Laboratory of Immunology

Videos from LBS Research

Movie 1: T cells exit HEVs in limited locations (Quicktime)

A single z slice from an intravital four-dimensional data set showing numerous T cells (red) exiting HEV (green) in a lymph node via lucent areas that appear to be gaps in the FRC sheath (“exit ramps”). The playback speed is 300x for both the main and zoomed image. See Bajénoff M, Egen JG, Koo LY, Laugier JP, Brau F, Glaichenhaus N, Germain RN. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity. 2006 Dec;25(6):989-1001.

Movie 2: T cells migrate along the FRC network (Quicktime)

Dynamic image of T-cell (red) migration along the FRC network (green). The trails of three of the T cells in this intravital dataset from a lymph node are highlighted in the second part of the movie with colored dots to help visualize the path taken along the fibers by a given T cell (z stack = 12 µM). The playback speed is 300x in the first part of the movie and 150x in the second part when the tracks are highlighted. See Bajénoff M, Egen JG, Koo LY, Laugier JP, Brau F, Glaichenhaus N, Germain RN. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity. 2006 Dec;25(6):989-1001.

Movie 3: Preferential interactions of naive CD8 T cells with CD4 T cell-engaged vs. non-engaged DCs (Quicktime)

Increased frequency of contact between polyclonal CD8+ T cells (green) and 323-pulsed DC (blue) as compared to that between CD8+ T cells and unpulsed DC (yellow) only a few microns away in the same imaging field within a lymph node. OT-2 (red) T cells can be seen in the CD8+ T cell – 323-pulsed DC clusters. The movie is edited to illustrate T-cell movement and contact with DCs. The tracks of polyclonal CD8+ T cells approaching and contacting the two indicated 323-pulsed DCs (blue arrows) are shown with blue dots for the tracks and a blue circle at the site of contact, while the track of a T cell approaching an unpulsed DC (yellow arrow) is shown with yellow dots and contact with a yellow circle. During this imaging session, a total of four and two contacts are made between CD8+ T cells and the two 323-pulsed DCs, while only one contact is made by a CD8+ T cell with the unpulsed DC. Total time: 34 minutes. Playback speed: 200x with pausing at the moment of contact. Scale bar: 20 µm. See Castellino F, Huang AY, Altan-Bonnet G, Stoll S, Scheinecker C, Germain RN. Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic interaction. Nature. 2006 Apr 13;440(7086):890-5.

Movie 4: Behaviors of recent immigrant B cells in and around peri-HEV DC arrays loaded with or without cognate Ag (Quicktime)

The video montage shows results of intravital imaging of four different combinations of MD4 or non-tg B cells (red) and HEL- or OVA-DCs (blue) in the peri-HEV region of a lymph node. HEVs (green) in all four scenes can be identified by the intravascular attachment of B cells. Scale bar: 30 µm. See Qi H, Egen JG, Huang AY, Germain RN. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science. 2006 Jun 16;312(5780):1672-6.

Movie 5: Distinct behaviors of MD4 and non-tg B cells in an LN containing HEL-DCs (Quicktime)

In this intravital lymph node data set, two MD4 B cells (red) remain in prolonged contact with HEL-DCs (blue) and show little migration. In contrast, non-tg B cells (green) rapidly move away toward a B follicle (not seen) without substantial attachment to the DCs. Scale bar: 20 µm.

Movie 6: Differential adhesion of Sap +/+ and Sap -/- T cells to antigen-presenting B cells (Quicktime)

Sap +/+ OT-2 T cells (red), Sap -/- OT-2 T cells (green), and MD4 B cells (blue) were co-transferred into mice that had previously been immunized with HEL-OVA conjugate antigen. Intravital imaging of the draining LNs was performed 24 to 36 hours after cell transfer. As compared to their Sap -/- counterparts and typified in this example, Sap +/+ OT-2 T cells were engaged in more stable interactions with cognate MD4 B cells. Playback speed: 450×. See Qi H, Cannons JL, Klauschen F, Schwartzberg PL, Germain RN. SAP-controlled T-B cell interactions underlie germinal centre formation. Nature. 2008 Oct 9;455(7214):764-9.

Movie 7: Sap -/- OT-2 T cells fail to stably interact with B cells in the absence of competition from Sap +/+ OT-2 in vivo (Quicktime)

GFP-expressing Sap +/+ or Sap -/-/+ OT-2 T cells engaged B cells in long-lasting conjugates; Sap -/- OT-2 T cells predominantly exhibited “hit-and-run” contacts with B cells. Imaging data in this movie are rendered in three-dimensional mode. Playback speed: 450×. See Qi H, Cannons JL, Klauschen F, Schwartzberg PL, Germain RN. SAP-controlled T-B cell interactions underlie germinal centre formation. Nature. 2008 Oct 9;455(7214):764-9.

Movie 8: Sap -/- T cells fail to be recruited and retained in nascent GC (Quicktime)

CFP-expressing Sap +/+ OT-2 T cells (red), GFP-expressing Sap -/- OT-2 T cells (green), and non-fluorescent MD4 B cells were co-transferred into mice that were subsequently immunized with HEL-OVA. Intravital imaging of draining LNs was performed six to eight days post-immunization. CMTPX-labeled polyclonal naïve B cells (blue) were transferred one or two days prior to imaging to help demarcate the follicular mantle and GC. The dotted line at the beginning of the movie approximates the GC border in this maximum intensity projection. Playback speed: 450×. See Qi H, Cannons JL, Klauschen F, Schwartzberg PL, Germain RN. SAP-controlled T-B cell interactions underlie germinal centre formation. Nature. 2008 Oct 9;455(7214):764-9.

Movie 9: Three-dimensional reconstruction of DC extensions across the epithelial layer of the terminal ileum (Windows Media Player)

Serial optical sections of an excised terminal ileum from a CD11c-EGFP mouse were obtained using two-photon microscopy and used to create a three-dimensional image of “balloon bodies” (BBs) (green) extending across the intact epithelial layers of the terminal ileum (red). The three-dimensional reconstruction encompasses a total of 65 optical sections at 1.5 µm/z-section to include a total depth of 97.5 µm. See Chieppa M, Rescigno M, Huang AY, Germain RN. Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement. J Exp Med. 2006 Dec 25;203(13):2841-51.

Movie 10: Different shape of trans-epithelial DC extensions (Quicktime)

An MHC CII-EGFP mouse was given Salmonella orally. The nuclei of all the cells in region of the terminal ileum imaged by intravital two-photon microscopy were labeled with Hoechst 33342 (blue), and the epithelium was stained with SNARF (red). Two different DCs (green) can be seen extending dendritic processes across the epithelium. See Chieppa M, Rescigno M, Huang AY, Germain RN. Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement. J Exp Med. 2006 Dec 25;203(13):2841-51.

Movie 11: Rapid association of blood-borne BCG with Kupffer cells observed in MHCII-EGFP mice (Quicktime)

Two-dimensional time-lapse image series from an MHCII-EGFP mouse that was infected with 1 x 107 BCG-RFP bacteria through an intravenous catheter one minute after beginning imaging. EGFP-expressing cells are shown in green, BCG-RFP fluorescence is shown in white, and a fluorescent blood tracer is shown in blue. Scale bar represents 20 µm. Playback speed is 30×. See Egen JG, Rothfuchs AG, Feng CG, Winter N, Sher A, Germain RN. Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas. Immunity. 2008 Feb;28(2):271-84.

Movie 12: T cells appear to migrate along macrophage cell bodies at the granuloma periphery (Quicktime)

Four-dimensional intravital image series from a LysM-EGFP mouse that had been infected with BCG three weeks earlier. Animals received in vitro stimulated, dye-labeled OT-2 TCR transgenic T cells 12 hours prior to imaging. EGFP-expressing cells are shown in green and T cells are shown in red. Tracks of an individual T cell approaching the granuloma border are shown in blue. Playback speed is 180x. Dimensions of the imaging field are 63x63x42. See Egen JG, Rothfuchs AG, Feng CG, Winter N, Sher A, Germain RN. Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas. Immunity. 2008 Feb;28(2):271-84.

Movie 13: Directional extravasation of neutrophils from blood vessels toward the site of skin damage and parasite deposition (Quicktime)

Four-dimensional intravital image series showing neutrophil extravasation from a blood vessel in the ventral ear pinnae of a Lys-eGFP mouse that had been inoculated with 1x104 L. major-RFP parasites 30 minutes prior to the start of imaging. EGFP-expressing cells are shown in green and L. major-RFP is shown in red. Playback speed is 600x. Scale bar, 30µm. See Peters NC, Egen JG, Secundino N, Debrabant A, Kimblin N, Kamhawi S, Lawyer P, Fay MP, Germain RN, Sacks D. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science. 2008 Aug 15;321(5891):970-4.

Movie 14: Magnified view of a portion of Movie 13 (Quicktime)

Shows the exit of neutrophils (green) from a blood vessel and the directional movement of these cells toward a region of damage and infection.

Movie 15: Sequential migration of neutrophils from the epidermis/dermis into sites of sand fly proboscis penetration through the stratum corneum (Quicktime)

Four-dimensional intravital image series from a Lys-eGFP mouse that had been bitten on the ventral ear pinnae by L. major infected sand flies immediately prior to imaging. Shown are XY, XZ, and YZ projection images. EGFP-expressing cells are shown in green, and the two-photon second harmonic signal is shown in blue. Playback speed is 600x. Scale bar is 30µm. See Peters NC, Egen JG, Secundino N, Debrabant A, Kimblin N, Kamhawi S, Lawyer P, Fay MP, Germain RN, Sacks D. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science. 2008 Aug 15;321(5891):970-4.

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