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The impact of negative selection on thymocyte migration in the medulla


Developing thymocytes are screened for self-reactivity before they exit the thymus, but how thymocytes scan the medulla for self antigens is unclear. Using two-photon microscopy, we observed that medullary thymocytes migrated rapidly and made frequent, transient contacts with dendritic cells. In the presence of a negative selecting ligand, thymocytes slowed, became confined to areas of approximately 30 μm in diameter and had increased contact with dendritic cells surrounding confinement zones. One third of polyclonal medullary thymocytes also showed confined, slower migration and may correspond to autoreactive thymocytes. Our data suggest that many autoreactive thymocytes do not undergo immediate arrest and death after encountering a negative selecting ligand but instead adopt an altered migration program while remaining in the medullary microenvironment.

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Figure 1: Sectioning the thymus facilitates imaging of the medulla.
Figure 2: Thymocyte migration in the medulla is rapid and confined.
Figure 3: The impact of negative selection on thymocyte migration.
Figure 4: Increased thymocyte-DC interactions during negative selection.
Figure 5: A subset of polyclonal medullary thymocytes shows slower, more confined migration.


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We thank H. Nolla for assistance with flow cytometry; the University of California Berkeley Molecular Imaging Center for microscopy; M. Anderson (University of California, San Francisco) for RIPmOVA mice; L. Ehrlich and R. Lewis for communication of unpublished data; and P. Bousso, B.J. Fowlkes, and members of the Robey laboratory for comments on this manuscript.

Author information

Authors and Affiliations



M.L.B. and E.L. designed and did experiments, analyzed and interpreted results and wrote the manuscript; I.D. analyzed the migration of Foxp3-GFP+ cells; P.H. provided technical support for imaging; Y.F.L. did manual scoring and edited tracks; A.K.C. provided guidance for quantitative analysis of cell migration data; and E.A.R. directed the study and wrote the manuscript.

Corresponding author

Correspondence to Ellen A Robey.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Table 1 (PDF 2960 kb)

Supplementary Movie 1

Thymocyte migration in the cortex and medulla of a cut thymic lobe. Representative runs in the cortex (left) and the medulla (right) of a cut thymic preparation of a WT+OT1→WT chimera showing DCs (CD11c-YFP, orange). The migratory path of 3 representative WT thymocytes (blue) is shown as a time-coded track (blue-red-yellow-white) with the duration of 10.4 min. Yellow arrow points to moving dendrites in the cortex of a cut thymus preparation. Dimensions for each imaging volume: 172 × 143 ×60 μm3 ×10.4 min. White bar represents 30 μm. (MOV 3959 kb)

Supplementary Movie 2

Thymocyte migrating in the medulla during negative selection. Representative runs in the medulla of OT1 thymocytes (green) and CD11c-YFP+ DCs nge) in a WT+OT1→-OVA chimera (left) and a WT+OT1→+OVA chimera (right). Tracks of 3 representative OT1 thymocytes with a duration of 12.5 min (green) are shown. Dimensions for both imaging volumes: 172 × 143 × 62 μm3 × 12.5 min. White bar represents 30 μm. (MOV 3551 kb)

Supplementary Movie 3

Thymocytes are confined to smaller areas in the presence of their negative-selecting ligands. A 3D rotation showing WT thymocyte tracks (blue) and OT1 thymocyte tracks (red) in the medulla of a WT+OT1→+OVA chimera. Dimensions: 172 × 143 ×60 μm3 ×14.6 min. (MOV 1804 kb)

Supplementary Movie 4

Self-reactive thymocytes rarely enter or leave confinement zones. Multiple examples of clusters of auto-reactive OT1 thymocyte tracks. Most of the thymocytes stay in the same confinement zones (colored tracks) during the time of the run (example 1) with some tracks that do not appear to be associated with a cluster (gray tracks). Only a few of cells (red tracks, examples 2-5) are seen entering (yellow circle, examples 3-5) or leaving a cluster of tracks (white circle, examples 4 and 5) while the spending most of the time in a cluster (cyan circle, examples 3-5). Areas highlighted with a white square are magnified to show examples of tracks entering or leaving a confinement zone, with OT1 thymocytes in green and CD11c-YFP+ DCs in orange. Dimensions: 172 × 143 × 60 μm3. White bar represents 30 μm. (MOV 6801 kb)

Supplementary Movie 5

Auto-reactive thymocyte tracks lie between DCs. Two representative 3D rotations showing that most auto-reactive OT1 thymocyte tracks (red) lie between the cell bodies of DCs (orange) in the medulla of a WT+OT1→+OVA chimera. Dimensions: 172 × 143 × 60 μm3. (MOV 3626 kb)

Supplementary Movie 6

Interactions of positive-selecting thymocytes with DCs in the medulla. An example of a single OT1 thymocyte (green) making successive contacts (white circles) with multiple DCs (orange) in the medulla of an OT1→-OVA chimera. In some case, the thymocyte associates closely with the DC body, occasionally turning after contacting the DC. The migratory path is color-coded to indicate time (blue-red-yellow-white). Dimensions: 71 × 72 ×30 μm3 × 20.5 min (MOV 2839 kb)

Supplementary Movie 7

Thymocyte-DC interactions in the medulla during negative selection. An example of a single OT1 thymocyte (green) making successive encounters (circles) with multiple DCs (orange) in the medulla of an OT1→+OVA chimera. The second, longer-lasting contact spans 8.5 min or 56.7% of time. Dimensions: 44.75 × 44.75 × 58 μm3 × 15 min. (MOV 2012 kb)

Supplementary Movie 8

Slow polyclonal thymocytes, like self-reactive OT1, are confined to smaller areas than fast polyclonal thymocytes. 3D rotations showing tracks for all wild-type thymocyte tracks (blue) that are separated into groups of fast (≥8 μm/min) and slow (≥8 μm/min) WT thymocyte tracks (cyan and purple, respectively), as well as OT1 thymocyte tracks (red) in the medulla of a WT+OT1• +OVA chimera. An overlapping zone of confinement containing slow WT tracks and auto-reactive OT1 tracks is highlighted with a white circle while a non-overlapping zone is shown with a yellow circle. Dimensions: 172 × 143 × 60 μm3. (MOV 2787 kb)

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Le Borgne, M., Ladi, E., Dzhagalov, I. et al. The impact of negative selection on thymocyte migration in the medulla. Nat Immunol 10, 823–830 (2009).

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