Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.

Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. We show here that Fas kills activated primary T cells efficiently in the absence of active caspases, which results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor kappa B activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP.

Correlations between imatinib pharmacokinetics, pharmacodynamics, adherence, and clinical response in advanced metastatic gastrointestinal stromal tumor (GIST): An emerging role for drug blood level testing?

Imatinib is the standard of care for patients with advanced metastatic gastrointestinal stromal tumors (GIST), and is also approved for adjuvant treatment in patients at substantial risk of relapse. Studies have shown that maximizing benefit from imatinib depends on long-term administration at recommended doses. Pharmacokinetic (PK) and pharmacodynamic factors, adherence, and drug-drug interactions can affect exposure to imatinib and impact clinical outcomes. This article reviews the relevance of these factors to imatinib's clinical activity and response in the context of what has been demonstrated in chronic myelogenous leukemia (CML), and in light of new data correlating imatinib exposure to response in patients with GIST. Because of the wide inter-patient variability in drug exposure with imatinib in both CML and GIST, blood level testing (BLT) may play a role in investigating instances of suboptimal response, unusually severe toxicities, drug-drug interactions, and suspected non-adherence. Published clinical data in CML and in GIST were considered, including data from a PK substudy of the B2222 trial correlating imatinib blood levels with clinical responses in patients with GIST. Imatinib trough plasma levels <1100ng/mL were associated with lower rates of objective response and faster development of progressive disease in patients with GIST. These findings have been supported by other analyses correlating free imatinib (unbound) levels with response. These results suggest a future application for imatinib BLT in predicting and optimizing therapeutic response. Nevertheless, early estimates of threshold imatinib blood levels must be confirmed prospectively in future studies and elaborated for different patient subgroups.

Increased numbers of circulating polyfunctional Th17 memory cells in patients with seronegative spondylarthritides

OBJECTIVE: A distinct subset of proinflammatory CD4+ T cells that produce interleukin-17 was recently identified. These cells are implicated in different autoimmune disease models, such as experimental autoimmune encephalomyelitis and collagen-induced arthritis, but their involvement in human autoimmune disease has not yet been clearly established. The purpose of this study was to assess the frequency and functional properties of Th17 cells in healthy donors and in patients with different autoimmune diseases. METHODS: Peripheral blood was obtained from 10 psoriatic arthritis (PsA), 10 ankylosing spondylitis (AS), 10 rheumatoid arthritis (RA), and 5 vitiligo patients, as well as from 25 healthy donors. Synovial tissue samples from a separate group of patients were also evaluated (obtained as paraffin-embedded sections). Peripheral blood cells were analyzed by multiparameter flow cytometry and immunohistochemistry. Cytokine production was examined by enzyme-linked immunosorbent assay and intracellular cytokine staining using specific monoclonal antibodies. Synovial tissue was examined for infiltrating T cells by immunohistochemical analysis. RESULTS: We found increased numbers of circulating Th17 cells in the peripheral blood of patients with seronegative spondylarthritides (PsA and AS), but not in patients with RA or vitiligo. In addition, Th17 cells from the spondylarthritis patients showed advanced differentiation and were polyfunctional in terms of T cell receptor-driven cytokine production. CONCLUSION: These observations suggest a role of Th17 cells in the pathogenesis of certain human autoimmune disorders, in particular the seronegative spondylarthritides.

Monitoring of human CD4 T cell responses in metastatic melanoma and arthritic patients

SUMMARY : Detailed knowledge of the different components of the immune system is required for the development of new immunotherapeutic strategies. CD4 T lymphocytes represent a highly heterogeneous group of cells characterized by various profiles of cytokine production and effector vs. regulatory functions. They are central players in orchestrating adaptive immune responses: unbalances between the different subtypes can lead either to aggressive autoimmune disorders or can favour the uncontrolled growth of malignancies. In this study we focused on the characterization of human CD4 T cells in advanced stage melanoma patients as well as in patients affected by various forms of autoimmune inflammatory spondyloarthropathies. In melanoma patients we report that a population of FOXP3 CD4 T cells, known as regulatory T cells, is overrepresented in peripheral blood, and even more in tumor-infitrated lymph nodes as well as at tumor sites, as compared to healthy donors. In tumor-infiltrated lymph nodes, but not in normal lymph nodes or in peripheral blood, FOXP3 CD4 T cells feature a highly differentiated phenotype (CD45RA-CCR7+/-), which suggests for a recent encounter with their cognate antigen. FOXP3 CD4 T cells have been described to be an important component of the several known immune escape mechanisms. We demonstrated that FOXP3 CD4 T cells isolated from melanoma patients exert an in vitro suppressive action on autologous CD4 T cells, thus possibly inhibiting an efficient anti-tumor response. Next, we aimed to analyse CD4 T cells at antigen-specific level. In advanced stage melanoma patients, we identified for the first time, using pMHCII multimers, circulating CD4 T cells specific for the melanoma antigen Melan-A, presented by HLA-DQB1 *0602. Interestingly, in a cohort of melanoma patients enrolled in an immunotherapy trails consisting of injection of a Melan-A derived peptide, we did not observe signif cant variations in the ex vivo frequencies of Melan-A specific CD4 T cells, but important differences in the quality of the specific CD4 T cells. In fact, up to 50% of the ex vivo Melan-A/DQ6 specific CD4 T cells displayed a regulatory phenotype and were hypoproliferative before vaccination, while more effector, cytokine-secreting Melan-A/DQ6 specific CD4 T cells were observed after immunization. These observations suggest that peptide vaccination may favourably modify the balance between regulatory and effector tumor-specific CD4 T cells. Finally, we identified another subset of CD4 T cells as possible mediator of pathology in a group of human autoimmune spondyloarthropathies, namely Th17 cells. These cells were recently described to play a critical role in the pathogenesis of some marine models of autommunity. We document an elevated presence of circulating Th17 cells in two members of seronegative spondyloarthropathies, e.g. psoriatic arthritis and ankylosing spondylitis, while we do not observe increased frequencies of Th17 cells in peripheral blood of rheumatoid arthritic patients. In addition, Th17 cells with a more advanced differentiation state (CD45RA-CCR7-CD27-) and polyfunctionality (concomitant secretion of IL-17, IL-2 and TNFα) were observed exclusively in patients with seronegative spondylarthropathies. Together, our observations emphasize the importance of CD4 T cells in various diseases and suggest that immunotherapeutic approaches considering CD4 T cells as targets should be evaluated in the future.

Immunosuppresseurs: usages systémiques et neurologiques [Systemic and neurological uses of immunosuppressive agents].

Involvement of the central or peripheral nervous system, frequently present in systemic inflammatory immune disorders, has to be considered a severe threat and requires aggressive immunosuppressive treatment to achieve rapid remission. This is usually obtained with high-dose systemic corticosteroids combined with cyclophosphamide. Once remission is obtained, immunosuppressive agents with a more favorable safety profile are needed to exert a corticosteroid-sparing effect and minimize adverse events. New therapeutic approaches are currently developed to treat autoimmune diseases, mostly linked to the definition of new indications for biological agents such as TNF-alpha antagonists and rituximab.

One case, 3 rare simultaneous findings: intramyocardial bronchogenic cyst, P.H558R variant of SCN5A gene, and granular cell tumor of the esophagus.

We describe the sudden death of a 42-year-old white man. The decedent was a healthy young man with a short clinical history of chest pain, fatigue, dizziness, and pyrosis. Two weeks before his death, he underwent medical evaluation for the aforementioned symptoms. Electrocardiogram, chest x-ray, and serum troponin were all within normal limits. Gastroesophageal reflux disease was suspected, and the decedent was treated with omeprazole. Medicolegal autopsy disclosed an incidental intramyocardial bronchogenic cyst and p.H558R variant of the SCN5A gene. The cyst was located between the epicardium and myocardium of the posterior face of the left superior ventricular wall, adjacent to the base of the heart. An incidental granular cell tumor of the esophagus was also identified, which was likely unrelated to death.

Role of Myeloid-Derived Suppressor Cells in tumor-associated pregnancy

Cancer progression is dependent, in part, on interactions between tumor cells and the host microenvironment. During pregnancy, physiological changes occur that include inflammation and reduced immunity, both of which can promote tumor growth. Accordingly, tumors are observed to be more aggressive and to have greater proclivity toward metastasis during pregnancy. In this work, myeloid-derived suppressor cells (MDSC), a population of heterogeneous and pluripotent cells that can down-regulate immune responses during pathological conditions, were studied in the context of mouse and human gestation. The gene expression profile of mouse MDSC has been shown to differ in pregnant and virgin mice, and the profile in pregnant animals bears similarity to that of MDSC associated with the tumor microenvironment. Common induced genes include Fibronectin1 and Olfactomedin4, which are known to be involved in extracellular matrix remodeling and tissue permissiveness to tumor cells implantation. Our observations suggest that mouse MDSC may represent a shared regulatory mechanism of tissue permissiveness that occurs during the physiological state of gestation and tumor growth. Pregnancy-associated changes in immunosuppressive myeloid cell activity have also been studied in humans. We show that CD33+ myeloid cells isolated from PBMC (peripheral blood mononuclear cells) of pregnant women are more strongly immunosuppressive on T cells than CD33+ cells removed from non-pregnant subjects. During murine gestation, decreased natural killer (NK) cell activity is responsible, at least in part, for the increase in experimental metastasis. However, although peripheral blood NK cell numbers and cytotoxicity were slightly reduced in pregnant women, neither appeared to be regulated by CD33+ cells. Nevertheless, based on its observed suppression of T cell responses, the CD33+ PBMC subset appears to be an appropriate myeloid cell population to study in order to elucidate mechanisms of immune regulation that occur during human pregnancy. Our findings regarding the immunosuppressive function of CD33+cells and the role of NK cells during human pregnancy are consistent with the notion that changes in the function of the immune system participate in the constitution of a permissive soil for tumour progression.

Identification of a new murine tumor necrosis factor receptor locus that contains two novel murine receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFkappaB activation, we propose that the mTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed them mDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event.

cFLIP protein prevents tumor necrosis factor-alpha-mediated induction of caspase-8-dependent apoptosis in insulin-secreting betaTc-Tet cells.

Type 1 diabetes is characterized by the infiltration of activated leukocytes within the pancreatic islets, leading to beta-cell dysfunction and destruction. The exact role played by interferon-gamma, tumor necrosis factor (TNF)-alpha, and interleukin-1beta in this pathogenic process is still only partially understood. To study cytokine action at the cellular level, we are working with the highly differentiated insulin-secreting cell line, betaTc-Tet. We previously reported that it was susceptible to apoptosis induced by TNF-alpha, in combination with interleukin-1beta and interferon-gamma. Here, we report that cytokine-induced apoptosis was correlated with the activation of caspase-8. We show that in betaTc-Tet cells, overexpression of cFLIP, the cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein, completely abolished cytokine-dependent activation of caspase-8 and protected the cells against apoptosis. Furthermore, cFLIP overexpression increased the basal and interleukin-1beta-mediated transcriptional activity of nuclear factor (NF)-kappaB, whereas it did not change cytokine-induced inducible nitric oxide synthase gene transcription and nitric oxide secretion. The presence of cFLIP prevented the weak TNF-alpha-induced reduction in cellular insulin content and secretion; however, it did not prevent the decrease in glucose-stimulated insulin secretion induced by the combined cytokines, in agreement with our previous data demonstrating that interferon-gamma alone could induce these beta-cell dysfunctions. Together, our data demonstrate that overexpression of cFLIP protects mouse beta-cells against TNF-alpha-induced caspase-8 activation and apoptosis and is correlated with enhanced NF-kappaB transcriptional activity, suggesting that cFLIP may have an impact on the outcome of death receptor-triggered responses by directing the intracellular signals from beta-cell death to beta-cell survival.

Mixed testicular germ cell tumor presenting as metastatic pure choriocarcinoma involving the maxillary gingiva.

Gingival metastases are infrequent and invariably associated with a widespread disease and a poor prognosis. Because of their unremarkable clinical appearance, they can be difficult to distinguish from more common gingival hyperplastic or reactive lesions, such as pyogenic granuloma, peripheral giant cell granuloma, and peripheral ossifying granuloma. We are reporting here an unusual case of a 36-year-old man with a mixed testicular germ cell tumor presenting as a metastatic pure choriocarcinoma involving the maxillary gingiva, extending from the first left premolar to the left second maxillary molar, mimicking a 'benign looking' gingival mass. Gingival metastases may be the first manifestation of a widespread metastatic disease and therefore particular attention must be paid to gingival lesions associated with atypical clinical symptoms and/or signs.

Ocular and systemic bio-distribution of rhodamine-conjugated liposomes loaded with VIP injected into the vitreous of Lewis rats.

PURPOSE: Local delivery of therapeutic molecules encapsulated within liposomes is a promising method to treat ocular inflammation. The purpose of the present study was to define the biodistribution of rhodamine-conjugated liposomes loaded with vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, following their intravitreal (IVT) injection in normal rats. METHODS: Healthy seven- to eight-week-old Lewis male rats were injected into the vitreous with empty rhodamine-conjugated liposomes (Rh-Lip) or with VIP-loaded Rh-Lip (VIP-Rh-Lip; 50 mM of lipids with an encapsulation efficiency of 3.0+/-0.4 mmol VIP/mol lipids). Twenty-four h after IVT injection, the eyes, the cervical, mesenteric, and inguinal lymph nodes (LN), and spleen were collected. The phenotype and distribution of cells internalizing Rh-Lip and VIP-Rh-Lip were studied. Determination of VIP expression in ocular tissues and lymphoid organs and interactions with T cells in cervical LN was performed on whole mounted tissues and frozen tissue sections by immunofluorescence and confocal microscopy. RESULTS: In the eye, 24 h following IVT injection, fluorescent liposomes (Rh-Lip and VIP-Rh-Lip) were detected mainly in the posterior segment of the eye (vitreous, inner layer of the retina) and to a lesser extent at the level of the iris root and ciliary body. Liposomes were internalized by activated retinal Müller glial cells, ocular tissue resident macrophages, and rare infiltrating activated macrophages. In addition, fluorescent liposomes were found in the episclera and conjunctiva where free VIP expression was also detected. In lymphoid organs, Rh-Lip and VIP-Rh-Lip were distributed almost exclusively in the cervical lymph nodes (LN) with only a few Rh-Lip-positive cells detected in the spleen and mesenteric LN and none in the inguinal LN. In the cervical LN, Rh-Lip were internalized by resident ED3-positive macrophages adjacent to CD4 and CD8-positive T lymphocytes. Some of these T lymphocytes in close contact with macrophages containing VIP-Rh-Lip expressed VIP. CONCLUSIONS: Liposomes are specifically internalized by retinal Müller glial cells and resident macrophages in the eye. A limited passage of fluorescent liposomes from the vitreous to the spleen via the conventional outflow pathway and the venous circulation was detected. The majority of fluorescent liposomes deposited in the conjunctiva following IVT injection reached the subcapsular sinus of the cervical LN via conjuntival lymphatics. In the cervical LN, Rh-Lip were internalized by resident subcapsular sinus macrophages adjacent to T lymphocytes. Detection of VIP in both macrophages and T cells in cervical LN suggests that IVT injection of VIP-Rh-Lip may increase ocular immune privilege by modulating the loco-regional immune environment. In conclusion, our observations suggest that IVT injection of VIP-loaded liposomes is a promising therapeutic strategy to dampen ocular inflammation by modulating macrophage and T cell activation mainly in the loco-regional immune system.

Degradation of ZAP-70 following antigenic stimulation in human T lymphocytes: role of calpain proteolytic pathway.

T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of zeta-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.

Increase of [(18)F]FLT tumor uptake in vivo mediated by FdUrd: toward improving cell proliferation positron emission tomography.

PURPOSE: 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT), a cell proliferation positron emission tomography (PET) tracer, has been shown in numerous tumors to be more specific than 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) but less sensitive. We studied the capacity of a nontoxic concentration of 5-fluoro-2'-deoxyuridine (FdUrd), a thymidine synthesis inhibitor, to increase uptake of [(18)F]FLT in tumor xenografts. METHODS: The duration of the FdUrd effect in vivo on tumor cell cycling and thymidine analogue uptake was studied by varying FdUrd pretreatment timing and holding constant the timing of subsequent flow cytometry and 5-[(125)I]iodo-2'-deoxyuridine biodistribution measurements. In [(18)F]FLT studies, FdUrd pretreatment was generally performed 1 h before radiotracer injection. [(18)F]FLT biodistributions were measured 1 to 3 h after radiotracer injection of mice grafted with five different human tumors and pretreated or not with FdUrd and compared with [(18)F]FDG tumor uptake. Using microPET, the dynamic distribution of [(18)F]FLT was followed for 1.5 h in FdUrd pretreated mice. High-field T2-weighted magnetic resonance imaging (MRI) and histology were used comparatively in assessing tumor viability and proliferation. RESULTS: FdUrd induced an immediate increase in tumor uptake of 5-[(125)I]iodo-2'-deoxyuridine, that vanished after 6 h, as also confirmed by flow cytometry. Biodistribution measurements showed that FdUrd pretreatment increased [(18)F]FLT uptake in all tumors by factors of 3.2 to 7.8 compared with controls, while [(18)F]FDG tumor uptake was about fourfold and sixfold lower in breast cancers and lymphoma. Dynamic PET in FdUrd pretreated mice showed that [(18)F]FLT uptake in all tumors increased steadily up to 1.5 h. MRI showed a well-vascularized homogenous lymphoma with high [(18)F]FLT uptake, while in breast cancer, a central necrosis shown by MRI was inactive in PET, consistent with the histomorphological analysis. CONCLUSION: We showed a reliable and significant uptake increase of [(18)F]FLT in different tumor xenografts after low-dose FdUrd pretreatment. These results show promise for a clinical application of FdUrd aimed at increasing the sensitivity of [(18)F]FLT PET.

Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors.

We describe a new mechanism regulating the tumor endothelial barrier and T cell infiltration into tumors. We detected selective expression of the death mediator Fas ligand (FasL, also called CD95L) in the vasculature of human and mouse solid tumors but not in normal vasculature. In these tumors, FasL expression was associated with scarce CD8(+) infiltration and a predominance of FoxP3(+) T regulatory (Treg) cells. Tumor-derived vascular endothelial growth factor A (VEGF-A), interleukin 10 (IL-10) and prostaglandin E2 (PGE2) cooperatively induced FasL expression in endothelial cells, which acquired the ability to kill effector CD8(+) T cells but not Treg cells because of higher levels of c-FLIP expression in Treg cells. In mice, genetic or pharmacologic suppression of FasL produced a substantial increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells. Pharmacologic inhibition of VEGF and PGE2 produced a marked increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells that was dependent on attenuation of FasL expression and led to CD8-dependent tumor growth suppression. Thus, tumor paracrine mechanisms establish a tumor endothelial death barrier, which has a critical role in establishing immune tolerance and determining the fate of tumors.

Toward improved immunocompetence of adoptively transferred CD8+ T cells.

Adoptive transfer of autologous or allogenic T cells to patients is being used with increased frequency as a therapy for infectious diseases and cancer. However, many questions remain with regard to defining optimized procedures for preparation and selection of T cell populations for transfer. In a new study in this issue of the JCI, Gattinoni and colleagues used a TCR transgenic mouse model to examine in vitro-generated tumor antigen-specific CD8+ T cells at various stages of differentiation for their efficacy in adoptive immunotherapy against transplantable melanoma. The results confirm that CD8+ T cells progressively lose immunocompetence with prolonged in vitro cultivation and suggest that effector CD8+ T cells alone may be considerably less potent at protecting hosts with advanced tumors than are less differentiated T cells.