Tord Labuda

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.

IKKβ Is Required for Peripheral B Cell Survival and Proliferation

NF-κB activity in mammalian cells is regulated through the IκB kinase (IKK) complex, consisting of two catalytic subunits (IKKα and IKKβ) and a regulatory subunit (IKKγ). Targeted deletion of Ikkβ results in early embryonic lethality, thus complicating the examination of IKKβ function in adult tissues. Here we describe the role of IKKβ in B lymphocytes made possible by generation of a mouse strain that expresses a conditional Ikkβ allele. We find that the loss of IKKβ results in a dramatic reduction in all peripheral B cell subsets due to associated defects in cell survival. IKKβ-deficient B cells are also impaired in mitogenic responses to LPS, anti-CD40, and anti-IgM, indicating a general defect in the ability to activate the canonical NF-κB signaling pathway. These findings are consistent with a failure to mount effective Ab responses to T cell-dependent and independent Ags. Thus, IKKβ provides a requisite role in B cell activation and maintenance and thus is a key determinant of humoral immunity.

Jak3- and JNK-dependent vascular endothelial growth factor expression in cutaneous T-cell lymphoma

Biopsies from patients with cutaneous T-cell lymphoma (CTCL) exhibit stage-dependent increase in angiogenesis. However, the molecular mechanisms responsible for the increased angiogenesis are unknown. Here we show that malignant CTCL T cells spontaneously produce the potent angiogenic protein, vascular endothelial growth factor (VEGF). Dermal infiltrates of CTCL lesions show frequent and intense staining with anti-VEGF antibody, indicating a steady, high production of VEGF in vivo. Moreover, the VEGF production is associated with constitutive activity of Janus kinase 3 (Jak3) and the c-Jun N-terminal kinases (JNKs). Sp600125, an inhibitor of JNK activity and activator protein-1 (AP-1) binding to the VEGF promoter, downregulates the VEGF production without affecting Jak3 activity. Similarly, inhibitors of Jak3 inhibit the VEGF production without affecting JNK activity. Downregulation of Stat3 with small interfering RNA has no effect, whereas curcumin, an inhibitor of both Jak3 and the JNKs, almost completely blocks the VEGF production. In conclusion, we provide evidence of VEGF production in CTCL, which is promoted by aberrant activation of Jak3 and the JNKs. Inhibition of VEGF-inducing pathways or neutralization of VEGF itself could represent novel therapeutic modalities in CTCL.

A novel xenograft model of cutaneous T-cell lymphoma.

Please cite this paper as: A novel xenograft model of cutaneous T‐cell lymphoma. Experimental Dermatology 2010; 19: 1096–1102.

MEK kinase 1 is a negative regulator of virus-specific CD8+ T cells

MEK kinase 1 (MEKK1) is a potent JNK‐activating kinase, a regulator of T helper cell differentiation, cytokine production and proliferation in vitro. Using mice deficient for MEKK1 activity (Mekk1ΔKD) exclusively in their hematopoietic system, we show that MEKK1 has a negative regulatory role in the generation of a virus‐specific immune response. Mekk1ΔKD mice challenged with vesicular stomatitis virus (VSV) showed a fourfold increase in splenic CD8+ T cell numbers. In contrast, the number of splenic T cells in infected WT mice was only marginally increased. The CD8+ T cell expansion in Mekk1ΔKD mice following VSV infection is virus‐specific and the frequency of virus‐specific T cells is significantly higher (more than threefold) in Mekk1ΔKD as compared to WT animals. Moreover, the hyper‐expansion of T cells seen in Mekk1ΔKD mice after VSV infection is a result of increased proliferation, since a significantly higher percentage of virus‐specific Mekk1ΔKD CD8+ T cells incorporated BrdU as compared to virus‐specific WT CD8+ T cells. In contrast, similar levels of apoptosis were detected in Mekk1ΔKD and WT T cells following VSV infection. These results strongly suggest that MEKK1 plays a negative regulatory role in the expansion of virus‐specific CD8+ T cells in vivo.

JNK1, but Not JNK2, Is Required in Two Mechanistically Distinct Models of Inflammatory Arthritis

The roles of the c-Jun N-terminal kinases (JNKs) in inflammatory arthritis have been investigated; however, the roles of each isotype (ie, JNK1 and JNK2) in rheumatoid arthritis and conclusions about whether inhibition of one or both is necessary for amelioration of disease are unclear. By using JNK1- or JNK2-deficient mice in the collagen-induced arthritis and the KRN T-cell receptor transgenic mouse on C57BL/6 nonobese diabetic (K/BxN) serum transfer arthritis models, we demonstrate that JNK1 deficiency results in protection from arthritis, as judged by clinical score and histological evaluation in both models of inflammatory arthritis. In contrast, abrogation of JNK2 exacerbates disease. In collagen-induced arthritis, the distinct roles of the JNK isotypes can, at least in part, be explained by altered regulation of CD86 expression in JNK1- or JNK2-deficient macrophages in response to microbial products, thereby affecting T-cell-mediated immunity. The protection from K/BxN serum-induced arthritis in Jnk1(-/-) mice can also be explained by inept macrophage function because adoptive transfer of wild-type macrophages to Jnk1(-/-) mice restored disease susceptibility. Thus, our results provide a possible explanation for the modest therapeutic effects of broad JNK inhibitors and suggest that future therapies should selectively target the JNK1 isoform.