MHC I Expression Regulates Co-clustering and Mobility of Interleukin-2 and -15 Receptors in T Cells

Abstract

MHC glycoproteins form supramolecular clusters with interleukin-2 and -15 receptors in lipid rafts of T cells. The role of highly expressed MHC I in maintaining these clusters is unknown. We knocked down MHC I in FT7.10 human T cells, and studied protein clustering at two hierarchic levels: molecular aggregations and mobility by Förster resonance energy transfer and fluorescence correlation spectroscopy; and segregation into larger domains or superclusters by superresolution stimulated emission depletion microscopy. Fluorescence correlation spectroscopy-based molecular brightness analysis revealed that the studied molecules diffused as tight aggregates of several proteins of a kind. Knockdown reduced the number of MHC I containing molecular aggregates and their average MHC I content, and decreased the heteroassociation of MHC I with IL-2Rα/IL-15Rα. The mobility of not only MHC I but also that of IL-2Rα/IL-15Rα increased, corroborating the general size decrease of tight aggregates. A multifaceted analysis of stimulated emission depletion images revealed that the diameter of MHC I superclusters diminished from 400-600 to 200-300 nm, whereas those of IL-2Rα/IL-15Rα hardly changed. MHC I and IL-2Rα/IL-15Rα colocalized with GM1 ganglioside-rich lipid rafts, but MHC I clusters retracted to smaller subsets of GM1- and IL-2Rα/IL-15Rα-rich areas upon knockdown. Our results prove that changes in expression level may significantly alter the organization and mobility of interacting membrane proteins.

Figure 1

Protein expression in control and MHC I knockdown cells determined by flow cytometry. Quantification of IL-2Rα molecules per cell was performed by using QIFIKIT beads (Dako North America). Expression of MHC I, MHC II, and IL-15Rα proteins was assessed by comparing the mean fluorescence intensities of directly labeled epitopes (targeted by Alexa 546-tagged W6/32, L243, and anti-FLAG mAbs) to that of directly labeled IL-2Rα (Alexa 546-anti-Tac mAb). Fluorescence intensities were normalized by the dye/protein antibody labeling ratios determined by absorption photometry. Error bars are SD values of averages of triplicate samples (n ∼ 10⁴ cells per sample).

Figure 2

FCS analysis of molecular mobility, concentration (N), aggregation (molecular brightness), and bleached fraction upon MHC I knockdown. (A and B) Normalized autocorrelation functions of MHC I and IL-2Rα. (C) Average diffusion coefficients. (D) Average particle numbers in the FCS detection volume. (E) Specific molecular brightness, F/N, normalized by the F/N values of Fab fragments in solution. (F) Median bleached fractions of molecules calculated from the intensities after 5, 20, and 100 s (after the first, fourth, and 20th runs) of the 100-s illumination period. The following Alexa 488-conjugated Fab fragments were used for labeling: W6/32 for MHC I, anti-Tac for IL-2Rα, anti-FLAG for IL-15Rα, and L243 for MHC II. Distributions were compared with Kolmogorov-Smirnov tests, ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.1; boxes represent 25 and 75%; whiskers, 10 and 90%. n = 25–35 cells per sample.

Figure 3

FRET analysis of the effect of MHC I knockdown on molecular proximities. (A) Means of average FRET efficiencies of duplicate flow cytometric measurements (N > 10,000 cells) between IL-2/15Rα subunits and MHC I. Proteins were labeled with different pairs of donor (Alexa 546)- and acceptor (Alexa 647)-tagged antibodies (IL-2Rα, IL-15Rα, and MHC I were labeled with Alexa 546-anti-Tac, Alexa 546-7A4 24. and Alexa 647-W6/32 mAbs). Positive control: FRET between the light chain (β2m, labeled with Alexa546-L368) and heavy chain of MHC I. Negative control: transferrin receptor (TfR, located in coated pits, labeled with Alexa 546-MEM75 mAb) and GPI-anchored CD48 (enriched in lipid rafts, targeted by Alexa647-MEM102 mAb). Error bars are SD values. (B) Distribution of pixelwise FRET efficiencies between IL-2Rβ + IL-2Rα measured by the acceptor photobleaching method using confocal microscopy (n > 25 cells per sample; IL-2Rβ and IL-2Rα were labeled with Alexa 546-Mikβ3 and Alexa 647-7G7-B6). Mean FRET efficiency values were 9 and 10% in control and KD cells. Cells were fixed with formaldehyde.

Figure 4

Two-color STED images of cell surface protein codistributions on FT7.10 T lymphoma cells. (A) MHC I and IL-2Rα and (B) MHC I and IL-15Rα. MHC I was labeled with Alexa 594-W6/32 mAb (green, top row), IL-2Rα and IL-15Rα with ATTO 647N-anti-Tac and ATTO 647N-anti-FLAG mAbs (red, middle row), and fixed with formaldehyde. In the overlay images (bottom row), the yellow color shows the overlapping regions of MHC I and IL-R distributions. Because of the large differences in gray values between raw intensity data of MHC knockdown and control samples, intensities were rescaled. (Yellow squares) Areas displayed in the high-magnification images next to the low-magnification image. Scale bar in the low- and high-magnification images represent 7 μm and 0.7 μm, respectively.

Figure 5

Cluster-size distributions of MHC I, IL-2Rα, and IL-15Rα determined from STED microscopy images. The schemes in the top row illustrate the calculation of each parameter shown below. (A) Effect of MHC I knockdown (KD) on cluster size determined from Gaussian fits to intensity peaks in STED images. Pixel size is 20 nm. (A1) Direction-averaged FWHM of intensity peaks. Each peak represents a receptor cluster (see Materials and Methods); FWHM values are measures of average cluster size. (A2–A4) Histograms display the probability distributions of peak widths of MHC I, IL-2Rα, and IL-15Rα clusters in control and MHC I knockdown cells. (A5) Mean values of FWHMs. ^∗∗∗∗p < 0.0005, ^∗p < 0.05 (Kolmogorov-Smirnov test), n = 10–15 cells per sample. (B) Spatial autocorrelation analysis of protein cluster sizes. (B1) Scheme of calculating radial autocorrelation functions for radii of 1–100 pixels. (B2–B4) Average radial autocorrelation functions of MHC I, IL-2Rα, and IL-15Rα in control and MHC KD cells. Radii were measured in pixels. (B5) Estimated mean cluster sizes of MHC I, IL-2Rα, and IL-15Rα based on a triexponential fit of the correlation curves. The shortest correlation radius is related to the PSF; the medium one (shown in the figure) corresponds to the average cluster size; and the largest one probably to intercluster distances. ^∗p < 0.05 (unpaired t-test). (C) Image segmentation analysis of protein clusters. (C1) Images were intensity-thresholded and the number of pixels in contiguous clusters was determined. (C2–C4) Probability distributions of cluster areas (number of pixels in contiguous areas above threshold) in control or MHC I KD cells. (C5) Mean values of cluster areas. ^∗∗∗∗p < 0.0005 (Kolmogorov-Smirnov test). (D) Analysis of relative protein content of clusters. (D1) Total fluorescence intensities of clusters defined in (C) were calculated and sorted according to cluster size. (D2–D4) Histograms represent probability distributions of protein content versus cluster area. (D5) Most populated cluster sizes for MHC I, IL-2Rα, and IL-15Rα. ^∗∗∗∗p < 0.0005 (Kolmogorov-Smirnov test).

Figure 6

Colocalization analysis of MHC I with IL-2Rα and IL-15Rα in STED images. (A) Fractions of IL-2Rα and IL-15Rα molecules residing in common clusters with MHC I (and vice versa) derived from STED images of doubly labeled samples (referred to, e.g., as IL-2Rα near MHC I). (B) Pearson’s correlation coefficients of MHC I-IL-2Rα and MHC I-IL-15Rα codistributions from STED images (n = 10–15 cells per sample). Samples were compared with two-tailed t-tests, ^∗∗∗p < 0.001, ^∗∗p < 0.01.

Figure 7

A possible visualization of the effects of MHC I knockdown on the clustering of the IL-2/IL-15 receptor complex and MHC I molecules. Encircled areas mark protein superclusters as discerned by STED microscopy (red dashed line for MHC I and gray dotted line for IL-R superclusters), which contain several distinct tight aggregates of codiffusing proteins. Some of these aggregates are mobile, while others have little or no mobility. Upon MHC I KD, the MHC I content of the aggregates decreased, leading to a decrease of their size and an increase of their mobility. MHC I supercluster size decreased upon KD from 400–600 to 200–300 nm (marked by the decreased area of MHC I-containing regions encircled by red dashed lines), whereas the mean geometry of IL-2/IL-15 receptor superclusters did not change significantly. To see this figure in color, go online.