Carsten Geisler

Vitamin D controls T cell antigen receptor signaling and activation of human T cells

Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-γ1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering led to an upregulation of ∼75-fold in PLC-γ1 expression, which correlated with greater TCR responsiveness. Induction of PLC-γ1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-γ1, which are required for subsequent classical TCR signaling and T cell activation.

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.

Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down‐regulated following activation of protein kinase C (PKC). Among other substrates the activated PKC in T cells phosphorylates the CD3 gamma subunit of the TCR. To investigate the role of CD3 gamma phosphorylation in PKC‐mediated TCR down‐regulation, point mutated CD3 gamma cDNA was transfected into the CD3 gamma‐negative T cell line JGN and CD3 gamma transfectants were analysed. Phosphorylation at S126 but not S123 in the cytoplasmic tail of CD3 gamma was required for PKC‐mediated down‐regulation of the TCR. Furthermore, analysis of a series of CD3 gamma truncation mutants indicated that in addition to S126 phosphorylation a motif C‐terminal of S126 was required for TCR down‐regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane‐proximal di‐leucine motif (L131 and L132) in the cytoplasmic tail of CD3 gamma was required for PKC‐mediated TCR down‐regulation in addition to phosphorylation at S126. Incubation of T cells in hypertonic medium known to disrupt normal clathrin lattices severely inhibited PKC‐mediated TCR down‐regulation in non‐mutated T cells, indicating that the TCR was down‐regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di‐leucine‐ or tyrosine‐based motifs is proposed.

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.

Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL)

Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that the most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) miRNAs distinguish CTCL from benign skin diseases with > 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Quantitative (q)RT-PCR analysis of 103 patients with CTCL and benign skin disorders validates differential expression of 4 of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A qRT-PCR-based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity and sensitivity, and with a classification accuracy of 95%, indicating that miRNAs have a high diagnostic potential in CTCL.

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.

The adjuvant mechanism of cationic dimethyldioctadecylammonium liposomes

Cationic liposomes are being used increasingly as efficient adjuvants for subunit vaccines but their precise mechanism of action is still unknown. Here, we investigated the adjuvant mechanism of cationic liposomes based on the synthetic amphiphile dimethyldioctadecylammonium (DDA). The liposomes did not have an effect on the maturation of murine bone‐marrow‐derived dendritic cells (BM‐DCs) related to the surface expression of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86. We found that ovalbumin (OVA) readily associated with the liposomes (> 90%) when mixed in equal concentrations. This efficient adsorption onto the liposomes led to an enhanced uptake of OVA by BM‐DCs as assessed by flow cytometry and confocal fluorescence laser‐scanning microscopy. This was an active process, which was arrested at 4° and by an inhibitor of actin‐dependent endocytosis, cytochalasin D. In vivo studies confirmed the observed effect because adsorption of OVA onto DDA liposomes enhanced the uptake of the antigen by peritoneal exudate cells after intraperitoneal injection. The liposomes targeted antigen preferentially to antigen‐presenting cells because we only observed a minimal uptake by T cells in mixed splenocyte cultures. The adsorption of antigen onto the liposomes increased the efficiency of antigen presentation more than 100 times in a responder assay with MHC class II‐restricted OVA‐specific T‐cell receptor transgenic DO11.10 T cells. Our data therefore suggest that the primary adjuvant mechanism of cationic DDA liposomes is to target the cell membrane of antigen‐presenting cells, which subsequently leads to enhanced uptake and presentation of antigen.

IL-23 and TH17-mediated inflammation in human allergic contact dermatitis

BACKGROUND IL-17-producing T(H) (T(H)17) cells are key mediators of chronic inflammation in mice. Recent studies have implicated T(H)17-mediated inflammation in the pathogenesis of human autoimmune diseases; however, the involvement of T(H)17 cells in allergic disorders remains largely elusive. OBJECTIVE To investigate T(H)17-mediated inflammation in human beings with allergic contact dermatitis; in particular, the innate response of keratinocytes to contact allergen, the induction of allergen-specific T(H)17 cells, and the presence of T(H)17-related effector cells in inflamed skin. METHODS Human keratinocytes were stimulated with nickel in vitro followed by measurements of IL-23 and IL-12 production by quantitative PCR and ELISA. Allergen-specific memory T cells from the blood of individuals with nickel allergy and healthy controls were identified and characterized by using a short-term ex vivo assay. Nickel patch test lesions and normal skin were analyzed for the expression of T(H)17-related cells and molecules by using immunohistochemistry. RESULTS Keratinocytes were found to produce IL-23, but no detectable IL-12, in a response to nickel stimulation. Memory T cells isolated from peripheral blood of individuals with nickel allergy, but not healthy controls, contained T(H)17 and T(H)1 cells proliferating in response to nickel-pulsed DCs. Inflamed skin of nickel-challenged allergic individuals contained infiltrating neutrophils and cells expressing IL-17, IL-22, CCR6, and IL-22R. CONCLUSION Our results demonstrate the involvement of T(H)17-mediated immunopathology in human allergic contact dermatitis, including both innate and adaptive immune responses to contact allergens.

The Vitamin D Receptor and T Cell Function

The vitamin D receptor (VDR) is a nuclear, ligand-dependent transcription factor that in complex with hormonally active vitamin D, 1,25(OH)2D3, regulates the expression of more than 900 genes involved in a wide array of physiological functions. The impact of 1,25(OH)2D3-VDR signaling on immune function has been the focus of many recent studies as a link between 1,25(OH)2D3 and susceptibility to various infections and to development of a variety of inflammatory diseases has been suggested. It is also becoming increasingly clear that microbes slow down immune reactivity by dysregulating the VDR ultimately to increase their chance of survival. Immune modulatory therapies that enhance VDR expression and activity are therefore considered in the clinic today to a greater extent. As T cells are of great importance for both protective immunity and development of inflammatory diseases a variety of studies have been engaged investigating the impact of VDR expression in T cells and found that VDR expression and activity plays an important role in both T cell development, differentiation and effector function. In this review we will analyze current knowledge of VDR regulation and function in T cells and discuss its importance for immune activity.

Malignant Tregs express low molecular splice forms of FOXP3 in Sézary syndrome

Sézary syndrome (SS) is an aggressive variant of cutaneous T-cell lymphoma. During disease progression, immunodeficiency develops; however, the underlying molecular and cellular mechanisms are not fully understood. Here, we study the regulatory T cell (Treg) function and the expression of FOXP3 in SS. We demonstrate that malignant T cells in 8 of 15 patients stain positive with an anti-FOXP3 antibody. Western blotting analysis shows expression of two low molecular splice forms of FOXP3, but not of wild-type (wt) FOXP3. The malignant T cells produce interleukin-10 and TGF-β and suppress the growth of non-malignant T cells. The Treg phenotype and the production of suppressive cytokines are driven by aberrant activation of Jak3 independent of the FOXP3 splice forms. In contrast to wt FOXP3, the low molecular splice forms of FOXP3 have no inhibitory effect on nuclear factor-κB (NF-κB) activity in reporter assays which is in keeping with a constitutive NF-κB activity in the malignant T cells. In conclusion, we show that the malignant T cells express low molecular splice forms of FOXP3 and function as Tregs. Furthermore, we provide evidence that FOXP3 splice forms are functionally different from wt FOXP3 and not involved in the execution of the suppressive function. Thus, this is the first description of FOXP3 splice forms in human disease.

Malignant cutaneous T-cell lymphoma cells express IL-17 utilizing the Jak3/Stat3 signaling pathway.

IL-17 is a proinflammatory cytokine that is crucial for the hosts protection against a range of extracellular pathogens. However, inappropriately regulated expression of IL-17 is associated with the development of inflammatory diseases and cancer. In cutaneous T-cell lymphoma (CTCL), malignant T cells gradually accumulate in skin lesions characterized by massive chronic inflammation, suggesting that IL-17 could be involved in the pathogenesis. In this study we show that IL-17 protein is present in 10 of 13 examined skin lesions but not in sera from 28 CTCL patients. Importantly, IL-17 expression is primarily observed in atypical lymphocytes with characteristic neoplastic cell morphology. In accordance, malignant T-cell lines from CTCL patients produce IL-17 and the synthesis is selectively increased by IL-2 receptor β chain cytokines. Small-molecule inhibitors or small interfering RNA against Jak3 and signal transducer and activator of transcription 3 (Stat3) reduce the production of IL-17, showing that the Jak3/Stat3 pathway promotes the expression of the cytokine. In summary, our findings indicate that the malignant T cells in CTCL lesions express IL-17 and that this expression is promoted by the Jak3/Stat3 pathway.