CD4⁺CD25⁻ T cells activated with IL-4+TGF-β express more IL-9 than Th1, Th2, Th17, or iTregs.

<p>CD4⁺CD25⁻ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β or Th1-, Th2-, Th17-, iTreg-polarizing condition for 96hrs. Cells were harvested, gated on CD4⁺ T cells, and were analyzed for IL-9⁺ cells by flow cytometry or were used to quantitate IL-9 transcripts by real-time PCR. Data is expressed as the mean±SD. (A) 2% of Th2 cells, 4% of iTregs, or 10% of cells treated with IL-4+TGF-β in combination were IL-9⁺, whereas Th1-, Th17-, or Th0-cells had negligible number of IL-9⁺ cells (<i>n = 7</i>); (B) Log-transformed ratios of IL-9 mRNA copies to 18S rRNA are shown. Cells treated with IL-4+TGF-β in combination had significantly higher levels of IL-9 mRNA as compared to polarized Th1-, Th2-, Th17-, iTreg-, or Th0-cells (<i>n</i> = 9).</p

IL-1β amplifies IL-4+TGF-β induced IL-9 production by memory CD4⁺CD25⁻CD45RO⁺ T cells.

<p>Resting memory CD4⁺CD25⁻CD45RO⁺ T cells (2.0×10⁵/ml in 96 well plates) activated with pbCD3/sCD28 alone or with IL-4+TGF-β, in the presence or absence of IL-1β, IL-2, IL-6, IL-12, and IL-21 for 96hrs and supernatants were collected. Influence of IL-1β, IL-2, IL-6, IL-12, and IL-21 on IL-9 production of IL-4+TGF-β treated CD4⁺CD25⁻CD45RO⁺ T cells was examined. IL-1β, IL-12 or IL-21 significantly elevated IL-4+TGF-β induced IL-9 production, but IL-1β had significantly higher influence compared to IL-12 or IL-21 (<i>n = 3</i>). Data is expressed as the mean±SD.</p

IL-4+TGF-β in presence of pbCD3/sCD28 activation induce generation of CD4⁺IL-9⁺ T cells.

<p><b>A)</b> CD4⁺CD25⁻ T cells (1.0×10⁶/ml in 24 well plates) were activated with plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) in presence or absence of IL-4 or TGF-β or IL-4+TGF-β for 96hrs and analyzed by flow cytometry for IL-9 expression. IL-4+TGF-β in combination induced significantly higher percentage of CD4⁺ T cells positive for IL-9, as compared to IL-4, TGF-β, or neither (<i>n = 14</i>). Data is expressed as the mean±SD. <b>B)</b> CD4⁺CD25⁻ T cells, CD4⁺CD25⁻CD45RA⁺ T cells (naïve T cells), CD4⁺CD25⁻CD45RO⁺ T cells (resting memory T cells) (2.0×10⁵/ml in 96 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β in a 96 well plate for 96hrs. Cells were surface stained for CD4 PerCP-Cy5.5 and intracellular stained for IL-9 PE. IL-4+TGF-β in combination induced IL-9 expression by both naïve and memory T cells, but memory T cells expressed high levels of IL-9. Data are representative of six independent experiments (six different donors).</p

Cytokine and transcription factor profile of memory CD4⁺CD25⁻CD45RO⁺ T cells activated with IL-4+TGF-β.

<p><b>A)</b> Log-transformed quantities of cytokines (pg/ml) are shown. CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β. Supernatants were collected at 96hrs post activation and IFNγ, IL-2, IL-5, IL-9, IL-10, IL-13, and IL-17 were quantified by ELISA. IL-4+TGF-β treated CD4⁺ T cells produced significantly high IL-2 and IL-9, but significantly low IFNγ, IL-13, and IL-17, as compared to CD4⁺CD25⁻CD45RO⁺ T cells not treated with IL-4+TGF-β (<i>n = 3</i>). Data is expressed as the mean±SD. <b>B)</b> Log-transformed ratios of mRNA copies to GAPDH mRNA copies for GATA3, RORC, IL-9, and Tbet are shown. CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were harvested and single cell sorted. IL-9 transcripts were quantified by qt-RT-PCR. 10,000 cells comprising of total cell population (TC) was also taken and the gene expression was averaged for single cell for reference. Cells positive for IL-9 transcripts were further quantitated for GATA3, RORC, and Tbet (<i>n</i> = 3). As IL-9 is expressed in only 10% of all CD4⁺ T cells activated with pbCD3/sCD28 in presence of IL-4+TGF-β, average IL-9 mRNA copies of TC are always lower than that of a single IL-9⁺ cell. CD4⁺IL-9⁺ T cells expressed GATA3 and RORC, but not Tbet. <b>C)</b> CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were surface stained for CD4 and intracellular stained for IL-9 and FOXP3. Cells were gated for CD4 and then IL-9⁺ or/and FOXP3⁺ cells were analyzed. 25% of CD4⁺IL-9⁺ T cells were also FOXP3⁺. Data are representative of seven independent experiments. <b>D)</b> CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4 or TGF-β or IL-4 plus TGF-β for 96hrs and analyzed by flow cytometry for FOXP3 expression. IL-4 significantly inhibited TGF-β induced FOXP3 expression (<i>n = 3</i>). Data is expressed as the mean±SD.</p

IRMs from B6.g7, NOD, and NOR induce FoxP3+ Tregs in the diabetogenic BDC2.5 TCR transgenic clone.

<p>CellTrace Violet labeled CD4+CD25-CD44-CD62L^hi naïve T cells from B6.g7/BDC2.5 TCR transgenic mice were cultured for 96 h with IRMs or CD11c^hiCD11b^low splenic DCs from the indicated strain pulsed with 2.5μM of the indicated agonist peptide. (A) CD25 upregulation and proliferation are shown for gated live CD4+Vβ4+ transgenic BDC2.5 T cells stimulated with B6.g7 (H-2^g7) splenic DCs, B6.g7 (H-2^g7) IRMs, or B6 (H-2^b) IRMs pulsed with the agonistic 1068–56 peptide (left column) or no peptide (right column). The expansion index for the gated live activated CD4+Vβ4+CD25+ population was determined using FlowJo analysis software and is noted in the upper-left corner. (B) FoxP3 expression is shown for the gated live activated CD4+Vβ4+CD25+ population. (C) Percent of gated live CD4+Vβ4+CD25+ transgenic T cells that acquire FoxP3 expression after culture with IRMs from the indicated strain are shown from independent experiments (n = 3–4 per strain). A One Way ANOVA indicated no significant difference among groups with (p = 0.95) or without peptide (p = 0.64).</p

A decrease in the proportion of immunoregulatory islet resident macrophages relative to immunostimulatory islet resident DCs heralds T1D in NOD mice.

<p>(A) The ratio of immunoinflammatory IRDCs to immunoregulatory IRMs was calculated at the indicated time-points. Data from individual mice (n = 7–10 per group) were pooled from 3–4 independent experiments. Data were compared with an unpaired t test (**, p<0.01; ***, p<0.001). (B) The absolute number of IRMs (CD45+Ly6C-CD11c+F4/80+CD16/32+), IRDCs (CD45+Ly6C-CD11c+F4/80-CD16/32-), and islet-infiltrating macrophages (CD45+Ly6C+CD11b+) were calculated per islet for NOD and NOR control mice at the indicated ages. Means +/- S.D. are shown for the same cohorts displayed in panel A. NOD groups were compared to the NOR control group utilizing an unpaired t test (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Decreased CD39 protein expression by islet resident macrophages heralds T1D in NOD mice.

<p>The MFI of CD39 expression on IRMs from NOD and NOR mice (n = 5 per group pooled from 2 independent experiments) was analyzed by flow cytometry using the CD45+Ly6C-CD11c+F4/80+CD16/32+ gate. Since each time-point was acquired separately, inter-graph comparison of MFIs is not possible. NOD groups were compared to the NOR control group using an unpaired t test (*, p<0.05, **, p<0.01).</p

Islet resident macrophages induce Tregs through a mechanism that is dampened by TLR4 stimulation.

<p>FACS-sorted ABM CD4+FoxP3/GFP- TCR transgenic T cells were CellTrace violet labeled and cultured with FACS-sorted live IRMs (CD45+Ly6C-CD11c+F4/80+CD16/32+), IRDCs (CD45+Ly6C-CD11c+F4/80-CD16/32-), or splenic DCs (CD11c^hiCD11b^low) from bm12 (I-A^bm12) mice for 96 h. (A) Proliferation of gated live CD25+CD4+Vα2+Vβ8+ ABM TCR transgenic T cells stimulated with IRM (solid line), IRDC (long dashed line), or splenic DC (short dashed line) stimulators is depicted in a histogram. The shaded histogram represents the unstimulated control. Representative data from 3 independent experiments are shown. (B) FoxP3/GFP expression in gated live activated CD25+CD4+Vα2+Vβ8+ ABM TCR transgenic T cells is shown from a representative experiment. (C) FoxP3/GFP expression in gated live activated CD25+CD4+Vα2+Vβ8+ ABM TCR transgenic T cells from independent experiments (n = 4–6 per group) are shown in scatter plots. Groups were compared by ANOVA (p<0.01) and Bonferroni’s post-test (***, p<0.001). (D) Concentration of IL-6 (pg/ml) in the supernatant of independent MLR cultures (n = 4–6 per group) is shown in a scatter plot. Groups were compared by ANOVA (p<0.01) and Bonferroni’s post-test (***, p<0.001).</p

Islet resident macrophages possess an immunoregulatory phenotype that is compromised by TLR4 activation.

<p>(A) IRMs (solid line) and IRDCs (dashed line) were analyzed directly <i>ex vivo</i> by flow cytometry for CD39, CD73 and galectin-9 using the CD45+Ly6C-CD11c+F4/80+CD16/32+ gate. Shaded histograms are the FMO controls and MFIs are indicated parenthetically. Representative data are shown from one of three independent experiments. (B) FACS-sorted B6 IRMs were stimulated with 1 μg/ml of LPS or 10 μg/ml of agonistic anti-CD40. After 72 h, mRNA from cultured cells was analyzed by real-time PCR for <i>Entpd1</i> (CD39), <i>Ccl2</i>, <i>Il1b</i>, and <i>Il6</i> transcripts. Each point represents data from an independent experiment (n = 3–4 per group).</p

Intra-islet CD45+CD11c+Ly6C-F4/80+CD16/32+ cells from C57BL/6 mice exhibit a tissue-resident macrophage phenotype.

<p>Purified C57BL/6 pancreatic islets were enzymatically digested and stained with anti-CD45, anti-CD11c, anti-Ly6C, anti-F4/80, and anti-CD16/32. (A) Identification of live CD45+ (not shown) CD11c+Ly6C- (left panel) cells that exhibit an islet-resident macrophage (IRM, F4/80+CD16/32+) or DC (IRDC, F4/80-CD16/32-) phenotype (right panel) are shown. (B) FACS-sorted IRMs and IRDCs were analyzed for <i>Csf1r</i> transcript levels by real-time PCR. Data from independent experiments (n = 4 per group) and their mean are plotted; groups were compared with an unpaired t test. (C) Histograms show protein expression of CD169, TIM-4, and CX3CR1 on gated IRM (solid line) and IRDC (dashed line) subsets. Shaded histograms are the fluorescence minus one (FMO) controls. Geometric mean fluorescence intensities (MFIs) are indicated parenthetically. Data are representative of three independent experiments.</p