Figure 1.
Evaluation of the specificity of PLP 139–151 dextramers by confocal microscopy in vitro.
For dextramer staining, two cell sources were used: (a) PLP 139-151-sensitized LNCs obtained from immunized mice; and (b) PLP 139-151-specific T cell hybridoma clone (B8). Viable cells harvested from LNC cultures on day 6 poststimulation, and also hybridoma cells were stained with PLP 139–151 dextramers/anti-CD4 or TMEV 70–86 (control) dextramers/anti-CD4. After washing, cells were fixed with 0.5% paraformaldehyde and examined by LSCM. Top panels (panels, a and b): cells stained with PLP 139–151 dextramers and anti-CD4. Bottom panels (panels, a and b): cells stained with TMEV 70–86 dextramers (control) and anti-CD4. Left panels: CD4, green; Middle panels: dextramers, red; Right panels: merged. Dext+ CD4+ T cells are shown with arrows for LNCs, whereas in PLP 139-151-specific hybridoma cells, the dext+ CD4+ cells are shown as such throughout the field. Original magnification 600×; bar = 20 µm.
Figure 2.
Detection of PLP-specific T cells by in situ staining with PLP 139–151 dextramers.
EAE was induced in SJL mice by immunizing the animals with PLP 139–151 in CFA. At termination, cerebrums collected from EAE mice were embedded in 4% agarose, and the sections were made using vibratome. After staining with cocktails containing either PLP 139–151 dextramers/anti-CD4 or TMEV 70–86 dextramers (control)/anti-CD4 and fixing with 4% PBS-buffered paraformaldehyde, sections were washed and mounted for examination by LSCM. Top panels: sections stained with PLP 139–151 dextramers and anti-CD4. Bottom panels: sections stained with TMEV 70–86 dextramers and anti-CD4. Left panel: CD4, green; Middle panel: dextramers, red; Right panel: merged (circles, dext+ CD4+ T cells; insets represent enlarged views of dext+ CD4+ T cells). Original magnification 1000×; bar = 20 µm.
Figure 3.
Quantitative analysis of PLP-specific CD4 T cells detected by in situ staining with PLP 139–151 dextramers in EAEmice.
Three paired cerebral sections, each 200 µm thick, were made from brains obtained from EAEmice using vibratome by cutting the sections from the dorsal to the ventral surface with 1.5 mm intervals between pairs. One section from each pair was stained with either PLP 139–151 or TMEV 70–86 (control) dextramers together with anti-CD4. After fixing and mounting, the sections were examined by LSCM (60X), and each inflammatory focus was subjected to a set of ‘Z’ serial images (15 to 25) taken sequentially. In total, 10 to 15 sets of such ‘Z’ serial images representing as many inflammatory foci were acquired from each section. In all the images, cells positive for both PLP 139–151 dextramers and CD4 or CD4 alone were counted using ‘ImageJ’ software. Finally, the cells counted in all the ‘Z’ serial images obtained from all the inflammatory foci were added to obtain the total number per section.
Table 1.
Quantitative analysis of PLP 139–151-dext+ CD4+ T cells in the brains of EAE mice by confocal microscopy.
Figure 4.
Detection of cardiac myosin-specific CD4 T cells by in situ staining.
EAM was induced in A/J mice by immunizing the animals with Myhc 334–352 in CFA; at termination on day 21, hearts were collected after perfusion, and the tissue blocks were prepared using 4% PBS-buffered agarose. Eight paired sections of 200 µm thickness each were made using vibratome. One section from each pair (a total of eight sections) were stained with mixtures containing either Myhc 334–352 dextramers/anti-CD4 or RNase 43–56 (control) dextramers/anti-CD4. After fixing with 4% PBS-buffered paraformaldehyde, the sections were washed and mounted for examination by LSCM. A collage of images showing the cells positive for both dextramers and CD4 or CD4 alone is shown. Top panels: sections stained with Myhc 334–352 dextramers and anti-CD4. Bottom panels: sections stained with RNase 43–56 dextramers and anti-CD4. Left panel: CD4, green; middle panel: dextramers, red; right panel: merged. (arrows, dext+ CD4+ T cells). Original magnification 1000×; bar = 20 µm.
Table 2.
Quantitative analysis of Myhc 334–352 dext+ CD4+ T cells in the hearts of EAM mice by confocal microscopy.