Journal: The Journal of Experimental Medicine

Article Title: In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain

doi: 10.1084/jem.20060420

Figure Lengend Snippet: Homogenous distribution of LFA-1 and TCR immunoreactivity along the plasma membrane of T cells not in contact with infected target cells. LFA-1 and TCR show homogeneous distribution along the plasma membrane of T cells neither in close apposition of target infected cells, nor involved in the formation of immunological synapses. Top panels (A–E) show a LFA-1 positive cell (B) not in contact with a TK-immunoreactive process (D), showing a uniform distribution of LFA-1. Panels F-J show a T cell with no LFA-1 expression (G) with a homogeneous distribution of TCR (H). Panels K-O show a T cell expressing LFA-1 (L) and TCR (M) not contacting a TK positive cell (N) showing no specific polarization and distribution in clusters. Panels P–T illustrate a CD8 + T cell expressing LFA-1 in a nonpolarized fashion in a cell that does not contact any infected cell. Bar, 25 μm.

Article Snippet: 50-μm coronal brain sections were cut serially through the striatum on a Leica vibratome, and immunofluorescence or DAB detection was performed as described previously , using the following primary antibodies recognizing: CD8 (1:500, mouse, Serotec), CD4 (1:100, mouse, Serotec), TK (1:10,000, rabbit, custom made), NeuN (1:1,000, mouse, Chemicon), GFAP (1:500, guinea pig, Advanced Immunochemical), phosphorylated Lck (1:50, rabbit, Cell Signaling), LFA-1 (1:500, mouse, IgG2a, BD Biosciences), TCR (1:100, mouse, IgG 1 , BD Biosciences), phosphorylated ZAP-70 (1:100, rabbit, Cell Signaling), and MHC-I (1:1,000, mouse, Serotec).

Techniques: Infection, Expressing

Journal: The Journal of Experimental Medicine

Article Title: In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain

doi: 10.1084/jem.20060420

Figure Lengend Snippet: Formation of c-SMAC and p-SMAC in vivo. TCR polarization and clustering within c-SMAC (A) and LFA-1 distribution to the p-SMAC (B). TCR staining is polarized to membrane areas displaying close contacts between T cells and infected brain cells, and clusters at sites where immunological synapses form (two different immunological synapses are shown in A and C). Three-dimensional reconstructions allow visualization of the formation of c-SMAC at the interface of lymphocytes and infected cells (A, under “Interface”); the quantification of the clustering of the TCR at the c-SMAC of the immunological synapses shownin A is illustrated in C (yellow arrow and intensity graph). LFA-1 forms a ring (p-SMAC) that surrounds c-SMAC, but is reduced within c-SMAC itself (two different immunological synapses are shown in B and D). Three-dimensional reconstructions show the LFA-1 immunoreactive p-SMAC ring at the interface between the T cells and target infected brain cells (B, under “Interface”); the quantification of the distribution of LFA-1 immunoreactivity at the p-SMAC of immunological synapses shown in B is illustrated in D (yellow arrow and intensity graph). Bars, 25 μm.

Article Snippet: 50-μm coronal brain sections were cut serially through the striatum on a Leica vibratome, and immunofluorescence or DAB detection was performed as described previously , using the following primary antibodies recognizing: CD8 (1:500, mouse, Serotec), CD4 (1:100, mouse, Serotec), TK (1:10,000, rabbit, custom made), NeuN (1:1,000, mouse, Chemicon), GFAP (1:500, guinea pig, Advanced Immunochemical), phosphorylated Lck (1:50, rabbit, Cell Signaling), LFA-1 (1:500, mouse, IgG2a, BD Biosciences), TCR (1:100, mouse, IgG 1 , BD Biosciences), phosphorylated ZAP-70 (1:100, rabbit, Cell Signaling), and MHC-I (1:1,000, mouse, Serotec).

Techniques: In Vivo, Staining, Infection

Journal: The Journal of Experimental Medicine

Article Title: In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain

doi: 10.1084/jem.20060420

Figure Lengend Snippet: SMAC formation at immunological synapses in vivo, between T cells and infected astrocytes in the brain. Two mature immunological synapses displaying typical and characteristic SMAC formations, are shown in A and B. The row of upper panels in A and B illustrate images captured in the given channels, from left to right: DAPI (blue), LFA-1 (red), TCR (green), LFA1+TCR, LFA1+TCR+DAPI (MERGE 1), the virally infected cell (TK; in white), and LFA1+TCR+DAPI+TK (MERGE 2). Notice that these mature immunological synapses are characterized by the specific distribution of LFA-1 and TCR contacting the virally infected cell. In the views shown in the top panels (side views of the immunological synapses), LFA-1 immunoreactivity displays high relative fluorescence density within areas of the membrane lateral to the close apposition of membranes of the lymphocyte with the infected cell, thus displaying the typical central region of lower density (see arrows in red channel) at the site of highest density of immunoreactivity for TCR (see arrows in the green channel and in the MERGE 1 and 2 images). The yellow asterisk indicates, in the white channel, the anatomical location of the T cell. The row of bottom panels in A and B illustrate, respectively, low (a) and high (b) magnifications of the close apposition between the T cell and the infected cell. These images were reconstructed using three-dimensional visualization software to illustrate the characteristic structure of the p-SMAC (LFA-1 ring) and c-SMAC (TCR cluster) at the interface of the immunological synapses seen in en face views (c and d). To produce the three-dimensional image of the interface shown in c and d, the three-dimensional reconstruction was rotated so that the plane of the interface of the immunological synapse (b, broken arrow) could be observed from above (white arrow in b shows the angle of vision of c and d). At the interface, typical “bull's eye” structures, characteristic of p-SMAC (LFA-1 outer ring) and c-SMAC (central TCR cluster) can be clearly recognized (c and d). In C, the intensity of fluorescence (I) was measured at the interface (yellow line) of the immunological synapse in a 0.5-μm confocal layer taken through the interface. The small graphs to the right of each synapse show the relative intensity values of fluorescence of LFA-1 (in red) and TCR (in green). Note the maximum of intensity for LFA-1 was in the periphery of the synapse (p-SMAC), whereas the TCR is clustered toward the center (c-SMAC). (D) A diagrammatic view of a T cell contacting an antigen-expressing astrocyte illustrating the localization of molecules involved in the immunological synapse and the polarization and phosphorylation of key tyrosine kinases. (E) A characteristic activated T cell nucleus displaying the polarized arched conformation opening toward the immunological synaptic interface. Bars, 15 μm.

Article Snippet: 50-μm coronal brain sections were cut serially through the striatum on a Leica vibratome, and immunofluorescence or DAB detection was performed as described previously , using the following primary antibodies recognizing: CD8 (1:500, mouse, Serotec), CD4 (1:100, mouse, Serotec), TK (1:10,000, rabbit, custom made), NeuN (1:1,000, mouse, Chemicon), GFAP (1:500, guinea pig, Advanced Immunochemical), phosphorylated Lck (1:50, rabbit, Cell Signaling), LFA-1 (1:500, mouse, IgG2a, BD Biosciences), TCR (1:100, mouse, IgG 1 , BD Biosciences), phosphorylated ZAP-70 (1:100, rabbit, Cell Signaling), and MHC-I (1:1,000, mouse, Serotec).

Techniques: In Vivo, Infection, Fluorescence, Software, Expressing

Journal: The Journal of Experimental Medicine

Article Title: In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain

doi: 10.1084/jem.20060420

Figure Lengend Snippet: Criteria used in the selection of optical planes from three-dimensional reconstructions of mature immunological synapses, for the illustration of SMAC formation in vivo. 90° rotations of three-dimensional reconstructions were made to observe the xz optical planes 1, 2, and 3 through axis y. Images A1, A2, and A3 show xz optical planes from image A. The immunological synaptic interface seen in optical plane xz is illustrated in A1; this optical plane was selected as the one in which the T cell's nucleus could not be visualized. Optical planes 2 and 3 (illustrated in A2 and A3) show the T cell's nucleus, demonstrating that these optical planes are at a distance from the synaptic interface and thus do not allow observation of the SMAC. Note that the optical plane 1 is the only one that shows the SMAC. yz optical planes 4, 5, and 6 were obtained from a 90° rotation of image B through the x axis. In plane 5 (illustrated in A5) the TCR is closely apposed to the infected cell without any intervening LFA-1; this plane is taken through the center of the c-SMAC. In images of planes 4 and 6 (illustrated in A4 and A6) LFA-1 immunoreactivity, rather than TCR, is in close apposition of the infected cell, indicating that these planes are taken through the p-SMAC. Note that this detailed three-dimensional reconstruction analysis was performed on the synapse illustrated in .

Article Snippet: 50-μm coronal brain sections were cut serially through the striatum on a Leica vibratome, and immunofluorescence or DAB detection was performed as described previously , using the following primary antibodies recognizing: CD8 (1:500, mouse, Serotec), CD4 (1:100, mouse, Serotec), TK (1:10,000, rabbit, custom made), NeuN (1:1,000, mouse, Chemicon), GFAP (1:500, guinea pig, Advanced Immunochemical), phosphorylated Lck (1:50, rabbit, Cell Signaling), LFA-1 (1:500, mouse, IgG2a, BD Biosciences), TCR (1:100, mouse, IgG 1 , BD Biosciences), phosphorylated ZAP-70 (1:100, rabbit, Cell Signaling), and MHC-I (1:1,000, mouse, Serotec).

Techniques: Selection, In Vivo, Infection

Journal: The Journal of Experimental Medicine

Article Title: In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain

doi: 10.1084/jem.20060420

Figure Lengend Snippet: Homogenous distribution of LFA-1 and TCR immunoreactivity along the plasma membrane of T cells not in contact with infected target cells. LFA-1 and TCR show homogeneous distribution along the plasma membrane of T cells neither in close apposition of target infected cells, nor involved in the formation of immunological synapses. Top panels (A–E) show a LFA-1 positive cell (B) not in contact with a TK-immunoreactive process (D), showing a uniform distribution of LFA-1. Panels F-J show a T cell with no LFA-1 expression (G) with a homogeneous distribution of TCR (H). Panels K-O show a T cell expressing LFA-1 (L) and TCR (M) not contacting a TK positive cell (N) showing no specific polarization and distribution in clusters. Panels P–T illustrate a CD8 + T cell expressing LFA-1 in a nonpolarized fashion in a cell that does not contact any infected cell. Bar, 25 μm.

Article Snippet: 50-μm coronal brain sections were cut serially through the striatum on a Leica vibratome, and immunofluorescence or DAB detection was performed as described previously , using the following primary antibodies recognizing: CD8 (1:500, mouse, Serotec), CD4 (1:100, mouse, Serotec), TK (1:10,000, rabbit, custom made), NeuN (1:1,000, mouse, Chemicon), GFAP (1:500, guinea pig, Advanced Immunochemical), phosphorylated Lck (1:50, rabbit, Cell Signaling), LFA-1 (1:500, mouse, IgG2a, BD Biosciences), TCR (1:100, mouse, IgG 1 , BD Biosciences), phosphorylated ZAP-70 (1:100, rabbit, Cell Signaling), and MHC-I (1:1,000, mouse, Serotec).

Techniques: Infection, Expressing

Journal: The Journal of Experimental Medicine

Article Title: In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain

doi: 10.1084/jem.20060420

Figure Lengend Snippet: Formation of c-SMAC and p-SMAC in vivo. TCR polarization and clustering within c-SMAC (A) and LFA-1 distribution to the p-SMAC (B). TCR staining is polarized to membrane areas displaying close contacts between T cells and infected brain cells, and clusters at sites where immunological synapses form (two different immunological synapses are shown in A and C). Three-dimensional reconstructions allow visualization of the formation of c-SMAC at the interface of lymphocytes and infected cells (A, under “Interface”); the quantification of the clustering of the TCR at the c-SMAC of the immunological synapses shownin A is illustrated in C (yellow arrow and intensity graph). LFA-1 forms a ring (p-SMAC) that surrounds c-SMAC, but is reduced within c-SMAC itself (two different immunological synapses are shown in B and D). Three-dimensional reconstructions show the LFA-1 immunoreactive p-SMAC ring at the interface between the T cells and target infected brain cells (B, under “Interface”); the quantification of the distribution of LFA-1 immunoreactivity at the p-SMAC of immunological synapses shown in B is illustrated in D (yellow arrow and intensity graph). Bars, 25 μm.

Article Snippet: 50-μm coronal brain sections were cut serially through the striatum on a Leica vibratome, and immunofluorescence or DAB detection was performed as described previously , using the following primary antibodies recognizing: CD8 (1:500, mouse, Serotec), CD4 (1:100, mouse, Serotec), TK (1:10,000, rabbit, custom made), NeuN (1:1,000, mouse, Chemicon), GFAP (1:500, guinea pig, Advanced Immunochemical), phosphorylated Lck (1:50, rabbit, Cell Signaling), LFA-1 (1:500, mouse, IgG2a, BD Biosciences), TCR (1:100, mouse, IgG 1 , BD Biosciences), phosphorylated ZAP-70 (1:100, rabbit, Cell Signaling), and MHC-I (1:1,000, mouse, Serotec).

Techniques: In Vivo, Staining, Infection

Journal: The Journal of Experimental Medicine

Article Title: In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain

doi: 10.1084/jem.20060420

Figure Lengend Snippet: SMAC formation at immunological synapses in vivo, between T cells and infected astrocytes in the brain. Two mature immunological synapses displaying typical and characteristic SMAC formations, are shown in A and B. The row of upper panels in A and B illustrate images captured in the given channels, from left to right: DAPI (blue), LFA-1 (red), TCR (green), LFA1+TCR, LFA1+TCR+DAPI (MERGE 1), the virally infected cell (TK; in white), and LFA1+TCR+DAPI+TK (MERGE 2). Notice that these mature immunological synapses are characterized by the specific distribution of LFA-1 and TCR contacting the virally infected cell. In the views shown in the top panels (side views of the immunological synapses), LFA-1 immunoreactivity displays high relative fluorescence density within areas of the membrane lateral to the close apposition of membranes of the lymphocyte with the infected cell, thus displaying the typical central region of lower density (see arrows in red channel) at the site of highest density of immunoreactivity for TCR (see arrows in the green channel and in the MERGE 1 and 2 images). The yellow asterisk indicates, in the white channel, the anatomical location of the T cell. The row of bottom panels in A and B illustrate, respectively, low (a) and high (b) magnifications of the close apposition between the T cell and the infected cell. These images were reconstructed using three-dimensional visualization software to illustrate the characteristic structure of the p-SMAC (LFA-1 ring) and c-SMAC (TCR cluster) at the interface of the immunological synapses seen in en face views (c and d). To produce the three-dimensional image of the interface shown in c and d, the three-dimensional reconstruction was rotated so that the plane of the interface of the immunological synapse (b, broken arrow) could be observed from above (white arrow in b shows the angle of vision of c and d). At the interface, typical “bull's eye” structures, characteristic of p-SMAC (LFA-1 outer ring) and c-SMAC (central TCR cluster) can be clearly recognized (c and d). In C, the intensity of fluorescence (I) was measured at the interface (yellow line) of the immunological synapse in a 0.5-μm confocal layer taken through the interface. The small graphs to the right of each synapse show the relative intensity values of fluorescence of LFA-1 (in red) and TCR (in green). Note the maximum of intensity for LFA-1 was in the periphery of the synapse (p-SMAC), whereas the TCR is clustered toward the center (c-SMAC). (D) A diagrammatic view of a T cell contacting an antigen-expressing astrocyte illustrating the localization of molecules involved in the immunological synapse and the polarization and phosphorylation of key tyrosine kinases. (E) A characteristic activated T cell nucleus displaying the polarized arched conformation opening toward the immunological synaptic interface. Bars, 15 μm.

Article Snippet: 50-μm coronal brain sections were cut serially through the striatum on a Leica vibratome, and immunofluorescence or DAB detection was performed as described previously , using the following primary antibodies recognizing: CD8 (1:500, mouse, Serotec), CD4 (1:100, mouse, Serotec), TK (1:10,000, rabbit, custom made), NeuN (1:1,000, mouse, Chemicon), GFAP (1:500, guinea pig, Advanced Immunochemical), phosphorylated Lck (1:50, rabbit, Cell Signaling), LFA-1 (1:500, mouse, IgG2a, BD Biosciences), TCR (1:100, mouse, IgG 1 , BD Biosciences), phosphorylated ZAP-70 (1:100, rabbit, Cell Signaling), and MHC-I (1:1,000, mouse, Serotec).

Techniques: In Vivo, Infection, Fluorescence, Software, Expressing

Journal: The Journal of Experimental Medicine

Article Title: In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain

doi: 10.1084/jem.20060420

Figure Lengend Snippet: Criteria used in the selection of optical planes from three-dimensional reconstructions of mature immunological synapses, for the illustration of SMAC formation in vivo. 90° rotations of three-dimensional reconstructions were made to observe the xz optical planes 1, 2, and 3 through axis y. Images A1, A2, and A3 show xz optical planes from image A. The immunological synaptic interface seen in optical plane xz is illustrated in A1; this optical plane was selected as the one in which the T cell's nucleus could not be visualized. Optical planes 2 and 3 (illustrated in A2 and A3) show the T cell's nucleus, demonstrating that these optical planes are at a distance from the synaptic interface and thus do not allow observation of the SMAC. Note that the optical plane 1 is the only one that shows the SMAC. yz optical planes 4, 5, and 6 were obtained from a 90° rotation of image B through the x axis. In plane 5 (illustrated in A5) the TCR is closely apposed to the infected cell without any intervening LFA-1; this plane is taken through the center of the c-SMAC. In images of planes 4 and 6 (illustrated in A4 and A6) LFA-1 immunoreactivity, rather than TCR, is in close apposition of the infected cell, indicating that these planes are taken through the p-SMAC. Note that this detailed three-dimensional reconstruction analysis was performed on the synapse illustrated in .

Article Snippet: 50-μm coronal brain sections were cut serially through the striatum on a Leica vibratome, and immunofluorescence or DAB detection was performed as described previously , using the following primary antibodies recognizing: CD8 (1:500, mouse, Serotec), CD4 (1:100, mouse, Serotec), TK (1:10,000, rabbit, custom made), NeuN (1:1,000, mouse, Chemicon), GFAP (1:500, guinea pig, Advanced Immunochemical), phosphorylated Lck (1:50, rabbit, Cell Signaling), LFA-1 (1:500, mouse, IgG2a, BD Biosciences), TCR (1:100, mouse, IgG 1 , BD Biosciences), phosphorylated ZAP-70 (1:100, rabbit, Cell Signaling), and MHC-I (1:1,000, mouse, Serotec).

Techniques: Selection, In Vivo, Infection

Journal: The Journal of Experimental Medicine

Article Title: In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain

doi: 10.1084/jem.20060420

Figure Lengend Snippet: Quantitative distribution of LFA-1 through the plasma membrane of T cells involved in formation of mature immunological synapses. The relative fluorescence of LFA-1 was measured in six T cells forming mature immunological synapses contacting virally infected cells (A). The red LFA-1 channel was selected [B] and the complete distribution of LFA-1 throughout the extent of the plasma membrane was delineated measuring the intensity of fluorescence in a linear graph (illustrated schematically in C). Panels D–I show the quantification of LFA-1 in six different cells forming mature immunological synapses. Small white arrows ‘1’ and ‘3’ indicate the p-SMAC and arrow ‘2’ the c-SMAC. Note that the level of LFA-1 is reduced (or absent) within the c-SMAC of all cells. Further LFA-1 levels are increased in the p-SMAC in some cells (illustrated in D, G, and I), while it does not increase in the p-SMAC of others [F, H, E]. Note that for clarity the target virally infected cells, found in apposition of the immunological synapses illustrated, are not shown; synapses shown in (D) and (E) are illustrated in further detail in , and synapse (F) in . Synapses H, G, and I are not illustrated further.

Article Snippet: 50-μm coronal brain sections were cut serially through the striatum on a Leica vibratome, and immunofluorescence or DAB detection was performed as described previously , using the following primary antibodies recognizing: CD8 (1:500, mouse, Serotec), CD4 (1:100, mouse, Serotec), TK (1:10,000, rabbit, custom made), NeuN (1:1,000, mouse, Chemicon), GFAP (1:500, guinea pig, Advanced Immunochemical), phosphorylated Lck (1:50, rabbit, Cell Signaling), LFA-1 (1:500, mouse, IgG2a, BD Biosciences), TCR (1:100, mouse, IgG 1 , BD Biosciences), phosphorylated ZAP-70 (1:100, rabbit, Cell Signaling), and MHC-I (1:1,000, mouse, Serotec).

Techniques: Fluorescence, Infection