BackgroundAlthough recent studies indicate a crucial role for IL-17A and IL-22 producing T cells in the pathogenesis of psoriasis, limited information is available on their frequency and heterogeneity and their distribution in skin in situ.Methodology/Principal FindingsBy spectral imaging analysis of double-stained skin sections we demonstrated that IL-17 was mainly expressed by mast cells and neutrophils and IL-22 by macrophages and dendritic cells. Only an occasional IL-17pos, but no IL-22pos T cell could be detected in psoriatic skin, whereas neither of these cytokines was expressed by T cells in normal skin. However, examination of in vitro-activated T cells by flow cytometry revealed that substantial percentages of skin-derived CD4 and CD8 T cells were able to produce IL-17A alone or together with IL-22 (i.e. Th17 and Tc17, respectively) or to produce IL-22 in absence of IL-17A and IFN-γ (i.e. Th22 and Tc22, respectively). Remarkably, a significant proportional rise in Tc17 and Tc22 cells, but not in Th17 and Th22 cells, was found in T cells isolated from psoriatic versus normal skin. Interestingly, we found IL-22 single-producers in many skin-derived IL-17Apos CD4 and CD8 T cell clones, suggesting that in vivo IL-22 single-producers may arise from IL-17Apos T cells as well.Conclusions/SignificanceThe increased presence of Tc17 and Tc22 cells in lesional psoriatic skin suggests that these types of CD8 T cells play a significant role in the pathogenesis of psoriasis. As part of the skin-derived IL-17Apos CD4 and CD8 T clones developed into IL-22 single-producers, this demonstrates plasticity in their cytokine production profile and suggests a developmental relationship between Th17 and Th22 cells and between Tc17 and Tc22 cells.

SUMMARYSimultaneous detection of two different antigens on paraffin-embedded and frozen tissues can be accomplished by double immunohistochemistry. However, many double chromogen systems suffer from signal overlap, precluding definite signal quantification. To separate and quantitatively analyze the different chromogens, we imported images into a Macintosh computer using a CCD camera attached to a diagnostic microscope and used Photoshop software for the recognition, selection, and separation of colors. We show here that Photoshop-based image analysis allows complete separation of chromogens not only on the basis of their RGB spectral characteristics, but also on the basis of information concerning saturation, hue, and luminosity intrinsic to the digitized images. We demonstrate that Photoshop-based image analysis provides superior results compared to color separation using bandpass filters. Quantification of the individual chromogens is then provided by Photoshop using the Histogram command, which supplies information on the luminosity (corresponding to gray levels of black-and-white images) and on the number of pixels as a measure of spatial distribution. (J Histochem Cytochem 47:119-125, 1999)

BackgroundT cell mediated inflammation contributes to atherogenesis and the onset of acute cardiovascular disease. Effector T cell functions are under a tight control of a specialized T cell subset, regulatory T cells (Treg). At present, nothing is known about the in situ presence of Treg in human atherosclerotic tissue. In the present study we investigated the frequency of naturally occurring Treg cells in all developmental stages of human atherosclerotic lesions including complicated thrombosed plaques.MethodologyNormal arteries, early lesions (American Heart Association classification types I, II, and III), fibrosclerotic plaques (types Vb and Vc) and ‘high risk’ plaques (types IV, Va and VI) were obtained at surgery and autopsy. Serial sections were immunostained for markers specific for regulatory T cells (FOXP3 and GITR) and the frequency of these cells was expressed as a percentage of the total numbers of CD3+ T cells. Results were compared with Treg counts in biopsies of normal and inflammatory skin lesions (psoriasis, spongiotic dermatitis and lichen planus).Principle findingsIn normal vessel fragments T cells were virtually absent. Treg were present in the intima during all stages of plaque development (0.5–5%). Also in the adventitia of atherosclerotic vessels Treg were encountered, in similar low amounts. High risk lesions contained significantly increased numbers of Treg compared to early lesions (mean: 3.9 and 1.2%, respectively). The frequency of FOXP3+ cells in high risk lesions was also higher compared to stable lesions (1.7%), but this difference was not significant. The mean numbers of intimal FOXP3 positive cells in atherosclerotic lesions (2.4%) was much lower than those in normal (24.3%) or inflammatory skin lesions (28%).ConclusionLow frequencies of Treg in all developmental stages of human plaque formation could explain the smoldering chronic inflammatory process that takes place throughout the longstanding course of atherosclerosis.