Mass Cytometric Analysis of Early-Stage Mycosis Fungoides

Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma. Early-stage disease is characterized by superficial infiltrates of small- to medium-sized atypical epidermotropic T lymphocytes that are clonal related. Nevertheless, the percentage of atypical T cells is low with many admixed reactive immune cells. Despite earlier studies, the composition and spatial characteristics of the cutaneous lymphocytic infiltrate has been incompletely characterized. Here, we applied mass cytometry to profile the immune system in skin biopsies of patients with early-stage MF and in normal skin from healthy individuals. Single-cell suspensions were prepared and labeled with a 43-antibody panel, and data were acquired on a Helios mass cytometer. Unbiased hierarchical clustering of the data identified the major immune lineages and heterogeneity therein. This revealed patient-unique cell clusters in both the CD4+ and myeloid cell compartments but also phenotypically distinct cell clusters that were shared by most patients. To characterize the immune compartment in the tissue context, we developed a 36-antibody panel and performed imaging mass cytometry on MF skin tissue. This visualized the structure of MF skin and the distribution of CD4+ T cells, regulatory T cells, CD8+ T cells, malignant T cells, and various myeloid cell subsets. We observed clusters of CD4+ T cells and multiple types of dendritic cells (DCs) identified through differential expression of CD11c, CD1a, and CD1c in the dermis. These results indicated substantial heterogeneity in the composition of the local immune infiltrate but suggest a prominent role for clustered CD4–DC interactions in disease pathogenesis. Probably, the local inhibition of such interactions may constitute an efficient treatment modality.</jats:p

Broad-Spectrum Sunscreens Offer Protection Against Urocanic Acid Photoisomerization by Artificial Ultraviolet Radiation in Human Skin1

SummaryCis-urocanic acid (UCA) has been indicated as an important mediator of ultraviolet (UV)-induced immunosuppression. In this study we describe a rapid, noninvasive method for the determination of the protective capacity of various sunscreens against the UV-induced isomerization of trans-UCA into its cis form. For this purpose we applied sunscreens prior to in vivo exposure of human volunteers with single or repeated broadband UVB irradiations of 100 mJ per cm2. We found significant but different levels of protection against UCA photoisomerization by all sunscreens that correlated with the sun protection factor. A comparison of various sunscreens with a sun protection factor of 10, showed that the best protection was offered by the sunscreens (containing organic UV filters or TiO2) with broad absorption spectra. The ability to inhibit cis-UCA formation was not influenced by the penetration characteristics of sunscreens, as determined by application of the sunscreen on quartz glass that was placed on the skin, preventing penetration of sunscreen in the skin. In addition ex vivo UV exposure of human skin was employed to permit other tests of immunomodulation, in this case the mixed epidermal cell lymphocyte reaction. The advantage of this ex vivo method is that there is no need to take biopsies from volunteers. Ex vivo irradiation of human skin with a single dose of 200 mJ per cm2 resulted in similar protection by the sunscreens against cis-UCA formation as in the in vivo system. Furthermore, the mixed epidermal cell lymphocyte reaction data correlated with the cis-UCA findings. We conclude that UCA isomerization is an excellent method to determine sunscreen efficacy and that broad-spectrum sunscreens offer good immunoprotection

Analysis of the Protective Effect of Topical Sunscreens on the UVB-Radiation – Induced Suppression of the Mixed-Lymphocyte Reaction

The mixed-lymphocyte reaction (MLR) was used to test the protective capacity of several sunscreens against UVB- radiation-induced suppression of the immune response. The MLR was performed using stimulator cells that had been exposed to 120 mJ/cm2 of an UVB source, whereas in simultaneous experiments a sunscreen-covered piece of quartz glass was placed in between the light source and the culture dish. The MLR response after UVB radiation was significantly decreased in comparison with the MLR response of non-radiated cells. This UV effect was partly inhibited by the tested sunscreens, whereas their vehicles alone showed hardly any effect. The protective capacity of sunscreens with similar protection factors, which were determined using the minimal erythematous dose (MED), showed a significant variation. These results suggest that the MED is not an accurate method to determine protection against UV-induced immunologic damage

Differential Effects of Sunscreens on UVB-Induced Immunomodulation in Humans

Ultraviolet radiation has been shown to suppress the (skin) immune system both in animal species and in humans. Whether sunscreens can prevent immunosuppression is a matter of debate. This study investigated the protective capacity of a commercial sunscreen lotion in humans. Part of the right arm of healthy volunteers was exposed to erythemagenic ultraviolet B doses of 160mJ per cm2 for four consecutive days. Before irradiation, sunscreen was applied either directly onto the skin or onto a piece of quartz fixed to the skin (to avoid penetration of the sunscreen in the epidermis where it cannot block the photoisomerization of trans-urocanic acid in cis-urocanic acid in the stratum corneum). The control group was irradiated without prior application of sunscreen. Four h after the last irradiation, epidermal sheets were obtained by the suction-blister method from both arms and epidermal cells were used as stimulator cells in the mixed epidermal cell lymphocyte reaction. Responses directed to epidermal cells derived from irradiated skin were expressed as percentages of responses directed to epidermal cells derived from the nonirradiated left arm. The mixed epidermal cell lymphocyte reaction responses in the control group were found to be significantly increased (205%). This enhancement of the mixed epidermal cell lymphocyte reaction responses was associated with an influx of CD36+DR+ macrophages in the irradiated skin. Application of the sunscreen, either onto a piece of quartz or directly onto the skin, prevented the increase of the mixed epidermal cell lymphocyte reaction responses and the influx of CD36+DR+ cells. In an earlier study, volunteers were exposed three times weekly to suberythemagenic doses of ultraviolet B over 4 wk, resulting in mixed epidermal cell lymphocyte reaction responses that were decreased to 20%. The same sunscreen was not able to prevent this suppression. These contradicting results indicate that the protective effect of sunscreens with respect to ultraviolet-induced immunomodulation is critically dependent on the choice of ultraviolet treatment

A comparison of CDKN2A mutation detection within the Melanoma Genetics Consortium (GenoMEL)

CDKN2A is the major melanoma susceptibility gene so far identified, but only 40% of three or more case families have identified mutations. A comparison of mutation detection rates was carried out by “blind” exchange of samples across GenoMEL, the Melanoma Genetics Consortium, to establish the false negative detection rates. Denaturing high performance liquid chromatography (DHPLC) screening results from 451 samples were compared to screening data from nine research groups in which the initial mutation screen had been done predominantly by sequencing. Three samples with mutations identified at the local centres were not detected by the DHPLC screen. No additional mutations were detected by DHPLC. Mutation detection across groups within GenoMEL is carried out to a consistently high standard. The relatively low rate of CDKN2A mutation detection is not due to failure to detect mutations and implies the existence of other high penetrance melanoma susceptibility genes

Features associated with germline CDKN2A mutations: a GenoMEL study of melanoma‐prone families from three continents

Background: The major factors individually reported to be associated with an increased frequency of CDKN2A mutations are increased number of patients with melanoma in a family, early age at melanoma diagnosis, and family members with multiple primary melanomas (MPM) or pancreatic cancer. Methods: These four features were examined in 385 families with [&gt;=]3 patients with melanoma pooled by 17 GenoMEL groups, and these attributes were compared across continents. Results: Overall, 39% of families had CDKN2A mutations ranging from 20% (32/162) in Australia to 45% (29/65) in North America to 57% (89/157) in Europe. All four features in each group, except pancreatic cancer in Australia (p = 0.38), individually showed significant associations with CDKN2A mutations, but the effects varied widely across continents. Multivariate examination also showed different predictors of mutation risk across continents. In Australian families, [&gt;=]2 patients with MPM, median age at melanoma diagnosis [&lt;=]40 years and [&gt;=]6 patients with melanoma in a family jointly predicted the mutation risk. In European families, all four factors concurrently predicted the risk, but with less stringent criteria than in Australia. In North American families, only [&gt;=]1 patient with MPM and age at diagnosis [&lt;=]40 years simultaneously predicted the mutation risk. Conclusions: The variation in CDKN2A mutations for the four features across continents is consistent with the lower melanoma incidence rates in Europe and higher rates of sporadic melanoma in Australia. The lack of a pancreatic cancer-CDKN2A mutation relationship in Australia probably reflects the divergent spectrum of mutations in families from Australia versus those from North America and Europe. GenoMEL is exploring candidate host, genetic and/or environmental risk factors to better understand the variation observe