1. Cell-to-cell contact between activated CD4+ T lymphocytes and unprimed monocytes interferes with a TH1 response 
Miriam Wittmann, MD, Mareike Alter, Tanja Stünkel, Alexander Kapp, PhD, Thomas Werfel, MD 
Journal of Allergy and Clinical Immunology 
Volume 114, Issue 4, Pages (October 2004)
DOI: /j.jaci
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

2. Fig 1
Activated T cells can both induce and suppress IL-12 production, depending on the activation state of the APC. Immunofluorescence was performed with human monocytes (3 × 105 cells/well) stimulated for 24 hours with IFN-γ (300 U/mL)+sCD40L (2 μg/mL) (stimulation; right panel) or with IFN-γ+3 × 105 paraformaldehyde-fixed T cells (left panel). T cells were either activated with 10 μg/mL PHA or left unstimulated (resting T cells). Preincubation (pre) was performed with activated or resting paraformaldehyde-fixed T cells. The percentages of cells positive for IL-12 (p40/70) were determined. Five independent experiments were performed, and mean values with SDs are given. Differences between resting and activated T cells were significant (P ≤ .002; t test).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

3. Fig 2
A, Dose-dependent suppression of IL-12 by activated T cells. Immunofluorescence was performed with human monocytes. All depicted monocytes apart from the medium control (upper left panel) were stimulated with IFN-γ (300 U/mL) and LPS (50 ng/mL). Preincubation (pre) followed by a change of culture medium was performed with a decreasing number of autologous, paraformaldehyde-fixed, PHA (10 μg/mL)–stimulated T cells (the ratio of monocytes to T cells is given in brackets) or with nonstimulated T cells (resting cells) for 16 hours. B, Effect of allergen-stimulated T cells on IL-12 production by autologous PBMCs. PBMCs of a grass-pollen allergic donor were stimulated with PHA (10 μg/mL), tetanus toxoid (10 μg/mL), or grass pollen (5 μg/mL) for 24 hours of left unstimulated. Subsequently, these cells (not paraformaldehyde-fixed) were used for a 16-hour preincubation with autologous nonstimulated PBMCs. At the end of the preincubation period, a change of culture medium was performed before stimulation with IFN-γ+LPS for 24 hours (apart from the medium control). As positive control, cells preincubated with sCD40L (2 μg/mL) are shown. Intracellular cytokine staining for IL-12 (p40/p70) of 1 representative out of 5 (A) or 3 (B) independent experiments is given.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

4. Fig 3
Effect of skin-derived T cells from acute eczematous lesions on IL-12 production. A, CD40L expression in inflamed skin. Immunohistochemistry was performed from an 48-hour eczematous lesion to potassium dichromate. CD40L expression was determined by staining with anti-CD40L mAb (right; left, negative control). B, IL-12 production by autologous PBMCs. Left, Negative control. Autologous PBMCs were stained for IL-12 (p40/p70). Middle, Positive control. Autologous PBMCs were stimulated with IFN-γ+LPS. Right, Cells were isolated from an acute patch test lesion and were incubated with autologous PBMCs. After a preincubation (pre) period of 16 hours, a change of culture medium was performed, and subsequently, cells were stimulated with IFN-γ+LPS for 24 hours. Intracellular cytokine staining for IL-12 (p40/p70) is shown. One out of 3 experiments is depicted. C, Evidence of involvement of the p44/42 pathway in IL-12 suppression by in vivo activated T cells. Purified monocytes were stimulated with IFN-γ+LPS. Preincubation followed by a change of culture medium was performed with in vivo activated skin-derived lymphocytes alone (middle panel) or in the presence of the MEK1/2 inhibitor PD98059 (60 μmol/L) for 16 hours (right panel). IL-12 (p40/p70) staining is shown. One representative out of 6 independent experiments is shown.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

5. Fig 4
Cell surface expression of T cells cocultured with distinctly stimulated monocytes. Monocytes were either preincubated with autologous activated T cells /sCD40L or not submitted to any preincubation. At the end of the preincubation, T cells were washed out. After stimulation of monocytes with IFN-γ+LPS, autologous T cells were again added. T cells were stimulated with antigen or anti-CD3 (1 μg/mL)+anti-CD28 (0.2 μg/mL) mAb. Cell surface expression of CD3+ T cells was determined after 48 hours. For CD54, mean fluorescence intensity values are shown; for IL-12Rβ2, activated cells (as determined by increases in the forward scatter) were gated and the proportion of IL-12Rβ2 cells determined by dot plot analysis. Differences between the 2 groups were significant as determined by paired t test analysis (P < .02).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

6. Fig 5
mRNA expression of T-bet and IFN-γ of T cells cocultured with distinctly stimulated monocytes. Autologous activated T cells were added to distinctly stimulated monocytes resulting in IL-12 high (not preincubated, left column) and low producers (preincubated with activated T cells, right column). mRNA of cocultured nonadherent cells was isolated and subjected to real-time PCR. The intensity of the fluorescence signal of the original data with corresponding melting curves is shown. Additional experiments were performed with optimized quantification conditions by using Relative Quantification Software.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions