John A. Carucci

Malignant melanoma: prevention, early detection, and treatment in the 21st century.

Malignant melanoma continues to present a significant public health problem as its incidence is rising faster than that of any other cancer in the US. At current rates, 1 in 74 Americans will develop melanoma during his or her lifetime.

Low Expression of the IL-23/Th17 Pathway in Atopic Dermatitis Compared to Psoriasis

The classical Th1/Th2 paradigm previously defining atopic dermatitis (AD) and psoriasis has recently been challenged with the discovery of Th17 T cells that synthesize IL-17 and IL-22. Although it is becoming evident that many Th1 diseases including psoriasis have a strong IL-17 signal, the importance of Th17 T cells in AD is still unclear. We examined and compared skin biopsies from AD and psoriasis patients by gene microarray, RT-PCR, immunohistochemistry, and immunofluorescence. We found a reduced genomic expression of IL-23, IL-17, and IFN-γ in AD compared with psoriasis. To define the effects of IL-17 and IL-22 on keratinocytes, we performed gene array studies with cytokine-treated keratinocytes. We found lipocalin 2 and numerous other innate defense genes to be selectively induced in keratinocytes by IL-17. IFN-γ had no effect on antimicrobial gene-expression in keratinocytes. In AD skin lesions, protein and mRNA expression of lipocalin 2 and other innate defense genes (hBD2, elafin, LL37) were reduced compared with psoriasis. Although AD has been framed by the Th1/Th2 paradigm as a Th2 polar disease, we present evidence that the IL-23/Th17 axis is largely absent, perhaps accounting for recurrent skin infections in this disease.

Human Langerhans cells induce distinct IL-22-producing CD4+ T cells lacking IL-17 production

IL-22 is a cytokine that acts mainly on epithelial cells. In the skin, it mediates keratinocyte proliferation and epidermal hyperplasia and is thought to play a central role in inflammatory diseases with marked epidermal acanthosis, such as psoriasis. Although IL-22 was initially considered a Th17 cytokine, increasing evidence suggests that T helper cells can produce IL-22 even without IL-17 expression. In addition, we have shown the existence of this unique IL-22-producing T cell in normal skin and in the skin of psoriasis and atopic dermatitis patients. In the present study, we investigated the ability of cutaneous resident dendritic cells (DCs) to differentiate IL-22-producing cells. Using FACS, we isolated Langerhans cells (LCs; HLA-DR+CD207+ cells) and dermal DCs (HLA-DRhiCD11c+BDCA-1+ cells) from normal human epidermis and dermis, respectively. Both LCs and dermal DCs significantly induced IL-22-producing CD4+ and CD8+ T cells from peripheral blood T cells and naive CD4+ T cells in mixed leukocyte reactions. LCs were more powerful in the induction of IL-22-producing cells than dermal DCs. Moreover, in vitro-generated LC-type DCs induced IL-22-producing cells more efficiently than monocyte-derived DCs. The induced IL-22 production was more correlated with IFN-γ than IL-17. Surprisingly, the majority of IL-22-producing cells induced by LCs and dermal DCs lacked the expression of IL-17, IFN-γ, and IL-4. Thus, LCs and dermal DCs preferentially induced helper T cells to produce only IL-22, possibly “Th22” cells. Our data indicate that cutaneous DCs, especially LCs, may control the generation of distinct IL-22 producing Th22 cells infiltrating into the skin.

Calcitonin Gene-Related Peptide Decreases Expression of HLA-DR and CD86 by Human Dendritic Cells and Dampens Dendritic Cell-Driven T Cell-Proliferative Responses Via the Type I Calcitonin Gene-Related Peptide Receptor

These studies were performed to establish whether functional receptors for calcitonin gene-related peptide (CGRP) are present on human dendritic cells (DCs) and to investigate potential immunomodulatory effects of CGRP on DCs other than Langerhans cells. Reverse transcriptase-PCR revealed expression of mRNA for a type 1 CGRP receptor by mature and immature blood-derived DCs. Sequence analysis confirmed the identity of the type 1 CGRP receptor (CGRP-R1). Addition of CGRP (10−7 M) to mature and immature DCs resulted in mobilization of intracellular calcium. Treatment of immature DCs with CGRP (10−7 M), before and after maturation in monocyte-conditioned medium, resulted in decreased cell surface expression of HLA-DR MHC class II and the costimulatory molecule, CD86. Treatment of immature DCs with CGRP (10−7 M) also resulted in decreased expression of CD86, but expression of HLA-DR was unchanged. When CGRP-treated mature DCs were used to stimulate allogeneic T cells, proliferative responses were dampened (∼50%), especially at low DC:T cell ratios (1:360). This effect was not observed with CGRP-treated, immature DCs. In contrast, CGRP-treated mature or immature DCs were no less efficient than untreated DCs in driving syngeneic T cell-proliferative responses to staphylococcal enterotoxin B. We conclude that mature and immature DCs express type 1 CGRP receptors and that signaling through these receptors may dampen mature DC-driven T cell proliferation most likely via down-regulation of CD86 and HLA-DR.

The Human Cutaneous Squamous Cell Carcinoma Microenvironment Is Characterized by Increased Lymphatic Density and Enhanced Expression of Macrophage-Derived VEGF-C

Metastases from primary cutaneous squamous cell carcinoma (SCC) account for the majority of the ∼10,000 non-melanoma skin cancer deaths in the United States annually. We studied lymphangiogenesis in human SCC because of the potential link to metastasis. SCC samples were stained for lymphatic endothelial vessel marker LYVE-1 and positive cells were counted and compared with cells in normal skin. Gene set enrichment analysis and reverse transcription (RT)-PCR were performed on SCC, on adjacent non-tumor-bearing skin, and on normal skin to determine the differential expression of lymphangiogenesis-associated genes. Laser capture microdissection (LCM) was performed to isolate tumor cells and tumor-associated inflammatory cells for further gene expression analysis. Immunofluorescence was performed to determine the source of vascular endothelial growth factor-C (VEGF-C) in the tumor microenvironment. We found increased lymphatic density and reorganized lymphatic endothelial vessels in the dermis immediately adjacent to SCC nests. RT-PCR confirmed the presence of VEGF-C in skin immediately adjacent to SCC. LCM confirmed the increased expression of VEGF-C, the SCC inflammatory infiltrate. The presence of CD163(+)/CD68(+)/VEGFC(+) cells and absence of VEGF-C expression by CD3(+) or CD11C(+) cells suggested that VEGF-C is derived from tumor-associated macrophages. Clarification of mechanisms governing SCC-mediated lymphangiogenesis may identify potential targets for therapeutic intervention against aggressive or inoperable disease.

Lesional dendritic cells in patients with chronic atopic dermatitis and psoriasis exhibit parallel ability to activate T-cell subsets

BACKGROUND Atopic dermatitis (AD) and psoriasis represent polar immune diseases. AD is a T(H)2/T(H)22-dominant disease, whereas psoriasis is considered a T(H)1/T(H)17 disease. Local immune deviation is suggested to be regulated by dendritic cell (DC)-induced T-cell polarization and recruitment of specific T-cell subsets by chemokines. Although the role of chemokines is well documented, the actual contribution of DCs to activate polar T-cell subsets in human subjects is still a matter of speculation. OBJECTIVE We sought to elucidate the significance of each cutaneous DC subset in disease-specific T-cell immune deviation. METHODS We performed a comprehensive analysis of major cutaneous resident (Langerhans cells and blood dendritic cell antigen 1-positive dermal DCs) and inflammatory (inflammatory dendritic epidermal cells and blood dendritic cell antigen 1-negative dermal DCs) DC subsets directly isolated from the lesional skin of patients with AD and those with psoriasis. RESULTS The ability of each DC subset to expand T(H)1, T(H)2, T(H)17, and T(H)22 subsets was similar between the 2 diseases, despite the association of both with accumulation of resident and inflammatory DCs. We also confirmed differential upregulation of chemokine expression in patients with AD (CCL17, CCL18, and CCL22) and psoriasis (CXCL1, IL-8, and CCL20). The expression of CCL17 and CCL22 was higher in Langerhans cells from patients with AD than from patients with psoriasis, whereas the opposite was observed for CXCL9 and CXCL10. CONCLUSION Our results suggest that DC polarity does not directly drive differential T-cell subset responses. Alternatively, disease-specific chemokines might recruit specific memory T-cell subsets into the skin, which in turn might be activated and expanded by DCs at the site of inflammation, maintaining differential immune polarity in these diseases.

Myeloid Dendritic Cells from Human Cutaneous Squamous Cell Carcinoma Are Poor Stimulators of T-Cell Proliferation

To determine the phenotype and function of myeloid dendritic cells (DCs) from human cutaneous squamous-cell carcinoma (SCC), we studied their surface marker expression and allo-stimulatory potential ex vivo. There were abundant CD11c(+) myeloid DCs, as well as TNF and inducible nitric oxide synthase (iNOS)-producing DCs, in and around SCC tumor nests. Although myeloid DCs from SCC, adjacent non-tumor-bearing skin, and normal skin, were phenotypically similar by flow cytometry, and there was a pronounced genomic signature of mature DCs in SCC, they showed different T-cell stimulatory potential in an allogeneic mixed leukocyte reaction. Myeloid DCs from SCC were less potent stimulators of allogeneic T-cell proliferation than DCs from non-tumor-bearing skin. Culture with a DC-maturing cytokine cocktail (IL-1beta, IL-6, TNF-alpha, and PGE(2)) enhanced stimulatory potential in DCs from non-tumor-bearing skin, whereas SCC-associated DCs remained poor stimulators of T-cell proliferation. The microenvironment associated with SCC showed expression of TGF-beta, IL-10, and VEGF-A, factors capable of suppressing the DC function. These findings indicate that CD11c(+)/HLA-DR(hi) DCs from SCC are mature, but are not potent stimulators of T-cell proliferation compared with phenotypically similar DCs isolated from non-tumor-bearing skin. Identification of mechanisms responsible for suppression of tumor-associated DCs may provide insight into the evasion of immunosurveillance by SCC.

Increased Tc22 and Treg/CD8 Ratio Contribute to Aggressive Growth of Transplant Associated Squamous Cell Carcinoma

Immune suppressed organ transplant recipients suffer increased morbidity and mortality from primary cutaneous SCC. We studied tumor microenvironment in transplant-associated SCC (TSCC), immune-competent SCC and normal skin by IHC, IF and RT-PCR on surgical discard. We determined T cell polarization in TSCC and SCC by intracellular cytokine staining of T cell crawl outs from human skin explants. We studied the effects of IL-22, an inducer of keratinocyte proliferation, on SCC proliferation in vitro. SCC and TSCC are both associated with significantly higher numbers of CD3+ and CD8+ T cells compared to normal skin. TSCC showed a higher proportion of Foxp3+ T regs to CD8+ T cells compared to SCC and a lower percentage of IFN-γ producing CD4+ T cells. TSCC, however, had a higher percentage of IL-22 producing CD8+ T cells compared to SCC. TSCC showed more diffuse Ki67 and IL-22 receptor (IL-22R) expression by IHC. IL-22 induced SCC proliferation in vitro despite serum starvation. Diminished cytotoxic T cell function in TSCC due to decreased CD8/T-reg ratio may permit tumor progression. Increased IL-22 and IL-22R expression could accelerate tumor growth in transplant patients. IL-22 may be an attractive candidate for targeted therapy of SCC without endangering allograft survival.

Immunosuppression affects CD4+ mRNA expression and induces Th2 dominance in the microenvironment of cutaneous squamous cell carcinoma in organ transplant recipients.

Squamous cell carcinoma (SCC) is the most frequent cancer in organ transplant recipients (OTRs). The immune system plays a major role in the fight against SCC, however, little is known about the local inflammatory response in SCC at all. We analyzed quantity and quality of the perineoplastic inflammatory SCC microenvironment in immunocompetent patients and immmunosuppressed OTRs. RNA expression profile of SCC patients was analyzed for 8 different sets of genes relating to Th1 versus Th2 response using Gene Set Enrichment Analysis. SCC from immunocompetent patients and OTRs were analyzed by real-time polymerase chain reactions for CD4, CD8, TBET, GATA-3, FOXP3, RORC, IFN-γ, IL-4, TGF-&bgr;, IL-10, and IL-17A mRNA expression. Immunohistochemistry was carried out in SCC for CD3, CD4, CD8, and FOXP3 expression. Considerable inflammation was seen in both patient groups. SCC in immunocompetent patients and OTRs was associated with a mixed Th1 and Th2 gene expression signature. CD4+ mRNA was diminished in immunosuppression. Skin adjacent to SCC in OTRs showed Th2 expression pattern as compared with immunocompetent patients. T-BET and IFN-γ mRNA expression were decreased in the OTR group. Although Th17-weighted inflammation was unchanged, IL-17A mRNA level was markedly decreased with immunosuppression. Regulatory T cells, characterized by FOX-P3 and TGF-β mRNA level, were decreased in OTRs. Our findings support the hypothesis that nontumor-bearing skin adjacent to SCC in OTRs is not necessarily normal and that the local microenvironment may contribute to a field effect contributing to higher recurrence rates and more aggressive behavior observed in these patients.

Gene Expression Profiling of the Leading Edge of Cutaneous Squamous Cell Carcinoma: IL-24-Driven MMP-7

The precise mechanisms governing invasion at the leading edge of SCC and its subsequent metastasis are not fully understood. We aimed to define the cancer related molecular changes that distinguish non-invasive tumor from invasive SCC. To this end, we combined laser capture microdissection with cDNA microarray analysis. We defined invasion-associated genes as those differentially regulated only in invasive SCC nests, but not in actinic keratosis or in situ SCC, compared to normal epidermis. There were 383 up- and 354 down-regulated genes in the “invasion set.” SCC invasion was characterized by aberrant expression of various proteolytic molecules. We noted increased expression of MMP7 and IL-24 in invasive SCC. IL-24 induced the expression of MMP7 in SCC cells in culture. In addition, blocking of MMP7 by a specific antibody significantly delayed the migration of SCC cells in culture. These results suggest a possible contribution of IL-24 to SCC invasion via enhancing focal expression of MMP7, though IL-24 has been suggested to have anti-tumor growth effects in other cancer types. Identification of regional molecular changes that regulate cancer invasion may facilitate the development of new targeted treatments for aggressive cancer.