Morten M. Nielsen

Inhibition of constitutively activated Stat3 correlates with altered Bcl-2/Bax expression and induction of apoptosis in mycosis fungoides tumor cells

The Jak/Stat signaling pathway transmits signals from many cytokine and growth factor receptors to target genes in the nucleus. Constitutive activation of Stat3 has recently been observed in many tumor cells and dysregulation of the Stat signaling pathway has been proposed to be implicated in malignant transformation. In a previous study, we found constitutively tyrosine phosphorylated Stat3 in mycosis fungoides tumor cells. Here, we show that the Jak kinase inhibitor, Ag490, inhibits the constitutive binding of Stat3 to an oligonucleotide representing the Stat-binding sequence from the ICAM promotor. The decreased ability of Stat3 to bind DNA precedes dynamic alterations in the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins (decreased Bcl-2 expression and increased Bax expression) and induction of apoptosis. Thus, our data suggest that the involvement of Stat3 in oncogenic transformation could be mediated through regulation of survival signals.

Constitutive STAT3-activation in Sezary syndrome: tyrphostin AG490 inhibits STAT3-activation, interleukin-2 receptor expression and growth of leukemic Sezary cells.

Interleukin-2 (IL-2) is a growth factor which upon binding to high-affinity receptors (IL-2Rαβγ) triggers mitogenesis in T cells. IL-2Rα expression is restricted to T cells which have recently encountered antigen, and in healthy individuals the majority (>95%) of peripheral T cells are IL-2Rα negative. An aberrant expression of IL-2Rα has recently been described in cutaneous T-cell lymphoma (CTCL). Here, we study the regulation of IL-2Rα expression and STATs in a tumor cell line obtained from peripheral blood from a patient with Sezary syndrome (SS), a leukemic variant of CTCL. We show that (1) STAT3 (a transcription factor known to regulate IL-2Rα transcription) is constitutively tyrosine-phosphorylated in SS tumor cells, but not in non-malignant T cells; (2) STAT3 binds constitutively to a STAT-binding sequence in the promotor of the IL-2Rα gene; (3) the Janus kinase inhibitor, tyrphostine AG490, inhibits STAT3 activation, STAT3 DNA binding, and IL-2Rα mRNA and protein expression in parallel; and (4) tyrphostine AG490 inhibits IL-2 driven mitogenesis and triggers apoptosis in SS tumor cells. In conclusion, we provide the first example of a constitutive STAT3 activation in SS tumor cells. Moreover, our findings suggest that STAT3 activation might play an important role in the constitutive IL-2Rα expression, survival, and growth of malignant SS cells.

Enhanced sensitization and elicitation responses caused by mixtures of common fragrance allergens

Background. Perfumes are complex mixtures composed of many fragrance ingredients, many of which are known to be only weak allergens when tested individually. It is therefore surprising that fragrance contact allergy is one of the most common forms of contact allergy.

IL-1β–Dependent Activation of Dendritic Epidermal T Cells in Contact Hypersensitivity

Substances that penetrate the skin surface can act as allergens and induce a T cell–mediated inflammatory skin disease called contact hypersensitivity (CHS). IL-17 is a key cytokine in CHS and was originally thought to be produced solely by CD4+ T cells. However, it is now known that several cell types, including γδ T cells, can produce IL-17. In this study, we determine the role of γδ T cells, especially dendritic epidermal T cells (DETCs), in CHS. Using a well-established model for CHS in which 2,4-dinitrofluorobenzene (DNFB) is used as allergen, we found that γδ T cells are important players in CHS. Thus, more IL-17–producing DETCs appear in the skin following exposure to DNFB in wild-type mice, and DNFB-induced ear swelling is reduced by ∼50% in TCRδ−/− mice compared with wild-type mice. In accordance, DNFB-induced ear swelling was reduced by ∼50% in IL-17−/− mice. We show that DNFB triggers DETC activation and IL-1β production in the skin and that keratinocytes produce IL-1β when stimulated with DNFB. We find that DETCs activated in vitro by incubation with anti-CD3 and IL-1β produce IL-17. Importantly, we demonstrate that the IL-1R antagonist anakinra significantly reduces CHS responses, as measured by decreased ear swelling, inhibition of local DETC activation, and a reduction in the number of IL-17+ γδ T cells and DETCs in the draining lymph nodes. Taken together, we show that DETCs become activated and produce IL-17 in an IL-1β–dependent manner during CHS, suggesting a key role for DETCs in CHS.

γδ T cells in homeostasis and host defence of epithelial barrier tissues

Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body — namely, the epidermis and the intestine — and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity and repair, host homeostasis and protection in the context of the symbiotic relationship with the microbiome and during pathogen clearance. Finally, we describe epithelium-specific butyrophilin-like molecules and briefly review their emerging role in selectively shaping and regulating epidermal and intestinal γδ T cell repertoires.

NKG2D-dependent activation of dendritic epidermal T cells in contact hypersensitivity.

The interaction between keratinocytes (KC) and skin-resident immune cells plays an important role in induction of contact hypersensitivity (CHS). A specific subset of γδ T cells termed dendritic epidermal T cells (DETC) are located in mouse epidermis, and we have recently shown that DETC become activated and produce IL-17 in an IL-1β-dependent manner during CHS. Various receptors on DETC, including NKG2D, are involved in DETC responses against tumors and during wound healing. The ligands for NKG2D (NKG2DL) are stress-induced proteins such as Mult-1, H60, Rae-1 in mice and MICA, MICB and ULBP in humans. Here, we show that allergens up-regulate expression of the NKG2DL Mult-1, H60 and Rae-1 in cultured mouse KC and of MICA in primary human KC. We demonstrate that Mult-1 is expressed in mouse skin exposed to allergen. Furthermore, we find that the vast majority of DETC in murine epidermis and skin-homing cutaneous lymphocyte-associated antigen (CLA) positive γδ T cells in humans express NKG2D. Finally, we demonstrate that blocking of NKG2D partially inhibits allergen-induced DETC activation. These findings demonstrate that NKG2D and NKG2DL are involved in allergen-induced activation of DETC and indicate that the NKG2D/NKG2DL pathway might be a potential target for treatment of CHS.

Rapid allergen‐induced interleukin‐17 and interferon‐γ secretion by skin‐resident memory CD8+ T cells

Skin‐resident memory T (TRM) cells are associated with immunological memory in the skin. Whether immunological memory responses to allergens in the skin are solely localized to previously allergen‐exposed sites or are present globally in the skin is not clear. Furthermore, the mechanisms whereby TRM cells induce rapid recall responses need further investigation.

Nickel acts as an adjuvant during cobalt sensitization

Metal allergy is the most frequent form of contact allergy with nickel and cobalt being the main culprits. Typically, exposure comes from metal‐alloys where nickel and cobalt co‐exist. Importantly, very little is known about how co‐exposure to nickel and cobalt affects the immune system. We investigated these effects by using a recently developed mouse model. Mice were epicutaneously sensitized with i) nickel alone, ii) nickel in the presence of cobalt, iii) cobalt alone, or iv) cobalt in the presence of nickel, and then followed by challenge with either nickel or cobalt alone. We found that sensitization with nickel alone induced more local inflammation than cobalt alone as measured by increased ear‐swelling. Furthermore, the presence of nickel during sensitization to cobalt led to a stronger challenge response to cobalt as seen by increased ear‐swelling and increased B and T cell responses in the draining lymph nodes compared to mice sensitized with cobalt alone. In contrast, the presence of cobalt during nickel sensitization only induced an increased CD8+ T cell proliferation during challenge to nickel. Thus, the presence of nickel during cobalt sensitization potentiated the challenge response against cobalt more than the presence of cobalt during sensitization to nickel affected the challenge response against nickel. Taken together, our study demonstrates that sensitization with a mixture of nickel and cobalt leads to an increased immune response to both nickel and cobalt, especially to cobalt, and furthermore that the adjuvant effect appears to correlate with the inflammatory properties of the allergen..

Epicutaneous exposure to nickel induces nickel allergy in mice via a MyD88-dependent and interleukin-1-dependent pathway

Several attempts to establish a model in mice that reflects nickel allergy in humans have been made. Most models use intradermal injection of nickel in combination with adjuvant to induce nickel allergy. However, such models poorly reflect induction of nickel allergy following long‐lasting epicutaneous exposure to nickel.

TCR down-regulation controls T cell homeostasis

TCR and cytokine receptor signaling play key roles in the complex homeostatic mechanisms that maintain a relative stable number of T cells throughout life. Despite the homeostatic mechanisms, a slow decline in naive T cells is typically observed with age. The CD3γ di-leucine-based motif controls TCR down-regulation and plays a central role in fine-tuning TCR expression and signaling in T cells. In this study, we show that the age-associated decline of naive T cells is strongly accelerated in CD3γLLAA knock-in mice homozygous for a double leucine to alanine mutation in the CD3γ di-leucine-based motif, whereas the number of memory T cells is unaffected by the mutation. This results in premature T cell population senescence with a severe dominance of memory T cells and very few naive T cells in middle-aged to old CD3γ mutant mice. The reduced number of naive T cells in CD3γ mutant mice was caused by the combination of reduced thymic output, decreased T cell apoptosis, and increased transition of naive T cells to memory T cells. Experiments with bone marrow chimeric mice confirmed that the CD3γLLAA mutation exerted a T cell intrinsic effect on T cell homeostasis that resulted in an increased transition of CD3γLLAA naive T cells to memory T cells and a survival advantage of CD3γLLAA T cells compared with wild-type T cells. The experimental observations were further supported by mathematical modeling of T cell homeostasis. Our study thus identifies an important role of CD3γ-mediated TCR down-regulation in T cell homeostasis.