Macropinocytosis drives T cell growth by sustaining the activation of mTORC1

Macropinocytosis is an evolutionarily-conserved, large-scale, fluid-phase form of endocytosis that has been ascribed different functions including antigen presentation in macrophages and dendritic cells, regulation of receptor density in neurons, and regulation of tumor growth under nutrient-limiting conditions. However, whether macropinocytosis regulates the expansion of non-transformed mammalian cells is unknown. Here we show that primary mouse and human T cells engage in macropinocytosis that increases in magnitude upon T cell activation to support T cell growth even under amino acid (AA) replete conditions. Mechanistically, macropinocytosis in T cells provides access of extracellular AA to an endolysosomal compartment to sustain activation of the mechanistic target of rapamycin complex 1 (mTORC1) that promotes T cell growth. Our results thus implicate a function of macropinocytosis in mammalian cell growth beyond Ras-transformed tumor cells via sustained mTORC1 activation.

Ethyl Acetate Fraction from Dendropanax morbifera Leaves Increases T Cell Growth by Upregulating NF-AT-Mediated IL-2 Secretion

Dendropanax morbifera Leveille (Araliaceae) is an endemic species that grows in Southwestern Korea and has been used as a folk medicine. Several studies reported that D. morbifera leaves have diverse therapeutic potentials. We found that the water extract of D. morbifera leaves increased the growth of EL-4 T cells. The water extract was divided into five fractions: [Formula: see text]-hexane, chloroform, ethyl acetate, [Formula: see text]-butanol, and water layers. The ethyl acetate (W-EA) fraction showed a more significant effect than the other fractions on the growth of EL-4 T cells, splenocytes, and isolated murine CD4[Formula: see text] T cells. We evaluated the W-EA fraction for its immunomodulatory effects focusing on T cell functions. First, we tested the effect of the W-EA fraction on the regulation of interleukin-2 (IL-2), a potent T cell growth factor. The W-EA fraction significantly increased IL-2 secretion in EL-4 T cells activated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io). In addition, the W-EA fraction increased interferon-gamma (IFN-[Formula: see text] production in isolated murine splenocytes activated with Concanavalin A (ConA). Next, we examined the effect of the W-EA fraction on the regulation of transcriptional factors related to IL-2 production in T cells. The W-EA fraction significantly increased PMA/Io-induced promoter activity of a nuclear factor of activated T cells (NF-AT) in EL-4 T cells, but did not show any significant effects on the promoters of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-[Formula: see text]B). These results indicate that the W-EA fraction from water extract of D. morbifera leaves enhances IL-2 production at the transcriptional levels via the up-regulation of NF-AT in PMA/Io-activated EL-4 T cells.

Reinvestigation of the T-Cell Inactivation Achieved for the Prevention of TA GV HD By 2500 Gy of Gamma Irradiation, a Comparison with Pathogen Reduction

Background: Transfusion Associated Graft vs. Host Disease (TA GVHD) results in high morbidity and mortality, caused by contaminating T-cells present in transfused blood products. Gamma irradiation (GI) of platelet components (PC) with a dose of 2500 cGy is the approved methodology to prevent TA GVHD in high-risk patients. That methodology has been established based on in vitro Limiting Dilution Assays (LDA) that quantify the inactivation of T-cells. The limited natural abundance of T-cells in blood products and the number of T-cells that can be reproducibly cultured in single wells have defined LDA assay sensitivity. LDA assays usually have a dynamic range of 4-5 log10. Even though that methodology has been used for decades with presumable success, cases of GVHD recognized as transfusion associated, have to be connected temporally and phenotypically to the blood product in use, in order to be characterized as such. That leaves open the possibility that some atypical cases of GVHD in blood product recipients may be miss-assigned and go unrecognized. An alternative methodology to prevent TA GVHD is the photochemical treatment of PC with amotosalen/UVA (PCT; INTERCEPTTM Blood System). PCT has replaced GI in Europe for pathoen inactivated PC for more than 10 years. In this study, we evaluated both GI and PCT on inactivation of T-cells using a highly sensitive approach that includes the use of higher numbers of PBMCs isolated by leukapheresis, and an increased number of cells cultured in a single well (107/well) by using larger wells and optimizing culture conditions. Methods: PBMCs harvested by leukapheresis from individual donors (AllCells, Alameda, CA) were spiked (106/mL) into identical units of human plasma and inactivated using either GI with 2500 cGy, PCT, or were retained as untreated control. For the LDA assay, PCT- or GI-treated cells isolated post treatment, were incubated in individual wells for 14 days in the presence of pooled allostimulator cells from 10 unrelated donors (5X106 treated with 7500 cGy) and growth stimulating factors (PHA and IL-2) under standard culture conditions. Proliferation was assessed by tritiated thymidine ([3H]TdR6.7 Ci/mmol) incorporation into PBMC, as well as by the observation of bright large clusters that were clearly observed at 40x magnification. Results: Initial experiments showed that 107PBMCs/well or 106PBMCs/well resulted in proliferation after GI at 2500 cGy, while 105 PBMCs/well did not. On the other hand, 107 PCT treated-PBMCs/well were found neither to proliferate, nor to incorporate [3H]TdRabove background. T-cell precursor frequency was measured for each donor by incubation of serial dilutions of viable PBMCs (50 - 1 /well; 12 wells per dilution) in the presence of 107 inactivated PBMCs. The experiment was repeated 10 times with PBMCs from different donors. For GI, [3H]TdR incorporation above background indicative of T-cell growth, as well as T-cell proliferating colonies were observed in 4 of 10 experiments when 106PBMCs/ well were cultured. No T-cell growth was detected by [3H]TdR incorporation when 105 GI PBMCs/well were cultured, while proliferating colonies were observed in 1 of 10 experiments. No T-cell proliferation was detected by either criterion, when 107 PCT-treated PBMCs/well were cultured. Conclusions: The accepted dose of 2500 cGy gamma irradiation for prevention of TA GVHD results in more than 4.2log10 but less than 6.2 log10 T-cell inactivation. However, it still allows the proliferation of T-cell clones from some donors. Treatment of T-cells with amotosalen/UVA results in complete inactivation of T-cells (>6.2 log10) with no break though proliferation detected. The data in this study have not been reviewed by the FDA. Disclosures Merkel: Cerus Corporation: Other: fee for service with Cerus Corporation. Giclas:Cerus Corporation: Other: fee for service with Cerus Corporation. Gibula:Cerus Corporation: Other: fee for service with Cerus Corporation. Andersen:Cerus Corporation: Other: fee for service with Cerus Corporation. Knight:Cerus Corporation: Other: fee for service with Cerus Corporation. Lin:Cerus corp: Employment. Castro:Cerus Corporation: Employment. Green:Cerus Corporation: Employment. Stassinopoulos:Cerus Corporation: Employment.

PI3K pathway inhibitor LY294002 alters Jurkat T cell biobehaviours via ERK1/2-ICBP90 mediation

Little is known about whether there is a relationshipbetweenPI3K/AKT, ERK1/2 and an inverted CCAAT box binding protein (ICBP90) in biological behaviours of tumour cells. The aim of this study was to determine thisusing Jurkat T cells. Compared to PD98059 (an ERK1/2 signaling inhibitor), DAPT (a Notch signaling inhibitor) or adriamycin (a classical anti-tumour drug), the inhibition of Jurkat T cell growth by LY294002 (a PI3K/Akt signaling inhibitor) was more obvious. LY294002 combined with adriamycin appeared to have a synergistic effect. LY294002 strongly blocked Jurkat T cells at each phase of cell cycle with a decrease of DNA content, superior to adriamycin. Consistent with these changes, the expression of phosphorylated ERK1/2 was markedly decreased in the LY294002-treated Jurkat T cells, leading to the reduction of ICBP90 production, followed by moderate attenuation of TGF-β secretion. The results suggest that deactivation of PI3K/Akt signalling can surpress Jurkat T cell growth through inhibiting cell proliferation and blocking the cell cycle. ICBP90 may mediate the PI3K/AKT-ERK1/2 signalling to regulate leukemia cell growth.