Stable T cell–dendritic cell interactions precede the development of both tolerance and immunity in vivo

Abstract

The maturation status of dendritic cells (DCs) determines whether they prime or tolerize T cells. We targeted ovalbumin peptide exclusively to DCs in situ using an antibody to DEC-205 and studied the interaction of DCs with naive CD4+ T cells in tolerizing or priming conditions. We used two-photon microscopy to simultaneously track antigen-specific OT-II T cells, nonspecific T cells and DCs in lymph nodes of living mice. In both tolerance and immunity, OT-II cells arrested on DCs near high endothelial venules beginning shortly after extravasation and regained their baseline speed by 18 h. Thus, early antigen-dependent T cell arrest on DCs is a shared feature of tolerance and priming associated with activation and proliferation.

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Figure 1: OT-II T cell activation and tolerance induced by α-DEC–OVA.
Figure 2: During early stages of priming and tolerance, antigen-specific T cells are located closer to HEVs in the superficial paracortex than are nonspecific T cells.
Figure 3: Tracking of antigen-specific and nonspecific T cells in the lymph nodes of living mice.
Figure 4: Mean cell velocities in tolerizing and priming conditions.
Figure 5: Early T cell arrest in tolerizing and priming conditions.
Figure 6: Directionality of antigen-specific and nonspecific T cells.

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Acknowledgements

We thank R. Steinman for discussions; E. Besmer for help with the manuscript; W. Gan for guidance on two-photon microscopy; S. Boscardin for α-DEC-CSP; and R. Masalimani for α-DEC–OVA plasmid. M.C.N. and M.L.D. contributed equally to this work. Supported by the National Institutes of Health (AI055037 to M.L.D. and AI051573 to M.C.N.), Irene Diamond Foundation (M.L.D.), Howard Hughes Medical Institute (M.C.N.), Rothschild Foundation (G.S.), Medical Scientist Training Program (GM07739 to R.L.L.) and German Research Foundation (DU 548/1-1 to D.D.).

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Correspondence to Michel C Nussenzweig or Michael L Dustin.

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Supplementary information

Supplementary Fig. 1

DC maturation induced by α-CD40 antibody. (PDF 304 kb)

Supplementary Fig. 2

Timeline for imaging T cell tolerance and priming. (PDF 1002 kb)

Supplementary Fig. 3

There is no systematic association between cell depth and average cell speed. (PDF 529 kb)

Supplementary Fig. 4

Instantaneous velocities of specific and non-specific cells. (PDF 116 kb)

Supplementary Fig. 5

Speeds of antigen-specific cells normalized to speed of control cells in the same imaging field. (PDF 59 kb)

Supplementary Video 1

Two-photon visualization of 3 types of cells and the vasculature in live lymph nodes (MOV 3182 kb)

This and all movies below are maximum intensity projections of a 50 μm-thick volume. Lymph node vasculature can be visualized with Q-dots that emit light at 655 nm. When excited at 910 nm, Q-dots are bright enough to visualize even capillaries, which was not possible with the small molecule-conjugated dextrans. HEVs can be identified by the morphology of the endothelial cells and the shadows of rolling lymphocytes. Antigen-specific EGFP-OT-II T cells (cyan) and non-specific ECFP-T cells (blue) move similarly in this field.

Supplementary Video 2

T cell extravasation in the lymph node of a living mouse (MOV 3160 kb)

T cells (blue, cyan) are visible moving throughout the vasculature (red), visualized by i.v. injection of Q-dots. Many vessels are partially in contact with dendritic cells (yellow). A single T cell is visible extravasating in the lower right quadrant, indicated by an arrow – it appears in the middle of the HEV at 8:11, remains relatively stationary for ten minutes and moves to the left and up over the next ten minutes

Supplementary Video 3

Phototoxicity interferes with dendrite probing and cell migration (MOV 3021 kb)

Even before phototoxicity is manifested in cell morphology, it leads to cellular immobility. Lymphocytes (red) stop moving and DCs (green) stop the probing motions of their dendrites. The use of both dendrite movement and non-specific T cells as internal controls permits sensitive detection of damage to the imaging field. In the upper right corner of the field, which was not affected, both T cell crawling and dendrite probing is visible.

Supplementary Video 4

Tolerizing interactions 1-6 h after T cell transfer (MOV 2598 kb)

Antigen-specific T cells (orange) move more slowly than non-specific T cells (red), and are more frequently arrested on dendritic cells (green). Tracks of specific EGFP-OT-II cells are cyan, and tracks of nonspecific ECFP-T cells are purple.

Supplementary Video 5

Arrest of antigen-specific T cells near HEVs (MOV 2119 kb)

Antigen-specific EGFP-T cells (cyan) found arrested on the basal surface HEVs. Non-specific T cells (blue) move normally. Tracks of EGFP-OT-II cells are green, tracks of ECFP-T cells are blue, and DCs are green. HEVs (red) were visualized by i.v. injection of Alexa 594-labeled MECA-79 antibody, which targets peripheral node addressin (PNAd).

Supplementary Video 6

Tolerizing interactions 6-12 h after T cell transfer (MOV 1722 kb)

Antigen-specific T cells (orange) move more slowly than non-specific T cells (red), and are more frequently arrested on dendritic cells (green). Tracks of specific EGFP-OT-II cells are cyan, and tracks of nonspecific ECFP-T cells are purple. Antigen-specific T cells are less arrested than at 1-6 h.

Supplementary Video 7

Tolerizing interactions 12-18 h after T cell transfer. (MOV 2894 kb)

Antigen-specific T cells (orange) and non-specific T cells (red) interact with DCs (green) as they move throughout the T cell area. Tracks of specific EGFP-OT-II cells are cyan, and tracks of nonspecific ECFP-T cells are purple. Antigen-specific T cells have largely recovered in speed, but still move slightly slower than nonspecific T cells.

Supplementary Video 8

Priming interactions 1-6 h after T cell transfer. (MOV 1851 kb)

Antigen-specific T cells (orange) move more slowly than nonspecific T cells (red) and are more frequently arrested on DCs (green). Tracks of specific EGFP-OT-II cells are cyan, and tracks of nonspecific ECFP-T cells are purple.

Supplementary Video 9

Priming interactions 6-12 h after T cell transfer. (MOV 2408 kb)

Antigen-specific T cells (orange) move more slowly than nonspecific T cells (red) and are more frequently arrested on DCs (green). Tracks of specific EGFP-OT-II cells are cyan, and tracks of nonspecific ECFP-T cells are purple. EGFP-OT-II cells are equally arrested at 6-12 h as they are at 1-6 h after transfer.

Supplementary Video 10

Priming interactions 12-18 h after T cell transfer. (MOV 2606 kb)

Antigen-specific T cells (orange) move more slowly than nonspecific T cells (red) and are more frequently arrested on DCs (green). Tracks of specific EGFP-OT-II cells are cyan, and tracks of nonspecific ECFP-T cells are purple. EGFP-OT-II cells have partially recovered in speed, but are still more arrested than the nonspecific ECFP-T cells.

Supplementary Video 11

T cell cluster formation (MOV 3205 kb)

This phenomenon is relatively rare with antigen targeted to DEC-205, as compared to cell-bound antigen delivered by a small fraction of APCs in the lymph node. The cluster seen here is composed of antigen-specific EGFP-OT-II T cells (orange) and not wild-type ECFP T cells (red).

Supplementary Video 12

T cell arrest on fluorescent and non-fluorescent dendritic cells (MOV 1556 kb)

T cell arrest on EYFP- cells was often observed. This may reflect DCs that express relatively low levels of EYFP or processes of EYFP+ DCs too thin to be visualized by multiphoton microscopy. The rightmost T cell is seen contacting an EYFP+ DC, then detaching and arresting nearby, possibly on the surface of a non-fluorescent APC.

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Shakhar, G., Lindquist, R., Skokos, D. et al. Stable T cell–dendritic cell interactions precede the development of both tolerance and immunity in vivo. Nat Immunol 6, 707–714 (2005). https://doi.org/10.1038/ni1210

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