Detection of perforin and granzyme B mRNA expressing cells in lichen sclerosus

Granzyme B and perforin messenger RNA (mRNA) expression has been shown to be a specific in vivo activation marker for cytotoxic cells. The aim of this study was to assess the contribution of cell-mediated cytotoxicity in the pathogenesis of lichen sclerosus. In situ hybridization and immunohistochemistry were performed on serial tissue sections of lesional skin biopsies and normal skin as control. Immunohistochemical staining showed that the cellular infiltrate of diseased skin consisted predominantly of T cells (CD3+) and some B cells (CD20+). Among T cells CD4+ and CD8+ cells were found in about equal numbers. In normal skin samples perforin and granzyme B mRNA expressing cells were only rarely found. In contrast, in biopsies from diseased skin a high percentage of infiltrating cells expressed mRNA for perforin and granzyme B. The perforin and granzyme B expressing cells were found in the dermal infiltrate and intraepidermally in close proximity to keratinocytes suggesting in situ activation of these cells. These findings provide evidence that cell-mediated cytotoxicity plays a significant role in tissue destruction in lichen sclerosus.

Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells

OBJECTIVE: Apoptosis of pancreatic beta-cells is critical in both diabetes development and failure of islet transplantation. The role in these processes of pro- and antiapoptotic Bcl-2 family proteins, which regulate apoptosis by controlling mitochondrial integrity, remains poorly understood. We investigated the role of the BH3-only protein Bid and the multi-BH domain proapoptotic Bax and Bak, as well as prosurvival Bcl-2, in beta-cell apoptosis. RESEARCH DESIGN AND METHODS: We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-alpha, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, gamma-radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry. RESULTS: Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand-induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF-alpha plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor-induced apoptosis. CONCLUSIONS: These results demonstrate that Bid is essential for death receptor-induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which beta-cells are destroyed.

Cytotoxic diacetylenic spiroketal enol ethers from Plagius flosculosus

Three new diacetylenic spiroketal enol ethers named flosculins A (1), B (2), and C (3), along with five known compounds (4-8) of the same structural class, were isolated from the leaves of Plagius flosculosus. The structures were deduced by extensive 1D and 2D NMR spectroscopy and mass spectrometry. All isolated compounds exhibited significant cytotoxic activity against leukemia cells (Jurkat T and HL-60). Compounds 5-8 induced apoptosis in HL-60 cells with corresponding IC(50) values ranging from 4 to 6 microM.

New cytotoxic saturated and unsaturated cyclohexanones from Anthemis maritima

Two new cyclohexenones (antheminones A and B) and a new cyclohexanone, (antheminone C) along with five known compounds were isolated from the leaves of Anthemis maritima L. The structures were mainly deduced from extensive 1D and 2D NMR spectroscopy and mass spectrometry. The new compounds were tested in vitro for their cytotoxic activity against adherent and non-adherent cancer cell lines. Antheminones A and C exhibited significant antiproliferative activity against leukemia cells with IC(50) values ranging from 3.2 to 14 microM.

Cytotoxic effects of camptothecin and cisplatin combined with tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in a model of primary culture of non-small cell lung cancer

The cytokine tumor-necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has been shown to preferentially induce apoptosis in cancer cells. A previous study of our group demonstrated that non-small cell lung cancer cell lines can be sensitized to Apo2L/TRAIL-induced apoptosis by chemotherapeutic agents. The aim of the present study was the evaluation of these results in a model of primary culture of non-small cell lung cancer.

AKT/mTOR pathway activation and BCL-2 family proteins modulate the sensitivity of human small cell lung cancer cells to RAD001

PURPOSE: The Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancers and plays an important role in small cell lung cancer (SCLC) biology. We investigated the potential of targeting mTOR signaling as a novel antitumor approach in SCLC. EXPERIMENTAL DESIGN: The expression of mTOR in patient specimens and in a panel of SCLC cell lines was analyzed. The effects on SCLC cell survival and downstream signaling were determined following mTOR inhibition by the rapamycin derivative RAD001 (Everolimus) or down-regulation by small interfering RNA. RESULTS: We found elevated expression of mTOR in patient specimens and SCLC cell lines, compared with normal lung tissue and normal lung epithelial cells. RAD001 treatment impaired basal and growth factor-stimulated cell growth in a panel of SCLC cell lines. Cells with increased Akt pathway activation were more sensitive to RAD001. Accordingly, a constitutive activation of the Akt/mTOR pathway was sufficient to sensitize resistant SCLC cells to the cytotoxic effect of RAD001. In the sensitive cells, RAD001 showed a strong additive effect to the proapoptotic action of the chemotherapeutic agent etoposide. Intriguingly, we observed low Bcl-2 family proteins levels in the SCLC cells with a constitutive Akt pathway activation, whereas an increased expression was detected in the RAD001-resistant SCLC cells. An antisense construct targeting Bcl-2 or a Bcl-2-specific inhibitor was able to sensitize resistant SCLC cells to RAD001. Moreover, SCLC tumor growth in vivo was significantly inhibited by RAD001. CONCLUSION: Together, our data show that inhibiting mTOR signaling with RAD001 potently disrupts growth and survival signaling in human SCLC cells.

Cathepsin W expressed exclusively in CD8+ T cells and NK cells, is secreted during target cell killing but is not essential for cytotoxicity in human CTLs

OBJECTIVE: Cathepsin W (CatW, lymphopain) is a putative cysteine protease with restricted expression to natural killer (NK) cells and CD8(+) T cells and so far unknown function and properties. Here, we characterize in detail, the regulation of human CatW during T-cell development in response to different stimuli and its functional involvement in cytotoxic lymphocyte effector function. MATERIALS AND METHODS: Western blots and real time polymerase chain reaction of sorted, unstimulated, and stimulated cell subsets (thymocytes, T cells, NK cells) and their culture supernatants were used to study regulation and expression of CatW. Primary CD8(+) T cells and short-term T-cell lines were transfected with small interfering RNA to study the involvement of CatW in effector function such as target cell killing and interferon-gamma production. RESULTS: Levels of CatW expression correlate closely with cytotoxic capacity both during development and in response to factors influencing cytotoxicity. Furthermore, CatW is secreted during specific target cell killing. However, knockdown of CatW expression by small interfering RNA neither influences target cell killing nor interferon-gamma production. CONCLUSION: Despite being expressed in the effector subset of CD8(+) and NK cells and of being released during target cell killing, our functional inhibition studies exclude an essential role of CatW in the process of cytotoxicity.

Programmed death 1 signaling on chronic myeloid leukemia-specific T cells results in T-cell exhaustion and disease progression

Chronic myeloid leukemia (CML) is a malignant myeloproliferative disease with a characteristic chronic phase (cp) of several years before progression to blast crisis (bc). The immune system may contribute to disease control in CML. We analyzed leukemia-specific immune responses in cpCML and bcCML in a retroviral-induced murine CML model. In the presence of cpCML and bcCML expressing the glycoprotein of lymphocytic choriomeningitis virus as a model leukemia antigen, leukemia-specific cytotoxic T lymphocytes (CTLs) became exhausted. They maintained only limited cytotoxic activity, and did not produce interferon-gamma or tumor necrosis factor-alpha or expand after restimulation. CML-specific CTLs were characterized by high expression of programmed death 1 (PD-1), whereas CML cells expressed PD-ligand 1 (PD-L1). Blocking the PD-1/PD-L1 interaction by generating bcCML in PD-1-deficient mice or by repetitive administration of alphaPD-L1 antibody prolonged survival. In addition, we found that PD-1 is up-regulated on CD8(+) T cells from CML patients. Taken together, our results suggest that blocking the PD-1/PD-L1 interaction may restore the function of CML-specific CTLs and may represent a novel therapeutic approach for CML.

Protective autophagy is involved in resistance towards MET inhibitors in human gastric adenocarcinoma cells

MET, also known as hepatocyte growth factor receptor (HGFR), is a receptor tyrosine kinase with an important role, both in normal cellular function as well as in oncogenesis. In many cancer types, abnormal activation of MET is related to poor prognosis and various strategies to inhibit its function, including small molecule inhibitors, are currently in preclinical and clinical evaluation. Autophagy, a self-digesting recycling mechanism with cytoprotective functions, is induced by cellular stress. This process is also induced upon cytotoxic drug treatment of cancer cells and partially allows these cells to escape cell death. Thus, since autophagy protects different tumor cells from chemotherapy-induced cell death, current clinical trials aim at combining autophagy inhibitors with different cancer treatments. We found that in a gastric adenocarcinoma cell line GTL-16, where MET activity is deregulated due to receptor overexpression, two different MET inhibitors PHA665752 and EMD1214063 lead to cell death paralleled by the induction of autophagy. A combined treatment of MET inhibitors together with the autophagy inhibitor 3-MA or genetically impairing autophagy by knocking down the key autophagy gene ATG7 further decreased cell viability of gastric cancer cells. In general, we observed the induction of cytoprotective autophagy in MET expressing cells upon MET inhibition and a combination of MET and autophagy inhibition resulted in significantly decreased cell viability in gastric cancer cells.

1.59 Reduced inflammatory potential of circulating gammadelta T cells in pregnancy induced improvement of Rheumatoid Arthritis

BACKGROUND AND OBJECTIVES During pregnancy, gammadelta T cells expand at the fetomaternal interface where they induce a tolerogenic milieu. Patients with rheumatoid arthritis (RA) experience a spontaneous improvement of their disease during pregnancy and a postpartum aggravation. By contrast, pregnant patients with ankylosing spondylitis (AS) often experience persistent active disease. We hypothesised that the pregnancy related modulation of disease activity in RA patients versus AS patients is associated with numerical and functional changes of circulating gammadelta T cells. MATERIAL AND METHODS The frequency of surface markers and the intracellular cytokine profile of freshly isolated gammadelta T cells from RA (n = 54) and AS (n = 26) patients and healthy controls (n = 40) were analysed at each trimester during pregnancy and 6-8 weeks postpartum by flow cytometry. RESULTS Very discrete changes of Vdelta1 or Vdelta2 frequency were seen during pregnancy and postpartum in healthy controls and AS patients. In RA, however, the frequency of Vdelta2 cells decreased in the third trimester when disease activity was low. Low percentages of Vdelta 2 cells were also found in non-pregnant RA patients with active arthritis, yet only pregnant RA patients showed reduced percentages of Vdelta2 cells positive for the activation marker CD69 and the intracellular cytokine TNFalpha. Similarly, Vdelta1 + TNFalpha + cells were lower in pregnant RA patients compared to non-pregnant RA patients. The percentage of Vdelta2 + TNFalpha + cells, Vdelta2+ CD69+ and Vdelta1+ CD69+ cells correlated with disease activity in RA. As for the receptors which modulate cytotoxicity, RA patients showed a rise of the anti-cytotoxic receptor NKG2A on Vdelta1 cells in the 2(nd) trimester and a decrease postpartum. Since the pro-cytotoxic receptor NKG2D remained unchanged, the NKG2D/NKG2A ratio on Vdelta1 cells was reduced in RA patients during pregnancy. In AS patients, persistent disease activity during pregnancy was reflected by an increased frequency of Vdelta2+ CD69+ cells and an unchanged frequency of Vdelta2+ TNFalpha+ cells. In addition, pregnant AS patients showed an increased frequency of Vdelta1+CD161+ cells. CONCLUSIONS Disease amelioration of RA during pregnancy correlates with changes of cell activation, pro-inflammatory cytokines and anti-cytotoxic receptors of gammadelta T cells. By contrast, active disease during pregnancy as found in AS is associated with unchanged inflammatory responses of gammadelta T cells. Since gammadelta T cells remain unchanged in healthy pregnant controls, the modulation of gammadelta T cells in RA rather seems to be an effect of improved disease than of pregnancy itself.

Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8(+) memory T cells.

Protection against malaria can be achieved by induction of a strong CD8(+) T-cell response against the Plasmodium circumsporozoite protein (CSP), but most subunit vaccines suffer from insufficient memory responses. In the present study, we analyzed the impact of postimmunization sporozoite challenge on the development of long-lasting immunity. BALB/c mice were immunized by a heterologous prime/boost regimen against Plasmodium berghei CSP that induces a strong CD8(+) T-cell response and sterile protection, which is short-lived. Here, we show that protective immunity is prolonged by a sporozoite challenge after immunization. Repeated challenges induced sporozoite-specific antibodies that showed protective capacity. The numbers of CSP-specific CD8(+) T cells were not substantially enhanced by sporozoite infections; however, CSP-specific memory CD8(+) T cells of challenged mice displayed a higher cytotoxic activity than memory T cells of immunized-only mice. CD4(+) T cells contributed to protection as well; but CD8(+) memory T cells were found to be the central mediator of sterile protection. Based on these data, we suggest that prolonged protective immunity observed after immunization and infection is composed of different antiparasitic mechanisms including CD8(+) effector-memory T cells with increased cytotoxic activity as well as CD4(+) memory T cells and neutralizing antibodies.

Depressed type 1 cytokine synthesis by superantigen-activated CD4+ T cells of women with human papillomavirus-related high-grade squamous intraepithelial lesions.

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4(+) T and CD8(+) T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4(+) T cells that produced IL-2 (P = 0.045), IFN-gamma (P = 0.040), and TNF-alpha (P = 0.015) and a significantly lower percentage of activated CD8(+) T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4(+) T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8(+) T cells.

CD8+ T cells and NK cells from blister fluid of an EEM/SJS overlap highly express Fas ligand and Granulysin

INTRODUCTION Erythema exsudativum multiforme majus (EEMM) and Stevens-Johnson Syndrome (SJS) are severe cutaneous reaction patterns caused by infections or drug hypersensitivity. The mechanism by which widespread keratinocyte death is mediated by the immune system in EEMM/SJS are still to be elucidated. Here, we characterized the blister cells isolated from a patient with EEMM/SJS overlap and investigated its cause. METHODS Clinical classification of the cutaneous eruption was done according to the consensus definition of severe blistering skin reactions and histological analysis. Common infectious causes of EEMM were investigated using standard clinical techniques. T cell reactivity for potentially causative drugs was assessed by lymphocyte transformation tests (LTT). Lymphocytes isolated from blister fluid were analyzed for their expression of activation markers and cytotoxic molecules using flow cytometry. RESULTS The healthy 58 year-old woman suffered from mild respiratory tract infection and therefore started treatment with the secretolytic drug Ambroxol. One week later, she presented with large palmar and plantar blisters, painful mucosal erosions, and flat atypical target lesions and maculae on the trunc, thus showing the clinical picture of an EEMM/SJS overlap (Fig. 1). This diagnosis was supported by histology, where also eosinophils were found to infiltrate the upper dermis, thus pointing towards a cutaneous adverse drug reaction (cADR). Analysis of blister cells showed that they mainly consisted of CD8+ and CD4+ T cells and a smaller population of NK cells. Both the CD8+ T cells and the NK cells were highly activated and expressed Fas ligand and the cytotoxic molecule granulysin (Fig. 2). In addition, in comparison to NK cells from PBMC, NK cells in blister fluids strongly upregulated the expression of the skin-homing chemokine receptor CCR4 (Fig 4). Surprisingly, the LTT performed on PBMCs in the acute phase was positive for Ambroxol (SI=2.9) whereas a LTT from a healthy but exposed individual did not show unspecific proliferation. Laboratory tests for common infectious causes of EEMM were negative (HSV-1/-2, M. pneumoniae, Parvovirus B19). However, 6 weeks later, specific proliferation to Ambroxol could no longer be observed in the LTT (Fig 4.).

Treatment of solid organ transplant recipients with autologous Epstein Barr virus-specific cytotoxic T lymphocytes (CTLs).

We have investigated the in vivo safety, efficacy, and persistence of autologous Epstein Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) for the treatment of solid organ transplant (SOT) recipients at high risk for EBV-associated posttransplantation lymphoproliferative disease (PTLD). EBV-CTLs generated from 35 patients expanded with normal kinetics contained both CD8 and CD4 lymphocytes and produced significant specific killing of autologous EBV-transformed B lymphoblastoid cell lines (LCLs). Twelve SOT recipients at high risk for PTLD, or with active disease, received autologous CTL infusions without toxicity. Real-time polymerase chain reaction (PCR) monitoring of EBV-DNA showed a transient increase in plasma EBV-DNA suggestive of lysis of EBV-infected cells, although there was no consistent decrease in virus load in peripheral-blood mononuclear cells. Interferon-gamma enzyme-linked immunospot (ELISPOT) assay and tetramer analysis showed an increase in the frequency of EBV-responsive T cells, which returned to preinfusion levels after 2 to 6 months. None of the treated patients developed PTLD. One patient with liver PTLD showed a complete response, and one with ocular disease has had a partial response stable for over one year. These data are consistent with an expansion and persistence of adoptively transferred EBV-CTLs that is limited in the presence of continued immunosuppression but that nonetheless produces clinically useful antiviral activity.

Promotion of liver regeneration by natural killer cells in a murine model is dependent on extracellular adenosine triphosphate phosphohydrolysis

Nucleotides, such as adenosine triphosphate (ATP), are released by cellular injury, bind to purinergic receptors expressed on hepatic parenchymal and nonparenchymal cells, and modulate cellular crosstalk. Liver resection and resulting cellular stress initiate such purinergic signaling responses between hepatocytes and innate immune cells, which regulate and ultimately drive liver regeneration. We studied a murine model of partial hepatectomy using immunodeficient mice to determine the effects of natural killer (NK) cell-mediated purinergic signaling on liver regeneration. We noted first that liver NK cells undergo phenotypic changes post-partial hepatectomy (PH) in vivo, including increased cytotoxicity and more immature phenotype manifested by alterations in the expression of CD107a, CD27, CD11b, and CD16. Hepatocellular proliferation is significantly decreased in Rag2/common gamma-null mice (lacking T, B, and NK cells) when compared to wildtype and Rag1-null mice (lacking T and B cells but retaining NK cells). Extracellular ATP levels are elevated post-PH and NK cell cytotoxicity is substantively increased in vivo in response to hydrolysis of extracellular ATP levels by apyrase (soluble NTPDase). Moreover, liver regeneration is significantly increased by the scavenging of extracellular ATP in wildtype mice and in Rag2/common gamma-null mice after adoptive transfer of NK cells. Blockade of NKG2D-dependent interactions significantly decreased hepatocellular proliferation. In vitro, NK cell cytotoxicity is inhibited by extracellular ATP in a manner dependent on P2Y1, P2Y2, and P2X3 receptor activation. Conclusion: We propose that hepatic NK cells are activated and cytotoxic post-PH and support hepatocellular proliferation. NK cell cytotoxicity is, however, attenuated by hepatic release of extracellular ATP by way of the activation of specific P2 receptors. Clearance of extracellular ATP elevates NK cell cytotoxicity and boosts liver regeneration.