T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node

Abstract

Dendritic cells (DCs) ingest antigens in peripheral tissues and migrate to lymph nodes where they present MHC class II-bound antigen to CD4(+) T cells. We used two-photon microscopy to image the single-cell dynamics of interactions between DCs and T cells within intact lymph nodes in the absence of relevant antigen. DCs were fluorescently labeled in vivo by cutaneous injection of alum adjuvant including carboxyfluorescein diacetate succinimidyl ester (CFSE). CFSE-positive DCs (CD11c(+), CD11b(+), and low-to-intermediate CD8(+)) were observed in draining lymph nodes 24-72 h later. Labeled DCs meandered slowly (2-3 microm x min(-1)) in the T cell zone near B cell follicles but vigorously extended long agile dendrites. Encounters between T cells and DCs arose as T cells moved autonomously along random paths. Moreover, T cells did not accumulate around DCs, and their relative velocities approaching and departing DCs were equivalent, implying that T cells are not attracted toward DCs by chemotactic gradients but rather encounter them by chance. T cell/DC contacts occurred primarily on dendrites at arm's length from the DC soma and typically lasted approximately 3 min, enabling an individual DC to interact with up to 5000 T cells per hour. We conclude that dynamic DC gesticulation and random T cell motility together enhance the stochastic scanning of the T cell repertoire, thereby enabling rapid initiation of the immune response.

Fig. 1.

CFSE labeling at the depot and subsequent trafficking of labeled cells to draining lymph nodes. (A) Imaging the alum depot 1.5 h after intradermal ear injection of adjuvant mixture containing CFSE and fluorescent Ova. Yellow scan line represents 1.26 mm. (B) Distribution of CFSE (green) and fluorescent Ova (red) along the line-scan near the edge of the adjuvant depot in skin. (C) The kinetics of CFSE⁺ cell trafficking to the lymph node. Draining cervical lymph nodes were harvested at varying times after adjuvant/CFSE injection and evaluated for CFSE⁺ cells by flow cytometry.

Fig. 2.

Characterization and localization of in vivo-labeled DCs in draining lymph nodes. (A) Surface expression of CD11c, CD11b, and CD8a on CFSE⁺ cells. CD8a staining was on average 4-fold lower than on CD8⁺ T cells. (B) Fluorescence-activated cell sorter analysis of cells isolated from lymph node 24 h after injection of adjuvant containing CFSE and fluorescent Ova. (Upper) Shown is the rare population (≈0.4% of total cells) that contains both CFSE and fluorescent Ova. (Lower) Histogram showing the percentage of CFSE⁺ cells that contain fluorescent Ova. (C) Two-photon image of a DC in a draining lymph node showing ingested fluorescent Ova (arrow) 24 h after injection of adjuvant containing CFSE and fluorescent Ova. (Bar = 15 μm.) (D) Image showing localization of CFSE⁺ DC in the T cell zone (TZ) near a B cell follicle (BF) in top (left) and side (right) views. (Bar = 50 μm.) Dotted line indicates approximate follicle boundary.

Fig. 3.

Dynamics of DC movement. (A) High-resolution monochrome and depth-encoded z-projections of a DC, illustrating elaborate dendrite morphology shown at indicated times (min:sec); note rapid changes in dendritic morphology (see also Movie 1). (Insets) Shown are enlarged views of a selected region. (Bar = 25 μm.) Arrowheads mark individual dendrites. (B) Histogram of instantaneous DC velocities (mean = 2.7 μm·min^–1) derived by tracking the center of fluorescence of 15 DCs. (C) Tracks of 15 DCs in the x, y plane normalized to their starting positions. (D) Superimposed images of a single DC, depth-encoded, to illustrate the swept volume over a 12-min period.

Fig. 4.

T cells approach DCs along random trajectories. (A–D) Cumulative fluorescence images of T cells (red) and DC (green) at indicated times during recording (min:sec). (Bar = 50 μm.) Frames present top (Upper) and side (Lower) views as maximum intensity projections of each orthogonal view across sequential time-lapse sequences (see also Movie 2). (E and F) Color separation of frames to illustrate the area covered by the DC (green) and the even dispersion of T cell tracks (red). (G) Profiles of green and red fluorescence intensity along the x, y, and z axes, marked by crosshairs in E and F showing that T cell density (red trace) does not increase near the DC (green trace). (H) Histogram of T cell velocities (n = 8,700) along vectors normal to DCs, showing an equal distribution of negative and positive velocities.

Fig. 5.

T cell/DC contact morphology and duration. (A) Time-lapse image sequence (times indicated min:sec) illustrating brief contacts between T cells and a DC in the absence of antigen. (Bar = 25 μm.) Top frames are true color (red, T cells; green, DC) projections along the z axis. Frames below are depth-encoded enlarged views of the same images (see also Movie 3). Multiple discrete contacts are indicated (arrows) where the DCs (green) and T cells (red) colocalize in the x-y plane and occupy the same z-position (display the same color) as seen in the pseudocolored image. (B) Contacts between T cells and DCs, rendered in three dimensions (see Movies 4 and 5). Contacts between T cells and DC were dynamic, changing rapidly in size over tens of seconds. Detailed morphometric analysis of several contacts: The average surface area in the contact region was 7.8 μm² (SE = 2.0, n = 32), ranging from more than one-half (>70 μm²) of the T cell's total surface to as little as 1 μm². Contacts took place an average of 16.8 μm (SE = 1.1 μm, n = 26) from the DC center of mass. (C) Histogram of T cell/DC contact durations (89 contacts: median duration = 3.4 min).