Superresolution imaging reveals nanometer- and micrometer-scale spatial distributions of T-cell receptors in lymph nodes

Abstract

T cells become activated when T-cell receptors (TCRs) recognize agonist peptides bound to major histocompatibility complex molecules on antigen-presenting cells. T-cell activation critically relies on the spatiotemporal arrangements of TCRs on the plasma membrane. However, the molecular organizations of TCRs on lymph node-resident T cells have not yet been determined, owing to the diffraction limit of light. Here we visualized nanometer- and micrometer-scale TCR distributions in lymph nodes by light sheet direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM). This dSTORM and SIM approach provides the first evidence, to our knowledge, of multiscale reorganization of TCRs during in vivo immune responses. We observed nanometer-scale plasma membrane domains, known as protein islands, on naïve T cells. These protein islands were enriched within micrometer-sized surface areas that we call territories. In vivo T-cell activation caused the TCR territories to contract, leading to the coalescence of protein islands and formation of stable TCR microclusters.

Keywords: T-cell activation; T-cell receptor; plasma membrane; protein cluster; superresolution.

Fig. 1.

Superresolution imaging of TCRs on naïve lymph node-resident T cells. (A) Light sheet dSTORM image of TCRs on naïve T cells in a lymph node section. (B) Cluster detection of single molecules of H57^AF647 (Left) vs. TCR islands labeled with H57^AF647 (Right) using TIRF-based dSTORM. Circles highlight detection events produced by single antibodies. (C) Characterization of the number of detected events per single antibody (red) and TCR protein island (black). The computed number of TCRs was based on the average number of events per single H57 antibody (∼20) and labeling efficiency of TCRs (∼30%) by immunostaining.

Fig. 2.

Light sheet dSTORM imaging of TCRs in lymph nodes before and after in vivo T-cell activation. (A) Representative superresolution images of T cells from mice without peptide injection (naïve), and in mice at 2, 5, and 24 h after peptide injection. (B) High-magnification view of the TCR organizations in boxed regions in A. (C) Intensity profiles of lines in B. Arrows mark local intensity peaks that represent protein islands. Reduced distances between adjacent protein islands cause an overlapping of intensity peaks and a broadening of the structure (w₁ = 240, w₂ = 507, w₃ = 533, and w₄ = 753 nm).

Fig. 3.

SIM imaging of TCRs in lymph nodes before and after in vivo T cell activation. (A) Low-magnification view of naïve T cells. (B) Medium-magnification view of the boxed region in A showing protein islands. (C) High-magnification view of the boxed region in B showing a TCR territory and protein islands within it. (D) Low-magnification view of in vivo activated T cells at 5 h after peptide injection. (E) Medium-magnification view of the boxed region in D showing areas with high TCR density on the plasma membrane. (F) High-magnification view of the boxed region in E showing a TCR microcluster.

Fig. 4.

Two-level organization model for TCR distribution in the plasma membrane. The illustration demonstrates the rearrangement of TCRs from naïve T cells (Upper) to activated T cells (Lower). The rearrangement is facilitated by the contraction of TCR territories (yellow) enriched with protein islands (small circles). The contraction can be initiated by the presence of agonist peptides (purple) and facilitated by endogenous peptides (gray).