 Dynabeads. Four days later, the frequency of de novo induced Foxp3 + cells and CD4 + T cell <t>proliferation</t> was determined by flow cytometry, as shown in representative dot plots from a single experiment representative of seven (B) or six (C) independent experiments with one mLN‐ and one pLN‐iFRCs per experiment. Numbers in gates indicate frequencies. Each dot within scatterplot depicts mean of technical replicates, and each line represents mean of depicted dots within respective groups. Data were pooled from seven (B) or six (C) independent experiments with one mLN‐ and one pLN‐iFRCs per experiment; p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; **** p < 0.0001. med, medium.

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1) Product Images from "Mesenteric lymph node stromal cell‐derived extracellular vesicles contribute to peripheral de novo induction of Foxp3 + regulatory T cells"

Article Title: Mesenteric lymph node stromal cell‐derived extracellular vesicles contribute to peripheral de novo induction of Foxp3 + regulatory T cells

Journal: European Journal of Immunology

doi: 10.1002/eji.201746960

Figure Legend Snippet: mLN‐iFRCs possess superior de novo Treg‐inducing capacity. (A) FRCs were ex vivo isolated from skin‐draining pLNs and gut‐draining mLNs and immortalized via lentiviral transduction. After doxycycline‐induced expansion, pLN‐ and mLN‐iFRCs were phenotyped by flow cytometry. Representative histograms depict expression of gp38 among CD45 − CD24 − CD31 − iFRCs originating from mLN and pLN. Histogram is from a single experiment representative of three independent experiments with one mLN‐ and one pLN‐iFRCs per experiment. (B, C) CTV‐labeled naïve CD4 + T cells from Foxp3 hCD2 xRag2 ‐/‐ xDO11.10 mice were added to iFRCs (B) or iFRC‐derived SNs (C) in presence of IL‐2 and anti‐CD3/CD28 Dynabeads. Four days later, the frequency of de novo induced Foxp3 + cells and CD4 + T cell proliferation was determined by flow cytometry, as shown in representative dot plots from a single experiment representative of seven (B) or six (C) independent experiments with one mLN‐ and one pLN‐iFRCs per experiment. Numbers in gates indicate frequencies. Each dot within scatterplot depicts mean of technical replicates, and each line represents mean of depicted dots within respective groups. Data were pooled from seven (B) or six (C) independent experiments with one mLN‐ and one pLN‐iFRCs per experiment; p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; **** p < 0.0001. med, medium.

Techniques Used: Ex Vivo, Isolation, Transduction, Flow Cytometry, Expressing, Labeling, Derivative Assay

MV‐associated TGF‐β is responsible for the tolerogenic phenotype of mLN‐iFRCs. MVs were isolated from SN of mLN‐ and pLN‐iFRCs by differential centrifugation and gravity‐driven filtration. (A, B) CTV‐labeled naïve CD4 + T cells from Foxp3 hCD2 xRag2 ‐/‐ xDO11.10 mice were cultured in presence of IL‐2, anti‐CD3/CD28 Dynabeads, and in part of the cultures MVs derived from either mLN‐ or pLN‐iFRCs were added. Four days later, the frequency of de novo induced Foxp3 + cells and CD4 + T cell proliferation was determined by flow cytometry, as shown in representative dot plots (A). Numbers in gates indicate frequencies. (B) Each dot within scatterplot depicts mean of technical replicates, and each line represents mean of depicted dots within respective groups. Data were pooled from seven independent experiments with single cultures of mLN‐ and pLN‐iFRCs per experiment; p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; ** p < 0.01; *** p < 0.001. (C) TGF‐β1 concentration in MVs of mLN‐ and pLN‐iFRCs was determined by ELISA. Each dot within scatterplot depicts mean of technical replicates, and lines represent mean of depicted dots within respective groups. Data were pooled from five independent experiments; p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; ** p < 0.01; *** p < 0.001. (D) Naïve T cells were stimulated as described above. In part of the cultures, neutralizing antibodies directed against TGF‐β1,2,3 were added. Each dot within scatterplot depicts mean of technical replicates, and dotted lines connect corresponding mean values from the four independently performed experiments. Each line represents mean of depicted dots within respective groups. p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; * p < 0.05; ** p < 0.01. med, medium.

Figure Legend Snippet: MV‐associated TGF‐β is responsible for the tolerogenic phenotype of mLN‐iFRCs. MVs were isolated from SN of mLN‐ and pLN‐iFRCs by differential centrifugation and gravity‐driven filtration. (A, B) CTV‐labeled naïve CD4 + T cells from Foxp3 hCD2 xRag2 ‐/‐ xDO11.10 mice were cultured in presence of IL‐2, anti‐CD3/CD28 Dynabeads, and in part of the cultures MVs derived from either mLN‐ or pLN‐iFRCs were added. Four days later, the frequency of de novo induced Foxp3 + cells and CD4 + T cell proliferation was determined by flow cytometry, as shown in representative dot plots (A). Numbers in gates indicate frequencies. (B) Each dot within scatterplot depicts mean of technical replicates, and each line represents mean of depicted dots within respective groups. Data were pooled from seven independent experiments with single cultures of mLN‐ and pLN‐iFRCs per experiment; p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; ** p < 0.01; *** p < 0.001. (C) TGF‐β1 concentration in MVs of mLN‐ and pLN‐iFRCs was determined by ELISA. Each dot within scatterplot depicts mean of technical replicates, and lines represent mean of depicted dots within respective groups. Data were pooled from five independent experiments; p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; ** p < 0.01; *** p < 0.001. (D) Naïve T cells were stimulated as described above. In part of the cultures, neutralizing antibodies directed against TGF‐β1,2,3 were added. Each dot within scatterplot depicts mean of technical replicates, and dotted lines connect corresponding mean values from the four independently performed experiments. Each line represents mean of depicted dots within respective groups. p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; * p < 0.05; ** p < 0.01. med, medium.

Techniques Used: Isolation, Centrifugation, Filtration, Labeling, Cell Culture, Derivative Assay, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay

other:

Article Title: Salt Sensing by Serum/Glucocorticoid-Regulated Kinase 1 Promotes Th17-like Inflammatory Adaptation of Foxp3 + Regulatory T Cells.
Article Snippet: 5 3 104 TEFF were plated in 96-well flat-bottomed plates together with 2 3 10 5 mitomycin-treated CD4- feeder cells in the presence of 1 mg/ml of soluble a-CD3ε (BD Bioscience). tTREG cells were pre-treated with control or high salt (+60 mM) medium in previously described TH17 polarizing conditions for 3 days, then FACS-purified again for CD4 + CD45.2+ GFP+ tTREG cells. iTREG cells were induced by TGFb in control medium or high salt (+60mM) medium for 3 days, then FACSpurified again for CD4+ CD45.2+ GFP+ iTREG cells.

Labeling:

Article Title: Salt Sensing by Serum/Glucocorticoid-Regulated Kinase 1 Promotes Th17-like Inflammatory Adaptation of Foxp3 + Regulatory T Cells
Article Snippet: .. Original T EFF cells were labeled with Cell Trace Violet (CTV) Proliferation Dye (Invitrogen) prior to co-culture with unlabelled original tT REG cells for post-co-culture identification of the ontogeny of any resultant cells, regardless of their Foxp3 expression. ..

Article Title: Salt Sensing by Serum/Glucocorticoid-Regulated Kinase 1 Promotes Th17-like Inflammatory Adaptation of Foxp3 + Regulatory T Cells
Article Snippet: Furosemide (Sigma Aldrich) was used at 100 μM, a concentration with no significant effect on cell survival and growth. .. FACS-sorted CD4 + CD45.1 + Foxp3 − (GFP − ) T EFF cells were labeled with Cell Trace Violet (CTV) Proliferation Dye (Invitrogen) and used as responder cells for the suppression assay. .. 5 × 10 4 T EFF were plated in 96-well flat-bottomed plates together with 2 × 10 5 mitomycin-treated CD4 − feeder cells in the presence of 1 μg/ml of soluble α-CD3ε (BD Bioscience). tT REG cells were pre-treated with control or high salt (+60 mM) medium in previously described T H 17 polarizing conditions for 3 days, then FACS-purified again for CD4 + CD45.2 + GFP + tT REG cells. iT REG cells were induced by TGFβ in control medium or high salt (+60 mM) medium for 3 days, then FACS-purified again for CD4 + CD45.2 + GFP + iT REG cells.

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Journal: European Journal of Immunology

Article Title: Mesenteric lymph node stromal cell‐derived extracellular vesicles contribute to peripheral de novo induction of Foxp3 + regulatory T cells

doi: 10.1002/eji.201746960

Figure Lengend Snippet: mLN‐iFRCs possess superior de novo Treg‐inducing capacity. (A) FRCs were ex vivo isolated from skin‐draining pLNs and gut‐draining mLNs and immortalized via lentiviral transduction. After doxycycline‐induced expansion, pLN‐ and mLN‐iFRCs were phenotyped by flow cytometry. Representative histograms depict expression of gp38 among CD45 − CD24 − CD31 − iFRCs originating from mLN and pLN. Histogram is from a single experiment representative of three independent experiments with one mLN‐ and one pLN‐iFRCs per experiment. (B, C) CTV‐labeled naïve CD4 + T cells from Foxp3 hCD2 xRag2 ‐/‐ xDO11.10 mice were added to iFRCs (B) or iFRC‐derived SNs (C) in presence of IL‐2 and anti‐CD3/CD28 Dynabeads. Four days later, the frequency of de novo induced Foxp3 + cells and CD4 + T cell proliferation was determined by flow cytometry, as shown in representative dot plots from a single experiment representative of seven (B) or six (C) independent experiments with one mLN‐ and one pLN‐iFRCs per experiment. Numbers in gates indicate frequencies. Each dot within scatterplot depicts mean of technical replicates, and each line represents mean of depicted dots within respective groups. Data were pooled from seven (B) or six (C) independent experiments with one mLN‐ and one pLN‐iFRCs per experiment; p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; **** p < 0.0001. med, medium.

Article Snippet: 10 5 naïve CD4 + T cells labeled with proliferation dye Cell Trace Violet (CTV, ThermoFisher Scientific) were added to iFRCs in 50 μL X‐VIVO containing 10 ng/mL IL‐2 and 0.5 × 10 5 Dynabeads mouse T activator CD3/CD28 (Thermo Fisher Scientific).

Techniques: Ex Vivo, Isolation, Transduction, Flow Cytometry, Expressing, Labeling, Derivative Assay

Journal: European Journal of Immunology

Article Title: Mesenteric lymph node stromal cell‐derived extracellular vesicles contribute to peripheral de novo induction of Foxp3 + regulatory T cells

doi: 10.1002/eji.201746960

Figure Lengend Snippet: MV‐associated TGF‐β is responsible for the tolerogenic phenotype of mLN‐iFRCs. MVs were isolated from SN of mLN‐ and pLN‐iFRCs by differential centrifugation and gravity‐driven filtration. (A, B) CTV‐labeled naïve CD4 + T cells from Foxp3 hCD2 xRag2 ‐/‐ xDO11.10 mice were cultured in presence of IL‐2, anti‐CD3/CD28 Dynabeads, and in part of the cultures MVs derived from either mLN‐ or pLN‐iFRCs were added. Four days later, the frequency of de novo induced Foxp3 + cells and CD4 + T cell proliferation was determined by flow cytometry, as shown in representative dot plots (A). Numbers in gates indicate frequencies. (B) Each dot within scatterplot depicts mean of technical replicates, and each line represents mean of depicted dots within respective groups. Data were pooled from seven independent experiments with single cultures of mLN‐ and pLN‐iFRCs per experiment; p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; ** p < 0.01; *** p < 0.001. (C) TGF‐β1 concentration in MVs of mLN‐ and pLN‐iFRCs was determined by ELISA. Each dot within scatterplot depicts mean of technical replicates, and lines represent mean of depicted dots within respective groups. Data were pooled from five independent experiments; p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; ** p < 0.01; *** p < 0.001. (D) Naïve T cells were stimulated as described above. In part of the cultures, neutralizing antibodies directed against TGF‐β1,2,3 were added. Each dot within scatterplot depicts mean of technical replicates, and dotted lines connect corresponding mean values from the four independently performed experiments. Each line represents mean of depicted dots within respective groups. p ‐values were calculated using one‐way ANOVA followed by Bonferroni's post‐test. ns, not significant; * p < 0.05; ** p < 0.01. med, medium.

Techniques: Isolation, Centrifugation, Filtration, Labeling, Cell Culture, Derivative Assay, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay

(A) Schematic diagram showing the in vitro co-culture system that enables the tracking of cell ontogeny (top panel). FACS-purified CD4 + GFP − Teff cells from B6.Foxp3 GFP mice were labeled with CellTrace Violet (CTV) proliferation dye and then combined with GFP + thymic Treg (tTreg) cells at a ratio of 6:1. After co-culture, cells of tTreg cell origin were identified as CTV − . Similarly, cells of original Teff cell origin were identified as CTV + . Representative flow cytometry plots showing the gating strategy to differentiate the cells of either tTreg or Teff cell origins (bottom panel). (B) tTreg and Teff cells were co-cultured (A) under increasing NaCl concentrations in the presence of Th17-polarizing cytokines for 72 h and in the presence or absence of a SGK1 inhibitor. Representative flow cytometry plots showing the expression of transcription factors RORγt and Helios in CTV − tTreg cells at 72 h. (C) Quantitation of the frequency of RORγt + cells in Foxp3 + CTV − tTreg cells. (D and E) Quantitative analyses of RORγt (D) and Foxp3 (E) protein expressions in tTreg cells by the assessment of median fluorescent intensity (MFI). (F) Representative flow cytometry plots showing the intracellular staining of IL-17A and RORγt in Teff cells or tTreg cells. (G) Assessment of the suppressive function of tTreg cells cultured in normal salt (NS) or high salt (HS) medium in a Th17-polarizing condition, as described in (A), in an in vitro suppression assay using CD4 + GFP − Teff cells as responders. Representative flow cytometry histograms of the proliferation of Teff by the dilution of the CTV proliferation dye. (H) Quantification of percent suppression at different Treg/Teff cell ratios. (I) Quantitative analysis of the frequency of RORγt + cells in Helios + and Helios − subsets of tTreg cells. Data are representative of three independent experiments with triplicates of each condition. Error bars represent mean ± standard deviation.

Journal: Cell reports

Article Title: Salt Sensing by Serum/Glucocorticoid-Regulated Kinase 1 Promotes Th17-like Inflammatory Adaptation of Foxp3 + Regulatory T Cells

doi: 10.1016/j.celrep.2020.01.002

Figure Lengend Snippet: (A) Schematic diagram showing the in vitro co-culture system that enables the tracking of cell ontogeny (top panel). FACS-purified CD4 + GFP − Teff cells from B6.Foxp3 GFP mice were labeled with CellTrace Violet (CTV) proliferation dye and then combined with GFP + thymic Treg (tTreg) cells at a ratio of 6:1. After co-culture, cells of tTreg cell origin were identified as CTV − . Similarly, cells of original Teff cell origin were identified as CTV + . Representative flow cytometry plots showing the gating strategy to differentiate the cells of either tTreg or Teff cell origins (bottom panel). (B) tTreg and Teff cells were co-cultured (A) under increasing NaCl concentrations in the presence of Th17-polarizing cytokines for 72 h and in the presence or absence of a SGK1 inhibitor. Representative flow cytometry plots showing the expression of transcription factors RORγt and Helios in CTV − tTreg cells at 72 h. (C) Quantitation of the frequency of RORγt + cells in Foxp3 + CTV − tTreg cells. (D and E) Quantitative analyses of RORγt (D) and Foxp3 (E) protein expressions in tTreg cells by the assessment of median fluorescent intensity (MFI). (F) Representative flow cytometry plots showing the intracellular staining of IL-17A and RORγt in Teff cells or tTreg cells. (G) Assessment of the suppressive function of tTreg cells cultured in normal salt (NS) or high salt (HS) medium in a Th17-polarizing condition, as described in (A), in an in vitro suppression assay using CD4 + GFP − Teff cells as responders. Representative flow cytometry histograms of the proliferation of Teff by the dilution of the CTV proliferation dye. (H) Quantification of percent suppression at different Treg/Teff cell ratios. (I) Quantitative analysis of the frequency of RORγt + cells in Helios + and Helios − subsets of tTreg cells. Data are representative of three independent experiments with triplicates of each condition. Error bars represent mean ± standard deviation.

Article Snippet: FACS-sorted CD4 + CD45.1 + Foxp3 − (GFP − ) T EFF cells were labeled with Cell Trace Violet (CTV) Proliferation Dye (Invitrogen) and used as responder cells for the suppression assay.

Techniques: In Vitro, Co-Culture Assay, Purification, Labeling, Flow Cytometry, Cell Culture, Expressing, Quantitation Assay, Staining, Suppression Assay, Standard Deviation

A. AI4 Splenocytes were adoptively transferred into NOD or NOD-Idd22 recipients and mice were monitored daily for development of T1D as before. AI4 splenocytes induced diabetes in >90% of NOD recipients by day 12 post transfer but failed to induce T1D in 90% of NOD-Idd22 mice through 30 days post transfer (P<0.001, logrank survival curve analysis). B. IS-CD8+ T cells (107) were adoptively transferred into recipient mice and monitored daily for development of T1D. IS-CD8 T cells failed to induce disease in NOD-Idd22 recipients by 28 days post transfer, whereas 100% of all NOD recipients developed T1D by 25 days post transfer (P<0.001, logrank survival curve analysis). C. Cell trace violet (CTV) labeled NOD.AI4α/β-Thy1α/β splenocytes were adoptively transferred into NOD-Idd22 and NOD recipients and pancreatic draining lymph nodes were biopsied three days later and analyzed via flow cytometery. A representative gating strategy for identification of transferred AI4 cytotoxic T lymphocytes (CTLs) in the pancreatic lymph node (PLN) is shown. Cells are initially selected on forward and side scatter, followed by exclusion of dead cells. AI4 cells are identified as CD8+ Thy1α+. Representative histograms show the extent of CTV dye dilution in NOD (top) and NOD-Idd22 (bottom) recipients. D. Thy1a+ frequency, number and proliferation index are identical in NOD and NOD-Idd22 recipients. Data points graphed are individual recipient mice (6 for NOD and 7 NOD-Idd22) and represent 3 individual experiments. E. The activation markers KLRG1, CD11a, CD43, and CD27 are all similar (or higher) in NOD-Idd22 recipients when compared to NOD, while, as expected, IL7Rα and CD62L expression was downregulated on proliferating cells (*P<0.05, Student’s t test). Data points graphed are individual recipient mice. Two independent experiments were performed. F. Representative micrographs of IHC stained pancreata from NOD (top row) and NOD-Idd22 (bottom row) recipients post transfer of 20×106 NOD.AI4α/β-Rag1−/−-Thy1α/β splenocytes at the indicated timepoints. NOD-Idd22 islets remain insulitis free with strong insulin staining through 9 days post transfer while NOD islets display increasing insulitis and reduction in insulin positive area and intensity through the same time period. Black scale bars indicate 200 um. G. NOD.AI4α/β-Rag1−/−-Thy1α/β splenocytes were adoptively transferred into NOD or NOD-Idd22 recipients. Pancreata were biopsied and fresh-frozen at indicated time points and stained via immunohistochemistry for insulin (Red) and Thy1α (Brown). Degree of infiltration of AI4 CTLs was determined in a similar fashion to insulitis scoring. Between 39 and 108 total islets from 5 recipient mice per group were scored and are listed in Supplemental Table.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Type 1 Diabetes-Resistance locus Idd22 Controls Trafficking of Autoreactive Cytotoxic T Lymphocytes into the Pancreatic Islets of NOD Mice 1

doi: 10.4049/jimmunol.1602037

Figure Lengend Snippet: A. AI4 Splenocytes were adoptively transferred into NOD or NOD-Idd22 recipients and mice were monitored daily for development of T1D as before. AI4 splenocytes induced diabetes in >90% of NOD recipients by day 12 post transfer but failed to induce T1D in 90% of NOD-Idd22 mice through 30 days post transfer (P<0.001, logrank survival curve analysis). B. IS-CD8+ T cells (107) were adoptively transferred into recipient mice and monitored daily for development of T1D. IS-CD8 T cells failed to induce disease in NOD-Idd22 recipients by 28 days post transfer, whereas 100% of all NOD recipients developed T1D by 25 days post transfer (P<0.001, logrank survival curve analysis). C. Cell trace violet (CTV) labeled NOD.AI4α/β-Thy1α/β splenocytes were adoptively transferred into NOD-Idd22 and NOD recipients and pancreatic draining lymph nodes were biopsied three days later and analyzed via flow cytometery. A representative gating strategy for identification of transferred AI4 cytotoxic T lymphocytes (CTLs) in the pancreatic lymph node (PLN) is shown. Cells are initially selected on forward and side scatter, followed by exclusion of dead cells. AI4 cells are identified as CD8+ Thy1α+. Representative histograms show the extent of CTV dye dilution in NOD (top) and NOD-Idd22 (bottom) recipients. D. Thy1a+ frequency, number and proliferation index are identical in NOD and NOD-Idd22 recipients. Data points graphed are individual recipient mice (6 for NOD and 7 NOD-Idd22) and represent 3 individual experiments. E. The activation markers KLRG1, CD11a, CD43, and CD27 are all similar (or higher) in NOD-Idd22 recipients when compared to NOD, while, as expected, IL7Rα and CD62L expression was downregulated on proliferating cells (*P<0.05, Student’s t test). Data points graphed are individual recipient mice. Two independent experiments were performed. F. Representative micrographs of IHC stained pancreata from NOD (top row) and NOD-Idd22 (bottom row) recipients post transfer of 20×106 NOD.AI4α/β-Rag1−/−-Thy1α/β splenocytes at the indicated timepoints. NOD-Idd22 islets remain insulitis free with strong insulin staining through 9 days post transfer while NOD islets display increasing insulitis and reduction in insulin positive area and intensity through the same time period. Black scale bars indicate 200 um. G. NOD.AI4α/β-Rag1−/−-Thy1α/β splenocytes were adoptively transferred into NOD or NOD-Idd22 recipients. Pancreata were biopsied and fresh-frozen at indicated time points and stained via immunohistochemistry for insulin (Red) and Thy1α (Brown). Degree of infiltration of AI4 CTLs was determined in a similar fashion to insulitis scoring. Between 39 and 108 total islets from 5 recipient mice per group were scored and are listed in Supplemental Table.

Article Snippet: Splenocytes from NOD.AI4α/β-Thy1α/β mice were isolated as described above and stained with Cell Trace Violet (CTV) cell proliferation dye (Life Technologies) according to manufacturer’s instructions.

Techniques: Labeling, Activation Assay, Expressing, Staining, Immunohistochemistry

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proliferation dye cell trace violet ctv (Thermo Fisher)

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