Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Influence of physical activity on the immune system in breast cancer patients during chemotherapy.

PURPOSE: Physical activity can impact the immune system in different ways, e.g. by alteration of the humoral and cellular immune response. Physical activity at medium intensity enhances numbers of cytotoxic T cells, NK cells and macrophages in healthy people. The aim of this study was to compare the effects of endurance and resistance training on the immune system in breast cancer patients during adjuvant chemotherapy. METHODS: In a prospective, controlled and randomized intervention exploratory trial, 12-week supervised endurance or resistance training were compared with usual care twice a week. Endpoints were the absolute numbers of the immune cells such as CD3+ T lymphocytes including CD4+- and CD8+, αß T cells, γδT cells, CD3-/CD16+/56+ NK cells and CD19+ B cells, before and after 12 weeks of treatment. Cell numbers were analyzed using fluorescence-activated cell sorting. RESULTS: Despite different physical interventions in all groups immune cell count decreased in CD3 T cells including TCR αß and CD4 T cells, NK cells and CD19 B cells 12 weeks after initiation of chemotherapy and start of the physical intervention program, while the reduction of γδ T cells and CD8 T cells is less prominent in the RT and UC group. CONCLUSION: Chemotherapy led to a decrease in nearly all measured immune cells. In this study, physical intervention with endurance or resistance training did not suppress cellular immunity any further. Larger multicenter trials are needed to evaluate the exact impact of sports intervention on immune cell subpopulations.

TLR-induced immunomodulatory cytokine expression by human gingival stem/progenitor cells.

During therapeutic application, mesenchymal stem cells (MSCs) may interact with their environment via their expressed toll-like-receptors (TLRs) leading to pro- or anti-inflammatory immune responses. The present study aimed to describe the gingival margin-derived stem/progenitor cells' (G-MSCs) TLR-induced immune regulatory response to specific TLR agonists. Gingival cells were obtained, immunomagnetically sorted via anti-STRO-1 antibodies and seeded out to achieve colony forming units (CFUs). G-MSCs were investigated for stem cell characteristics and TLR expression. Specific TLR agonists were applied and m-RNA expression of pro- and anti-inflammatory factors was analyzed via real-time polymerase chain reaction. G-MSCs showed all characteristics of stem/progenitor cells. All TLR agonists induced pro-inflammatory cytokines, except for the TLR3 agonist, which significantly promoted the anti-inflammatory response. (p⩽0.05, Wilcoxon-Signed-Ranks-Test). TLR-induced immunomodulation by G-MSCs could impact their therapeutic potential in vivo. Two distinctive pro-inflammatory and an anti-inflammatory TLR-induced phenotypes of G-MSCs become noticeable in this study.

Monitoring and functional characterization of the lymphocytic compartment in pancreatic ductal adenocarcinoma patients.

BACKGROUND/OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) still has a poor prognosis and current treatments including immunotherapy often fail. This might be due to the pronounced immunosuppressive milieu impairing infiltration and function of immune effector cells. This study aimed at a comprehensive analysis of immune cells in PDAC patients by determining absolute and relative peripheral blood cell numbers of immune cell subsets along with their functional capacity. METHODS: Whole blood cells or isolated peripheral blood mononuclear cells were characterized by flow cytometry. PDAC tissues were analyzed by immunohistochemistry. Anti-tumor activity of immune effector cells was determined by RTCA system. RESULTS: Our data demonstrate that relative CD4+ memory- and regulatory T cell numbers were enhanced, whereas determination of absolute cell numbers revealed generally lower immune cell numbers in PDAC patients compared to healthy controls. γδ T cells accumulated at higher numbers compared to αß T cells in the malignant ductal epithelium of PDAC tissues indicating that γδ T cells infiltrate into the tumor. Cytotoxicity against tumor cells of even small numbers of T- and NK cells could be induced by a bispecific antibody targeting CD3+ T cells to human epidermal growth factor receptor (HER)2 expressing PDAC cells or Trastuzumab. Importantly, a critical number of γδ T cells was required for significant tumor cell killing by a bispecific antibody engaging the γδ T cell receptor on γδ T cells and HER2 on tumor cells. CONCLUSION: Monitoring immune cells along with the determination of their functional capacity provides a comprehensive assessment as a prerequisite for a personalized immunotherapeutic PDAC treatment.

Dyrk1a regulates the cardiomyocyte cell cycle via D-cyclin-dependent Rb/E2f-signalling.

AIMS: Down syndrome-associated dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A) is a ubiquitously expressed protein kinase. Up to date a variety of targets have been identified, establishing a key role for Dyrk1a in selected signalling pathways. In cardiomyocytes, Dyrk1a acts as a negative regulator of hypertrophy by phosphorylating transcription factors of the NFAT family, but its mechanistic function in the heart remains poorly understood. This study was designed to investigate a potential protective role of Dyrk1a in cardiac hypertrophy in vivo. METHODS AND RESULTS: We generated transgenic mice with cardiac-specific overexpression of Dyrk1a. Counterintuitively, these mice developed severe dilated cardiomyopathy associated with congestive heart failure and premature death. In search for the cause of this unexpected phenotype, we found that Dyrk1a interacts with all members of the D-cyclin family and represses their protein levels in vitro and in vivo. Particularly, forced expression of Dyrk1a leads to increased phosphorylation of Ccnd2 on Thr280 and promotes its subsequent proteasomal degradation. Accordingly, cardiomyocytes overexpressing Dyrk1a display hypo-phosphorylated Rb1, suppression of Rb/E2f-signalling, and reduced expression of E2f-target genes, which ultimately results in impaired cell cycle progression. CONCLUSIONS: We identified Dyrk1a as a novel negative regulator of D-cyclin-mediated Rb/E2f-signalling. As dysregulation of this pathway with impaired cardiomyocyte proliferation leads to cardiomyopathy, dose-specific Dyrk1a expression and activity appears to be critical for the hyperplastic and hypertrophic growth of the developing heart.

TLR expression profile of human gingival margin-derived stem progenitor cells

BACKGROUND: Gingival margin-derived stem/progenitor cells (G-MSCs) show remarkable periodontal regenerative potential in vivo. During regeneration, G-MSCs may interact with their inflammatory environment via toll-likereceptors (TLRs). The present study aimed to depict the G-MSCs TLRs expression profile. MATERIAL AND METHODS: Cells were isolated from free gingival margins, STRO-1-immunomagnetically sorted and seeded to obtain single colony forming units (CFUs). G-MSCs were characterized for CD14, CD34, CD45, CD73, CD90, CD105, CD146 and STRO-1 expression, and for multilineage differentiation potential. Following G-MSCsˇŻ incubation in basic or inflammatory medium (IL-1¦Â, IFN-¦Ă, IFN-¦Á, TNF-¦Á) a TLR expression profile was generated. RESULTS: G-MSCs showed all stem/progenitor cellsˇŻ characteristics. In basic medium G-MSCs expressed TLRs 1, 2, 3, 4, 5, 6, 7, and 10. The inflammatory medium significantly up-regulated TLRs 1, 2, 4, 5, 7 and 10 and diminished TLR 6 (pˇÜ0.05, Wilcoxon-Signed-Ranks-Test). CONCLUSIONS: The current study describes for the first time the distinctive TLRs expression profile of G-MSCs under uninflamed and inflamed conditions

TlR expression profile of human gingival margin-derived stem progenitor cells.

BACKGROUND: Gingival margin-derived stem/progenitor cells (G-MSCs) show remarkable periodontal regenerative potential in vivo. During regeneration, G-MSCs may interact with their inflammatory environment via toll-like-receptors (TLRs). The present study aimed to depict the G-MSCs TLRs expression profile. MATERIAL AND METHODS: Cells were isolated from free gingival margins, STRO-1-immunomagnetically sorted and seeded to obtain single colony forming units (CFUs). G-MSCs were characterized for CD14, CD34, CD45, CD73, CD90, CD105, CD146 and STRO-1 expression, and for multilineage differentiation potential. Following G-MSCs' incubation in basic or inflammatory medium (IL-1ß, IFN-γ, IFN-α, TNF-α) a TLR expression profile was generated. RESULTS: G-MSCs showed all stem/progenitor cells' characteristics. In basic medium G-MSCs expressed TLRs 1, 2, 3, 4, 5, 6, 7, and 10. The inflammatory medium significantly up-regulated TLRs 1, 2, 4, 5, 7 and 10 and diminished TLR 6 (p≤0.05, Wilcoxon-Signed-Ranks-Test). CONCLUSIONS: The current study describes for the first time the distinctive TLRs expression profile of G-MSCs under uninflamed and inflamed conditions.

Novel bispecific antibodies increase γδ T-cell cytotoxicity against pancreatic cancer cells.

The ability of human γδ T cells from healthy donors to kill pancreatic ductal adenocarcinoma (PDAC) in vitro and in vivo in immunocompromised mice requires the addition of γδ T-cell-stimulating antigens. In this study, we demonstrate that γδ T cells isolated from patients with PDAC tumor infiltrates lyse pancreatic tumor cells after selective stimulation with phosphorylated antigens. We determined the absolute numbers of γδ T-cell subsets in patient whole blood and applied a real-time cell analyzer to measure their cytotoxic effector function over prolonged time periods. Because phosphorylated antigens did not optimally enhance γδ T-cell cytotoxicity, we designed bispecific antibodies that bind CD3 or Vγ9 on γδ T cells and Her2/neu (ERBB2) expressed by pancreatic tumor cells. Both antibodies enhanced γδ T-cell cytotoxicity with the Her2/Vγ9 antibody also selectively enhancing release of granzyme B and perforin. Supporting these observations, adoptive transfer of γδ T cells with the Her2/Vγ9 antibody reduced growth of pancreatic tumors grafted into SCID-Beige immunocompromised mice. Taken together, our results show how bispecific antibodies that selectively recruit γδ T cells to tumor antigens expressed by cancer cells illustrate the tractable use of endogenous γδ T cells for immunotherapy.

Monitoring Circulating γδ T Cells in Cancer Patients to Optimize γδ T Cell-Based Immunotherapy.

The success of γδ T cell-based immunotherapy, where the cytotoxic activity of circulating γδ T lymphocytes is activated by nitrogen-containing bisphosphonates (n-BP), or possibly by bispecific antibodies or the combination of both, requires a profound knowledge of patients' γδ T cells. A possible influence of radio- or chemotherapy on γδ T cells as well as their reported exhaustion after repetitive treatment with n-BP or their lack of response to various cancers can be easily determined by the monitoring assays described in this perspective article. Monitoring the absolute cell numbers of circulating γδ T cell subpopulations in small volumes of whole blood from cancer patients and determining γδ T cell cytotoxicity using the Real-Time Cell Analyzer can give a more comprehensive assessment of a personalized tumor treatment. Possible future directions such as the combined usage of n-BP or phosphorylated antigens together with bispecific antibodies that selectively target γδ T cells to tumor-associated antigens, will be discussed. Such strategies induce expansion and enhance γδ T cell cytotoxicity and might possibly avoid their exhaustion and overcome the immunosuppressive tumor microenvironment.

18:1/18:1-Dioleoyl-phosphatidylglycerol prevents alveolar epithelial apoptosis and profibrotic stimulus in a neonatal piglet model of acute respiratory distress syndrome.

BACKGROUND: 18:1/18:1-Dioleoyl-phosphatidylgycerol (DOPG) is a surfactant phospholipid that is nearly non-detectable in neonatal surfactant films. When alveolar macrophages are exposed to DOPG in vitro, secretory phospholipase A2 (sPLA2) production is blocked, resulting in suppressed macrophage activity and improved surfactant function. We investigated whether the addition of DOPG to a commercially available surfactant preparation would improve lung function in a neonatal piglet model of acute respiratory distress syndrome. MATERIALS AND METHODS: Respiratory failure was achieved by triple-hit lung injury (repeated broncho-alveolar lavage, injurious ventilation, tracheal lipopolysaccharide instillation, each intervention 24 h apart) in twenty-four domestic piglets aged 2-6 days and subject to mechanical ventilation. Following each lung injury protocol the piglets were treated with surfactant alone or with surfactant + DOPG. RESULTS: Within 72 h of mechanical ventilation, we observed significantly improved gas exchange (oxygenation and ventilation), lung mechanics (compliance and resistance of the respiratory system), and pulmonary oedema (extra-vascular lung water index) in the surfactant + DOPG group. This favourable clinical effect could be attributed to improved surfactant function, reduced sPLA2 secretion, inhibition of macrophage migration, reduced alveolar epithelial apoptosis, and suppression of amphiregulin and TGF-ß1 expression in pulmonary tissues as a prerequisite for fibrous lung repair. CONCLUSIONS: We conclude that surfactant fortified by DOPG preserves lung function, and prevents alveolar epithelial injury and fibrous stimulus by reduction of sPLA2 in a neonatal model of acute respiratory distress syndrome without any relevant discernable side effects. Hence, DOPG supplementation in a neonatal lung exerts important function protecting effects and seems to be justified in cases of overwhelming pulmonary inflammation.

Vδ2 T cell deficiency in granulomatosis with polyangiitis (Wegener's granulomatosis).

Previous studies have characterized phenotypic and functional alterations within T-cell receptor αß-expressing T cells in patients with granulomatosis with polyangiitis (GPA). We analyzed the frequency, subset composition and in vitro activation of blood γδ T cells in GPA patients. We observed a significant reduction of γδ T cell numbers, due to the selective depletion of the Vδ2 subset which remained consistent over time upon repeated analysis. The loss of Vδ2 T cells was not due to migration into the inflamed lesions as very few γδ T cells were detected in inflammatory infiltrates. The memory subset distribution did not differ among Vδ2 T cells from healthy donors and GPA patients. Importantly, the remaining Vδ2 T cells were capable of responding to phosphoantigen stimulation in vitro. The marked depletion of blood Vδ2 T cells in GPA suggests cellular exhaustion, possibly due to chronic exposure to and continuous overstimulation by microbial phosphoantigens.

Down-regulation of the cancer/testis antigen 45 (CT45) is associated with altered tumor cell morphology, adhesion and migration.

BACKGROUND: Due to their restricted expression in male germ cells and certain tumors, cancer/testis (CT) antigens are regarded as promising targets for tumor therapy. CT45 is a recently identified nuclear CT antigen that was associated with a severe disease score in Hodgkin's lymphoma and poor prognosis in multiple myeloma. As for many CT antigens, the biological function of CT45 in developing germ cells and in tumor cells is largely unknown. METHODS: CT45 expression was down-regulated in CT45-positive Hodgkin's lymphoma (L428), fibrosarcoma (HT1080) and myeloma (U266B1) cells using RNA interference. An efficient CT45 knock-down was confirmed by immunofluorescence staining and/or Western blotting. These cellular systems allowed us to analyze the impact of CT45 down-regulation on proliferation, cell cycle progression, morphology, adhesion, migration and invasive capacity of tumor cells. RESULTS: Reduced levels of CT45 did not coincide with changes in cell cycle progression or proliferation. However, we observed alterations in cell adherence, morphology and migration/invasion after CT45 down-regulation. Significant changes in the distribution of cytoskeleton-associated proteins were detected by confocal imaging. Changes in cell adherence were recorded in real-time using the xCelligence system with control and siRNA-treated cells. Altered migratory and invasive capacity of CT45 siRNA-treated cells were visualized in 3D migration and invasion assays. Moreover, we found that CT45 down-regulation altered the level of the heterogeneous nuclear ribonucleoprotein syncrip (hnRNP-Q1) which is known to be involved in the control of focal adhesion formation and cell motility. CONCLUSIONS: Providing first evidence of a cell biological function of CT45, we suggest that this cancer/testis antigen is involved in the modulation of cell morphology, cell adherence and cell motility. Enhanced motility and/or invasiveness of CT45-positive cells could contribute to the more severe disease progression that is correlated to CT45-positivity in several malignancies.

Aurora kinase inhibitor AZD1152 has an additional effect of platinum on a sequential application at the human ovarian cancer cell line SKOV3.

PURPOSE: The treatment of ovarian tumors is carried out with platinum medicine which can lead to incompatibilities or resistances. Thus, it is of great interest to check new medicine suitability for its application. AZD1152 is an Aurora kinase inhibitor predominantly works against Aurora kinase B involved in the chromosome segregation. Cells become polyploidy and reduce the proliferation by this impairment. To investigate whether AZD1152, may play a role in the treatment of ovarian carcinoma we serving it to the cisplatinum-resistant cell line SKOV3 alone and in combination with platinum. METHODS: We look at the proliferation, the ploidy, the phases of cell cycle and the apoptosis activity of the cells. RESULTS AND CONCLUSION: We could show that the combination of both medicines in the preclinical experiment produces a working advantage.

Recurrent mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcriptome sequencing.

Burkitt lymphoma is a mature aggressive B-cell lymphoma derived from germinal center B cells. Its cytogenetic hallmark is the Burkitt translocation t(8;14)(q24;q32) and its variants, which juxtapose the MYC oncogene with one of the three immunoglobulin loci. Consequently, MYC is deregulated, resulting in massive perturbation of gene expression. Nevertheless, MYC deregulation alone seems not to be sufficient to drive Burkitt lymphomagenesis. By whole-genome, whole-exome and transcriptome sequencing of four prototypical Burkitt lymphomas with immunoglobulin gene (IG)-MYC translocation, we identified seven recurrently mutated genes. One of these genes, ID3, mapped to a region of focal homozygous loss in Burkitt lymphoma. In an extended cohort, 36 of 53 molecularly defined Burkitt lymphomas (68%) carried potentially damaging mutations of ID3. These were strongly enriched at somatic hypermutation motifs. Only 6 of 47 other B-cell lymphomas with the IG-MYC translocation (13%) carried ID3 mutations. These findings suggest that cooperation between ID3 inactivation and IG-MYC translocation is a hallmark of Burkitt lymphomagenesis.

Topical application of phosphatidyl-inositol-3,5-bisphosphate for acute lung injury in neonatal swine.

Hypoxemic respiratory failure of the neonatal organism involves increased acid sphingomyelinase (aSMase) activity and production of ceramide, a second messenger of a pro-inflammatory pathway that promotes increased vascular permeability, surfactant alterations and alveolar epithelial apoptosis. We comparatively assessed the benefits of topical aSMase inhibition by either imipramine (Imi) or phosphatidylinositol-3,5-bisphosphate (PIP2) when administered into the airways together with surfactant (S) for fortification. In this translational study, a triple-hit acute lung injury model was used that entails repeated airway lavage, injurious ventilation and tracheal lipopolysaccharide instillation in newborn piglets subject to mechanical ventilation for 72 hrs. After randomization, we administered an air bolus (control), S, S+Imi, or S+PIP2. Only in the latter two groups we observed significantly improved oxygenation and ventilation, dynamic compliance and pulmonary oedema. S+Imi caused systemic aSMase suppression and ceramide reduction, whereas the S+PIP2 effect remained compartmentalized in the airways because of the molecule's bulky structure. The surfactant surface tensions improved by S+Imi and S+PIP2 interventions, but only to a minor extent by S alone. S+PIP2 inhibited the migration of monocyte-derived macrophages and granulocytes into airways by the reduction of CD14/CD18 expression on cell membranes and the expression of epidermal growth factors (amphiregulin and TGF-ß1) and interleukin-6 as pro-fibrotic factors. Finally we observed reduced alveolar epithelial apoptosis, which was most apparent in S+PIP2 lungs. Exogenous surfactant "fortified" by PIP2, a naturally occurring surfactant component, improves lung function by topical suppression of aSMase, providing a potential treatment concept for neonates with hypoxemic respiratory failure.

poly(I:C) costimulation induces a stronger antiviral chemokine and granzyme B release in human CD4 T cells than CD28 costimulation.

dsRNA is frequently associated with viral replication. Here, we compared the costimulatory effect of the synthetic analog of dsRNA, poly(I:C), and the agonistic anti-CD28 mAb on anti-CD3 mAb-activated, freshly isolated human CD4 T cells. We tested the hyphothesis that poly(I:C) and anti-CD28 mAb costimulation differ in their effect on the CD4 T cell immune response. Our study shows that costimulation of CD4 T cells by poly(I:C) enhanced CD3-induced production of IP-10, MIP1-α/ß, RANTES, and granzyme B involved in antiviral activity more than anti-CD28 mAb. poly(I:C) stimulation, on its own, activated the transcription of IRF7 in human CD4 T cells. Combined CD3 and poly(I:C) stimulation significantly enhanced the transcription of IRF7 and additionally, NF-κBp65 phosphorylation, which might be involved in the induction of antiviral chemokines and the enhanced cytotoxic activity of poly(I:C)-treated CD4 T cells. In comparison with poly(I:C), anti-CD28 mAb as a costimulus induced a stronger proinflammatory response, as indicated by enhanced TNF-α secretion. poly(I:C) had a costimulatory effect on Akt phosphorylation, whereas anti-CD28 mAb only slightly enhanced Akt phosphorylation. In contrast to poly(I:C), anti-CD28 mAb was essential for proliferation of anti-CD3-stimulated CD4 T cells; however, poly(I:C) further increased the anti-CD28/anti-CD3-mediated proliferation. These results indicate that poly(I:C)- and anti-CD28 mAb-induced signaling differ in their costimulatory effect on the CD3-driven, antiviral chemokine release and proinflammatory cytokine secretion in freshly isolated human CD4 T cells.

Inositol-trisphosphate reduces alveolar apoptosis and pulmonary edema in neonatal lung injury.

D-myo-inositol-1,2,6-trisphosphate (IP3) is an isomer of the naturally occurring second messenger D-myo-inositol-1,4,5-trisphosphate, and exerts anti-inflammatory and antiedematous effects in the lung. Myo-inositol (Inos) is a component of IP3, and is thought to play an important role in the prevention of neonatal pulmonary diseases such as bronchopulmonary dysplasia and neonatal acute lung injury (nALI). Inflammatory lung diseases are characterized by augmented acid sphingomyelinase (aSMase) activity leading to ceramide production, a pathway that promotes increased vascular permeability, apoptosis, and surfactant alterations. A novel, clinically relevant triple-hit model of nALI was developed, consisting of repeated airway lavage, injurious ventilation, and lipopolysaccharide instillation into the airways, every 24 hours. Thirty-five piglets were randomized to one of four treatment protocols: control (no intervention), surfactant alone, surfactant + Inos, and surfactant + IP3. After 72 hours of mechanical ventilation, lungs were excised from the thorax for subsequent analyses. Clinically, oxygenation and ventilation improved, and extravascular lung water decreased significantly with the S + IP3 intervention. In pulmonary tissue, we observed decreased aSMase activity and ceramide concentrations, decreased caspase-8 concentrations, reduced alveolar epithelial apoptosis, the reduced expression of interleukin-6, transforming growth factor-ß1, and amphiregulin (an epithelial growth factor), reduced migration of blood-borne cells and particularly of CD14(+)/18(+) cells (macrophages) into the airspaces, and lower surfactant surface tensions in S + IP3-treated but not in S + Inos-treated piglets. We conclude that the admixture of IP3 to surfactant, but not of Inos, improves gas exchange and edema in our nALI model by the suppression of the governing enzyme aSMase, and that this treatment deserves clinical evaluation.

Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes.

We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression. The active forms of all three enzymes, caspase-8, caspase-7 and A-SMase, but not caspase-3, colocalize in internalized TNF receptosomes. While caspase-8 and caspase-3 are unable to induce activation of purified pro-A-SMase, we found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72-kDa pro-A-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule. Caspase-7 down modulation revealed the functional link between caspase-7 and A-SMase, confirming proteolytic cleavage as one further mode of A-SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase.

Novel splice variants of human IKKε negatively regulate IKKε-induced IRF3 and NF-kB activation.

The inhibitor of κB kinase ε (IKKε) is pivotal for an efficient innate immune response to viral infections and has been recognized as breast cancer oncogene. The antiviral function of IKKε involves activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-κB, thus inducing the expression of type I IFN. Here, we have identified two novel splice variants of human IKKε, designated IKKε-sv1 and IKKε-sv2, respectively. Interestingly, RT-PCR revealed quantitatively different isoform expression in PBMC from different individuals. Moreover, we found cell type- and stimulus-specific protein expression of the various splice variants. Overexpression of full-length wt IKKε (IKKε-wt) leads to the activation of NF-κB- as well as IRF3-driven luciferase reporter genes. Although none of the splice variants activates IRF3, IKKε-sv1 still activates NF-κB, whereas IKKε-sv2 is also defective in NF-κB activation. Both splice variants form dimers with IKKε-wt and inhibit IKKε-wt-induced IRF3 signaling including the antiviral activity in a dominant-negative manner. The lack of IRF3 activation is likely caused by the failure of the splice variants to interact with the adapter proteins TANK, NAP1, and/or SINTBAD. Taken together, our data suggest alternative splicing as a novel regulatory mechanism suitable to shift the balance between different functions of IKKε.

gammadelta T-cells: basic features and potential role in vasculitis.

gammadelta T-cells are a numerically small subset of T-cells with distinct features. They recognise antigens that are not seen by other immune cells. At the functional level, gammadelta T-cells share some features with alphabeta T-cells but also exert functions that are otherwise performed by specialised subsets of alphabeta T-cells (e.g. IL-17 production, regulatory activity). We discuss the potential role of gammadelta T-cells in various clinical forms of vasculitis.