Renata B. Filler

Acute upregulation of an NKG2D ligand promotes rapid reorganization of a local immune compartment with pleiotropic effects on carcinogenesis

The self-encoded ligands MICA (human) and Rae-1 (mouse) for the cytotoxic lymphocyte activating receptor NKG2D are highly expressed in carcinomas and inflammatory lesions and have been linked to immunosurveillance and graft rejection. However, whether NKG2D ligands have an intrinsic ability to acutely regulate tissue-associated immune compartments is not known. Here we show that epidermis-specific upregulation of Rae-1 induced rapid, coincident and reversible changes in the organization of tissue-resident Vγ5Vδ1 TCRγδ+ intraepithelial T cells and Langerhans cells, swiftly followed by epithelial infiltration by unconventional αβ T cells. Whereas local Vγ5Vδ1+ T cells limited carcinogenesis, Langerhans cells unexpectedly promoted it. These results provide unique insight into the early phases of tissue immunosurveillance and indicate that acute changes in NKG2D ligands may alone initiate a rapid, multifaceted immunosurveillance response in vivo.

Resident Skin-specific γδ T Cells Provide Local, Nonredundant Regulation of Cutaneous Inflammation

The function of the intraepithelial lymphocyte (IEL) network of T cell receptor (TCR) γδ+ (Vγ5+) dendritic epidermal T cells (DETC) was evaluated by examining several mouse strains genetically deficient in γδ T cells (δ−/− mice), and in δ−/− mice reconstituted with DETC or with different γδ cell subpopulations. NOD.δ−/− and FVB.δ−/− mice spontaneously developed localized, chronic dermatitis, whereas interestingly, the commonly used C57BL/6.δ−/− strain did not. Genetic analyses indicated a single autosomal recessive gene controlled the dermatitis susceptibility of NOD.δ−/− mice. Furthermore, allergic and irritant contact dermatitis reactions were exaggerated in FVB.δ−/−, but not in C57BL/6.δ−/− mice. Neither spontaneous nor augmented irritant dermatitis was observed in FVB.β−/− δ−/− mice lacking all T cells, indicating that αβ T cell–mediated inflammation is the target for γδ-mediated down-regulation. Reconstitution studies demonstrated that both spontaneous and augmented irritant dermatitis in FVB.δ−/− mice were down-regulated by Vγ5+ DETC, but not by epidermal T cells expressing other γδ TCRs. This study demonstrates that functional impairment at an epithelial interface can be specifically attributed to absence of the local TCR-γδ+ IEL subset and suggests that systemic inflammatory reactions may more generally be subject to substantial regulation by local IELs.

The Distinct Contributions of Murine T Cell Receptor (TCR)γδ+ and TCRαβ+ T Cells to Different Stages of Chemically Induced Skin Cancer

Epithelial tissues in which carcinomas develop often contain systemically derived T cell receptor (TCR)αβ+ cells and resident intraepithelial lymphocytes that are commonly enriched in TCRγδ+ cells. Recent studies have demonstrated that γδ cells protect the host against chemically induced cutaneous malignancy, but the role of αβ T cells has been enigmatic, with both protective and tumor-enhancing contributions being reported in different systems. This study aims to clarify the contributions of each T cell type to the regulation of squamous cell carcinoma induced in FVB mice by a two-stage regimen of 7,12-dimethylbenz[a]anthracene initiation followed by repetitive application of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. This protocol permits one to monitor the induction of papillomas and the progression of those papillomas to carcinomas. The results show that whereas γδ cells are strongly protective, the nonredundant contributions of αβ T cells to the hosts protection against papillomas are more modest. Furthermore, at both high and low doses of carcinogens, αβ T cells can contribute to rather than inhibit the progression of papillomas to carcinomas. As is likely to be the case in humans, this study also shows that the contribution of T cells to tumor immunosurveillance is regulated by modifier genes.

Langerhans Cells Facilitate Epithelial DNA Damage and Squamous Cell Carcinoma

The Dark Side of Langerhans Cells Several immune cell populations reside in the skin and are thought to provide a protective barrier against infections and to act as sentinels against malignant transformation. However, studies in mice that lack Langerhans cells, a subset of dendritic cells, have suggested that these cells may actually promote tumorigenesis. Using a mouse model of squamous cell carcinoma, Modi et al. (p. 104) now reveal how Langerhans cells may promote the transformation of skin epithelial cells. In response to the carcinogen 7,12-dimethylbenz[α]anthracene (DMBA), Langerhans cells increased their expression of the cytochrome P-450 enzyme CYP1B1, which can metabolize DMBA to the mutagenic DMBA-trans-3,4-diol. Thus, besides their functions in regulating the adaptive immune response, Langerhans cells may participate in the metabolism of environmental carcinogens. A specialized immune cell population in the skin promotes tumorigenesis by metabolizing environmental carcinogens. Polyaromatic hydrocarbons (PAHs) are prevalent, potent carcinogens, and 7,12-dimethylbenz[a]anthracene (DMBA) is a model PAH widely used to study tumorigenesis. Mice lacking Langerhans cells (LCs), a signatory epidermal dendritic cell (DC), are protected from cutaneous chemical carcinogenesis, independent of T cell immunity. Investigation of the underlying mechanism revealed that LC-deficient skin was relatively resistant to DMBA-induced DNA damage. LCs efficiently metabolized DMBA to DMBA-trans-3,4-diol, an intermediate proximal to oncogenic Hras mutation, and DMBA-treated LC-deficient skin contained significantly fewer Hras mutations. Moreover, DMBA-trans-3,4-diol application bypassed tumor resistance in LC-deficient mice. Additionally, the genotoxic impact of DMBA on human keratinocytes was significantly increased by prior incubation with human-derived LC. Thus, tissue-associated DC can enhance chemical carcinogenesis via PAH metabolism, highlighting the complex relation between immune cells and carcinogenesis.

Characterizing tumor-promoting T cells in chemically induced cutaneous carcinogenesis.

There is a longstanding but poorly understood epidemiologic link between inflammation and cancer. Consistent with this, we previously showed that αβ T cell deficiency can increase resistance to chemical carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene and promoted by phorbol 12-myristate 13-acetate. This provoked the hypothesis that αβ T cell deficiency removed T regulatory cells that limit the anti-tumor response or removed a specific tumor-promoting (T-pro) T cell population. Here we provide evidence for the latter, identifying a novel CD8+ subset that is a candidate for T-pro cells. We demonstrate that CD8 cell-deficient mice show substantially less tumor incidence and progression to carcinoma, whereas susceptibility is restored by CD8+ cell reconstitution. To characterize the putative T-pro cells, tumor-infiltrating lymphocytes were isolated from normal and CD4−/− mice, revealing an activated population of T cell receptor αβ+CD8+CD44+CD62L− cells expressing the inflammatory mediators IFNγ, TNFα, and cyclooxygenase-2, but deficient in perforin, relative to recirculating cells of equivalent phenotype. This novel population of CD8+ T cells has intriguing similarities with other lymphocytes that have been associated with tissue growth and invasiveness and has implications for inflammation-associated carcinogenesis, models of cancer immunosurveillance, and immunotherapeutic strategies.

Rapid generation of maturationally synchronized human dendritic cells: contribution to the clinical efficacy of extracorporeal photochemotherapy

Extracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T-cell lymphoma, graft-versus-host disease, and allografted organ rejection. Its clinical and experimental efficacy in cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the antigen-presenting dendritic cell (DC) differentiation pathway, within a single day, without added cytokines, as determined by enhanced expression of relevant genes. The resulting DCs are capable of processing and presentation of exogenous and endogenous antigen and are largely maturationally synchronized, as assessed by the level of expression of costimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome reveals that activation or suppression of more than 1100 genes produces a reproducible distinctive molecular signature, common to ECP-processed monocytes from normal subjects, and those from patients. Because ECP induces normal monocytes to enter the DC differentiation pathway, this phenomenon is independent of disease state. The efficiency with which ECP stimulates new functional DCs supports the possibility that these cells participate prominently in the clinical successes of the treatment. Appropriately modified by future advances, ECP may potentially offer a general source of therapeutic DCs.

Anti‐inflammatory effects in the skin of thymosin‐β4 splice‐variants

The intraepithelial lymphocyte (IEL) network of T‐cell receptor γδ+ (Vγ5+) dendritic epidermal T cells (DETC) in murine skin down‐regulates cutaneous inflammation, although the mechanism is unknown. Thymosin‐β4 (Tβ4), identified by serial analysis of gene expression as a predominant transcript in gut IEL, encodes both a ubiquitous actin‐binding protein (UTβ4) with demonstrated capacity to inhibit neutrophilic infiltration, and a splice‐variant limited to lymphoid tissue (LTβ4) with unknown bioactivity. Freshly isolated Vγ5+ DETCs expressed both forms, while only LTβ4 was preferentially up‐regulated after cellular activation in vitro. To compare the anti‐inflammatory properties of LTβ4 and UTβ4 in the skin in vivo, the biological activities of synthesized polypeptides were assessed using three different strategies: neutrophil infiltration by footpad λ‐carrageenan injection; irritant contact dermatitis to 12‐O‐tetradecanoylphorbol 13‐acetate; and allergic contact dermatitis to 2,4‐dinitrofluorobenzene. These studies clearly showed that the anti‐inflammatory activities of LTβ4 were broader and most often stronger than those of UTβ4. Thus, the activation‐responsive expression of the lymph‐specific form of Tβ4 may be one mechanism by which DETC, and possibly other IELs, down‐regulate local inflammation.

IL-9 Regulates Allergen-Specific Th1 Responses in Allergic Contact Dermatitis

The cytokine IL-9, derived primarily from T-helper (Th)-9 lymphocytes, promotes expansion of the Th2 subset and is implicated in the mechanisms of allergic asthma. We hypothesize that IL-9 also plays a role in human allergic contact dermatitis (ACD). To investigate this hypothesis, skin biopsy specimens of positive patch test sites from non-atopic patients were assayed using qPCR and immunohistochemistry. Along with Th2 associated cytokines, IFN-γ, IL-4, and IL-17A, expression of IL-9, and PU.1, a Th9-associated transcription factor, were elevated when compared to paired normal skin. Immunohistochemistry on ACD skin biopsies identified PU.1+CD3+, and PU.1+CD4+ cells, consistent with Th9 lymphocytes, in the inflammatory infiltrate. PBMC from nickel-allergic patients, but not non-allergic controls, show significant IL-9 production in response to nickel. Blocking studies with monoclonal antibodies to HLA-DR (but not HLA-A, B, C) or chloroquine significantly reduced this nickel-specific IL-9 production. Additionally, blockade of IL-9 or IL-4 enhanced allergen-specific IFN-γ production. A contact hypersensitivity model using IL-9−/− mice, shows enhanced Th1 lymphocyte immune responses, when compared to WT mice, consistent with our human in vitro data. This study demonstrates that IL-9, through its direct effects on Th1 and ability to promote IL-4 secretion, has a regulatory role for Th1 lymphocytes in ACD.

Cutaneous Two-Stage Chemical Carcinogenesis

INTRODUCTIONThe induction of cutaneous carcinogenesis in mice by topical administration of chemicals enables the local, systemic, and environmental factors that influence tumor susceptibility, growth, and progression to be studied in the laboratory. Chemical carcinogenesis in mouse skin has been used for several decades, and continues to help in the identification of important molecular and immunological pathways involved in cutaneous malignancy. This protocol describes the two-stage application of chemicals to the skin for the initiation and promotion of cutaneous tumors. In the two-stage model of induction, a single application of the chemical initiator mutagen 7,12-dimethylbenz[a]anthracene (DMBA) is followed by repeated applications of a pro-inflammatory phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; otherwise known as phorbol 12-myristate 13-acetate [PMA]). Resulting tumors may develop in the form of benign papillomas that regress or progress to SCC, or SCC may arise without an apparent precursor lesion. Thus, two-stage chemical carcinogenesis provides an opportunity to monitor early and late events in cancer development and progression.

Induction of monocyte-to-dendritic cell maturation by extracorporeal photochemotherapy: initiation via direct platelet signaling.

Extracorporeal Photochemotherapy (ECP) is a widely used therapy for cutaneous T cell lymphoma (CTCL). Although the mechanism of clinical action of ECP is not precisely established, previous studies have shown evidence of induction of dendritic cells (DCs). Here we show that, under flow conditions similar to those in post-capillary venules, ECP promotes platelet immobilization and activation, initiating stepwise receptor-ligand interactions with monocytes, which then differentiate into DC. These findings clarify how ECP directly stimulates DC maturation; suggest a new clinically applicable approach to the obtainment of DC; and identify a novel mechanism that may reflect physiological induction of DC.