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Please cite this paper as: Enhanced T-cell activation by immature dendritic cells loaded with HSP70-expressing heat-killed melanoma cells. Experimental Dermatology 2010; 19: 108–116. Vaccination protocols that utilize dendritic cells (DCs) to elicit therapeutic immunity against tumors are the subject of intense research. Given that the capacity of DCs to cross-present antigens is physiologically low, there is considerable interest to develop strategies that enhance that pathway. In order to best exploit the enhanced cross-presentation of antigens bound to heat shock protein 70 (HSP70), we analysed melanoma cell preparations for their HSP70 expression. Western blotting revealed strong upregulation of HSP70 after heat-killing in contrast to UV-B irradiation. When the uptake of heat-killed necrotic cells by DCs at various levels of maturation was assessed, 61 ± 7% of immature DCs (iDCs) internalized fluorescence-labelled necrotic material. Apoptotic material from UV-B-irradiated cells was internalized by only 48 ± 5% of iDCs. Maturation-inducing cytokines did not affect the uptake when added simultaneously with the tumor cell preparations. Loading DCs with heat-necrotic or apoptotic melanoma cells slightly reduced CD83 expression while leaving CD208 (DC-LAMP) expression unchanged. As determined by IFN-γ-detecting enzyme-linked-immunospot assays, iDCs loaded with heat-killed melanoma cells activated autologous T cells most effectively when used without any further maturation, whereas DCs loaded with apoptotic material required maturation. In conclusion, HSP70-expressing melanoma cells could be generated by heat-killing. Loading iDCs with heat-killed melanoma cells resulted in a superior priming of autologous T cells in vitro. [ABSTRACT FROM AUTHOR]

Dieckhoff K, Graf P, Beinhauer B, Schwaerzler C, Carballido JM, Neumann C, Zachmann K, Jung T. Deficient translocation of c-Rel is associated with impaired Th1 cytokine production in T cells from atopic dermatitis patients.Decreased production of T helper type 1 (Th1) cytokines, such as interferon-γ (IFN-γ) or interleukin-2 (IL-2), is a hallmark of atopic diseases. While accessory signals from antigen-presenting cells may be missing, T cells themselves may be suppressed in their ability to produce substantial amounts of Th1 cytokines. We show, in this study, that T cell receptor (TCR)-activated T cells from atopic dermatitis (AD) patients proliferate less than control T cells and produce lower amounts of IFN-γ and IL-2, but comparable amounts of IL-4. Because mice lacking the nuclear factor kappa B (NF-κB) transcription factors– p65 or c-Rel– show reduced Th1, but undisturbed Th2 responses, we investigated the role of c-Rel and p65 for Th1 cytokine production in T cells from healthy and severe AD patients. TCR-activated primary T cells from healthy donors treated with c-Rel antisense oligonucleotides produced lower levels of IL-2 and IFN-γ and proliferated less efficiently than the corresponding control T cells. Moreover, transfection of primary T cells with c-Rel or p65 enhanced proliferation and production of IL-2 and IFN-γ. Nuclear extracts of activated primary T cells from AD donors bound weakly to NF-κB-specific oligonucleotides, compared to extracts from healthy control T cells. Western blotting studies revealed that nuclear, but not cytosolic, extracts from T cells of AD patients lacked significant amounts of c-Rel and p65. T cell clones derived from AD patients failed to sufficiently translocate c-Rel and p65 into the nucleus following activation. Thus, impaired nuclear translocation of c-Rel and p65 may determine an impaired Th1 cytokine response in AD. [ABSTRACT FROM AUTHOR]

Lippert U, Zachmann K, Henz BM, Neumann C. Human T lymphocytes and mast cells differentially express and regulate extra- and intracellular CXCR1 and CXCR2. CXCL8 plays a major role in cell recruitment to sites of inflammation. Apart from neutrophils, little is known, however, about the cellular distribution and regulation of CXCL8 receptors in cells involved in acquired and adaptive immune responses. In previous studies, we have demonstrated the extracellular expression and function of CXCR1/2 on mast cells and also detected an intracellular pool of CXCR1/2. Here, we have addressed the question of receptor regulation during stimulation of human mast cells (HMC-1 cell line) and have studied T cells in comparison. Cell permeabilization was performed to detect both surface and possible intracellular receptor pools. HMC-1 cells stained positive for both receptors on the cell surface (CXCR1, 50%; CXCR2, 51%) and also after cell permeabilization (CXCR1, 86%; CXCR2, 74%). Similarly, T cells exhibited both cell-surface receptor expression (CXCR1, 30%; CXCR2, 23%) and higher total receptor expression (CXCR1, 50%; CXCR2, 36%), although overall values were lower than that in HMC-1 cells. On immunoblot, molecular weights of extra- and intracellular receptors on mast cells were the same, excluding altered receptor glycosylation. On stimulation with phorbol 12-myristate 13-acetate plus calcium ionophore, a time-dependent decrease of surface-membrane receptors was observed in both cell types, while total receptor remained the same, suggesting that receptor shedding is not involved. The kinetics of membrane receptor internalization and replenishment differed for the two cell types. Furthermore, receptor internalization was associated with decreased F-actin polymerization, a basic prerequisite for cell migration. These findings demonstrate for the first time the expression of extra- and intracellular CXCR1/2 receptors on T cells and delineate the dynamics of CXCR1/2 receptors on mast cells and T cells. Furthermore, they suggest a cell-type-specific and finely tuned regulation of chemokine responses at the receptor level in the context of inflammation. [ABSTRACT FROM AUTHOR]

Background: Sensitization to aeroallergens is a frequent event in patients with atopic dermatitis (AD). The role of allergen-specific T cells in the pathogenesis of AD is, however, not fully clear. A detailed immunological characterization of allergen-specific T cells able to migrate to inflamed skin might therefore be helpful in determining the relevance of allergen-specific T cells to clinical symptoms. Objective: To establish a simple assay to simultaneously monitor the expression of skin-related homing molecules and their proliferative response to an allergen on peripheral T cells. To evaluate whether this assay is able to identify AD patients in whom exposure to an allergen induces dermatitis. Methods: The expression of CLA, CD7 and CD4 was simultaneously measured by flow cytometry with the DNA content as a parameter of proliferation at the single cell level. House dust mite antigen (DerP) and tetanus toxoid (TT) were used as antigens. Results: CD4+ CLA+ CD7–, but not CLA– T cells from patients with AD proliferated and expanded following DerP stimulation. In contrast, TT stimulation resulted in proliferation of both, CLA+ and CLA– T cells from both, atopic and healthy donors. Conclusions: Single cell analysis revealed a predominant localization of allergen-reactive T cells within the CD7– CLA+ CD4+ T cell subset in AD. Thus, this simple assay not only confirms previously published results, but also extends our knowledge on the biology of CLA and CD7 cells in AD. Ultimately, this will be helpful in classifying those patients who might benefit from allergen avoidance therapies.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Recent studies of the cytokine pattern in skin lesions of patients with cutaneous T-cell lymphoma (CTCL) have shown that interleukin-4 (IL-4) and Il-10, both cytokines produced by T-helper type 2 cells, dominate in these lesions. Also, in single studies, interferon-γ (IFN-γ), a major cytokine of Th-1-cells, has been found to be absent. Consequently, it has been hypothesized that immune-suppressive Th-2 cytokines may promote local growth of the malignant lymphocyte clone. However, there is so far no evidence for T-cells as the source of the Th-2 cytokines in CTCL skin lesions nor have these cytokines been investigated at a clonal T-cell level. We established a total of 120 T-cell clones (TCCs) from lesional skin and 54 TCCs from the blood of four patients with mycosis fungoides. Epidermal TCCs (mostly CD8-positive) and dermal TCCs (mostly CD4-positive) were stimulated by the mitogen concanavalin A and, seeking a polarized cytokine pattern, the supernatants were assessed by ELISA. We showed that the vast majority of TCCs were able to secrete IFN-γ and IL-4. IFN-γ-deficient TCCs occurred only in the epidermis. Some (18) TCCs were found to be either negative for IL-10 production or to produce low levels only. No significant differences were observed between blood- and skin-derived TCCs. Thus a polarized Th-2 cytokine pattern was not detectable among cultured skin-infiltrating nonmalignant T-cells (TILs) isolated from early mycosis fungoides. It therefore appears unlikely that Th-2-mediated immune suppression is a major mechanism operating in early CTCL. However, this does not exclude its role in late-stage disease. [ABSTRACT FROM AUTHOR]

Background: Soluble interleukin 4 receptors (sIL–4R) are present in biological fluids. In contrast to mice, in man no distinct mRNA coding for sIL–4R has been described, suggesting that human sIL–4R is exclusively produced by proteolytic cleavage of the cell surface receptor. It is not known whether human sIL–4R is actively produced during an immune response. Methods: Human purified T cells, CD4+, CD8+, CD45RA+ and CD45R0+ T cell subpopulations were activated in vitro. sIL–4R was determined in the supernatants, cell surface IL–4R was measured by flow cytometry and RT–PCR. Results: Recombinant sIL–4R inhibited IL–4–mediated proliferation and IL–5 upregulation by T cells. sIL–4R could be detected at low levels in supernatants of nonactivated T cells, but at high levels following TCR engagement. This response was paralleled by enhanced transcription and de novo synthesis of the human cell surface IL–4R. Both, activated naive CD45RA+ and memory CD45R0+ T cells, produced sIL–4R with long–lasting kinetics. IL–4 increased sIL–4R production by activated CD45RA+, but there was less of an increase by CD45R0+ T cells. In addition, interferon–γ enhanced sIL–4R production. Cycloheximide and dexamethasone inhibited sIL–4R production by activated T cells, but did not abolish constitutive release of sIL–4R. Phosphoramidon and 1,10–phenanthroline dose–dependently inhibited shedding of the IL–4R, even in nonactivated T cells. Conclusion: The production of human sIL–4R by T cells is regulated by TCR stimuli, IL–4 and IFN–γ and needs the activity of metalloproteinases. Thus, sIL–4R should be regarded as inducible and due to its IL–4–antagonizing activity an immunoregulatory molecule. [ABSTRACT FROM AUTHOR]

We have reported previously on the generation of a monoclonal antibody against human macrophage migration inhibitory factor (MIF), which is a mediator of cellular immunity. Macrophage migration inhibitory factor activity in the migration assay was closely correlated with antibody reactivity. Using this antibody called 1C5/B, we are now able to study the expression of MIF in situ. Here, we report on the detection of MIF in blood lymphocytes and skin of a patient with a leukemic cutaneous T-cell lymphoma with the characteristics of Sézary syndrome. Ninety percent of the patient's Ficoll Hypaque-isolated peripheral white blood cells were of the helper phenotype. By conventional immunoperoxidase method, 94% reacted strongly positive with the antibody 1C5/B. In contrast, using the immunofluorescence method only 25% reacted positive. This indicates that the majority of the tumor cells did not express the molecule on their membrane but only in the cytoplasma. No other marker, such as interleukin 2 receptor, HLA-DR antigen, or interferon-gamma could be related to the expression of MIF. Also the cellular infiltrate in the skin was composed mainly of T helper cells and reacted positive with 1C5/B. As less than 3% of normal blood lymphocytes reacted with 1C5/B we suggest that the conversion to positivity may be a characteristic feature of the leukemic T-cell phenotype in Sézary syndrome. [ABSTRACT FROM AUTHOR]

The article focuses on the inhalant allergens in the pathogenesis of atopic dermatitis (AD). It cites studies of immunoglobulin E (IgE) in the pathogenesis of AD, and its role for allergic manifestations. It highlights the allergen-specific T cells in the IgE response. It suggests that the reduction of the environmental inhalant allergen load should be beneficial for AD patients.

In atopic individuals, allergen-specific CD4+ T lymphocytes often belong to the T-helper 2 (Th2) subset as they secrete the marker cytokines interleukin-4 (IL-4) and IL-5 but not interferon-γ (INF-γ). IL-10 is a cytokine the production of which, in the mouse system has been described to be restricted to the Th2 subset, but in the human was found to be produced by both Th1 and Th2 T cell clones (TCC). We have recently shown that house dust mite antigen (Dermatophagoides pteronyssinus)-specific TCC isolated from skin of patients with atopic dermatitis have a more polarized Th2 cytokine production profile than TCC obtained from the peripheral blood of these patients. In this study, we report that skin-derived TCC secrete more IL-10, IL-4 and IL-5, than TCC isolated from the blood of the same individual (pIn atopic individuals, allergen-specific CD4+ T lymphocytes often belong to the T-helper 2 (Th2) subset as they secrete the marker cytokines interleukin-4 (IL-4) and IL-5 but not interferon-γ (INF-γ). IL-10 is a cytokine the production of which, in the mouse system has been described to be restricted to the Th2 subset, but in the human was found to be produced by both Th1 and Th2 T cell clones (TCC). We have recently shown that house dust mite antigen (Dermatophagoides pteronyssinus)-specific TCC isolated from skin of patients with atopic dermatitis have a more polarized Th2 cytokine production profile than TCC obtained from the peripheral blood of these patients. In this study, we report that skin-derived TCC secrete more IL-10, IL-4 and IL-5, than TCC isolated from the blood of the same individual (p<0.05). The difference was more significant with specific TCC than with non-specific TCC. Furthermore, there was a positive correlation between the production of IL-10 and that of IL-4 and IL-5, respectively. In addition, the amount of IL-4 and IL-5 secreted by specific TCC from the skin correlated positively. These results were confirmed by the detection of mRNA by PCR. Finally, our data confirm that in human blood-derived TCC IL-10 secretion is not related to a particular cytokine production profile. We suggest that the skin of AD provides an unique environment for the development of aTh2-like secretion pattern not only with respect to IL-4 and IL-5 but also regarding IL-10. [ABSTRACT FROM AUTHOR]

Using a contact hypersensitivity model in BALB/c mice we have previously been able to show that Corynehacterium parvum or C. parvum serum (C.p.s.) of mice treated 24 hr before with C. parvum inhibited the suppressor T-lyrnphocyte (Tscell) response induced by epicutaneous antigen overload with 2,4-dinitrofluorobenzene (DNFB) or by i.v. injection of 2,4-dinitrobenzene sulfonic acid (DNBSO3) without inhibition of the T effector cell response (TDH-cell). In the present investigation we further analyzed the factor responsible for Ts-cell inhibition. Treatment of the C.p.s. with sheep-antimouse interferon (IFN) globulin neutralized its Ts-cell inhibitory effect. Intravenous (i.v.) injection of a crude mouse fibroblast IFN (340 U per mouse) 2 hr after i.v. application of a dose of DNBSO3 inducing tolerance had a similar Ts-cell inhibitory effect as observed with C.p.s. Injection of an electrophoretically pure α- and β-IFN preparation (1000 U per mouse) increased contact sensitivity in mice sensitized with an antigen overload and inhibited the induction of s-cells by DNBSO3 i.v. This result is highly suggestive that the Ts-cell inhibitory factor in serum of C. parum-treated mice is IFN and it shows that Ts-cells as compared to TDH-cells are susceptible to the inhibitory effect of highly purified IFN. This finding suggests that IFN may be an important immunoregulatory factor of delayed hypersensitivity not only in contact allergy but also in bacterial and viral defense. [ABSTRACT FROM AUTHOR]