Translating clinical activity and gene expression signatures of etanercept and ciclosporin to the psoriasis xenograft SCID mouse model.

BACKGROUND: The psoriasis xenograft severe combined immunodeficiency (SCID) mouse model is used in drug discovery to obtain preclinical proof-of-principle of new antipsoriatic drug candidates. Validation of this model by antipsoriatic therapeutic agents in clinical use is important to understand its utility as well as its limitations. The effects of the clinically efficacious antitumour necrosis factor-α biologics have not yet been demonstrated in the psoriasis xenograft SCID mouse model. OBJECTIVES: To investigate the effect of etanercept and to explore the time-dependent changes induced by ciclosporin on psoriatic biomarkers at the gene expression level in the psoriasis xenograft SCID mouse model. METHODS: Xenografted SCID mice were treated either with etanercept and vehicle for 2 weeks or with ciclosporin and vehicle for 2 and 4 weeks, respectively. Treatment-induced changes in the psoriatic grafts were assessed by gene expression analysis and compared with published clinical microarray data. The grafts were further evaluated by histology and immunohistochemistry. RESULTS: Etanercept induced normalization of gene expression, which correlated with a significant reduction in epidermal thickness as well as a decrease in the number of proliferative cells. Anti-inflammatory activity induced by ciclosporin preceded the reduction in epidermal hyperplasia. Comparison of the etanercept- and ciclosporin-induced gene expression signatures with clinical microarray data showed significant correlations. CONCLUSIONS: Efficacy of etanercept and ciclosporin could be translated to the psoriasis xenograft SCID mouse model.

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.

Identification of the target of monoclonal antibody A6H as dipeptidyl peptidase IV/CD26 by LC MS\MS.

The monoclonal antibody (MAb) A6H, originally developed to fetal renal tissues, was found to be highly reactive to renal cell carcinoma and was subsequently demonstrated to co-stimulate a subpopulation of T cells. The A6H antigen had not been identified heretofore. Antigen from detergent extracts of renal cell carcinoma cells (7860) was immunoabsorbed with A6H-agarose, and the resin-bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen had a molecular weight of approximately 120 kDa as determined by Western blots. The 120-kDa protein band was excised and subjected to in-gel tryptic digestion, and the resulting peptides were separated and analyzed by liquid chromatography tandem mass spectrometry (LC MS\MS). The tandem mass spectra of the eluting peptides were used in combination with the SEQUEST computer program to search a human National Cancer Institute (NCI) protein database for the identity of the protein. The target antigen was shown to be dipeptidyl peptidase IV (DPP IV), which is also known as the cluster differentiation antigen CD26. Flow analysis of the expression of the A6H antigen and of CD26 on 7860 cells and on peripheral blood lymphocytes supported the identification of the A6H antigen as DPP IV. Recognition that the A6H antigen is DPP IV/CD26 afforded the opportunity to compare previous studies on A6H with those on other anti-CD26 antibodies in terms of expression in cancer cell lines and various tissues and as co-stimulators of T-cell activation.

Carbohydrate groups of alpha1-microglobulin are important for secretion and tissue localization but not for immunological properties.

The role of the carbohydrates for the structure and functions of the plasma and tissue protein alpha1-microglobulin (alpha1m) was investigated by deletion of the sites for N-glycosylation by site-directed mutagenesis (N17,96-->Q). The mutated cDNA was expressed in a baculovirus-insect cell system resulting in a nonglycosylated protein. The biochemical properties of N17,96Q-alpha1m were compared to nonmutated alpha1m, which carries two short non-sialylated N-linked oligosaccharides when expressed in the same system. Both proteins carried a yellow-brown chromophore and were heterogeneous in charge. Circular dichroism spectra and antibody binding indicated a similar overall structure. However, the secretion of N17,96Q-alpha1m was significantly reduced and approximately 75% of the protein were found accumulated intracellularly. The in vitro immunological effects of recombinant nonmutated alpha1m and N17,96Q-alpha1m were compared to the effects of alpha1m isolated from plasma, which is sialylated and carries an additional O-linked oligosaccharide. All three alpha1m variants bound to human peripheral lymphocytes and mouse T cell hybridomas to the same extent. They also inhibited the antigen-stimulated proliferation of peripheral lymphocytes and antigen-stimulated interleukin 2-secretion of T cell hybridomas in a similar manner. After injection of rats intravenously, the blood clearance of recombinant nonmutated and N17,96Q-alpha1m was faster than that of plasma alpha1m. Nonmutated alpha1m was located primarily to the liver, most likely via binding to asialoglycoprotein receptors, and N17,96Q-alpha1m was located mainly to the kidneys. It is concluded that the carbohydrates of alpha1m are important for the secretion and the in vivo turnover of the protein, but not for the structure or immunological properties.

Selective induction of p38 mitogen-activated protein kinase activity following A6H co-stimulation in primary human CD4(+) T cells.

We have recently described the novel A6H antigen expressed on human peripheral blood T cells and on renal cell carcinoma cells. Cross-linking of the A6H antigen results in co-stimulation of human CD4(+) T cells, characterized by induction of the transcription factor activator protein-1 (AP-1), proliferation and prominent IFN-gamma production, but low levels of IL-2. The proximal signaling events associated with A6H ligation include protein tyrosine kinase phosphorylation and association of p56 Lck, ZAP-70 and the TCR zeta chain. In this study we show that A6H co-stimulation selectively induced activation of the p38 mitogen-activated protein kinase (MAPK) pathway, whereas no significant c-Jun N-terminal kinases (JNK) activity was observed. In contrast, CD28 co-stimulation resulted in both p38 and JNK MAPK activities. Human CD4(+) T cells co-stimulated with A6H up-regulated AP-1 binding proteins reactive with a proximal AP-1 binding site in the human IFN-gamma promoter and a consensus AP-1 binding site. Moreover, preincubation of the T cells with the specific p38 MAPK inhibitor SB203580 resulted in decreased AP-1 binding following A6H or CD28 co-stimulation. This suggests that the p38 MAPK pathway is required for induction of full AP-1 binding activity in human CD4(+) T cells co-stimulated with A6H or CD28. Blocking the p38 MAPK pathway by SB203580 completely inhibited IFN-gamma production from A6H co-stimulated T cells and radically reduced IFN-gamma production from T cells co-stimulated with anti-CD28. In contrast, no significant inhibition of IL-2 production was seen after blocking of the p38 MAPK in either A6H or CD28 co-stimulated T cells. Since the p38 MAPK recently has been shown to be critically involved in regulation of IFN-gamma production from T(h)1 cells, we propose that A6H co-stimulation induces a specific pathway, mediated via p38 and AP-1 activation, for induction of a T(h)1 profile in human CD4(+) T cells.

Cutting edge: TCR stimulation by antibody and bacterial superantigen induces Stat3 activation in human T cells.

Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.

Rapid tyrosine phosphorylation of Lck following ligation of the tumor-associated cell surface molecule A6H.

We have recently described the A6H antigen as a novel 120-140 kDa molecule which is co-expressed on human peripheral blood T cells and renal cell carcinoma cells. Engagement of the A6H antigen results in co-stimulation of CD4+ T cells but it remained unknown how cross-talk between the A6H antigen and the TCR-CD3 complex takes place and which signaling pathway might be involved. Here we show that ligation of the A6H antigen with mAb induces tyrosine phosphorylation of the Lck protein tyrosine kinase (PTK). Co-ligation of the A6H antigen with CD3 resulted in augmented Lck phosphorylation and mitogenesis. In addition, A6H ligation induced an up-regulation of CD3-mediated phosphorylation of the 23 kDa high mol. wt form of TCR zeta and the zeta-associated protein, ZAP-70. Co-precipitation of Lck and ZAP-70 was only seen in T cells activated by combined A6H and anti-CD3 stimulation. In contrast, another Src family PTK, Fyn, was not affected by A6H ligation. In conclusion, we now demonstrate, for the first time, that A6H ligation triggers Lck phosphorylation, and that cross-talk between A6H and the TCR-CD3 complex involves Lck, ZAP-70 and the slow migrating isoform of TCR zeta. These results further suggests that A6H ligation is sufficient for triggering some of the early events in T cell activation, whereas full activation of the T cell, characterized by proliferation and cytokine production, requires co-ligation of the TCR-CD3 complex.

ICAM-1 costimulation induces IL-2 but inhibits IL-10 production in superantigen-activated human CD4+ T cells.

We have previously reported that costimulatory pathways including B7-CD28 and lymphocyte function-associated antigen-3 (LFA-3)-CD2 shape distinct activation profiles in human CD4+ T cells. We now show that superantigen (SAg), in combination with intracellular adhesion molecule-1 (ICAM-1) costimulation drives a proliferative response accompanied by high levels of interleukin-2 (IL-2) and moderate levels of interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF). This response profile differs from that observed in B7 or LFA-3 costimulated T cells because our previous results showed that B7-CD28 costimulation was accompanied by high levels of IL-2, IFN-gamma and TNF, whereas LFA-3 was a potent inducer of IFN-gamma and TNF, but had little influence on IL-2 production. The ICAM-1-induced IL-2 production could efficiently be abrogated with monoclonal antibody (mAb) against ICAM-1 or LFA-1, showing that the activation is dependent of a functional ICAM-1-LFA-1 pathway. SAg-induced IL-2, IFN-gamma and TNF were detected in both CD4+ and CD8+ T cells, whereas production of IL-10 was restricted to CD4+ T cells. A major finding in the present study was that ICAM-1 costimulation strongly inhibits IL-10 production in CD4+ T cells. Our data demonstrate that ICAM-1 costimulation is sufficient to induce large amounts of IL-2. The presence of ICAM-1 results in suppression of IL-10 production in T helper (Th) cells, which may favour the development of Th1 and not Th2 T cells.

The tumour associated cell surface antigen A6H is costimulatory for human CD4+ but not CD8+ T cells.

The A6H monoclonal antibody (mAb) recognizes a 120,000-140,000 MW antigen that is expressed at similar densities on 85-90% of human CD4+ and CD8+ T cells and on renal cell carcinomas. The binding of the A6H mAb induced a costimulatory signal in anti-CD3 activated T cells. In the present report, we show that A6H costimulated cell proliferation and cytokine production in purified CD4+ T cells. Unexpectedly, the CD8+ T-cell subpopulation failed to respond. CD4+ T cells costimulated with the A6H mAb upregulated CD80, CD86, CD71, interleukin-2 (IL-2)R alpha, IL-2R beta and IL-2R gamma, while no corresponding up-regulation of these cell surface molecules was seen in CD8+ T cells. In order to investigate the nature of the A6H mAb costimulus at the transcriptional level we have examined induction of the transcription factors OCT-1, AP-1 and NF-kappa B which are known to be transcriptional regulators of several cytokine and cytokine receptor genes, including the IL-2 and IL-2R genes. Co-ligation of the A6H antigen and the CD3 complex induced expression of the transcription factor AP-1 in CD4+ T cells, whereas no increase in NF-kappa B and octamer-binding (Oct) proteins was seen compared to T cells stimulated with anti-CD3 alone. Furthermore, no induction of AP-1 was seen in A6H costimulated CD8+ T cells. These results suggests that both proximal steps in CD8+ T-cell activation as well as the later phases are unresponsive to A6H ligation. Molecular differences of the A6H molecule or distinct regulation of the A6H transduced AP-1 activation pathway may exist in CD4+ and CD8+ T cell subpopulations.

Fusion of a signal sequence to the interleukin-1 beta gene directs the protein from cytoplasmic accumulation to extracellular release.

Interleukin (IL)-1 differs from most other cytokines by the lack of a signal sequence, which results in the retention of the immature proform intracellularly (i.c.). Several cell types have the capacity to produce IL-1, but release has been shown to be restricted predominantly to monocytes/macrophages and associated with apoptosis of the producer cell. These features have limited the studies on IL-1 in early T cell-APC interactions. To develop a model for studying the biological effects of IL-1 beta release during long-lasting immune responses, we have established cells transfected with IL-1 beta cDNA constructs. To construct a hybrid gene for IL-1 beta release, the signal sequence from the related IL-1 receptor antagonist was fused to the gene encoding the 17-kDa mature form of IL-1 beta. A murine fibroblast cell line was transduced with retroviral technique and analyzed for the expression of human IL-1 beta, with or without a signal sequence (ssIL-1 beta and IL-1 beta, respectively). The fibroblasts transduced with either IL-1 beta or ssIL-1 beta expressed similar levels of human IL-1 beta mRNA. High levels of IL-1 bioactivity were recorded in freeze-thaw extracts from cells expressing the IL-1 beta protein i.c., and in supernatants of ssIL-1 beta-transduced cells, which indicates that the initial formation of a proform of IL-1 beta is not required for correct folding of the protein. Treatment of ssIL-1 beta-transduced cells with Brefeldin A (BFA), an inhibitor of protein transport in the endoplasmatic reticulum, induced accumulation of the protein i.c. BFA treatment did not affect IL-1 beta-transduced cells, while lipopolysaccharide-activated human monocytes increased the secretion of IL-1 beta. Cytoplasmic staining of single cells demonstrated that expression of the ssIL-1 beta gene directed the protein to a perinuclear Golgi-like compartment, whereas cells transduced with IL-1 beta cDNA showed a diffuse cytoplasmic distribution pattern. Secretion of IL-1 beta from human monocytes was under certain conditions accompanied by cell death. In contrast, in the fibroblast cell line transduced to secrete IL-1 beta, no accompanying cell death could be detected. Gene targeting of IL-1 to the secretory or cytoplasmic pathway may be useful for elucidating the role of IL-1 in T cell-APC interactions, avoiding cell death of the producer cells.

A novel co-stimulatory T cell antigen co-expressed on renal cell carcinoma.

The A6H mAb raised primarily against human renal cell carcinoma (RCC) has previously been shown to bind strongly to RCC, to some degree to colon carcinoma but only marginally to a variety of normal tissues. Immunohistochemical analysis or RCC tissues containing tumor-infiltrating lymphocytes revealed that A6H stained both tumor cells and lymphocytes. FACS analysis of human peripheral blood cells demonstrated that A6H stained both tumor cells and lymphocytes. FACS analysis of human peripheral blood cells demonstrated that A6H mAb stained 85-90% of both CD4+ and CD8+ T cells, but not granulocytes, monocytes, NK cells or B cells. Furthermore, 85-90% of naive and memory T helper cells were stained with A6H suggesting that the A6H mAb defines unique subsets within these T cell populations. Dual staining showed that A6H mAb bind to an antigen that is clearly distinct from other cell surface molecules on T cells, including CD28, CD29, CD26, CD44 and ICAM-2. A6H mAb binding induced a second signal in anti-CD3 mAb activated T cells, resulting in cell proliferation, IL-2 receptor expression and vigorous production of IFN-gamma and TNF, and production of minor amounts of IL-2. Immunoprecipitation with A6H mAb indicated a molecular weight of 120-140 kDa on both T cells and RCC. We suggest that the A6H mAb defines a unique T cell surface antigen which is involved in signal transduction and is expressed on subsets of human T cells. The co-expression of A6H on T cells and tumor cells suggests a possible function related to common properties of these cells.