Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons. Consequently, Pin1-deficient cells and mice failed to mount TLR-mediated, interferon-dependent innate and adaptive immune responses. Given the critical role of aberrant activation of IRAK1 and type I interferons in various immune diseases, controlling IRAK1 activation via inhibition of Pin1 may represent a useful therapeutic approach.

Defective NKT cell activation by CD1d+ TRAMP prostate tumor cells is corrected by interleukin-12 with α-galactosylceramide.

Numerical and functional defects of invariant natural killer T cells (iNKT) have been documented in human and mouse cancers, resulting in a defect in IFN production in several malignancies. iNKT cells recognize glycolipids presented on CD1d molecules by dendritic and related cells, leading to their activation and thereby regulating immune reactions. Activated iNKT cells cytokine secretion and cytotoxicity can inhibit existing and spontaneous tumor growth, progression, and metastasis. We have identified functional iNKT cell defects in the murine TRAMP prostate cancer model. We found that iNKT cells show the ability to migrate into TRAMP prostate tumors. This infiltration was mediated through CCL2: CCR5 chemokine: receptor interaction. Prostate tumor cells expressing CD1d partially activated iNKT cells, as appreciated by up-regulation of CD25, PD-1 and the IL-12R. However, despite inducing up-regulation of these activation markers and, hence, delivering positive signals, prostate tumor cells inhibited the IL-12-induced STAT4 phosphorylation in a cell-cell contact dependent but CD1d-independent manner. Consequently, tumor cells did not induce secretion of IFNgamma by iNKT cells. Blocking the inhibitory Ly49 receptor on iNKT cells in the presence of alpha-GalCer restored their IFNgamma production in vivo and in vitro. However, Ly49 blockade alone was not sufficient. Importantly, this defect could be also be reversed into vigorous secretion of IFNgamma by the addition of both IL-12 and the exogenous CD1d ligand alpha-galactosylceramide, but not by IL-12 alone, both in vivo and in vitro. These data underscore the potential to optimize iNKT-based therapeutic approaches.

Essential role of Pin1 in the regulation of TRF1 stability and telomere maintenance.

Telomeres are essential for maintaining cellular proliferative capacity and their loss has been implicated in ageing. A key regulator in telomere maintenance is the telomeric protein TRF1, which was also identified as Pin2 in a screen for Pin1. Pin1 is a unique prolyl isomerase that regulates protein conformation and function after phosphorylation. However, little is known about the role of Pin1 in telomere regulation or the modulation of TRF1 by upstream signals. Here we identify TRF1 as a major conserved substrate for Pin1 during telomere maintenance and ageing. Pin1 inhibition renders TRF1 resistant to protein degradation, enhances TRF1 binding to telomeres, and leads to gradual telomere loss in human cells and in mice. Pin1-deficient mice also show widespread premature ageing phenotypes within just one generation, similar to those in telomerase-deficient mice after 4-5 consecutive generations. Thus, Pin1 is an essential regulator of TRF1 stability, telomere maintenance and ageing.

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha.

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.

Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.

Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.

Negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase Pin1.

Recognition of double-stranded RNA activates interferon-regulatory factor 3 (IRF3)-dependent expression of antiviral factors. Although the molecular mechanisms underlying the activation of IRF3 have been studied, the mechanisms by which IRF3 activity is reduced have not. Here we report that activation of IRF3 is negatively regulated by the peptidyl-prolyl isomerase Pin1. After stimulation by double-stranded RNA, induced phosphorylation of the Ser339-Pro340 motif of IRF3 led to its interaction with Pin1 and finally polyubiquitination and then proteasome-dependent degradation of IRF3. Suppression of Pin1 by RNA interference or genetic deletion resulted in enhanced IRF-3-dependent production of interferon-beta, with consequent reduction of virus replication. These results elucidate a previously unknown mechanism for controlling innate antiviral responses by negatively regulating IRF3 activity via Pin1.

Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production.

Psoriasis is one of the most common T cell-mediated autoimmune diseases in humans. Although a role for the innate immune system in driving the autoimmune T cell cascade has been proposed, its nature remains elusive. We show that plasmacytoid predendritic cells (PDCs), the natural interferon (IFN)-alpha-producing cells, infiltrate the skin of psoriatic patients and become activated to produce IFN-alpha early during disease formation. In a xenograft model of human psoriasis, we demonstrate that blocking IFN-alpha signaling or inhibiting the ability of PDCs to produce IFN-alpha prevented the T cell-dependent development of psoriasis. Furthermore, IFN-alpha reconstitution experiments demonstrated that PDC-derived IFN-alpha is essential to drive the development of psoriasis in vivo. These findings uncover a novel innate immune pathway for triggering a common human autoimmune disease and suggest that PDCs and PDC-derived IFN-alpha represent potential early targets for the treatment of psoriasis.

Metastatic melanoma secreted IL-10 down-regulates CD1 molecules on dendritic cells in metastatic tumor lesions.

CD1 molecules are expressed by antigen-presenting cells such as dendritic cells and mediate primary immune responses to lipids and glycolipids which have been shown to be expressed by various tumors. Glycolipids are expressed by melanoma cells but, despite their immunogenicity, no efficient spontaneous immune responses are elicited. As IL-10 has previously been shown to down-regulate CD1a on dendritic cells and is known to be expressed by various melanoma cell lines, we investigated if melanoma-derived IL-10 could down-regulate CD1 molecule expression on dendritic cells as a possible way to circumvent immune recognition. We found that CD1a, CD1b, CD1c, and CD1d were significantly down-regulated on dendritic cells in metastatic (n = 10) but not in primary melanoma lesions (n = 10). We further detected significantly higher IL-10 protein levels in metastatic than in primary melanomas. Moreover, supernatants from metastatic melanomas were significantly more effective in down-regulating CD1 molecules on dendritic cells than supernatants from primary melanoma cultures. This effect was blocked using a neutralizing IL-10 antibody in a dose dependent manner. Our findings suggest that metastatic but not primary melanomas can down-regulate CD1 molecules on infiltrating dendritic cells by secreting IL-10 which may represent a novel way to escape the immune response directed against the tumor.

Vaccination of patients with cutaneous T-cell lymphoma using intranodal injection of autologous tumor-lysate-pulsed dendritic cells.

Cutaneous T-cell lymphoma (CTCL) is a lymphoproliferative skin disease with limited therapeutic options. Ten CTCL patients were treated with once-weekly intranodal injection of 1 x 106 mature monocyte-derived dendritic cells (DCs) pulsed with 100 microg/mL tumor lysate protein equivalent and keyhole limpet hemocyanin (50 microg/mL). Tumor-specific delayed-type hypersensitivity (DTH) reactions developed in 8 of 8 patients challenged with tumor-lysate-pulsed DCs and in 3 of 8 patients challenged with tumor lysate alone. Three of 5 patients showed significant tumor-lysate-specific increases of in vitro peripheral blood lymphocyte proliferation coinciding with increased interferon-alpha (IFN-alpha) production. Five of 10 (50%) patients had objective responses. Four patients had partial responses (PRs). Two are still in PR, and the other 2 patients had a mean PR duration of 10.5 months. One patient had a complete response (CR) for 19 months that is ongoing. The remaining 5 patients had progressive disease. In the 5 responder patients, 6.8 +/- 1.4 vaccinations were necessary to induce an objective clinical response. Response was associated with low tumor burden. Continuation of vaccinations with new tumor lysate derived from progressive lesions reinduced treatment responses in 2 patients in PR. Selected patients had massive infiltration of CD8+ and TIA+ cytotoxic T cells at the site of regressing lesions and molecular remission after therapy. Intranodal injection of autologous tumor-lysate-pulsed DCs is well-tolerated and achieves immunologic and objective clinical responses in selected CTCL patients.

Functional expression of the eotaxin receptor CCR3 in CD30+ cutaneous T-cell lymphoma.

Little is known about mechanisms involved in skin-specific homing of cutaneous T-cell lymphoma (CTCL). Chemokine/chemokine receptor interactions have been implicated in the homing of lymphoma cells to various tissue sites. We investigated tissue samples and tumor cell suspensions of patients with CD30(+) CTCL (n = 8) and CD30(-) CTCL (mycosis fungoides, n = 6; Sézary syndrome, n = 6) for expression of the chemokine receptors CCR3, CCR4, and CCR8 and the CCR3 ligands eotaxin/CCL11, monocyte chemoattractant protein 3 (MCP-3)/CCL7, and RANTES (regulated on activation, normal T expressed and secreted)/CCL5. Of 8 CD30(+) CTCLs, 7 expressed CCR3, 4 CCR4, and none CCR8. CCR3 expression was not found in skin tissue samples from 12 CD30(-) CTCLs. Coexpression of CCR3 and CD30 was demonstrated by flow cytometry in tumor cell suspensions. Internalization experiments demonstrated functionality of CCR3 expressed by freshly isolated tumor cells. Actin polymerization as well as migration in response to eotaxin was demonstrated in a CD30(+) cutaneous lymphoma cell line. CCR3 ligand eotaxin/CCL11 was detected in lesional skin of CD30(+) CTCL by immunohistochemistry, preferentially in tumor cells. Eotaxin/CCL11 expression in tumor cells was confirmed by intracellular immunofluorescence. Analysis of cytokine expression pattern of CCR3-bearing infiltrating cells showed a predominance of interleukin-4 (IL-4) but not interferon-gamma (IFN-gamma) protein expression,1 consistent with a T-helper 2 (Th-2) profile. These results suggest that expression of CCR3 and its ligand eotaxin/CCL11 plays a role in the recruitment and retention of CD30(+) malignant T cells to the skin.

Expression of CXC chemokine receptor-4 enhances the pulmonary metastatic potential of murine B16 melanoma cells.

The chemokine receptors CC chemokine receptor (CCR) 7 and CXC chemokine receptor (CXCR) 4 have been implicated in cancer metastasis. To evaluate whether CXCR4 is sufficient to increase tumor metastasis in an organ-specific manner, we transduced murine B16 melanoma cells with CXCR4 (CXCR4-B16) and followed the metastatic fate of the transduced cells in both i.v. and s.c. inoculation models of metastasis. CXCR4-B16 cells demonstrated marked increases (>10-fold) in pulmonary metastasis compared with vector (pLNCX2)-B16 after i.v. and s.c. inoculation of tumor cells. The increase in metastasis could be completely inhibited by T22, a small peptide antagonist of CXCR4. As early as 24 and 48 h after i.v. injection, CXCR4-B16 cells were significantly increased in the lung compared with control B16 cells by 5- and 10-fold (P < 0.05), respectively. CXCR4-B16 cells adhered better to both dermal and pulmonary microvascular endothelial cells relative to control B16 cells. Moreover, CXCL12 promoted the growth of CXCR4-B16 cells in vitro. Whereas expression of CXCR4 in B16 cells dramatically enhanced pulmonary metastasis, metastasis to the lymph nodes, liver, and kidney was rare. Immunohistochemical staining of both primary human cutaneous melanoma and pulmonary metastases revealed CXCR4 expression. Thus, CXCR4 plays a potentially important role in promoting organ-selective metastasis, possibly by stimulating tumor adhesion to microvascular endothelial cells and by enhancing the growth of tumor cells under stress.

Functional detection of epithelial cell adhesion molecule specific cytotoxic T lymphocytes in patients with lung cancer, colorectal cancer and in healthy donors.

Epithelial cell adhesion molecule (Ep-CAM) derived antigenic peptides have been identified that can be recognized by cytotoxic T lymphocytes (CTL) in a major histocompatibility complex (MHC) class I restricted fashion. Thus, altered expression of Ep-CAM in a variety of human tumors might render a potential target for T cell mediated therapy. We have examined, whether the novel HLA-A*0201 restricted peptide ILYENNVIT (184-192) corresponding to Ep-CAM and one heteroclitic modified variant peptide previously demonstrated to be immunogenic in the human system can elicit antigen specific CTL responses in HLA-A2 positive patients with history of Ep-CAM expressing cancer of lung and colon. Specific CTL recognition of T2 target cells pulsed with the native peptide as well as of the lung cancer cell line A549 indicates that an appropriate T cell repertoire can be expanded from peripheral blood from patients in clinical remission and with advanced cancer. Despite an overall low frequency, peptide specific precursor CTLs could be readily expanded from peripheral blood from 6/8 patients that were diagnosed previously with Ep-CAM expressing lung cancer and 4/8 control individuals (2/5 healthy donors and 2/3 colon cancer patients). CTLs from three of five lung cancer patients tested also lyzed the HLA-A2(+) and Ep-CAM expressing lung cancer cell line A549. We did not detect an increased frequency of pCTLs after peripheral blood monocytes (PBMCs) were stimulated with the heteroclitic compound peptide. The results of our study indicate that Ep-CAM specific precursor CTL can be expanded in vitro and a specific T cell response against this epitope can be elicited in patients at various stages of lung cancer.