PI3Kδ is essential for tumor clearance mediated by cytotoxic T lymphocytes.

BackgroundPI3Kδ is a lipid kinase of the phosphoinositide 3-kinase class 1A family and involved in early signaling events of leukocytes regulating proliferation, differentiation and survival. Currently, several inhibitors of PI3Kδ are under investigation for the treatment of hematopoietic malignancies. In contrast to the beneficial effect of inhibiting PI3Kδ in tumor cells, several studies reported the requirement of PI3Kδ for the function of immune cells, such as natural killer and T helper cells. Cytotoxic T lymphocytes (CTLs) are essential for tumor surveillance. The scope of this study is to clarify the potential impact of PI3Kδ inhibition on the function of CTLs with emphasis on tumor surveillance.Principal findingsPI3Kδ-deficient mice develop significantly bigger tumors when challenged with MC38 colon adenocarcinoma cells. This defect is accounted for by the fact that PI3Kδ controls the secretory perforin-granzyme pathway as well as the death-receptor pathway of CTL-mediated cytotoxicity, leading to severely diminished cytotoxicity against target cells in vitro and in vivo in the absence of PI3Kδ expression. PI3Kδ-deficient CTLs express low mRNA levels of important components of the cytotoxic machinery, e.g. prf1, grzmA, grzmB, fasl and trail. Accordingly, PI3Kδ-deficient tumor-infiltrating CTLs display a phenotype reminiscent of naïve T cells (CD69(low)CD62L(high)). In addition, electrophysiological capacitance measurements confirmed a fundamental degranulation defect of PI3Kδ-/- CTLs.ConclusionOur results demonstrate that CTL-mediated tumor surveillance is severely impaired in the absence of PI3Kδ and predict that impaired immunosurveillance may limit the effectiveness of PI3Kδ inhibitors in long-term treatment

CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

The transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance

Reduced expression of cytotoxic components in <i>PI3Kδ−/−</i> CTLs.

<p>WT and <i>PI3Kδ−/−</i> splenocytes were activated for 3 days with aCD3ε and cultured in T cell medium. A. mRNA expression of <i>grzmA</i> (WT: 0.097±0.036; versus <i>PI3Kδ−/−</i>: 0.014±0.01, n = 6, <i>p = 0.0003</i>), g<i>rzmB</i> (WT: 1±0.076; versus <i>PI3Kδ−/−</i>: 0.419±0.075, n = 6, <i>p<0.0001</i>) and <i>prf1</i> (WT: 0.0433±0.004; versus <i>PI3Kδ−/−</i>: 0.013±0.003, n = 6, <i>p<0.0001</i>) was quantified by qRT-PCR and normalized to the house-keeping gene g<i>apdh</i>. B. Similarly, under standard culturing conditions mRNA levels of <i>trail</i> (WT: 0.0049±0.0012; versus <i>PI3Kδ−/−</i>: 0.0026±0.0007, n = 4, <i>p = 0.0178</i>) and <i>fasl</i> (WT: 0.0138±0.0015; versus <i>PI3Kδ−/−</i>: 0.0039±0.0001, n = 4, <i>p<0.0001</i>) were measured. C. <i>Ifng</i> mRNA was quantified by qRT-PCR under standard culturing conditions (WT: 0.019±0.0005; versus <i>PI3Kδ−/−</i>: 0.004±0.001, n = 6, <i>p<0.0001</i>) and after stimulation with 5 ng/ml IL-12 for 4 h (WT: 0.241±0.06; versus <i>PI3Kδ−/−</i>: 0.087±0.016, n = 6, <i>p = 0.0002</i>). D. To quantify IFN-γ protein levels, WT and <i>PI3Kδ−/−</i> splenocytes were stimulated with ConA. After 48 h supernatants were harvested and IFN-γ release was measured by ELISA (WT: 6515±2061 pg/ml; versus <i>PI3Kδ−/−</i>: 1359±1147 pg/ml, n = 3, <i>p = 0.0193</i>). IFN-γ release of unstimulated controls of WT and <i>PI3Kδ−/−</i> splenocytes was below detection limit of the assay (<10 pg/ml). Statistics were calculated with an unpaired Student’s <i>t</i>-test, and values represent mean±SD. One out of two independently performed experiments with comparable results is shown.</p

PI3Kδ is indispensable for CTL degranulation.

<p>A. Using the whole cell patch clamp technique, the cellular capacitance of aCD3-activated and in T cell medium cultivated WT and <i>PI3Kδ−/−</i> CTLs was determined. Membrane capacitances before (ctr) and after stimulation with PMA and ionomycin (PMA/iono) at the single cell level are summarized from two representative experiments (WT: ctr: 6.3±1.2pF, PMA/iono: 9±1.8pF, n = 10, <i>p = 0.01</i>; versus <i>PI3Kδ−/−</i>: ctr: 6.7±1.7pF, PMA/iono: 6.6±1.8, n = 9, <i>p = 0.4</i>; paired <i>t</i>-test; values represent mean±SEM). B. Fold increase in membrane capacitance due to stimulation with PMA/iono is calculated (WT: 1.46±0.13, n = 10, <i>p = 0.0064</i>; versus <i>PI3Kδ−/−</i>: 0.94±0.06%, n = 9, <i>p = 0.3</i>). Additionally, WT CTLs were pre-incubated with the PI3Kδ isoform-specific inhibitor IC-87114 (1 µM, 4 µM, 1 hour), DMSO-treatment served as control (DMSO: 2.14±0.18, n = 11, <i>p<0.0001</i>; 1 µM IC-87114: 0.98±0.03, n = 11, <i>p = 0.49</i>; 4 µM IC-87114: 0.98±0.01, n = 9, <i>p = 0.18</i>; values representing mean±SEM, One sample <i>t</i>-test). C. <i>In vitro</i> cultivated WT and <i>PI3Kδ−/−</i> CTLs were challenged with PMA and ionomycin. The percentage of CD107a+ T cells was measured <i>via</i> flow cytometry before and after the stimulus (WT: ctr: 15.5±2.4%, PMA/iono: 41.2±1.3%, <i>p<0.0001</i>; versus <i>PI3Kδ−/−</i>: ctr: 24.6±2.7%, PMA/iono: 29.3±1.7%, <i>p = 0.085</i>; n = 8, paired <i>t</i>-test, values represent mean±SEM). D. Accordingly, the degranulation of WT CTLs upon pharmacological inhibition of PI3Kδ using CAL-101 was tested (ctr: 6±0.7%; +PMA/iono: DMSO: 23.3±2.4%, 0.1 µM: 18.1±2%, 0.5 µM: 16.6±2.1%, 1 µM: 13.6±1.3%, 5 µM: 11.7±1%, n≥6, values represent mean±SEM. One-Way ANOVA revealed p<0.0001, Tukey’s Post-Hoc test was significant <i>p<0.01</i> for 1 µM and highly significant <i>p<0.001</i> for 5 µM compared to DMSO control). E. WT and <i>PI3Kδ−/−</i> CTLs were cultivated in T cell medium and their expression of CD107a was measured <i>via</i> flow cytometry under basal conditions and after electrostimulation or electrostimulation plus the degranulation inhibitor Concanamycin A, respectively (WT: ctr: 11.5±1.2% CD107a+ cells, electrostimulation: 41±3.3% CD107a+ cells, <i>p = 0.0001</i>; versus <i>PI3Kδ−/−</i>: ctr: 11±1.3% CD107a+ cells, electrostimulation: 12±0.8% CD107a+ cells, <i>p = 0.54</i>, paired <i>t</i>-test, values represent mean±SEM). All experiments were performed at least two times independently and summarized in the depicted graphs.</p

Diminished reaction of <i>PI3Kδ−/−</i> CTLs to allogeneic mixed lymphocytes, but unaltered proliferation and Ca²⁺-response.

<p>A. WT and <i>PI3Kδ−/−</i> splenocytes were CFSE-labeled and cultured in the absence and presence of allogeneic (BALB/c), mitomycin C-treated splenocytes. At the indicated time points, cells were harvested and proliferation of responding CTLs was assessed by flow cytometry. Percentages of proliferating CFSE+CD8+ T cells with and without the stimulus of mixed lymphocytes are illustrated. Proliferating CD8+ T cells were discriminated from undivided T cells by the reduced levels of CFSE in daughter cells. B. WT splenocytes were CFSE-labeled and cultivated in analogy to A. Pharmacological inhibition of PI3Kδ was achieved by treatment with indicated concentrations of CAL-101 during the experimental procedure. DMSO-treatment served as negative control. C. Proliferation of WT and <i>PI3Kδ−/−</i> CTLs in response to aCD3ε treatment was assessed in a CFSE proliferation assay. D. Proliferation of WT and <i>PI3Kδ−/−</i> aCD3-activated T cells was assessed under standard T cell medium conditions (in the presence of IL-2) and after deprivation from IL-2 by performing an [³H]-thymidine incorporation assay over 48 hours (with IL-2: WT: 12097±491cpm; versus <i>PI3Kδ−/−</i>: 12413±501cpm; without IL-2: WT: 1392±381cpm; versus <i>PI3Kδ−/−</i>: 1140±160cpm, n = 6, values represent mean±SD). E., F. WT and <i>PI3Kδ−/−</i> splenocytes were stained with 1 µM Indo-1 AM. Ca²⁺ flux in response to aCD3ε followed by crosslinking with streptavidin (E) or thapsigargin (F) was measured in CD8+ T cells using flow cytometry. Treatment with ionomycin served as positive control. Three independent experiments were carried out and one representative experiment is shown, respectively.</p

Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice

Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage

Adaptive pathways: possible next steps for payers in preparation for their potential implementation

Medicines receiving a conditional marketing authorization through Medicines Adaptive Pathways to Patients (MAPPs) will be a challenge for payers. The “introduction” of MAPPs is already seen by the European Medicines Agency (EMA) as a fait accompli, with payers not consulted or involved. However, once medicines are approved through MAPPs, they will be evaluated for funding by payers through different activities. These include Health Technology Assessment (HTA) with often immature clinical data and high uncertainty, financial considerations, and negotiations through different types of agreements, which can require monitoring post launch. Payers have experience with new medicines approved through conditional approval, and the fact that MAPPs present additional challenges is a concern from their perspective. There may be some activities where payers can collaborate. The final decisions on whether to reimburse a new medicine via MAPPs will have more variation than for medicines licensed via conventional processes. This is due not only to increasing uncertainty associated with medicines authorized through MAPPs but also differences in legal frameworks between member states. Moreover, if the financial and side-effect burden from the period of conditional approval until granting full marketing authorization is shifted to the post-authorization phase, payers may have to bear such burdens. Collection of robust data during routine clinical use is challenging along with high prices for new medicines during data collection. This paper presents the concept of MAPPs and possible challenges. Concerns and potential ways forward are discussed and a number of recommendations are presented from the perspective of payers