The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

Abstract

CD4 molecules on the surface of T lymphocytes greatly augment the sensitivity and activation process of these cells, but how it functions is not fully understood. Here we studied the spatial organization of CD4, and its relationship to T-cell antigen receptor (TCR) and the active form of Src kinase p56lck (Lck) using single and dual-color photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). In nonactivated T cells, CD4 molecules are clustered in small protein islands, as are TCR and Lck. By dual-color imaging, we find that CD4, TCR, and Lck are localized in their separate clusters with limited interactions in the interfaces between them. Upon T-cell activation, the TCR and CD4 begin clustering together, developing into microclusters, and undergo a larger scale redistribution to form supramolecluar activation clusters (SMACs). CD4 and Lck localize in the inner TCR region of the SMAC, but this redistribution of disparate cluster structures results in enhanced segregation from each other. In nonactivated cells these preclustered structures and the limited interactions between them may serve to limit spontaneous and random activation events. However, the small sizes of these island structures also ensure large interfacial surfaces for potential interactions and signal amplification when activation is initiated. In the later activation stages, the increasingly larger clusters and their segregation from each other reduce the interfacial surfaces and could have a dampening effect. These highly differentiated spatial distributions of TCR, CD4, and Lck and their changes during activation suggest that there is a more complex hierarchy than previously thought.

Keywords: PALM; T-cell receptor; coreceptor CD4; dSTORM; superresolution microscopy.

Fig. 1.

Analysis of the spatial distribution of CD4 in live T-cell membranes by PALM. (A) Colormap probability density plots for CD4–PSCFP2 expressed in live T cells interfacing with nonactivating (PLL, Left) and activating (IE^k–MCC and B7.1, Right) immobilized surfaces. (Scale bar, 1 μm.) Color bar scale represents the relative molecular density. (B) Ripley’s K-function plot of CD4–PSCFP2 molecules on a nonactivated (red solid line) or an activated (blue solid line) surface with a 99% confidence interval (black dashed line). Peaks of Ripley’s K-function plots were analyzed for the maxima values of L(r) − r (C) and r_max (D) for multiple cells (n = 15 and 21 for nonactivating and activating condition, respectively). *P < 0.01 and **P < 0.0001 (Student t test). Data are representative (A and B) and collections (C and D) of experiments for the selected areas of 3 × 3 μm², in total 28 cells.

Fig. 2.

Analysis of relative spatial distribution of CD4 and TCR in live T-cell membranes by dual-color PALM. (A) Dual-color PALM images for CD3ζ–PSCFP2 (green) and CD4–PAmCherry (red) coexpressed in live T cells interfacing nonactivating (PLL, Left) and activated (IE^k–MCC and B7.1, Right) immobilized surfaces. Each color map represents the probability density distribution image of each fluorescence channel. Color bar scale represents the relative molecular density. Enlarged dual-color PALM images are 1 × 1 μm² areas of white Inset squares. (Scale bar, 1 μm.) (B and C) Bivariate pair-correlation function curves (red solid lines) of CD3ζ and CD4 on nonactivating (B) and activating (C) conditions. Blue dashed lines indicate 95% confidence intervals of a random labeling (100 Monte Carlo simulations). (D) The degree of mixing parameter between CD3ζ and CD4 (as defined in the text and Materials and Methods) compared between nonactivating and activating conditions for multiple cells (n = 4 for both conditions). *P < 0.0001 (Student t test). Data in A–C are representative of experiments for a total of 18 cells. Only the cells with the representative 3 × 3 μm² areas where both molecules were detected at densities bigger than 100 molecules per square micrometer were used for the determination of the degree of mixing parameter (D).

Fig. 3.

Analysis of relative spatial distribution of CD4 and TCR by dual-color TIRFM and dSTORM. (A) Dual-color TIRFM and dSTORM images of T cells fixed at 10 min after interfacing with nonactivating PLL (Upper) or activating LBL (Lower) surfaces. The T cells were stained with anti-CD3ε–AF488 (green) and anti-CD4–AF647 (red) antibodies. Each color map in dSTORM data represents the probability density distribution image of each fluorescence channel. Color bar scale represents the relative molecular density. (Scale bar, 5 μm.) (B) Enlarged dual-color dSTORM images of 3 × 3 μm² areas of white Inset squares in A. (C and D) Bivariate pair-correlation function curves (red solid lines) of CD3ε and CD4 on nonactivating (C) and activating (D) conditions. Blue dashed lines indicate 95% confidence intervals of a random labeling (100 Monte Carlo simulations). (E) The degree of mixing parameter between CD3ε and CD4 (as defined in the text and Materials and Methods) compared between nonactivating (PLL) and activating (LBL) conditions for multiple cells (n = 8 for both conditions). *P < 0.05 (Student t test). Data in A–D are representative of experiments for a total of 15 cells. Only the cells with the representative 3 × 3 μm² areas where both molecules were detected at densities bigger than 100 molecules per square micrometer were used for the determination of the degree of mixing parameter (E).

Fig. 4.

Analysis of relative spatial distribution of active Lck paired with CD4 or TCR by dual-color TIRFM and dSTORM. (A) Dual-color TIRFM (the first three columns from the Left) and dSTORM (the fourth column) images of T cells fixed at 10 min after interfacing with nonactivating PLL (the first and third rows from the Top) or activating LBL (the second and fourth rows) surfaces. Active Lck molecules were stained with anti-Lck–pY394 antibody (AF488, green) and TCR or CD4 were stained with anti-CD3ε (Upper two rows) or anti-CD4 (Lower two rows) antibodies (AF647, red). (Scale bar, 5 μm.) Dual-color dSTORM images (the fourth column) are enlarged for 3 × 3 μm² areas of white Inset squares in TIRFM images (the third column). (B) Relative fluorescence intensities of active Lck (green solid lines) paired with TCR or CD4 (red solid lines) for nonactivating (PLL, Left column) and activating (LBL, Right column) on the diagonal yellow dashed lines in TIRFM images in A. (C and D) Clustering analysis for the active Lck molecules by Ripley’s K function. Peaks of Ripley’s K-function plots (Fig. S11) were analyzed for the maxima values of L(r) − r (C) and r_max (D) for multiple cells (n = 32 for PLL, n = 31 for LBL). (E) The degree of mixing parameter between active Lck and CD3ε and between active Lck and CD4 were compared for nonactivating (PLL) and activating (LBL) conditions from the experiments of multiple cells (n = 9 for Lck–CDε on PLL, n = 5 for Lck–CDε on LBL, n = 11 for Lck–CD4 on PLL, and n = 8 for Lck–CD4 on LBL). NS, statistically nonsignificant. *P < 0.01 and **P < 0.001 (Student t test). Data in A and B are representative of experiments for a total of 33 cells.