Earl J. Glusac

Th2 responses induced by epicutaneous or inhalational protein exposure are differentially dependent on IL-4

Atopic individuals are predisposed to mounting vigorous Th2-type immune responses to environmental allergens. To determine the factors responsible, animal models that closely mimic natural modes of allergen exposure should prove most informative. Therefore, we investigated the role of IL-4, a known Th2-promoting cytokine, in generation of Th2 responses after exposure of either the skin or airway to soluble protein. Compared with wild-type (WT) mice, IL-4-deficient (IL-4(-/-)) mice showed markedly impaired Th2 activation after primary exposure to inhaled ovalbumin (OVA), with decreased OVA-specific IgG1 and IgE, and significantly fewer eosinophils in bronchoalveolar lavage (BAL) fluid after airway challenge. In contrast, IL-4(-/-) mice initially exposed to epicutaneous (e.c.) OVA mounted Th2 responses equivalent to responses in WT mice, with high numbers of eosinophils in BAL fluid. Because Th2 responses were not induced by e.c. OVA exposure in Stat6(-/-) mice (mice lacking signal transducer and activator of transcription 6), the role of IL-13 was tested. In vivo depletion of IL-13 prevented Th2 responses induced by e.c. OVA exposure in IL-4(-/-) mice. These data demonstrate a marked difference in the IL-4 dependence of Th2 responses generated at two anatomic sites of natural allergen encounter and identify the skin as a particularly potent site for Th2 sensitization.

Bone Marrow-Derived Cells Contribute to Epithelial Engraftment during Wound Healing

Recent findings suggest that bone marrow-derived cells (BMDC) may contribute to tissue maintenance throughout the body. However, it is not yet known whether marrow-derived epithelial cells are capable of undergoing proliferation. Our laboratory has shown that BMDC engraft as keratinocytes in the skin at low levels (</= 1%) in the absence of injury. Here we show that skin damage affects the degree of engraftment of BMDC as keratinocytes and that the keratinocytes are actively cycling. Female mice reconstituted with sex-mismatched BM were wounded by punch biopsy and incision. At the wound site, engraftment of BMDC as epidermal cells increased within 1 day, and continued to increase to approximately 4% by 3 weeks after injury. Using a Cre-lox system, fusion of BMDC with epithelial cells was ruled out. BMDC-derived epithelial cells at the wound edges expressed Ki67, a marker for actively cycling cells, and this proliferation correlated with an increase in the number of donor-derived cells within the wound. Donor-derived cytokeratin 5-expressing cells were rare, suggesting that BMDC do not engraft as epidermal stem cells, and the level of engraftment peaked and then decreased over time, further suggesting that BMDC may assist in early wound healing by engrafting as transit-amplifying cells, which then differentiate into keratinocytes.

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.

Reassessment of histologic parameters in the diagnosis of mycosis fungoides.

The histologic diagnosis of mycosis fungoides (MF) can be difficult to establish and is based on interpretation of numerous subtle changes, most of which may be present to some degree in many inflammatory and neoplastic cutaneous conditions. To reassess the diagnostic criteria for making a histologic diagnosis of MF, we retrospectively reviewed histologic sections from 64 patients with mycosis fungoides (MF+) and compared the findings with sections from 47 patients who were biopsied to exclude MF and were shown not to have the disease (MF-). Patients were selected as MF+ or MF- independent of histologic findings based on the clinical course with at least 3 years of follow-up and immunophenotyping results. Following patient selection, at least two observers reviewed each slide without knowledge of final diagnosis and graded the intensity of approximately 25 histologic parameters. On univariate analysis, the following parameters were significant at beyond the p = 0.01 level: Pautriers abscesses, haloed lymphocytes, exocytosis, disproportionate epidermotropism, epidermal lymphocytes larger than dermal lymphocytes, hyperconvoluted intraepidermal lymphocytes, and lymphocytes aligned within the basal layer. Haloed lymphocytes proved to be the most robust discriminator of MF from non-MF on multivariate analysis. These findings show that whereas many previously described features do discriminate between MF and inflammatory mimics, others are much less specific. Furthermore, few cases demonstrate all histologic features; for example, Pautriers microabscesses were seen in only 37.5% of our cases. We conclude that a combination of specific histologic parameters can be used to establish a microscopic diagnosis of MF without the necessity of confirmatory immunophenotyping in the vast majority of cases.

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.

Induction of human tumor-loaded dendritic cells

A preferred anti‐cancer vaccine would be tumor‐specific, simple to rapidly construct and safe to administer. It would permit immunization against a spectrum of the tumors distinctive antigens, without requiring their prior identification. Toward these goals, we describe a modification of standard extracorporeal photopheresis (ECP) which initiates, within a single day, both monocyte‐to‐dendritic cell (DC) differentiation and malignant cell apoptosis. The transition of monocytes to immature DCs was identified by the expression of cytoplasmic CD83 and membrane CD36 in the absence of membrane CD14 staining, as well as induction of membrane CD83 expression. Differentiating DCs were avidly phagocytic and engulfed apoptotic malignant T cells. Differentiating DCs were capable of stimulating significant proliferation of normal alloreactive lymphocyte responders, indicting increased expression of membrane MHC class II molecules. This approach provides a clinically practical means of developing tumor‐loaded cells that have initiated the transition to DCs without the requirement of exogenous cytokines, excessive cellular manipulation or isolation. Construction of DC vaccines using this methodology can be generalized to other diseases and may offer a novel approach for improved cancer immunotherapy.

Immunohistologic differential diagnosis of basal cell carcinoma, squamous cell carcinoma, and trichoepithelioma in small cutaneous biopsy specimens

The distinction between squamoid basal cell carcinoma and basaloid squamous cell carcinoma (or between BCC and trichoepithelioma variants) is usually made readily on the basis of defined histological criteria. However, these differential diagnoses occasionally can pose difficult morphological problems. The stated distinctions are clinically important because the risk of progressive disease is significantly higher with squamous carcinoma of the skin than with basal cell carcinoma (BCC), and a trichoepithelioma misinterpreted as BCC burdens the patient with an inaccurate diagnosis that may result in inappropriate surgery. Recent reports have suggested that reactivity with the monoclonal antibody Ber‐EP4 is capable of separating histologically similar basal cell and squamous carcinomas, and that ihe expression of bcl‐2 or CD34 antigen is able to distingtush BCC from trichoepithelioma. However, corroborative studies of these contentions are few in number. In order to investigate the usefulness of the stated immunostains in the above‐cited differential diagnoses, the authors analyzed 45 basal cell carcinomas and 22 squamous carcinomas, as well as 36 trichoepitheliomas. The monoclonal antibodies Ber‐EP4, My10 (CD34), and anti‐bcl‐2 were applied to formalin‐fixed paraffin sections in all cases, using a standard avidin‐biotin‐peroxidase complex method. Most BCCs demonstrated strong, diffuse cytoplasmic labeling with Ber‐EP4 and anti‐bcl‐2. In contrast, the squamous carcinomas were uniformly negative for the former marker and only focally reactive for the latter in four examples. ‘Peripheral’bcl‐2 staining of trichoepitheliomas was noted in 24 of 33 of the immunoreactive tumors, but the remainder were marked diffusely and similarly to most BCCs. Among the latter, immature trichoepitheliomas were diffusely reactive for this marker in 6 of 8 cases. Labeling of epithelium for CD34 failed to discriminate between any of the tumor types under evaluation, whereas staining of peritumoral stroma was characteristic of the majority of trichoepitheliomas and more than one‐third of metatypical basal cell carcinomas. These data support the suggestion that Ber‐EP4 and bcl‐2 are useful in the separation of BCC from squamous carcinomas. Nevertheless, they also serve to caution against reliance upon bcl‐2 and CD34 immunostains in attempting to distinguish BCC from trichoepithelioma in histologically enigmatic cases. There is currently no certain method other han conventional microscopy that can be applied successfully to the latter problem.

p63 is a useful marker for cutaneous spindle cell squamous cell carcinoma.

Background:  Cutaneous spindle cell squamous cell carcinoma (SCSCC) is a rare variant of SCC. This lesion is sometimes difficult to diagnose based purely on morphologic features. p63 is a member of the p53 gene family that can be identified in epithelial malignancies.

Primary Cutaneous B-Cell Lymphoma Treated With Radiotherapy: A Comparison of the European Organization for Research and Treatment of Cancer and the WHO Classification Systems

PURPOSE To determine the relationship between the WHO and European Organization for Research and Treatment of Cancer (EORTC) pathologic classifications for primary cutaneous B-cell lymphoma (CBCL) and the implication of this relationship on initial treatment. PATIENTS AND METHODS Patients with primary CBCL treated with radiotherapy were identified retrospectively. Initial biopsy specimens were reviewed by two dermatopathologists and classified according to the EORTC and WHO systems. Primary outcomes were recurrence-free and overall survival. RESULTS Thirty-four patients were identified; initial biopsy specimens were adequate for classification in 32 patients. Four different composite histopathologic subtypes of lymphoma were identified: 53% (17 of 32) follicle center cell by EORTC and diffuse large B-cell by WHO (FCC/DLB), 25% (eight of 32) follicle center cell by EORTC and follicular by WHO (FCC/Fol), 13% (four of 32) marginal zone by EORTC and WHO (MZ/MZ), and 9% (three of 32) large B-cell of the leg by EORTC and diffuse large B-cell by WHO (Leg/DLB). Five-year relapse-free survival ranged from 62% to 73% for FCC/DLB, FCC/Fol, and MZ/MZ but was 33% for Leg/DLB (P =.6). Five-year overall survival was 100% for FCC/DLB, FCC/Fol, and MZ/MZ but was 67% for Leg/DLB (P =.07). At 5 years, 21% of all patients had developed extracutaneous disease. CONCLUSION Two-thirds of primary cutaneous FCC lymphomas by EORTC criteria satisfy WHO criteria for DLB lymphoma. Unlike DLB lymphoma presenting in nodal or noncutaneous sites, these lesions are associated with an indolent course and may be treated with local radiotherapy alone.

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.