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Purpose: This study addresses the sensitivity of different peripheral CD4 + T-lymphocyte subsets to irradiation (IR) and identifies potential targets for the prevention or treatment of radiation-induced toxicity., Methods: This study was performed on peripheral blood mononuclear cells or sorted peripheral memory lymphocytes of CCR6 + mucosa-homing Th17/CCR6 neg Th and regulatory T subtypes of healthy volunteers. Cells were irradiated with a 2 Gy with or without pharmacologic inhibitors of different signaling pathways. Senescence of irradiated cells was assessed by resistance to apoptosis and determination of various senescence-associated biomarkers (senescence associated b-galactosidase activity, p16Ink4a-, p21Cdkn1a-, gH2A.X-, H2A.J expression). Cytokine production was measured in supernatants of irradiated cells by Luminex technology., Results: Not all CD4 + memory T lymphocyte subsets were equally radiosensitive. High sensitivity of CCR6 + Th17 lymphocytes to IR-induced senescence was shown by expression of the histone variant H2A.J, higher SA-b-Gal activity, and upregulation of p16 Ink4a and p21 Cdkn1a expression. Lower Annexin V staining and cleaved caspase-3, and higher expression of antiapoptotic genes Bcl-2 and Bcl-xL LF, showed that CCR6 + Th17 lymphocytes were more resistant to IR-induced apoptosis than CCR6 neg memory Th and regulatory T lymphocytes. After a 2 Gy IR, both CCR6 + Th17 and CCR6 neg cells acquired a moderate senescence-associated secretory phenotype, but only CCR6 + Th17 cells secreted interleukin 8 (IL-8) and vascular endothelial growth factor-A (VEGF-A). Pharmacologic targeting of reactive oxygen species (ROS), mitogen-activated protein kinases (MAPKs), and mammalian target of rapamycin (mTOR) signaling pathways prevented the expression of senescent markers and IL-8 and VEGF-A expression by CCR6 + Th17 cells after IR., Conclusions: This study suggests that IR induces senescence of CCR6+Th17 lymphocytes associated with secretion of IL-8 and VEGF-A that may be detrimental to the irradiated tissue. ROS-MAPKs signaling pathways are candidate targets to prevent this CCR6 + Th17-dependent radiation-induced potential toxicity. Finally, the ratio of circulating H2A.J + senescent CCR6 + Th17/CD4 + T lymphocytes may be a candidate marker of individual intrinsic radiosensitivity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Chronic exposure to environmental pollutants is often associated with systemic inflammation. As such, cigarette smoking contributes to inflammation and lung diseases by inducing senescence of pulmonary cells such as pneumocytes, fibroblasts, and endothelial cells. Yet, how smoking worsens evolution of chronic inflammatory disorders associated with Th17 lymphocytes, such as rheumatoid arthritis, psoriasis, Crohn's disease, and multiple sclerosis, is largely unknown. Results from human studies show an increase in inflammatory CD4 + Th17 lymphocytes at blood- and pulmonary level in smokers. The aim of the study was to evaluate the sensitivity of CD4 + Th17 lymphocytes to cigarette smoke-induced senescence. Mucosa-homing CCR6 + Th17- were compared to CCR6 neg -and regulatory T peripheral lymphocytes after exposure to cigarette smoke extract (CSE). Senescence sensitivity of CSE-exposed cells was assessed by determination of various senescence biomarkers (β-galactosidase activity, p16 Ink4a - and p21 expression) and cytokines production. CCR6 + Th17 cells showed a higher sensitivity to CSE-induced senescence compared to controls, which is associated to oxidative stress and higher VEGFα secretion. Pharmacological targeting of ROS- and ERK1/2 signalling pathways prevented CSE-induced senescence of CCR6 + Th17 lymphocytes as well as VEGFα secretion. Altogether, these results identify mechanisms by which pro-oxidant environmental pollutants contribute to pro-angiogenic and pathogenic CCR6 + Th17 cells, therefore potential targets for therapeutic purposes.

Ionizing radiation-exposure induces a variety of cellular reactions, such as senescence and apoptosis. Senescence is a permanent arrest state of the cell division, which can be beneficial or detrimental for normal tissue via an inflammatory response and senescence-associated secretion phenotype. Damage to healthy cells and their microenvironment is considered as an important source of early and late complications with an increased risk of morbidity in patients after radiotherapy (RT). In addition, the benefit/risk ratio may depend on the radiation technique/dose used for cancer eradication and the irradiated volume of healthy tissues. For radiation-induced fibrosis risk, the knowledge of mechanisms and potential prevention has become a crucial point to determining radiation parameters and patients' intrinsic radiosensitivity. This review summarizes our understanding of ionizing radiation-induced senescent cell in fibrogenesis. This mechanism may provide new insights for therapeutic modalities for better risk/benefit ratios after RT in the new era of personalized treatments., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Early diagnosis and prognosis monitoring for Stevens-Johnson syndrome/toxic epidermal necrolysis (TEN) still remain a challenge. This study aims to explore any cytokine/chemokine with prognostic potential in Stevens-Johnson syndrome/TEN. Through screening a panel of 28 serological factors, IL-6, IL-8, IL-15, tumor necrosis factor-α, and granulysin were upregulated in patients with Stevens-Johnson syndrome/TEN and selected for the further validation in total 155 patients with Stevens-Johnson syndrome/TEN, including 77 from Taiwan and 78 from the Registry of Severe Cutaneous Adverse Reactions. Among these factors evaluated, the levels of IL-15 (r = 0.401; P < 0.001) and granulysin (r = 0.223; P = 0.026) were significantly correlated with the disease severity in 112 samples after excluding patients with insufficient data to calculate the score of TEN. In addition, IL-15 was also associated with mortality (P = 0.002; odds ratio, 1.09; 95% confidence interval, 1.03-1.14; P = 0.001; adjusted odds ratio, 1.10; 95% confidence interval, 1.04-1.16). Consistent results were obtained after the exclusion of Taiwanese patients with sepsis to rule out possible confounders. Moreover, IL-15 was shown to enhance cytotoxicity of cultured natural killer cells and blister cells from patients with TEN. Our findings highlight a usefulness of IL-15 in prognosis monitoring and therapeutic intervention of this devastating condition., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The transcription factor p53 is overexpressed in the lung of patients with emphysema, but it remains unclear if it has a deleterious or protective effect in disease progression. We investigated the role of p53 in the elastase-induced emphysema model and the molecular underlining mechanisms. Wild-type (WT) and p53(-/-) mice were instilled with pancreatic porcine elastase. We quantified emphysema (morphometric analysis), chemokine (C-C motif) ligand 2 (CCL2), and TNF-α in bronchoalveolar lavage (BAL) (ELISA), oxidative stress markers [heme oxygenase 1 (HO1), NAD(P)H dehydrogenase quinone 1 (NQO1), and quantitative RT-PCR], matrix metalloproteinase 12 (MMP12) expression, and macrophage apoptosis (cleaved caspase-3, immunofluorescence). p53 gene expression was up-regulated in the lung of elastase-instilled mice. p53 deletion aggravated elastase-induced emphysema severity, pulmonary inflammation (macrophage and neutrophil numbers and CCL2 and TNF-α levels in BAL), and lung oxidative stress. These findings, except for the increase in CCL2, were reproduced in WT mice transplanted with p53(-/-) bone marrow cells. The increased number of macrophages in p53(-/-) mice was not a consequence of reduced apoptosis or an excess of chemotaxis toward CCL2. Macrophage expression of MMP12 was higher in p53(-/-) mice compared with WT mice after elastase instillation. These findings provide evidence that p53(-/-) mice and WT mice grafted with p53(-/-) bone marrow cells are more prone to developing elastase-induced emphysema, supporting a protective role of p53, and more precisely p53 expressed in macrophages, against emphysema development. The pivotal role played by macrophages in this phenomenon may involve the MMP12-TNF-α pathway.

TCR-dependent and costimulation signaling, cell division, and cytokine environment are major factors driving cytokines expression induced by CD4(+) T cell activation. PEA-15 15 (Protein Enriched in Astrocyte / 15 kDa) is an adaptor protein that regulates death receptor-induced apoptosis and proliferation signaling by binding to FADD and relocating ERK1/2 to the cytosol, respectively. By using PEA-15-deficient mice, we examined the role of PEA-15 in TCR-dependent cytokine production in CD4(+) T cells. TCR-stimulated PEA-15-deficient CD4(+) T cells exhibited defective progression through the cell cycle associated with impaired expression of cyclin E and phosphoRb, two ERK1/2-dependent proteins of the cell cycle. Accordingly, expression of the division cycle-dependent cytokines IL-2 and IFNγ, a Th1 cytokine, was reduced in stimulated PEA-15-deficient CD4(+) T cells. This was associated with abnormal subcellular compartmentalization of activated ERK1/2 in PEA-15-deficient T cells. Furthermore, in vitro TCR-dependent differentiation of naive CD4(+) CD62L(+) PEA-15-deficient T cells was associated with a lower production of the Th2 cytokine, IL-4, whereas expression of the Th17-associated molecule IL4I1 was enhanced. Finally, a defective humoral response was shown in PEA-15-deficient mice in a model of red blood cell alloimmunization performed with Poly IC, a classical adjuvant of Th1 response in vivo. Collectively, our data suggest that PEA-15 contributes to the specification of the cytokine pattern of activated Th cells, thus highlighting a potential new target to interfere with T cell functional polarization and subsequent immune response.

IL4I1 (interleukin-4-induced gene 1) is a phenylalanine oxidase produced mainly by APCs of myeloid origin, and converts phenylalanine (Phe) to phenylpyruvate, hydrogen peroxide, and ammonia. We have previously shown that IL4I1 is highly expressed by tumor-associated macrophages from various human cancers and facilitates immune evasion from the cytotoxic response in a murine tumor model. Indeed, IL4I1 inhibits T-cell proliferation via hydrogen peroxide toxicity on effector/memory T cells. Here, we explored the effect of IL4I1 on naïve CD4(+) T-cell differentiation. We show that IL4I1 stimulates the generation of Foxp3(+) regulatory T (Treg) cells in vitro from human and mouse T cells. This effect was observed with IL4I1 from different sources, including the naturally produced enzyme. Conversely, IL4I1 limits Th1 and Th2 polarization while modifying the Th17 phenotype, in particular, by inducing its own production. Analysis of Treg-cell induction under conditions of Phe deprivation and hydrogen peroxide addition suggests that Phe consumption by the enzyme participates in Treg-cell enrichment. In line with this hypothesis, IL4I1 inhibits mTORC1 signaling shortly after T-cell activation. Thus, the IL4I1 enzyme may act on T cells both by direct inhibition of effector cell proliferation and by indirect immunoregulation mediated by Treg-cell induction., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Alteration of functional regenerative properties of parenchymal lung fibroblasts is widely proposed as a pathogenic mechanism for chronic obstructive pulmonary disease (COPD). However, what these functions are and how they are impaired in COPD remain poorly understood. Apart from the role of fibroblasts in producing extracellular matrix, recent studies in organs different from the lung suggest that such cells might contribute to repair processes by acting like mesenchymal stem cells. In addition, several reports sustain that the Hedgehog pathway is altered in COPD patients thus aggravating the disease. Nevertheless, whether this pathway is dysregulated in COPD fibroblasts remains unknown., Objectives and Methods: We investigated the stem cell features and the expression of Hedgehog components in human lung fibroblasts isolated from histologically-normal parenchymal tissue from 25 patients--8 non-smokers/non-COPD, 8 smokers-non COPD and 9 smokers with COPD--who were undergoing surgery for lung tumor resection., Results: We found that lung fibroblasts resemble mesenchymal stem cells in terms of cell surface marker expression, differentiation ability and immunosuppressive potential and that these properties were altered in lung fibroblasts from smokers and even more in COPD patients. Furthermore, we showed that some of these phenotypic changes can be explained by an over activation of the Hedgehog signaling in smoker and COPD fibroblasts., Conclusions: Our study reveals that lung fibroblasts possess mesenchymal stem cell-features which are impaired in COPD via the contribution of an abnormal Hedgehog signaling. These processes should constitute a novel pathomechanism accounting for disease occurrence and progression.

Primary cutaneous aggressive epidermotropic T-cell lymphoma (PCAETCL) is a very rare lymphoma characterized by rapidly growing necrotic cutaneous lesions with an epidermotropic CD8+ T-cell neoplastic infiltrate observed histopathologically. It is associated with a very poor outcome, despite aggressive multi-agent chemotherapy. We report a 49-year-old human immunodeficiency virus (HIV)-infected patient who developed PCAETCL with associated marked vascular injury leading to diffuse purpuric and necrotic lesions complicated by recalcitrant hemophagocytic activation syndrome. The lymphoma strongly and diffusely expressed CD158k/KIR3DL2 at the protein and transcript level and NKp46 transcripts, in addition to CD8 and cytotoxic proteins. We observed a diffuse CD158k/KIR3DL2 protein expression in another case of PAETCL, not associated with immunodeficiency, which was used as a positive control. PCAETCL can develop in HIV-infected patients and may present in vasculitis-like fashion. The possible role of immunosuppression and/or HIV in oncogenesis can be postulated, as patients infected with HIV may develop anti-HIV cytotoxic CD8+ lymphoproliferations. The frequency of CD158k/KIR3DL2 and NKp46 expression in PCAECL remains to be studied in a series of cases, and may represent interesting targets for future treatments., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial anion channel. Morbidity is mainly due to lung disease, which is characterized by chronic neutrophilic inflammation. Deregulation of inflammatory pathways is observed in the airways of CF patients, as evidenced by exaggerated NF-κB activity, causing an increase in the local release of pro-inflammatory cytokines such as IL-8. COMMD1, a pleiotropic protein, was recently shown to interact with CFTR and to promote CFTR cell surface expression. The effect of COMMD1 on the NF-κB pathway was assessed in CF and non-CF bronchial epithelial cells by knockdown and overexpression experiments. Results showed that (i) COMMD1 knockdown induced NF-κB-dependent transcription, (ii) COMMD1 overexpression inhibited NF-κB activity and was associated with a decrease in IL-8 transcript level and protein secretion. These data demonstrate the anti-inflammatory properties of COMMD1 in bronchial epithelial cells and open new therapeutic avenues in CF., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with lung fibroblast senescence, a process characterized by the irreversible loss of replicative capacity associated with the secretion of inflammatory mediators. However, the mechanisms of this phenomenon remain poorly defined., Objectives: The aim of this study was to analyze the role of prostaglandin E2 (PGE2), a prostaglandin known to be increased in COPD lung fibroblasts, in inducing senescence and related inflammation in vitro in lung fibroblasts and in vivo in mice., Methods: Fibroblasts were isolated from patients with COPD and from smoker and nonsmoker control subjects. Senescence markers and inflammatory mediators were investigated in fibroblasts and in mice., Measurements and Main Results: Lung fibroblasts from patients with COPD exhibited higher expression of PGE2 receptors EP2 and EP4 as compared with nonsmoker and smoker control subjects. Compared with both nonsmoker and smoker control subjects, during long-term culture, COPD fibroblasts displayed increased senescent markers (increased senescence associated-β galactosidase activity, p16, and p53 expression and lower proliferative capacity), and an increased PGE2, IL-6, IL-8, growth-regulated oncogene (GRO), CX3CL1, and matrix metalloproteinase-2 protein and cyclooxygenase-2 and mPGES-1 mRNA expression. Using in vitro pharmacologic approaches and in vivo experiments in wild-type and p53(-/-) mice we demonstrated that PGE2 produced by senescent COPD fibroblasts is responsible for the increased senescence and related inflammation. PGE2 acts either in a paracrine or autocrine fashion by a pathway involving EP2 and EP4 prostaglandin receptors, cyclooxygenase-2-dependent reactive oxygen species production and signaling, and consecutive p53 activation., Conclusions: PGE2 is a critical component of an amplifying and self-perpetuating circle inducing senescence and inflammation in COPD fibroblasts. Modulating the described PGE2 signaling pathway could provide a new basis to dampen senescence and senescence-associated inflammation in COPD.

Exposure to titanium dioxide (TiO2) nanoparticles (NPs) is associated with lung remodeling, but the underlying mechanisms are unknown. Matrix metalloprotease (MMP)-1 is an important actor in matrix homeostasis and could therefore participate in TiO2 NP effects. Our aim was to evaluate the effects of TiO2 NPs on MMP-1 expression and activity in lung pulmonary fibroblasts and to understand the underlying mechanisms and assess the importance of the physicochemical characteristics of the particles in these effects. Human pulmonary fibroblasts (MRC-5 cell line and primary cells) were exposed to 10 or 100 μg/cm(2) TiO2 (two anatases, two anatase/rutile mix, one rutile NP, and one micrometric) and carbon black (CB) NPs for 6 to 48 hours. We examined cell viability, MMP-1 expression and activity, and the implication of oxidative stress, transforming growth factor (TGF)-β, extracellular MMP inducer, and IL-1β in MMP-1 expression. All TiO2 NPs induced MMP-1 (mRNA and protein expression), repression of procollagen-1, and α-actin expression, but only the two anatase/rutile mix induced MMP-1 activity. Micrometric TiO2 had smaller effects than TiO2 NPs, and CB NPs did not induce MMP-1. MMP-1 induction by TiO2 NPs was not related to TGF-β, oxidative stress, or EMPRIN expression but was related to IL-1β expression, which partly drives MMP-1 induction by two TiO2 NPs (one anatase/rutile mix and the rutile one). Taken together, our results show that TiO2 NPs are potent inducers and regulators of MMP-1 expression and activity, partly via an IL-1β-dependent mechanism. This may explain TiO2 lung remodeling effects.

By revisiting CD90, a GPI-anchored glycoprotein, we show that CD90 is expressed by a subset of CD4(+) and CD8(+) human T cells. CD4(+)CD90(+) cells share similarities with Th17 cells because they express the Th17-specific transcription factor RORC2 and produce IL-17A. CD4(+)CD90(+) cells are activated memory T cells that express the gut mucosal markers CCR6, CD161, and the α(4) and β(7) integrins. Compared with CD90-depleted CCR6(+) memory Th17 cells, CD4(+)CD90(+) cells express higher levels of IL-22 and proinflammatory cytokines (IL-6, TNF-α and GM-CSF), but they produce lower levels of IL-21 and no IL-9. Analyses of CD8(+)CD90(+) cells reveal that they express RORC2 and are able to produce higher levels of IL-17A, IL-22, and CCL20 compared with CD90-depleted CD8(+) cells. These data show that CD90 identifies Th17 and Tc17 cells with a peculiar cytokine profile. Studies of circulating CD90(+) cells in HIV patients show that CD90(+) cells are decreased with an imbalance of the CD4(+)CD90(+)/regulatory T cell ratio in nontreated patients compared with treated patients and healthy donors. Overall, human CD90 identifies a subset of Th17 and Tc17 cells within CD4(+) and CD8(+) T cells, respectively, which are depleted during HIV infection.

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation of unknown pathogenesis., Objectives: To investigate whether telomere dysfunction and senescence of pulmonary vascular endothelial cells (P-ECs) induce inflammation in COPD., Methods: Prospective comparison of patients with COPD and age- and sex-matched control smokers. Investigation of mice null for telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes., Measurements and Main Results: In situ lung specimen studies showed a higher percentage of senescent P-ECs stained for p16 and p21 in patients with COPD than in control subjects. Cultured P-ECs from patients with COPD exhibited early replicative senescence, with decreased cell-population doublings, a higher percentage of β-galactosidase-positive cells, reduced telomerase activity, shorter telomeres, and higher p16 and p21 mRNA levels at an early cell passage compared with control subjects. Senescent P-ECs released cytokines and mediators: the levels of IL-6, IL-8, monocyte chemotactic protein (MCP)-1, Hu-GRO, and soluble intercellular adhesion molecule (sICAM)-1 were elevated in the media of P-ECs from patients compared with control subjects at an early cell passage, in proportion to the senescent P-EC increase and telomere shortening. Up-regulation of MCP-1 and sICAM-1 led to increased monocyte adherence and migration. The elevated MCP-1, IL-8, Hu-GROα, and ICAM-1 levels measured in lungs from patients compared with control subjects correlated with P-EC senescence criteria and telomere length. In Tert(-/-) and/or Terc(-/-) mouse lungs, levels of the corresponding cytokines (MCP-1, IL-8, Hu-GROα, and ICAM-1) were also altered, despite the absence of external stimuli and in proportion to telomere dysfunction., Conclusions: Telomere dysfunction and premature P-EC senescence are major processes perpetuating lung inflammation in COPD.

Nitric oxide (NO) in combination with superoxide produces peroxynitrites and induces protein nitration, which participates in a number of chronic degenerative diseases. NO is produced at high levels in the human emphysematous lung, but its role in this disease is unknown. The aim of this study was to determine whether the NO synthases contribute to the development of elastase-induced emphysema in mice. nNOS, iNOS, and eNOS were quantified and immunolocalized in the lung after a tracheal instillation of elastase in mice. To determine whether eNOS or iNOS had a role in the development of emphysema, mice bearing a germline deletion of the eNOS and iNOS genes and mice treated with a pharmacological iNOS inhibitor were exposed to elastase. Protein nitration was determined by immunofluorescence, protein oxidation was determined by ELISA. Inflammation and MMP activity were quantified by cell counts, RT-PCR and zymography in bronchoalveolar lavage fluid. Cell proliferation was determined by Ki67 immunostaining. Emphysema was quantified morphometrically. iNOS and eNOS were diffusely upregulated in the lung of elastase-treated mice and a 12-fold increase in the number of 3-nitrotyrosine-expressing cells was observed. Over 80% of these cells were alveolar type 2 cells. In elastase-instilled mice, iNOS inactivation reduced protein nitration and increased protein oxidation but had no effect on inflammation, MMP activity, cell proliferation or the subsequent development of emphysema. eNOS inactivation had no effect. In conclusion, in the elastase-injured lung, iNOS mediates protein nitration in alveolar type 2 cells and alleviates oxidative injury. Neither eNOS nor iNOS are required for the development of elastase-induced emphysema.

We have shown that ObRb, the leptin receptor, is overexpressed in colorectal cancer cells, and that this may influence the patients' outcome. We investigated colonocytes as leptin targets and characterized their pivotal role in antitumor immune response. Cytokine and chemokine mRNAs in HT29 cells were measured by targeted arrays. In vitro, normal colonocytes and human colon cancer cells (HT29, Caco-2, SW480, and HCT116) were used to investigate ObRb transduction system and cytokine releases. Animal colonocytes and CD8 splenocytes and human HT29, HCT116, and CD8(+) cells from blood donors were used to investigate the lymphocyte response to the colonocytes when stimulated by leptin. Leptin-induced cytokine releases in the normal colonic mucosa and tumor growth and cytokine releases within tumors in vivo were measured in male rats and nude mice, respectively. Statistical analysis was done by Fisher's exact and Mann-Whitney U tests. Various cytokines and their receptors were produced in normal and tumoral colonocytes in response to leptin by increasing nuclear factor-kappaB activation. Interleukin-8 (IL-8) was the main cytokine produced in vitro. The levels of IL-8 and its receptor, CXCR1, were higher in tumors than in homologous normal mucosa. Systemic leptin enhanced the proinflammatory cytokines in normal colonocytes and in HT29 xenografted tumor colonocytes. Colonocyte-derived products after leptin treatment stimulated perforin and granzyme B expressions in normal CD8(+) T cells in vitro. Leptin triggers an inflammatory response in tumor tissue by directly stimulating colonocytes, which can recruit T cytotoxic cells in the tumor microenvironment.

The incidence of colorectal cancers (CRC) may be influenced by environmental factors, including nutrition. The role of peptides regulating food intake in controlling the growth and recurrence of human tumors is controversial. Leptin, a cytokine-like peptide, regulates food intake. We investigated the expression of leptin and its receptor in 171 consecutive patients (78 female and 93 male; 71 years) with CRC. Leptin concentrations in the serum (ELISA) were determined before tumor removal. ObRb was characterized in tumors and normal homologous tissues and culture cells (HT29, HCT116, and HCT116 with a transferred chromosome 3) by using immunocytochemistry, immunohistochemistry, reverse transcription-PCR (RT-PCR), and Western blotting. Microsatellite instability (MSI) phenotype was characterized by immunohistochemistry and pentaplex PCR. mRNAs of cytokines and chemokines were quantified in tumors and in normal homologous tissues (RT-PCR) in 43 patients. Adequate statistical tests, including multivariate analysis adjusted for pathologic tumor-node-metastasis (pTNM), MSI-H, and ObRb phenotypes, were used. Higher expression of ObRb in tumors compared with the homologous normal mucosa, pTNM staging but not leptin serum level, was associated with patients' progression-free survival (PFS). Tumor ObRb phenotype and pTNM were independent predictive factors of PFS. ObRb was more strongly expressed in HCT116 cells than in HCT116-Ch3 cells as well as in MSI-H tumors than in microsatellite stability and potentially associated with efficient cytotoxic antitumoral response as assessed by immunohistochemistry and RT-PCR measurements. We suggest that leptin receptor expression in tumors is involved in adaptive immune response in sporadic colon and rectal tumors likely via MSI-H phenotype orientation.

The distinction between Sézary syndrome (SS) and benign erythrodermic inflammatory diseases (EID) is difficult to make both clinically and on skin biopsies, since histomorphology can provide nonspecific results. New markers of circulating malignant Sézary cells have been recently described, especially CD158k/KIR3DL2 and T-plastin, but it has not been yet determined whether they could help in the diagnosis of erythroderma in skin samples. In this study, 13 frozen skin specimens from 10 SS patients and 26 from EID were analyzed for CD158k/KIR3DL2 expression using immunohistochemistry with AZ158 mAb, which also recognizes the monomeric CD158e/KIR3DL1 receptor. Although positive in all SS samples, immunohistochemistry appeared to not reliably discriminate between SS and EID. Therefore in all samples disclosing a significant staining with AZ158 mAb, CD158k/KIR3DL2, CD158e/KIR3DL1 and T-plastin mRNA expression were analyzed on the same skin specimen using conventional and/or quantitative real-time reverse transcription (RT)-PCR. Interestingly, only CD158k/KIR3DL2 transcripts were found to be significantly overexpressed in skin biopsies from patients with SS (P<0.0001), including when normalization to CD3 expression was achieved (P=0.0003). In light of these findings, CD158k/KIR3DL2 transcripts appear to be a unique molecular marker of SS in skin samples, allowing differential diagnosis with benign EID in routine practice.

The interpretation of cellular infiltrate from renal transplant recipients with borderline (BL) changes is still a challenging problem. To analyze the immune phenotype of such infiltrate, we quantified the mRNA expression of Foxp3 and interleukinL-10 and granzyme B (GB) in 15 kidney biopsies with BL changes. Controls were patients presenting type IA acute rejection and nonrejecting patients. Only levels of GB mRNA correlated significantly with response to antirejection therapy. Levels of Foxp3 mRNA in BL changes were intermediate between type IA acute rejection and nonrejecting controls. To determine the balance of alloagressive to graft-protecting T cells, we quantified the Foxp3/GB ratio. BL changes T cells infiltrate expressed a significantly higher Foxp3/GB ratio than that in IA acute rejection. These results suggest that T cell infiltrate from BL change exhibit a tolerogenic rather than a cytotoxic phenotype.

We investigated the causes of the altered functionality of T cells cultured under conditions designed for cell and gene therapy and the strategies to prevent their defects. We first showed that human T cells cultured for 6 days with anti-CD3 +/- anti-CD28 antibodies and interleukin-2 presented a 50% decrease of their proliferative responses to allogeneic or recall antigens. Similarly, day-6 cultured murine T cells completely lost their capacity to reject allogeneic skin grafts and to provoke graft-versus-host disease (GVHD) when infused into irradiated semi-allogeneic mice. Interestingly, injection of higher amounts of cultured T cells restored GVHD induction. Moreover, depletion of CD25+ cells prior to T-cell cultures can prevent these deficiencies both in mice and humans. Therefore, we demonstrated that culture conditions used for T-cell therapy preferentially activated and expanded regulatory T cells (Treg's). Thus, we showed that dividing cells sorted from T-cell cultures strongly suppressed the proliferation of autologous T cells in response to allogeneic stimulation. An increased detection of Foxp3 at mRNA and protein levels in the cultures confirmed the Treg expansion. Overall, we demonstrate that T-cell cultures promote Treg expansion over effector T cells, leading to deleterious immune functions, and that this imbalance can be prevented by an initial depletion of CD25+ cells.

Toxic epidermal necrolysis is a rare disease observed as a consequence of adverse reactions to drugs. It results in the widespread apoptosis of epidermal cells and has a high mortality rate. The mechanisms leading to this apoptosis are not yet elucidated. We investigated whether the cytokines present in the blister fluid, which accumulates under necrotic epidermis, originated from T lymphocytes and may play a role in the propagation of keratinocyte apoptosis. Interferon gamma (IFN-gamma), soluble tumor necrosis factor alpha (TNF-alpha), soluble Fas ligand (sFas-L) were present in much higher concentration in the blister fluids of 13 toxic epidermal necrolysis (TEN) patients than in control fluids from burns. The results of RT-PCR studies, however, indicated that only IFN-gamma and to a lesser extent interleukin (IL)-18 were produced by mononuclear cells present in the fluid. That suggests that the other cytokines also present (TNF-alpha, sFas-L, IL-10) rather originated from activated keratinocytes. Fas-L was indeed overexpressed on the membranes of keratinocytes in lesional skin in situ. The Th1 profile of T lymphocyte activation found in the blister fluid of patients with TEN is consistent with a key role for drug-specific cytotoxic T lymphocytes (CTL) as previously reported, the activation of keratinocytes by IFN-gamma making them sensitive to cell-mediated cytolysis. We propose the hypothesis that the production of Fas-L, TNF-alpha, and IL-10 by keratinocytes could be a defense mechanism against CTL rather than a way of propagating apoptosis among epidermal cells.

Background: Because histopathologic criteria cannot always predict the pathogenesis and response to curative antirejection therapy, new hope derives from the molecular analysis of intragraft immunologic markers. We studied whether the cutoff of intragraft expression level of T-cell activation markers may define subgroups of acute rejection differing either in type of rejection or clinical outcome., Methods: Forty-three human renal-allograft biopsies were quantified for mRNA expression of granzyme B, Fas ligand, interferon (IFN)gamma, interleukin (IL)-4, and IL-6 with a reverse-transcriptase real-time quantitative polymerase chain reaction (RT-PCR) method. Expression levels were correlated with the histopathologic rejection type according to the Banff 1997 classification criteria, and with the sensitivity to the antirejection immunosuppressive therapy, by means of receiver operating-characteristic (ROC) curves., Results: Granzyme B and Fas ligand mRNA expression up-regulation correlated with all allograft rejection types (P<0.01 for all). Moreover, granzyme B showed the highest sensitivity (90%) and specificity (78%) for the potential detection of histologic borderline changes that will require immunosuppressive therapy (area under the curve [AUC]=0.856, P<0.01). Curative antirejection-therapy resistance of overt, acute-rejection episode was significantly associated with higher Fas ligand gene expression (AUC=0.764, P<0.01, sensitivity [71%], specificity [99.5%])., Conclusions: Real-time RT-PCR quantification of the over-expression of the granzyme B gene in kidney-graft biopsies has proved to be as reliable in detecting acute rejection as histologic assessment. Furthermore, we demonstrate that the simultaneous measurement of the mRNA up-regulation of Fas ligand might represent an efficient new tool for the prediction of pejorative outcome of acute rejection.

Primary mediastinal large B-cell lymphoma (PMBL), currently recognized as a diffuse large B-cell lymphoma (DLBCL) subtype, shows increased expression of interleukin 4 (IL-4)/IL-13 signaling effectors and targets, suggesting constitutive activation of these pathways. We therefore investigated the functional state of the signal transducer and activator of transcription 6 (STAT6), mediating IL-4/IL-13 transcriptional effects. Constitutive STAT6 phosphorylation and DNA-binding activity were detected in PMBL cell lines but not DLBCL cell lines. Moreover, immunohistochemical analysis revealed nuclear phosphorylated STAT6 (P-STAT6) in 8 of 11 PMBL, compared with 1 of 10 DLBCL primary tumors (P =.01). IL-4 and IL-13 transcripts were absent in PMBL cell lines and expressed at low levels in tumors, indicating that, contrary to classical Hodgkin lymphoma (cHL), STAT6 activation is not due to an autocrine IL-4/IL-13 secretion. We demonstrated an amplification of the JAK2 gene in 2 of 6 PMBL cases, and showed higher JAK2 mRNA levels in PMBL compared with DLBCL (P =.005). The Janus kinase 2 (JAK2) was constitutively phosphorylated in the PMBL MedB1 cell line. MedB1 treatment with JAK2 inhibitor AG490 partially decreased STAT6 phosphorylation, suggesting that JAK2 is partially involved in STAT6 activation in these cells. Our findings highlight phosphorylated STAT6 as a characteristic distinguishing PMBL from DLBCL, but a common feature to PMBL and cHL, supporting the hypothesis of common pathogenic events in these 2 lymphomas.

Background: Acute rejections are scored according to three main criteria: vasculitis, tubulitis and interstitial infiltration as defined in the Banff classification. Typically, B cells account for <8% of the infiltrates and oedema is limited. The clinical significance of severe interstitial oedema and plasma cell-rich infiltrates (OPcR) are still a matter of debate., Methods: Kidney graft biopsies performed between 1991 and 1998 were retrospectively evaluated for these two criteria., Results: Among the 826 biopsies performed during the study period, 14 samples in 12 patients met these criteria; 11 were of Banff type I acute rejection and three were borderline. Based on clinical data, all were treated as acute rejections. OPcR occurred at a median of 187 days post-transplantation. All episodes were steroid resistant. Graft survival was 40% at 1 year following the rejection. Circulating antibodies reactive either to donor HLA or to endothelial cells were present in eight of 12 patients and widespread C4d deposit in peritubular capillary were present in three out of five patients studied. Level of gamma-interferon mRNA within the graft was significantly higher than in standard acute cellular rejection (ACR)., Conclusion: This study showed that OPcR rejections portend a poor outcome irrespective of the Banff score. Our data strongly support the hypothesis that a humoral component participated in the graft injuries. Altogether, the data suggest that OPcR rejection might represent a late and attenuated variant of acute humoral rejection that should be classified separately from ACR.

Mechanisms underlying the pathophysiology of minimal change nephrotic syndrome (MCNS), the most frequent of glomerular diseases in children, remain elusive, although recent arguments suggest that T cell dysfunction may be involved in the pathogenesis of this disease. Recently, we reported that activated T cells of these patients display a down-regulation of IL-12R beta2 chain, suggesting an early commitment toward Th2 phenotype. In this study, we show that the short form of the proto-oncogene c-maf, a known activator of the IL-4 gene, is highly induced in MCNS T cells during relapse, where it translocates to the nuclear compartment and binds to the DNA responsive element. Unexpectedly, the nuclear localization of c-maf did not promote the IL-4 gene transcription in relapse. Using several approaches, we show in this study that RelA blunts IL-4 induction in T cells during the relapse in these patients. We demonstrate that the ex vivo inhibition of proteasome activity in T cells from relapse, which blocks NF-kappaB activity, strongly increases the IL-4 mRNA levels. Overexpression of c-maf in T cells induces a high level of IL-4 promoter-driven luciferase activity. In contrast, coexpression of c-maf with NF-kappaB RelA/p50, or RelA, but not p50, inhibits the c-maf-dependent IL-4 promoter activity. Finally, we demonstrated that, in T cell overexpressing RelA and c-maf, RelA expelled c-maf from its DNA binding site on IL-4 gene promoter, which results in active inhibition of IL-4 gene transcription. Altogether, these results suggest that the involvement of c-maf in Th2 commitment in MCNS operates through IL-4-independent mechanisms.

We have recently established that enhancement of intracellular calcium cycling and contraction in response to beta2-adrenergic receptor (beta2-AR) stimulation exclusively relies on the activation of the cytosolic phospholipase A2 (cPLA2) and arachidonic acid production, via a pertussis toxin-sensitive G protein (possibly Gi), in embryonic chick cardiomyocytes. We aimed to investigate the relevance of the beta2-AR/Gi/cPLA2 pathway in the human myocardium. In left ventricular biopsies obtained from explanted hearts, beta2-AR stimulation exerted either an inhibition of cPLA2 that was insensitive to pertussis toxin (PTX) treatment, or an activation of cPLA2, sensitive to PTX treatment. In right atrial appendages from patients who were undergoing open heart surgery, we demonstrated that beta2-AR-induced activation of cPLA2 was favored in situations of altered beta1-AR and/or beta2-AR/adenylyl cyclase (AC) stimulations. Alterations were characterized by an increase in EC50value of norepinephrine and a decrease in the maximal AC activation in response to zinterol, respectively. Quantitative reverse transcription-polymerase chain reaction analyses highlighted a positive correlation between the expression of AC5 and AC6 mRNAs in human cardiac atria, which suggested that functional alterations in AC responses were unlikely to be related to changes in the AC5/AC6 mRNA ratio. In addition, the shift from the cyclic AMP to the arachidonic acid pathway was not supported at the transcriptional level by opposite regulation of AC and cPLA2mRNAs expression. This study gives the first evidence of the recruitment of cPLA2by beta2-ARs in the human heart and suggests that the Gi/cPLA2pathway could substitute for a deficient Gs/AC pathway in mediating beta2-AR responses.

We have previously described two cytotoxic T lymphocyte clones isolated from lymphocytes infiltrating a human major histocompatibility complex class II-/class I+, CD4+ cutaneous T cell lymphoma. These clones displayed a CD4+CD8dim+ (TC5) and CD4+ CD8- (TC7) phenotype and mediated a specific major histocompatibility complex class I-restricted cytotoxic activity toward Cou-LB autologous tumor cell line. Our studies were performed to elucidate the mechanism involved in T-cell-clone-mediated cytotoxicity and to determine the cytokine profile of both the lymphoma cell line and specific cytotoxic T lymphocyte clones. The results indicate that, despite surface expression of Fas receptor on Cou-LB and Fas ligand induction on TC5 and TC7 cell membranes, the CD4+ cytotoxic T lymphocyte clones do not use this cytotoxic mechanism to lyse their specific target. The TC7 clone uses instead a granzyme-perforin-dependent pathway. Furthermore, quantitative analysis of Th1 and Th2 cytokine mRNA expression in the cutaneous T cell lymphoma cell line as well as in TC5 and TC7 clones indicated that, whereas the tumor cells display a Th2-type profile (interleukin-4, interleukin-6, and interleukin-10), the cytotoxic T lymphocyte clones express Th1-type cytokines (interferon-gamma, granulocyte macrophage colony stimulating factor, and interleukin-2). In addition, preincubation of the tumor-infiltrating lymphocyte clones with autologous tumor cells induced their activation and subsequent amplification of the Th1-type response. These results indicate a direct contribution of the malignant cells in the Th1/Th2 imbalance observed frequently in cutaneous T cell lymphoma patients and suggest their potential role in depressed cell-mediated immunity. Identification of CD4+ Th1-type cytotoxic T lymphocyte clones, the tumor antigen they recognize, and optimization of their cytokine expression profile should be useful for the design of new immunotherapy protocols in cutaneous T cell lymphoma.

We investigated specific signaling events initiated after T cell triggering through the costimulatory surface receptors CD2 and CD28 as compared with activation via the Ag receptor (TCR/CD3). We therefore followed the phosphorylation of stathmin, a ubiquitous cytoplasmic phosphoprotein proposed as a general relay integrating diverse intracellular signaling pathways through the combinatorial phosphorylation of serines 16, 25, 38, and 63, the likely physiologic substrates for Ca2+/calmodulin (CaM)-dependent kinases, mitogen-activated protein (MAP) kinase, cyclin-dependent kinases (cdks), and protein kinase A, respectively. We addressed the specific protein kinase systems involved in the CD2 pathway of T cell activation through the analysis of stathmin phosphorylation patterns in exponentially growing Jurkat T cells, as revealed by phosphopeptide mapping. Stimulation via CD2 activated multiple signal transduction pathways, resulting in phosphorylation of distinct sites of stathmin, the combination of which only partially overlaps the CD3- and CD28-induced patterns. The partial redundancy of the three T cell activation pathways was evidenced by the phosphorylation of Ser25 and Ser38, substrates of MAP kinases and of the cdk family kinase(s), respectively. Conversely, the phosphorylation of Ser16 of stathmin was observed in response to both CD2 and CD28 triggering, but not CD3 triggering, with a kinetics compatible with the lasting activation of CaM kinase II in response to CD2 triggering. In vitro, Ser16 of recombinant human stathmin was phosphorylated also by purified CaM kinase II, and in vivo, CaM kinase II activity was indeed stimulated in CD2-triggered Jurkat cells. Altogether, our results favor an association of CaM kinase II activity with costimulatory signals of T lymphocyte activation and phosphorylation of stathmin on Ser16.

Background: Acute graft-versus-host disease (aGVHD) is still one of the main causes of morbidity and mortality after allogeneic bone marrow transplantation. Attempts to avoid GVHD are associated with an increased risk of relapse, probably because the graft-versus-leukemia effect is also abrogated. It was recently suggested that a high frequency of host-specific donor helper T cell precursors (HTLp) might be predictive of significant aGVHD (grade > or = II)., Methods: We retrospectively studied the frequency of HTLp by means of simplified limiting-dilution analysis to determine its predictive value for aGVHD and relapse. Pre-bone marrow transplantation, host-specific donor HLTp frequencies were analyzed in 32 patients who had received marrow from HLA-identical siblings for hematological malignancies, in terms of aGVHD and relapse., Results: HTLp frequencies were significantly higher in patients who had aGVHD > or = grade II (n=14) than in those without aGVHD (n=18) (P=0.007). Patients who relapsed (n=13) had significantly lower HTLp frequencies than those who did not relapse (n=19) (P<0.0001). The probabilities of relapse (Kaplan-Meier method) when the HTLp frequency was higher and lower than 1/200,000 were 0% and 88%, respectively (P<0.0001)., Conclusions: The definition of HTLp cut-off values predictive of aGVHD and relapse should contribute to donor selection and could open the way to protocols adapting immunomodulation to the likely risk of aGVHD and relapse.

Stathmin is a ubiquitous, highly conserved phosphoprotein, which most likely acts as an intracellular relay integrating various transduction pathways triggered by extracellular signals. Two post-translational isoforms (alpha and beta) have been previously identified whose increasingly phosphorylated forms migrate as a set of isoelectric variant spots (molecular mass 19 kDa; pI 6.2-5.6) on two-dimensional electrophoretic gels. In parallel with the phosphorylation of these forms of stathmin, two sets of three proteins migrating with slightly higher apparent molecular masses (21 and 23 kDa respectively) also incorporated radioactive phosphate in response to cell regulation through various transduction pathways. These phosphoproteins, previously referred to as proteins '16' and '17', share several biochemical properties with stathmin and are recognized by antibodies directed to stathmin or to stathmin peptides. Furthermore, when rat stathmin cDNA was transfected into mouse myogenic C2 cells, it directed the expression of protein sets 16 and 17 together with the 19 kDa forms of stathmin, as detected with a species-specific anti-stathmin antiserum. Proteins 16 and 17 are thus novel phosphorylated derivatives of stathmin, encoded by the same cDNA as its previously identified 19 kDa forms. These results increase the known complexity and diversity of stathmin patterns, which may yield the molecular support for its proposed role as a relay integrating various signals which regulate the proliferation, differentiation and functions of cells during development and adult life.

A human CD4+ T lymphocyte clone synthesized platelet-activating factor (PAF) acether when stimulated via the CD2 pathway. PAF-acether was characterized by biochemical and biophysical properties and precursor-product relationships (alkyl-acyl-sn-glycero-3-phosphocholine (GPC)----alkyl-lyso-GPC (lyso-PAF)----PAF-acether) were demonstrated. The clone contained substantial amounts of alkyl-acyl-GPC. i) Hydrolysis of alkyl-acyl-GPC upon CD2 stimulation was evidenced: [3H]alkyl-lyso-GPC was formed from [3H]alkyl-acyl-GPC in [3H] alkyl-labeled cells; alkyl-lyso-GPC production was also bioassayed after CD2 triggering. ii) The rate of arachidonate transfer from diacyl-GPC to alkyl-acyl-GPC increased after CD2 stimulation of the [3H]arachidonate-labeled P28D T cells, demonstrating alkyl-lyso-GPC formation. iii) Comparison of the molecular species of the produced PAF-acether with those of arachidonate-containing alkyl-acyl-GPC raises the possibility that the produced PAF-acether is related to alkyl-arachidonoyl-GPC.

CD2 triggering of human T lymphocyte activation has been associated with the activation of different interacting protein kinases, including protein kinase C (PKC). However the precise roles of its phosphorylated substrates are still unknown. We show here that PKC-dependent and -independent pathways are responsible for the CD2-induced phosphorylation of stathmin, a ubiquitous soluble phosphoprotein, most likely acting as a general intracellular relay integrating various second messenger pathways. The phosphorylated variants of stathmin provide a fingerprint reflecting the second messenger pathway(s) stimulated. The respective roles of both PKC and stathmin in the regulation of T lymphocyte proliferation are discussed.

In previous studies we demonstrated the triggering of the phospholipase C (PLC) pathway during the activation of an Ag-specific human CD4+ T lymphocyte clone by a mitogenic pair of CD2 (X11,D66) mAb. Similar conditions were applied to investigate a possible involvement of a phospholipase A2 (PLA2) acting as an additional alternative pathway during human T cell activation. Our results show that arachidonic acid or its derivatives are released after CD2 triggering. This release is largely independent of PLC activation and is mediated by a PLA2 because: 1) phosphatidylcholine is the preferential source of [3H]arachidonate release; 2) [3H]arachidonic acid release and phosphatidylcholine hydrolysis are blocked by two inhibitors of solubilized PLA2, mepacrine, and 4-p-bromophenacylbromide; and 3) we evidenced a PLA2 activity in cell homogenates. Extracellular calcium appears to play a critical role because the effects of CD2 mAb were inhibited in a Ca2(+)-depleted medium. In contrast, protein kinase C is not implicated since PMA, a protein kinase C activator, neither stimulated arachidonic acid release nor modulated CD2-induced arachidonic acid release. Cyclic AMP which has been proved to regulate the activity of the PLC in T lymphocytes does not appear to play an important role in the regulation of PLA2 activity since PGE2 has only a minimal effect on [3H]-arachidonate release. Altogether, these findings suggest that CD2 triggering stimulates a PLA2 activity in T lymphocytes via an extracellular Ca2(+)-dependent PLC protein kinase C independent mechanism.

Activation of human T lymphocytes via the CD2 molecule produces an enhanced turnover of phosphatidylinositol (PI) cycle-related phospholipids accompanied by the increased production of diacylglycerol (DG) and phosphorylated derivatives of inositol (IP). In this report we demonstrate that increased levels of intracellular cyclic AMP induced in human T lymphocytes by prostaglandin E2 or dibutyryl cAMP antagonize these early biochemical events of the CD2 activation process. Thus, a substantial inhibition of the CD2-induced increase in 32P-phosphatidic acid and 32P-PI values is observed. In parallel, both the DG production and the IP release triggered by the CD2 signal are strongly reduced contrasting with an almost conserved Ca2+ response. We also report here that cAMP does inhibit the CD2-induced proliferation in a dose-dependent manner while the proliferation generated independently of DG and IP production by a combination of Ca2+ ionophore A23187 and 12-O-tetradecanoylphorbol 13-acetate is not affected. These results therefore suggest that (a) intracellular cAMP levels may participate in the regulation of the PI cycle-related transduction pathway involved in the activation process of human T lymphocytes via the CD2 molecule; (b) the observed cAMP-mediated functional inhibitory effects are mainly related to an alteration of this cellular transduction signal; and (c) considering the putative critical second messenger role in the T cell proliferative response of DG and IP, respectively thought to activate the protein kinase C and to raise the intracellular free Ca2+, the lowering of DG production may be the key event responsible for this cAMP-mediated effect.

The effect of a mitogenic combination of two different anti-CD2 monoclonal antibodies on the PGE2-stimulated and basal cAMP production in human CD4+ T cell clones was investigated. The anti-CD2 stimulation strongly potentiates the PGE2-induced cAMP production while both PMA and A23187 produced a less potent effect. On the opposite the anti-CD2 treatment is without any effect on the basal cAMP level contrasting with a marked increase of intracellular cAMP concentrations with A23187 or the combination of A23187 and PMA. These results suggest that activation of CD4+ human T cells via the CD2 molecule significantly influences the cAMP-related transduction pathway. Although PMA and A23187 also modulate the activity of this pathway, their effect in this model is more likely mediated through an amplification of basal cAMP production.