Biophysical Characterization of CD6—TCR/CD3 Interplay in T Cells

Activation of the T cell receptor (TCR) on the T cell through ligation with antigen-MHC complex of an antigen-presenting cell (APC) is an essential process in the activation of T cells and induction of the subsequent adaptive immune response. Upon activation, the TCR, together with its associated co-receptor CD3 complex, assembles in signaling microclusters that are transported to the center of the organizational structure at the T cell-APC interface termed the immunological synapse (IS). During IS formation, local cell surface receptors and associated intracellular molecules are reorganized, ultimately creating the typical bull's eye-shaped pattern of the IS. CD6 is a surface glycoprotein receptor, which has been previously shown to associate with CD3 and co-localize to the center of the IS in static conditions or stable T cell-APC contacts. In this study, we report the use of different experimental set-ups analyzed with microscopy techniques to study the dynamics and stability of CD6-TCR/CD3 interaction dynamics and stability during IS formation in more detail. We exploited antibody spots, created with microcontact printing, and antibody-coated beads, and could demonstrate that CD6 and the TCR/CD3 complex co-localize and are recruited into a stimulatory cluster on the cell surface of T cells. Furthermore, we demonstrate, for the first time, that CD6 forms microclusters co-localizing with TCR/CD3 microclusters during IS formation on supported lipid bilayers. These co-localizing CD6 and TCR/CD3 microclusters are both radially transported toward the center of the IS formed in T cells, in an actin polymerization-dependent manner. Overall, our findings further substantiate the role of CD6 during IS formation and provide novel insight into the dynamic properties of this CD6-TCR/CD3 complex interplay. From a methodological point of view, the biophysical approaches used to characterize these receptors are complementary and amenable for investigation of the dynamic interactions of other membrane receptors

Evidence for Metabolic Provisioning by a Common Invertebrate Endosymbiont, Wolbachia pipientis, during Periods of Nutritional Stress

Wolbachia are ubiquitous inherited endosymbionts of invertebrates that invade host populations by modifying host reproductive systems. However, some strains lack the ability to impose reproductive modification and yet are still capable of successfully invading host populations. To explain this paradox, theory predicts that such strains should provide a fitness benefit, but to date none has been detected. Recently completed genome sequences of different Wolbachia strains show that these bacteria may have the genetic machinery to influence iron utilization of hosts. Here we show that Wolbachia infection can confer a positive fecundity benefit for Drosophila melanogaster reared on iron-restricted or -overloaded diets. Furthermore, iron levels measured from field-collected flies indicated that nutritional conditions in the field were overall comparable to those of flies reared in the laboratory on restricted diets. These data suggest that Wolbachia may play a previously unrecognized role as nutritional mutualists in insects

Mean fecundity measures of female <i>D. melanogaster</i> reared on low iron food (tea).

<p>The total number of eggs laid by a single female was counted over a three-day period and the average calculated. Standard error bars are indicated; replicate numbers are noted within the columns. Uninfected females are denoted by an open bar, <i>Wolbachia</i>-infected females by a filled bar. Female flies reared on cornmeal fly diet are described as “Control.” Mean fecundities that are significantly different are denoted by * (p<0.05; ANOVA) or ** (p<0.001; Mann-Whitney U Test).</p

Mean fecundity measures of female <i>D. melanogaster</i> reared on high iron food (FeCl₃).

<p>The total number of eggs laid by a single female was counted over a three-day period and the average calculated. Standard error bars are indicated; replicate numbers are noted within the columns. Uninfected females are denoted by an open bar, <i>Wolbachia</i>-infected females by a filled bar. Female flies reared on cornmeal fly diet are described as “Control.” Mean fecundities that are significantly different are denoted by *(p<0.05) or ** (p<0.001; Mann-Whitney U Test).</p

Mean fecundity measures of female <i>D. melanogaster</i> reared on low iron food (BPS).

<p>The total number of eggs laid by a single female was counted over a three-day period and the average calculated. Standard error bars are indicated; replicate numbers are noted within the columns. Uninfected females are denoted by an open bar, <i>Wolbachia</i>-infected females by a filled bar. Female flies reared on cornmeal fly diet are described as “Control.” Mean fecundities that are significantly different are denoted by * (p<0.05; ANOVA).</p

Effect of SHP2 depletion on IL2 expression.

<p>SHP2 KD and wt Jurkat E6.1 T cells were stimulated with PMA + ionomycin (+), αCD3 & αCD28, αCD3 alone, αCD28 alone or were left unstimulated (–) for 22 h. IL2 in the supernatants was quantified by sandwich ELISAs. Given are the absorption values ± SEM. The p-values are from a full factorial two-way ANOVA and represent the significance of the overall corrected model (corr m), the effect of CD28 expression (CD28 expr), the effect of the stimulus and the interaction factor (int fact) between stimuli and CD28 expression. For all conditions <i>n</i> = 3 samples, all from a single experiment representative of four independent experiments.</p

Quantification of the effect of CD28 expression on cell surface spreading and tyrosine phosphorylation.

<p>The original images of the experiment of Fig. 2 were quantified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079277#pone.0079277.s009" target="_blank">Macro S1</a>) and the values were normalized to the mean value of the measured property within that image. Normalized values of experiments with inverted stamp and overlay configurations were pooled. The graphs show the mean ± SEM. <i>A-C</i>) Cells stimulated with stripes containing αCD3 and stripes containing αCD28. (<i>n</i> = 10 images from two separate samples in which stamp and overlay stimuli were reversed (Fig. 2<i>B & C</i>) in total counting 1010 CD28 low and 127 CD28 high cells). <i>D-F</i>) Cells stimulated with stripes containing αCD3 and stripes containing unspecific IgG2a only. (<i>n</i> = 10 images from two separate samples in which stamp and overlay stimuli were reversed (Fig. 2<i>D & E</i>) in total counting 921 CD28 low and 97 CD28 high cells). <i>G-I</i>) Cells stimulated with stripes containing unspecific IgG2a only and stripes containing αCD28. (<i>n</i> = 10 images from two separate samples in which stamp and overlay stimuli were reversed (Fig. 2<i>F & G</i>) in total counting 1006 CD28 low and 165 CD28 high cells). <i>A, D & G</i>) The background-corrected, αphosphotyrosine intensity per surface area. Corrected model p-values were determined by two-way factorial ANOVAs in which no interaction terms were included. <i>B, E & H</i>) The contact surface area per cell. Two-sample T-tests were used to generate the p-values. C, <i>F & I</i>) The integrated, background-corrected, αphosphotyrosine intensity per cell (Two-sample T-tests).</p

Image processing of phosphoPLCγ1 signals and cluster formation.

<p>Overview of the image processing protocol as described in Materials and Methods and used for the analysis of the experiments described in Fig. 4. In order to resolve clusters in print, an enlarged segment of a microscopy image labeled with αphospho-PLCγ1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079277#pone.0079277.s003" target="_blank">Fig. S3</a>) is shown as an example. Image processing and quantification was done on a per image basis. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079277#pone.0079277.s010" target="_blank">Macro S2</a> describes the full procedure utilized to analyze the images. In short, the pPLCγ1 signal was thresholded to generate a binary mask of all cells. This image was inverted to generate a mask of the background signal. The CFSE image was thresholded and was used in combination with the mask of all cells to generate a mask of CFSE labeled cells and a mask of unlabeled cells. The image of the printed stripes was thresholded to generate a mask of the printed structures and inversed to also generate a mask of the overlaid areas. Combining the masks of the printed structures and overlaid areas with the masks of the cells formed the masks of the CFSE labeled cells on stamped stripes, the CFSE labeled cells on overlaid structures, the unlabeled cells on stamped stripes and the unlabeled cells on overlaid structures. These four masks were used to measure the surface areas the cells covered on both surfaces. Combining the stripe and overlay masks with the background mask enabled the measurement of surface areas not covered by cells. The last six generated masks were, in turn, applied to the original pPLCγ1 image and from the resulting images the total pPLCγ1 signal per condition could be determined. Together with the total surface areas of the specific condition, the signal intensity per µm² was calculated. Surface specific background corrections were applied. In addition, a binary cluster mask was generated from the pPLCγ1 image. This mask was segmented using the four masks of cells on surfaces creating four new masks. From these masks cluster numbers were counted and by applying them to the original pPLCγ1 image cluster intensities could be determined. Finally, the cell numbers per image were determined by eye using the original transmission images and the cell masks. The various colors correspond to the graphs in Fig. 6 and indicate which masks and images are required to produce the particular data.</p

Detection of the stimulus dependence of total tyrosine phosphorylation (<i>B</i>) and phosphoY783 PLCγ1 (<i>C</i>) in Jurkat cells and SHP2 KD cells.

<p><i>A</i>) For the side-by-side analysis of signaling in Wt and SHP2 KD Jurkat E6.1 T cells, one of the lines was labeled with the cell tracer CFSE. After overnight serum starvation the cells are pooled and incubated on micropatterned, stimulating surfaces for 10 min. Subsequently, the cells are fixed with 3% PFA, permeabilized and immunolabeled for the detection of signaling clusters. <i>B & C</i>) In the top panels, SHP2 KD cells are CFSE labeled and in the bottom panels, wt cells are labeled. Panels from left to right: transmission images; CFSE; immunofluorescence; overlay of the stamped pattern (blue) and the immunolabel (grayscale). In the overlay panels the contrast and brightness for both channels were adjusted proportionally for clarity. 12.5 µg/ml αCD3 + 12.5 µg/ml αCD28 coated stamps were used to generate a striped pattern which was overlaid with 5 µg/ml αCD3. CFSE channels were recorded with saturated signals to facilitate image processing. Scale bars 20 µm.</p

Quantification of the effects of CD28 costimulation and SHP2 deficiency.

<p>The values acquired through image segmentation as described in Fig. 5 were normalized to the mean value of the specific property for that image. The information of multiple images from multiple experiments was used for further analyses. The graphs depict the stimulus and SHP2 dependence of spreading and tyrosine phosphorylation showing the mean ± SEM (based on number of images) of the respective property. KD  =  SHP2 knock-down E6.1 Jurkat cells; wt  =  wild type E6.1 Jurkat cells; 3  =  stripes of αCD3 alone; 3+28  =  αCD3+αCD28-containing stripes (Fig. 4). The colored squares correspond to the colors bordering images and masks in Fig. 5 used to retrieve the data required for the graph in question. Corrected model p-values were determined by two-way factorial ANOVAs in which no interaction terms were included (<i>A-C & E-G</i>) or two-sample T-tests (D <i>& H-J</i>). <i>A-D</i>) Cells labeled with the αphosphotyrosine antibody (<i>n</i> = 15 images resulting from three separate experiments with varying CFSE/unlabeled and stamp/overlay conditions in total containing 861 KD and 615 wt cells). <i>E-H</i>) Cells labeled with the αphosphoY783-PLCγ1 antibody (<i>n</i> = 26 images resulting from five separate experiments with varying CFSE/unlabeled and stamp/overlay conditions in total containing 1804 KD and 1502 wt cells). <i>A & E</i>) Average, background-corrected, overall intensity per surface area. <i>B & F</i>) Average, background-corrected intensity of cluster pixels. <i>C & G</i>) Average number of clusters per surface area. <i>D & H</i>) Average number of clusters per cell. <i>I & J</i>) The average contact surface area per cell (<i>I</i>) and surface-preference-score (<i>J</i>, see text) were determined from pooled data from the phosphoTyr and phosphoY783 PLCγ1 experiments (<i>n</i> = 41 images from 8 experiments with varying CFSE/unlabeled and stamp/overlay conditions in total containing 2665 KD and 2117 wt cells).</p