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

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

Schematic model of nanoparticles binding to pegylated islet.

<p>Avidin groups on the nanoparticle surface mediate nanoparticle attachment to biotinylated PEG that coats the islet.</p

Prolonged functionality of encapsulated islets in vivo.

<p>Pancreatic islets from DBA/2 mice were grafted under the kidney capsule of C57BL/6 recipients: the islets were either untreated (Ctrl); or encapsulated in PEG alone (PEG alone); or with PEG decorated with empty nanoparticles (PEG+Empty Nano); or with PEG decorated with LIF-containing nanoparticles (PEG+LIF-Nano). The ability of these grafts to support normoglycemia over 100 d is shown as “% survival” in (A). Histology of grafts taken from recipients showing normoglycemia at 100 d revealed well-preserved β cells containing insulin, as illustrated in (B).</p

Long-term normoglycemia derived from pegylated DBA/2 islet grafts in C57BL/6 recipients.

<p>Grafts were placed under the kidney capsule. No immunosuppressive therapy was given.</p><p>“>100 d” indicates number of recipients reaching >100 days normoglycemia.“<100 d” indicates number of recipients failing to reach long-term normoglycemia.</p

Nanoparticle coating of mouse islets.

<p>(A) Islets incubated with PEG plus coumarin-6 (green)-labeled nanoparticles (Nano) observed under fluorescence (left) and confocal (right) microscopes. Scale bar, 50 µm. (B) Naked control islets (CTR), or pegylated islets coated with coumarin-6 labeled nanoparticles (Nano) imaged by SEM immediately after encapsulation. Scale bar, 100 µm. (C) Islets imaged at 21 days post culture: images e–g show naked islets (CTR), images h–j show islets draped with PEG plus coumarin-6-nano (Nano). The naked islets show degradation in marked contrast to the well-preserved nano-pegylated islets. Scale bar, 50 µm.</p

Prolonged viability of encapsulated islets in vitro.

<p>(A) Staining of viable (green) <i>versus</i> dead (red) cells in cultures of naked islets (CTR), pegylated islets (PEG), or pegylated plus empty-nanoparticle islets (Nano) at 1 d, 7 d, 14 d, and 21 d. (B) Percentages of viable cells in the different groups during culture. (C) Insulin staining in naked (CTR) and PEG-Nano-coated islets at 2 d and 14 d culture: more insulin positive cells were observed in islets encapsulated with nanoparticles (lower panels) compared to naked islets (upper panels). At least 10 islets were included in each group. Red represents insulin staining, blue staining (DAPI) represents nuclear staining in all cells. * p<0.05 and ** p<0.01.</p

Encapsulation does not affect islet function.

<p>(A) Insulin secretion (ng/mL/h/islet) was measured in naked (light grey bars, CTR) and nano-PEG-encapsulated (dark grey bars, Nano) islet cultures cultured overnight after encapsulation stimulated with 2.8 mM, or 28 mM glucose for 24 h. (B) Insulin stimulation index of the naked and nanoparticle-coated islets shown in (A). At least 20 islets were included in each group, and the data represents 3 individual experiments.</p