Lack of Functional P110δ Affects Expression of Activation Marker CD80 but Does Not Influence Functions of Neutrophils.

The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated Tyr-Xaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr --> Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.

Expression of activation markers on basophils in a controlled model of anaphylaxis

Thrombopoietin (TPO) stimulates a network of intracellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryocytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Czeta (PKCzeta) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCzeta and PI3K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCzeta in steady-state megakaryopoiesis.

Lisocabtagene maraleucel (liso-cel) is an investigational drug product composed of autologous CD8+ and CD4+ T cells expressing a CD19-specific chimeric antigen receptor (CAR), being evaluated in clinical trials for treatment of B cell malignancies. The CELMoD CC-122 is being evaluated in non-Hodgkin lymphoma (NHL) and can augment T cell function through Cereblon-mediated degradation of Ikaros and Aiolos. Combination approaches aimed at enhancing CAR T cell function may increase the rate, depth, and durability of clinical responses. Here we report pre-clinical results supporting the potential combination of CC-122 an liso-cel. To determine potentiating effects of CC-122 following acute activation (anti-idiotypic Ab or CD19+ targets), T cells expressing the CAR of liso-cel were cultured with CC-122 and assessed for intracellular signaling, cytolytic activity, cytokine production, and activation marker expression. CC-122 significantly increased NFκB, Nur77, and STAT5 as well as effector cytokine production and activation marker expression. Activation-induced exhaustion of CAR T may decrease durable responses in patients. We developed a chronic stimulation assay to render CAR T cells hypofunctional (reduced cytolysis and IL-2 secretion) and examined whether enhanced activation mediated by CC-122 would exhaust the CD19-directed CAR T cells more rapidly. CC-122 was added in one of two regimens: during chronic stimulation (“concurrent”) or during re-challenge (“rescue”). Effects on exhaustion in “concurrent” or “rescue” cultures were evaluated, including by RNAseq and measuring of effector function in re-challenge assays with CD19+ targets. Surprisingly, “concurrent” CC-122 partially reversed a gene signature associated with CAR T cell hypofunctionality and preserved more effector function relative to controls. In “rescue” assays, CC-122 dose-dependently increased cytokine production and restored cytolytic function of exhausted CAR T cells upon re-challenge with CD19+ tumor spheroids. In a disseminated tumor xenograft model, concurrent or delayed dosing of CC-122, in combination with two sub-curative doses of liso-cel, significantly increased median survival by 30 to 40 days. In some cases, survival endpoints were not reached with the combination. Taken together, CC-122 treatment of CD19-directed CAR T cells was shown to enhance CAR T activation in acute stimulation assays; limit CAR T exhaustion onset during chronic stimulation as assessed by gene expression; and reverse an exhausted, hypofunctional CAR T state induced by chronic stimulation. The data suggest that liso-cel pharmacologic activity may both be enhanced and extended by this combinatorial approach. These findings support an ongoing clinical investigation evaluating the addition of CC-122 to liso-cel in patients with R/R, aggressive NHL (NCT03310619). Citation Format: Heidi K. Jessup, Evan P. Thomas, Jim S. Qin, Yue Jiang, Oleksandr Baturevych, Neha Soni, Aye T. Chen, Ruth A. Salmon, Melissa G. Works, Michael O. Ports. Avadomide (CC-122) increases effector function and reverses exhaustion in chronically stimulated lisocabtagene maraleucel anti-CD19 CAR T drug product [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2320.

Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers

BackgroundTityus serrulatus scorpion venom (TsV) contains toxins that act on K+ and Na+ channels and account for the venom’s toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells.MethodsCytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations.ResultsTsV at concentrations of 25 to 100 μg/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4+ and CD8+ T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8+ CD25+ T lymphocyte subset. TsV alone, at 50 and 100 μg/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses.ConclusionsTsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.Electronic supplementary materialThe online version of this article (doi:10.1186/s40409-015-0046-3) contains supplementary material, which is available to authorized users.

Natural killer (NK) cells express an activating receptor, 2B4, that enhances cellular cytotoxicity. Upon NK cell activation by ligation of 2B4, the intracellular domain of 2B4 associates with the X-linked lymphoproliferative disease (XLP) gene product, signaling lymphocytic activation molecule-associated protein/SH2D1A (SAP/SH2D1A). Defective intracellular association of 2B4 with mutated SAP/SH2D1A is likely to underlie the defects in cytotoxicity observed in NK cells from patients with XLP. We report here a role for phosphoinositide 3-kinase (PI3K) in the recruitment and association of SAP/SH2D1A to 2B4 in human NK cells. The activation of normal NK cells by ligation of 2B4 leads to the phosphorylation of 2B4, recruitment of SAP/SH2D1A, and association of the p85 regulatory subunit of PI3K. The inhibition of PI3K enzymatic activity with either wortmannin or LY294002 prior to 2B4 ligation does not alter the association of 2B4 with the p85 subunit but prevents the recruitment of SAP/SH2D1A to 2B4. In addition, PI3K inhibitors significantly diminish the cytotoxic function of primary NK cells. This observed inhibition of cytotoxicity, present in normal NK cells, was less apparent or absent in NK cells derived from a patient with XLP. These data indicate that the cytotoxicity of activated NK cells is mediated by the association of 2B4 and SAP/SH2D1A, and that this association is dependent upon the activity of PI3K.

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin alpha(IIb)beta(3), necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin alpha(IIb)beta(3) activation (affinity modulation) primarily occurs downstream of G(i)-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin alpha(IIb)beta(3) bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110 beta isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin alpha(IIb)beta(3) activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin alpha(IIb)beta(3) association with the contractile cytoskeleton. Analysis of integrin alpha(IIb)beta(3) adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin alpha(IIb)beta(3) bonds. These studies demonstrate an important role for PI3K p110 beta in regulating the avidity of high affinity integrin alpha(IIb)beta(3) receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110 beta inhibitors.

Allele variants in the L-carnitine (LCAR) transporters OCTN1 (SLC22A4, 1672 C --> T) and OCTN2 (SLC22A5, -207 G --> C) have been implicated in susceptibility to Crohn's disease (CD). LCAR is consumed in the diet and transported actively from the intestinal lumen via the organic cation transporter OCTN2. While recognized mainly for its role in fatty acid metabolism, several lines of evidence suggest that LCAR may also display immunosuppressive properties. This study sought to investigate the immunomodulatory capacity of LCAR on antigen-presenting cell (APC) and CD4+ T cell function by examining cytokine production and the expression of activation markers in LCAR-supplemented and deficient cell culture systems. The therapeutic efficacy of its systemic administration was then evaluated during the establishment of colonic inflammation in vivo. LCAR treatment significantly inhibited both APC and CD4+ T cell function, as assessed by the expression of classical activation markers, proliferation and cytokine production. Carnitine deficiency resulted in the hyperactivation of CD4+ T cells and enhanced cytokine production. In vivo, protection from trinitrobenzene sulphonic acid colitis was observed in LCAR-treated mice and was attributed to the abrogation of both innate [interleukin (IL)-1beta and IL-6 production] and adaptive (T cell proliferation in draining lymph nodes) immune responses. LCAR therapy may therefore represent a novel alternative therapeutic strategy and highlights the role of diet in CD.

Influence of Lenalidomide Treatment on Immune Effector Cells From High-Risk Smoldering Multiple Myeloma (SMM) Patients,

Viral infections with cytomegalovirus (CMV) or human adenovirus (HAdV) after stem cell transplantation are still associated with a high morbidity and mortality. Transfer of T-cell immunity from a healthy individual to a stem cell transplant recipient, known as adoptive T-cell transfer, has been shown to be effective to prevent viral complications. Treatment efficacy will depend on the availability of functional T-cell lines with a strong T(helper)1 response. Ex vivo isolation of antigen-specific T cells could be performed on the basis of the cytokine capture technique or antigen-induced expression of activation markers. In this study, we compare the specificity, expansion/differentiation potential, and T(helper)1 response against CMV and HAdV after different isolation strategies. Antigen-specific T cells from healthy donors were isolated by antigen-induced expression of IFN-γ and/or CD137 after stimulation with the viral antigens hexon (HAdV) or pp65 (CMV). Isolation of antigen-specific T cells based on the expression of activation markers is feasible and less time consuming, but in contrast to isolation based on IFN-γ secretion, it leads to a reduction of T(helper)1 cells. Both isolated CD137(+) and isolated IFN-γ(+) T cells mainly consist of CD4(+) T(CentralMemory) and T(EffectorMemory) cells with high expansion potential and effective cytokine production. CD154(+) is mainly expressed on CD4(+)T cells and shows coexpression with IFN-γ on activated T cells, which cannot be found for CD137(+) cells. In conclusion, T-cell lines could be easily generated on the basis of IFN-γ(+) and/or expression of the activation marker CD137 but both approaches result in different T-cell populations, which may lead to divergent T-cell responses in vivo.

Heart failure affects approximately 1-3% of Western society. There is currently no cure and treatments largely delay disease progression. Consequently, there is great interest in identifying strategies that can improve cardiac function and reverse some of the negative consequences associated with heart failure. This thesis investigates the cardioprotective properties of a gene activated in the athlete’s heart [phosphoinositide 3-kinase (PI3K), p110α] in a setting of heart failure. Two double-transgenic mouse models were generated to assess the role of PI3K in a setting of cardiac stress (dilated cardiomyopathy, DCM). Mice either expressing a constitutively active mutant of PI3K (p110α) (caPI3K) or a dominant negative mutant of PI3K (p110α) (dnPI3K) were crossed with a transgenic mouse model of DCM [due to over-expression of mammalian sterile 20-like kinase 1 (Mst1)]. Increasing PI3K activity in the DCM model (caPI3K-Mst1) improved lifespan and cardiac function, whereas decreasing PI3K activity in the DCM model (dnPI3K-Mst1) had an adverse effect. The cardioprotective properties of PI3K (p110α) were mediated, at least in part, by the kinase Akt. Using the dnPI3K-Mst1 model, I was able to show that reduced PI3K (p110α) activity increases the heart’s susceptibility to atrial fibrillation (AF, the most common arrhythmia in cardiology departments worldwide). dnPI3K-Mst1 mice displayed overt atrial remodelling, varying degrees of conduction blockade and developed spontaneous AF. To assess a possible link between PI3K activity and AF in humans, PI3K (p110α) activity was measured in atrial appendages of patients with AF (acute or chronic) and compared to patients without AF. PI3K (p110α) activity was lower in patients with AF compared to patients in sinus rhythm. These results suggest that reduced PI3K (p110α) makes the heart more susceptible to the development of AF. Thus, strategies or agents that can activate PI3K (p110α) specifically in the heart may represent a useful therapeutic approach for AF. An unanticipated but novel finding was the observation that female dnPI3K-Mst1 mice showed faster disease progression than males. Prior to menopause, females are normally protected against cardiovascular disease compared with males. In contrast, in settings of aging, diabetes or hypertension [associated with depressed or defective PI3K (p110α) activity] females are more prone to cardiac disease than males. Taken together with my results, this suggests that there may be an interaction between PI3K (p110α) and estrogen, and that this interaction is essential for the cardioprotection seen in pre-menopausal women. Data obtained from dnPI3K-Mst1 mice suggests that PI3K (p110α) plays an important role in mediating cardioprotection in females. Unexpectedly, ovariectomy had a beneficial effect on the cardiac phenotype of Mst1 mice, but no significant effect in caPI3K-Mst1 or dnPI3K-Mst1 mice. The mechanisms responsible for these phenotypes will require further investigation. In summary, this thesis presents compelling evidence to support investigation into therapeutics that activate components of the PI3K (p110α) signalling pathway in a setting of cardiac stress.

A novel role for hepatic stellate cells in pathogenesis of visceral leishmaniasis.

Adipose tissue, adipokines, and inflammation

GoalTo evaluate the acute impact of a nutraceutical blend on immune surveillance.Study designA randomized, double-blind, placebo-controlled, cross-over trial was conducted in 11 healthy subjects. Blood samples were taken immediately before and at 1, 2, and 3 hours after consuming placebo or 500 mg of UP360, which is a blend of botanicals from Aloe vera, Poria cocos, and rosemary (APR extract). Immunophenotyping and flow cytometry quantified numbers of monocytes, NK cells, NKT cells, CD8+ cytotoxic T cells, γδT cells, and total T cells, and expression of CD25 and CD69 activation markers. Plasma was tested for cytokines, chemokines, growth factors, and enzymatic activity of superoxide dismutase and catalase.ResultsCompared to the placebo, consumption of APR extract triggered rapid increases in chemokine levels starting at 1 hour, including IP-10 (P<0.05) and MCP-1 (P<0.1), which peaked at 2 hours (P<0.01) and 3 hours (P<0.05), respectively. The stem cell-mobilizing growth factor G-CSF increased at 2 hours (P<0.05). Increased immune surveillance involved a transient effect for monocytes at 1 hour, followed by NKT cells, CD8+ cytotoxic T cells, and γδT cells at 2–3 hours. Increased immune cell alertness was seen at 1 hour by increased CD25 expression on monocytes (P<0.01), NKT cells (P<0.01), and T cells (P<0.05). NKT cells showed upregulation of CD69 at 2 hours (P<0.01). Increased enzymatic activity was seen at 2 hours for the antioxidant enzymes superoxide dismutase (P<0.05) and catalase (P<0.01).ConclusionConsumption of APR extract triggered acute changes to chemokine levels. In addition, immune alertness was increased via the expression of activation markers on multiple types of innate immune cells, followed by increased immune surveillance and antioxidant protection. This suggests a beneficial enhancement of natural immune surveillance, likely via a combination of gut-mediated cytokine release and vagus nerve communication, in combination with cellular protection from oxidative stress.