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PNAS 98 (19): 10805-10810

Copyright © 2001 by the National Academy of Sciences.

Single-cell analysis of signal transduction in CD4 T cells stimulated by antigen in vivo

Traci Zell*,{dagger},{ddagger}, Alexander Khoruts{dagger},{ddagger},§, Elizabeth Ingulli{dagger}, Jody L. Bonnevier{dagger}, Daniel L. Mueller{dagger},§, and Marc K. Jenkins*,{dagger},||

Departments of *Microbiology, §Medicine, and Pediatrics, and the {dagger}Center for Immunology, University of Minnesota, Minneapolis, MN 55455


Figure 1
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Figure 1 Comparison of phospho-c-jun detection by flow cytometry and JNK enzymatic activity in naive T cells stimulated in vitro with mAbs. Purified DO11.10 T cells were stimulated for 15 or 30 min with control hamster IgG, anti-CD3, or anti-CD3 and anti-CD28 mAbs. (A) Whole-cell lysates from stimulated cells were incubated with GST-c-jun beads and analyzed for JNK activity. (B) Intracellular staining of phosphorylated c-jun protein in cells stimulated with control IgG (dashed line), anti-CD3 (thin line), or anti-CD3 and anti-CD28 mAbs (bold line). (C) Intracellular staining of phosphorylated c-jun protein in normal or c-jun-deficient mouse embryo fibroblasts stimulated for 10 min with EGF (bold line) or nothing (dashed line).

 

Figure 2
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Figure 2 Comparison of c-jun phosphorylation after in vitro or in vivo stimulation of naive T cells with peptide antigen. (A) A representative flow cytometry dot plot showing CD4 and KJ1–26 staining of spleen cells from transferred recipients. The gating strategy discriminates transferred DO11.10 CD4+, KJ1–26+ cells (R3) from the recipient's CD4+, KJ1–26– cells (R2). (B) Antiphospho c-jun staining of DO11.10 (bold line) or recipient CD4 T cells (dashed line) from the same sample after initiation of in vitro culture with ovalbumin peptide-pulsed splenocytes (Left) or i.v. injection of ovalbumin peptide (Right). (C) Time course of c-jun phosphorylation after in vitro (open squares) or in vivo (filled squares) stimulation with ovalbumin peptide. The difference between the phospho-c-jun signal (mean fluorescence intensity, MFI) in DO11.10 and recipient CD4 T cells is shown.

 

Figure 3
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Figure 3 Naive CD4 T cells are located near I-Ad-expressing cells in vivo. The locations of DO11.10 CD4 T cells (shown green) and cells expressing anti-I-Ad (shown red in A) or anti-CD11c (shown red in B) in thin sections of the spleen are shown. A and B show adjacent sections containing the same white pulp chord with a perimeter of B-cell-rich follicles (F) surrounding a central periarteriolar lymphoid sheath (P). A magnified view of a periarteriolar lymphoid sheath is shown in C with DO11.10 T cells shown green and I-Ad-expressing cells shown red. The areas of yellow color indicate significant overlap between the two cell types. D shows the mean percentage of DO11.10 T cells (± the standard deviation) found interacting with cells stained with I-Ad, CD11c, or control Ig, as evidenced by the presence of yellow color. At least 200 cells from two to four white pulp chords were counted in each group.

 

Figure 4
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Figure 4 Phosphorylation of c-jun and p38 MAPK is CD28-independent in DO11.10 T cells. Mice were transferred with either naïve DO11.10 (squares) or CD28-deficient (circles) DO11.10 T cells. Phosphorylation of c-jun (A) or p38 MAPK (D) was assessed at the indicated times after i.v. injection of ovalbumin peptide. The difference between the signal (mean fluorescence intensity, MFI) in DO11.10 and recipient CD4 T cells is shown. C shows histograms of phospho-p38 MAPK staining in DO11.10 (bold line) or recipient (thin line) CD4 T cells at the indicated times after i.v. injection of ovalbumin peptide. Intracellular IL-2 was measured 2.5 h after i.v. injection of ovalbumin peptide (closed bars) or nothing (open bars) (B). These data are representative of two independent experiments.

 

Figure 5
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Figure 5 Effect of cyclosporin A treatment on c-jun and p38 MAPK phosphorylation. Recipients of naive DO11.10 CD4 T cells were either untreated (open squares) or treated with cyclosporin A for 4 days to achieve a pharmacological level of drug (closed squares). Phosphorylation of c-jun (A) and p38 MAPK (B) was then measured at the indicated times after i.v. injection of ovalbumin peptide. The difference between the signal (mean fluorescence intensity, MFI) in DO11.10 and recipient CD4 T cells ± SD is shown (n = 2). (C) IL-2 was measured 3 h after i.v. injection of ovalbumin peptide (closed bars) or nothing (open bars). These data are representative of two independent experiments.

 


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