T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node - PubMed (original) (raw)

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

Mark J Miller et al. Proc Natl Acad Sci U S A. 2004.

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.

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Figures

Fig. 1.

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.

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.

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.

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.

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).

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