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BACKGROUND - Mycosis fungoides (MF) is a rare, but potentially devastating malignancy. It classically presents with cutaneous patches and plaques and can progress to tumours on the skin with lymph node, blood and visceral involvement. While most patients with MF have a relatively benign disease course, a subset of patients will develop progressive disease that is often fatal.
OBJECTIVE - The aim of this study was to identify genetic markers in early MF limited to the skin (stages IA-IIA) that distinguish those patients who will have progressive disease from those who will not, so that early appropriate treatment may be instituted.
METHODS - The study includes 18 patients who were diagnosed with early stage MF at the time of biopsy and had follow-up to determine which patients developed progressive disease. RNA was extracted from skin biopsy specimens and analysed for expression of CD3, FOXP3, IFNγ, Interleukin (IL)-4, IL-13, KIR3DL2, MICB, PLS3 and STAT4 by quantitative real-time polymerase chain reaction.
RESULTS/CONCLUSIONS - Reduced expression of FOXP3 and STAT4 and increased expression of IL-4 relative to CD3 expression levels were significantly associated with MF progression. Further studies will be needed to fully assess the usefulness of these genetic markers to predict disease progression and guide treatment options in patients diagnosed with early MF.
© 2013 European Academy of Dermatology and Venereology.
The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect, mammalian, and human cells6 including primary human T cells. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency. Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized. Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry. PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb). Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.
Castleman disease is a rare hematologic disorder, closely linked to the HHV-8, and most commonly observed in immunocompromised individuals. Thirteen months following a liver transplant for CPS-1 defect, a 15-month-old boy presented with fevers, anemia, and growth retardation. Abdominal CT scan showed splenomegaly and generalized lymphadenopathy. Histology of chest wall lymph nodes revealed a mixed CD3+ T-cell and CD20+ B-cell population with atretic germinal centers consistent with multicentric Castleman disease. Qualitative DNA PCR detected HHV-8 in the resected lymph node and in the blood, supporting the diagnosis. Immunosuppression was tapered, and he was transitioned from tacrolimus to sirolimus. His graft function remained stable, and repeat imaging showed regression of the lymphadenopathy. The child is living one yr after Castleman disease diagnosis with a well-functioning graft. Castleman disease is a potential complication of solid organ transplant and HHV-8 infection. Reduction in immunosuppression and switch to sirolimus may be an effective strategy to treat this condition.
© 2011 John Wiley & Sons A/S.
Tetrahydrobiopterin (BH(4)) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O(2)(·-)) rather than NO. The rate-limiting enzyme for BH(4) production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH(4) levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH(4). Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH(4) levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH(4). Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH(4) accumulation, reduced NO production, and increased T cell O(2)(·-) production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH(2) polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH(4) in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH(4) levels, NO production, and modulating T cell cytokine production.
The immune system is complex, with multiple layers of regulation that serve to prevent the production of self-antigens. One layer of regulation involves regulatory T cells (Tregs) that play an essential role in maintaining peripheral self-tolerance. Patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis have decreased levels of HDL, suggesting that apoA-I concentrations may be important in preventing autoimmunity and the loss of self-tolerance. In published studies, hypercholesterolemic mice lacking HDL apoA-I or LDLr(-/-), apoA-I(-/-) (DKO), exhibit characteristics of autoimmunity in response to an atherogenic diet. This phenotype is characterized by enlarged cholesterol-enriched lymph nodes (LNs), as well as increased T cell activation, proliferation, and the production of autoantibodies in plasma. In this study, we investigated whether treatment of mice with lipid-free apoA-I could attenuate the autoimmune phenotype. To do this, DKO mice were first fed an atherogenic diet containing 0.1% cholesterol, 10% fat for 6 weeks, after which treatment with apoA-I was begun. Subcutaneous injections of 500 μg of lipid-free apoA-I was administered every 48 h during the treatment phase. These and control mice were maintained for an additional 6 weeks on the diet. At the end of the 12-week study, DKO mice showed decreased numbers of LN immune cells, whereas Tregs were proportionately increased. Accompanying this increase in Tregs was a decrease in the percentage of effector/effector memory T cells. Furthermore, lipid accumulation in LN and skin was reduced. These results suggest that treatment with apoA-I reduces inflammation in DKO mice by augmenting the effectiveness of the LN Treg response.
Macrophages and T-lymphocytes are known to accumulate in the white adipose tissue (WAT) of obese mice and humans, but the factors that cause this infiltration are not yet determined. Chemokines, which attract leukocytes to inflammatory sites, are candidates for this process. Macrophage inflammatory protein-1alpha (MIP-1alpha) expression is significantly elevated in WAT of obese mice and humans and positively correlates with fasting plasma insulin, but its potential role in leukocyte recruitment to WAT is unknown. MIP-1alpha-deficient, heterozygous, and wild-type mice were fed a Western diet (WD) for 16 wk. Plasma lipids, adipose tissue mass, energy expenditure, food intake, liver triglyceride content, and inflammatory cytokine expression were not different among genotypes. Gene expression of macrophage markers F4/80 and CD68, as well as T-lymphocyte marker CD3epsilon was increased in perigonadal WAT of obese WD-fed mice but was not influenced by MIP-1alpha expression level. Immunohistochemical analysis of WAT also showed no effect of MIP-1alpha on macrophage content. Two related chemokines, MIP-1beta and RANTES, had reduced expression in obese male MIP-1alpha-deficient mice compared with wild-type controls (P < or = 0.05). In mice fed the WD for 6 wk, WAT macrophage content was unchanged; however, CD8+ T-lymphocytes accumulated to a lesser extent in the MIP-1alpha-null mice. Therefore, expression of MIP-1alpha, as well as that of MIP-1beta and RANTES, increases as a consequence of weight gain, but these chemokines may not be required for the recruitment of monocytes to WAT during diet-induced obesity in mice and may impact T-lymphocyte recruitment only at early time points after WD feeding.
Antigen-presenting cells (APCs) control T-cell responses by multiple mechanisms, including the expression of co-stimulatory molecules and the production of cytokines and other mediators that control T-cell proliferation, survival and differentiation. Here, we demonstrate that soluble factor(s) produced by Toll-like receptor (TLR)-activated APCs suppress activation-induced cell death (AICD). This effect was observed in non-stimulated APCs, but it was significantly increased after lipopolysaccharide (LPS) treatment. Using different KO mice, we found that the LPS-induced protective factor is dependent on TLR4/MyD88. We identified the protective factor as prostaglandin E(2) (PGE(2)) and showed that both APC-derived supernatants and PGE(2) prevented CD95L upregulation in T cells in response to TCR/CD3 stimulation, thereby avoiding both AICD and activated T cell killing of target macrophages. The PGE(2) receptors, EP2 and EP4, appear to be involved since pharmacological stimulation of these receptors mimics the protective effect on T cells and their respective antagonists interfere with the protection induced by either APCs derived or synthetic PGE(2). Finally, the engagement of EP2 and EP4 synergistically activates protein kinase A (PKA) and exchange protein directly activated by cAMP pathways to prevent AICD. Taken together, these results indicate that APCs can regulate T-cell levels of CD95L by releasing PGE(2) in response to LPS through a TLR4/MyD88-dependent pathway, with consequences for both T cell and their own survival.
The T cell antigen receptor (TCR)-CD3 complex is unique in having ten cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). The physiological importance of this high TCR ITAM number is unclear. Here we generated 25 groups of mice expressing various combinations of wild-type and mutant ITAMs in TCR-CD3 complexes. Mice with fewer than seven wild-type CD3 ITAMs developed a lethal, multiorgan autoimmune disease caused by a breakdown in central rather than peripheral tolerance. Although there was a linear correlation between the number of wild-type CD3 ITAMs and T cell proliferation, cytokine production was unaffected by ITAM number. Thus, high ITAM number provides scalable signaling that can modulate proliferation yet ensure effective negative selection and prevention of autoimmunity.
Infection with HIV-1 perturbs homeostasis of human T cell subsets, leading to accelerated immunologic deterioration. While studying changes in CD4(+) memory and naïve T cells during HIV-1 infection, we found that a subset of CD4(+) effector memory T cells that are CCR7(-)CD45RO(-)CD45RA(+) (referred to as TEMRA cells), was significantly increased in some HIV-infected individuals. This T cell subset displayed a differentiated phenotype and skewed Th1-type cytokine production. Despite expressing high levels of CCR5, TEMRA cells were strikingly resistant to infection with CCR5 (R5)-tropic HIV-1, but remained highly susceptible to CXCR4 (X4)-tropic HIV-1. The resistance of TEMRA cells to R5-tropic viruses was determined to be post-entry of the virus and prior to early viral reverse transcription, suggesting a block at the uncoating stage. Remarkably, in a subset of the HIV-infected individuals, the relatively high proportion of TEMRA cells within effector T cells strongly correlated with higher CD4(+) T cell numbers. These data provide compelling evidence for selection of an HIV-1-resistant CD4(+) T cell population during the course of HIV-1 infection. Determining the host factors within TEMRA cells that restrict R5-tropic viruses and endow HIV-1-specific CD4(+) T cells with this ability may result in novel therapeutic strategies against HIV-1 infection.
The homing and differentiation mechanisms of endothelial progenitor cells (EPCs) at sites of vascular lesions are unclear. To investigate whether platelets play a role in the recruitment and differentiation of EPCs, we made use of a robust mouse embryonic EPC (eEPC) line that reliably differentiates to a mature endothelial phenotype. We found that platelets stimulate chemotaxis and migration of these murine eEPCs. Further, the substantial adhesion of murine eEPCs on immobilized platelets that occurs under dynamic flow conditions is inhibited by neutralizing anti-P-selectin glycoprotein ligand-1 and anti-VLA-4 (beta1-integrin) monoclonal antibodies but not by anti-CD11b (aM-integrin; macrophage antigen-1). Coincubation of murine eEPCs with platelets for 5 days induced differentiation of EPCs to mature endothelial cells as verified by positive von Willebrand factor immunofluorescence and detection of Weibel Palade bodies through electron microscopy. We conclude that platelets may play a critical part in the capture and subsequent differentiation of murine eEPCs at sites of vascular lesions, revealing a possible new role of platelets in neoendothelization after vascular injury.