T-cell receptor microclusters critical for T-cell activation are formed independently of lipid raft clustering

Abstract

We studied the function of lipid rafts in generation and signaling of T-cell receptor microclusters (TCR-MCs) and central supramolecular activation clusters (cSMACs) at immunological synapse (IS). It has been suggested that lipid raft accumulation creates a platform for recruitment of signaling molecules upon T-cell activation. However, several lipid raft probes did not accumulate at TCR-MCs or cSMACs even with costimulation and the fluorescence resonance energy transfer (FRET) between TCR or LAT and lipid raft probes was not induced at TCR-MCs under the condition of positive induction of FRET between CD3 zeta and ZAP-70. The analysis of LAT mutants revealed that raft association is essential for the membrane localization but dispensable for TCR-MC formation. Careful analysis of the accumulation of raft probes in the cell interface revealed that their accumulation occurred after cSMAC formation, probably due to membrane ruffling and/or endocytosis. These results suggest that lipid rafts control protein translocation to the membrane but are not involved in the clustering of raft-associated molecules and therefore that the lipid rafts do not serve as a platform for T-cell activation.

FIG. 1.

Images of lipid raft-associated proteins and lipid raft probes at TCR-MCs. (A) AND-Tg T cells expressing mLAT(wt)-GFP, mLAT(ΔCP)-GFP, Lck-GFP, or Lck(N10)-GFP together with CD3ζ-Halo, or cells expressing CD3ζ-Halo and stained with CTXB-Alexa 488, were loaded with TMR ligand and placed on the planar bilayer containing ICAM-I and I-E^k with 10 μM MCC peptide. Images were collected 2 min after attachment to the bilayer. The cells expressing Lck(N10)-GFP and CD28-CFP were fixed at 2 min on the CD80-containing bilayer and stained with anti-CD3ɛ-biotin and streptavidin-Alexa 566. The large bright area in cells with mLAT(ΔCP)-GFP reflects its presence within cytoplasmic organelles as detected in the x-z image (see Fig. S1 in the supplemental material). (B) The numbers of GFP and Halo tag clusters were counted objectively using software with Gaussian fitting and counting capability. Data are means ± standard deviations of 15 to 30 cells. (C) The cells used in panel A were incubated for 10 min, and images were collected. DIC, differential interference contrast.

FIG. 2.

FRET analysis for the interaction between CD3ζ or LAT and a lipid raft probe, Lck(N10). (A) Images of CFP, YPet, and FRET efficiency (FRET/CFP) of AND-TCR hybridoma cells expressing either CD3ζ-CFP or Lck(N10)-YPet or either pair CD3ζ-CFP/Lck(N10)-YPet or pair LAT-CFP/Lck(N10)-YPet at 2 min. The diagrams in the left column depict the labeled molecules under each experimental condition. (B) The intensity profiles at the peaks of CD3ζ-CFP or LAT-CFP were arranged according to peak position. The intensities were averages from a 10- × 41-pixel area of each peak. Data are means ± standard errors of the means of 40 to 50 peaks from 3 cells. Blue line, CFP; orange, YPet; pink, FRET/CFP. a.u., arbitrary units. (C) Kymograph analysis for time-lapse measurement of FRET/CFP between CD3ζ-CFP and Lck(N10)-YPet. Images were taken every 2.6 s after cellular attachment, and 5- × 348-pixel images were extracted and arranged in temporal order.

FIG. 3.

Expression and microcluster formation of LAT mutants on a planar bilayer. (A) AND-Tg T cells expressing CD3ζ-Halo and mLAT(wt)-GFP, mLAT(C29A)-GFP, mLAT(C26,29A)-GFP, or LAXhLAT-GFP were loaded with TMR ligand and placed on the bilayer for 2 min. (B) AND-Tg T cells expressing CD3ζ-Halo and hLAT(wt)-GFP, hLAT(Y132F)-GFP, or hLAT(3YF)-GFP were placed on the bilayer for 2 min. (C) Numbers of clusters of GFP or Halo tag were counted objectively by software as described for Fig. 1. Data are means ± standard deviations of 15 to 30 cells. DIC, differential interference contrast.

FIG. 4.

Endocytosis of lipid raft probes and CD3ζ at the interface between T cells and APC. (A) AND-Tg T cells expressing CD3ζ-Halo and stained with CTXB-Alexa 488 were loaded with TMR ligand and incubated with 5 μM MCC peptide-loaded LPS-activated B cells. Video images were collected 8 min after starting the incubation. Images taken at each time point revealed a different time course of CD3ζ-Halo and CTXB-Alexa 488 accumulation. (B) AND-Tg T cells expressing Lck(N10)-GFP or CD3ζ-GFP or stained with CTXB-Alexa 488 were incubated with the activated B cells for 20 min in the presence of 10 μM FM 4-64. White arrows indicate cSMAC (A) and endocytosed molecules (B). DIC, differential interference contrast.