γδ T cells have similar TCR threshold but enhanced ERK1/2 phosphorylation comparing to αβ T cells.

<p>(A) Percentages of BrdU⁺ proliferative cells are higher in the γδ T cell subset <i>in vivo</i>. Left: representative plots of BrdU⁺ populations (circled) in αβ and γδ T cells. Right: Graph of percentages of BrdU⁺ cells in αβ and γδ T cells. C57BL/6J mice received single dose of BrdU (1 mg) via intraperitoneal injection, and splenocytes were isolated 3 days later. BrdU⁺ cells were identified by fluorescently labeled anti-BrdU antibody and flow cytometry. Results were averaged from 8 mice, and shown in mean ± SEM. *** P<0.001. (B) Top: Splenocytes from C57BL/6J mice were labeled with CFSE (2.5 µM), and then stimulated with 0–10 µg/ml plate-bound αCD3 and 1 µg/ml soluble αCD28 for 24 hours. CFSE content in cells was measured by flow cytometry and graphed against αCD3 concentrations. Results were averaged from 5 mice, and shown in mean ± SEM. Lower: Log scaling of αCD3 concentration against % of maximum response was used to calculate EC₅₀. (C) CD3 expression in γδ T cells was higher than in αβ T cells under no or lower stimulation, but decreased more dramatically with stronger stimulation. The experimental condition was the same as in (B). (D) Western blotting shows high levels of phosphorylated ERK1/2 in γδ T cells, relative to αβ T cells, at baseline. αβ and γδ T cells were isolated from the same set of C57BL/6J mice via FACS sorting. Cells were either untreated, or incubated with αCD3 and αCD28 antibodies (20 µg/ml each) for 5 minutes and then with anti-hamster IgG (20 µg/ml) for additional 2 minutes at 37°C. Cells were lyzed, separated by SDS-PAGE, and immunoblotted with indicated antibodies. The image was representative of 3 independent experiments. (E) Inhibition of ERK1/2 significantly lowers proliferation in γδ T cells <i>in vitro</i>. Splenocytes from C57BL/6J mice were labeled with CFSE (2.5 µM), and then stimulated with 1 µg/ml plate-bound αCD3 and 1 µg/ml soluble αCD28 for 24 hours, in the absence (vehicle) or presence of 10 µM and 25 µM U0126. CFSE dilution in cells was measured by flow cytometry, and used to calculate cell proliferation. Results were averaged from 7–8 mice, and shown in mean ± SEM. ** P<0.01; *** P<0.001; ns not statistically significant.</p

γδ T cells are more activated <i>in vivo</i>.

<p>Cholesterol depletion reduces the activated phenotype of γδ T cells. Cholesterol addition significantly increases the activation status of αβ T cells. (A–B) Staining of T cell activation markers shows more γδ T cell are in the activated state <i>in vivo</i>, comparing to αβ T cells. Splenocytes were isolated from C57BL/6J mice. T cell activation was identified by (A) CD44^hi CD62L⁻ and (B) CD69⁺ cells. Representative flow cytometric plots are shown on the left and the percentages of activated T cells are graphed on the right. Results were averaged from 6 mice, and shown in mean ± SEM. (C–E) Splenocytes from C57BL/6J were stimulated with T-activator CD3/CD28 beads for 4 hours in the presence or absence of MβCD to remove cholesterol from the plasma membrane. (C) Reduction of lipid raft levels in αβ and γδ T cells with cholesterol depletion. The reduction was more significant in γδ T cells, and the difference in lipid raft level became insignificant in αβ and γδ T cells after MβCD treatment. Lipid raft content was expressed in median florescent intensity of CT-B staining. (D) The percentage of CD44^hi CD62L⁻ cells was significantly lower in γδ T cells after 4 hours of cholesterol depletion. (E) The difference in expression of early activation marker CD69 was not statistically significant in both αβ or γδ T cells after MβCD treatment for 4 hours. However, the reduction of CD69 was significant in γδ T cells at 2 hours (inset) Results were averaged from 8 mice. Results were shown in mean ± SEM. (F) C57BL/6J splenocytes were stimulated with T-activator CD3/CD28 beads in the presence or absence of 10 and 20 µg/ml cholesterol for 4 hours. The percentage of CD44^hiCD62L⁻ cells was significantly increased in αβ T cells in a dose-dependent manner of cholesterol addition. On the other hand, activated γδ T cells were reduced with excess cholesterol. Results were averaged from 8 mice and shown in mean ± SEM. * P<0.05; ** P<0.01; *** P<0.001; ns not statistically significant.</p

γδ T cells have increased intracellular neutral lipids, cholesterol, and lipid rafts.

<p>(A) Splenocytes were isolated from C57BL/6J mice. Intracellular lipid was quantified by Nile Red staining. Left: A representative plot shows the relative intensity of Nile Red staining on αβ (solid black line) and γδ (tinted shade) T cells. Right: Graph shows the median florescent intensity of Nile Red staining on αβ and γδ T cells. Results were averaged from 5 mice, and shown in mean ± SEM. (B) Total and free cholesterol was quantified in purified αβ (white) and γδ (black) T cells from C57BL/6J mouse spleens by gas chromatography. Cholesteryl ester was calculated as the difference between total and free cholesterol (multiplied by 1.67). Intracellular total cholesterol and cholesteryl ester content is significantly higher in γδ T cells. Cholesterol level was normalized to cell numbers. N = 5. (C) αβ and γδ T cells were isolated from splenocytes of C57BL/6J mice. Average cell diameters were obtained from a Multisizer IV Coulter Counter (Beckman Coulter) and showed no significant difference (ns) in αβ and γδ T cells. Results were averaged from 5 mice, and shown in mean ± SEM. (D) Staining for GM1 shows significantly higher lipid raft content on the plasma membrane of γδ T cells. Splenocytes were isolated from C57BL/6J mice. GM1 of lipid raft was identified by cholera toxin subunit B (CT-B) binding. Left: A representative plot shows the relative intensity of CT-B staining on αβ (solid black line) and γδ (tinted shade) T cells. Right: Graph shows the median florescent intensity of CT-B staining on αβ and γδ T cells. Results were averaged from 8 mice. Results were shown in mean ± SEM. * P<0.05, ** P<0.01, *** P<0.001. (E) Confocal microscopic images show increased lipid rafts staining on the membrane of γδ T cells (300×). Green: GM1.</p

[Ca²⁺]_i flux and insulin release patterns show mouse-to-mouse imprinting.

<p>(A) [Ca²⁺]_i flux and insulin release traces from islets taken from three different mice (labeled accordingly). Displayed oscillation frequency averages are 9 min (Mouse 1), 4.5 min (Mouse 2), and 15 s (Mouse 6). Periods were calculated using local minimum values. Insulin oscillations from mouse 6 were faster than the measured temporal resolution (22 s) of the chip, causing under sampling of secretion dynamics. (B) Comparison of average [Ca²⁺]_i and insulin oscillation periods from each animal. Data sets are n≥6 islets and error bars are ±1 standard deviation. (C) Plot of average [Ca²⁺]_i versus insulin for each mouse. The linear relationship of data points suggests good agreement of oscillation frequencies (R² = 0.98; p<0.0001).</p

Both inbred and outbred mice display islet imprinting.

<p>(A–B) Two representative C57BL/6J mice out of a group of 9 displayed very different [Ca²⁺]_i oscillation patterns. Three representative islets from Mouse 6 display slow oscillations (A, period: 3.9±0.2 minutes, n = 12 islets total) and three representative islets from Mouse 5 display fast oscillations (B, period: 0.9±0.6 minutes, n = 9 islets total). One trace shown in B (bottom) shows a clear ‘slow component’ that was representative of n = 4 islets from Mouse 5 (period: 5.4±0.1 minutes). (C–D) The variation in the period of [Ca²⁺]_i oscillations indicates distinct differences from mouse to mouse for the inbred B6 strain (C) and the outbred CD-1 strain (D), as shown by one-way ANOVA (p<1.0e-24). Boxes drawn around Mouse 6 and Mouse 5 in (C) are described above.</p

Effects of weight gain with age on imprinting.

<p>(A–B) Representative examples of islet [Ca²⁺]_i patterns in 11 mM glucose among lean mice weighing <30 g (A) and aged/large mice weighing >40 g (B). (C) Mean period of oscillations among 10 lean and 3 aged/large Swiss-Webster mice. Mean weight and mean period of islet [Ca²⁺]_i oscillations differed between groups (p<0.001).</p

Dispersed beta cells do not display frank imprinting.

<p>(A) Representative examples of oscillatory patterns from 3 individual beta cells (A) and 3 islets (B) taken from the same mouse (Mouse 8 as indicated by the box in C). (C–D) Mean period ± SEM from 12 sets of beta cells (C) and corresponding islets (D) from the same mouse. Beta cells displayed longer period and also a greater degree of variability in their periods as noted by the large standard deviations they exhibited compared to islets. A total of 137 beta cells and 109 islets were recorded among 12 mice. One-way ANOVA indicates differences among beta-cell periods (P<0.01) and substantial differences among islet periods (p<1.0e-25) from mouse to mouse. (E) Scatter plot showing the relationship between oscillatory periods of beta cells and islets among the 12 mice studied (R² = 0.39, p = 0.22).</p