Activation Of Murine Lymphocytes Research Articles

Characterization of murine lymphocyte activation and exhaustion markers by a 14-color flow cytometry panel.

Previously designed flow cytometry panels have provided a framework to analyze T-cell activation; however, few provide an extensive view of lymphocyte populations, and none are optimized for murine models. This article describes a panel designed specifically to assess the expression of activation and exhaustion markers in expanding lymphocyte populations in tumor-bearing mice across two distinct genetic backgrounds: BALB/c and C57BL/6. This comprehensive panel enables the assessment of multiple functional states and immune checkpoint markers across cytotoxic CD8+ T cells, helper and regulatory CD4+ T cells and NK cells in murine whole blood, lymph nodes and tumor.

Isolation and Activation of Murine Lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Isolation and Activation of Murine Lymphocytes

Isolation and Activation of Murine Lymphocytes

The classical NFκB pathway is required for phloroglucinol-induced activation of murine lymphocytes

Background Phloroglucinol (PG), a polyphenolic compound, has been proposed to show free radical scavenging, anti-tumor, and immunomodulation effects on immune cells. In this study, we investigated PG for its immunological activity and their molecular mechanisms, specifically focusing on the functional activation of nuclear factor-κB (NFκB), an important transcription factor involved in the activation and differentiation of lymphocytes, and subsequent recruitment of murine lymphocytes. Methods We tested whether PG may contribute to the activation of immune response through the NFκB pathway by 3H-thymidine incorporation assay, Western blot, intracellular cytokine assays and Electrophoretic mobility shift assay (EMSA) in murine lymphocytes. Results PG markedly enhanced the proliferation of lymphocytes by inducing the degradation and phosphorylation of IκB, which leads to the activation of NFκB p65. Also, PG induced the activation of mitogen-activated protein kinases (MAPKs) such as ERK1/2, JNK, and p38, upstream molecules of NFκB pathway. In addition, PG augmented the secretion of interleukin (IL)-2 in mature T lymphocytes and their production of IL-2. Furthermore, the application of TPCK significantly reduced the activation of lymphocytes by PG via inhibiting the NFκB activation. Conclusion These results suggest that PG induces the activation of lymphocytes by inducing the proliferative activity and secretion of IL-2 through the classical NFκB. General significance The effect of PG on lymphocyte activation was assayed for the first time. These results would also elucidate its underlying mechanism.

The Effects of Phyllanthus niruri Aqueous Extract on the Activation of Murine Lymphocytes and Bone Marrow-Derived Macrophages

Phyllanthus niruri L. (Euphorbiaceae) is acclaimed world-wide for its versatile ethnomedicinal uses. It features in recipes used by some herbalists to manage different diseases, including claims of efficacy against many life-threatening infections, such as HIV/AIDS and hepatitis. In order to understand the mechanisms and the involvement of the immune system in mediating these activities, the effects of the aqueous extract of P. niruri on the activation of murine lymphocytes and macrophages were investigated. The study showed that the extract of P. niruri is a potent murine lymphocytes mitogen, inducing significant (p < 0.01) increases in the expression of surface activation maker (CD69) and proliferation of B and T lymphocytes. The production of interferon-γ (IFN- γ) and interleukine-4 (IL-4) by P. niruri extract-stimulated naïve splenocytes cultures was also significantly (p < 0.05) increased in a concentration-dependent manner. Various indices of activation and functions murine bone marrow-derived macrophages were significantly (p < 0.05) enhanced by pre-treatment with the extract, including phagocytosis, lysosomal enzymes activity, and TNF-α release. Phyllanthus niruri extract was also shown to modulate nitric oxide release by macrophages. These activities suggest that stimulation of the immune system by the extracts of P. niruri could be partly responsible for the ethnomedicinal applications in the management of infectious diseases.

Activation of murine lymphocytes and modulation of macrophage functions by fractions of Alchornea cordifolia (Euphorbiaceae) leaf extract

The immune system is highly complex, intricately regulated group of cells whose integrated function is essential to health. Modulating the functions of these cells offers important pharmacological and therapeutic approaches in many disease conditions.This study reports on the in vitro immunostimulant activities of two flavonoid-rich fractions of Alchornea cordifolia (Euphorbiaceae) leaf extract: EAC and AAC, obtained by fractionating the methanol extract into ethylacetate and acetone soluble fractions, respectively.The lymphoproliferative effect of the fractions on naïve murine splenocytes and thymocytes as well as the modulatory effects on the phagocytic and lysosomal enzyme activities of elicited murine macrophages was investigated. A. cordifolia fractions, EAC and AAC, produced significant (P<0.05) and concentration-related (10–250 μg/ml) increases in the proliferation of splenocytes and thymocytes cultures which were comparable to the mitogenic effects of lipopolysaccharide, LPS (10 μg/ml) and concanavalin A, ConA (2 μg/ml) used as standard mitogens. EAC and AAC (15.6–250 μg/ml) significantly (P<0.05) increased phagocytosis and intracellular killing capacity measured as percentage increase in nitroblue tetrazolium (NBT) dye reduction. Lysosomal phosphatase activity of peritoneal macrophages, measured by p-nitrophenyl phosphate (p-NPP) hydrolysis, was also increased significantly (P<0.05) by EAC and AAC (15.6–250 μg/ml). Treatment of macrophage cultures with EAC and AAC (15.6–250 μg/ml) decreased the expression of nitric oxide significantly (P<0.05) in the supernatant. This study demonstrates strong immunomodulatory activities of A. cordifolia leaf extracts which could explain some of the therapeutic benefits attributed to the plant in traditional medicine and could also be exploited as a source of novel immunoregulating substances.

Activation of murine lymphocytes and modulation of macrophage functions by fractions ofAlchornea cordifolia(Euphorbiaceae) leaf extract

Activation of murine lymphocytes and modulation of macrophage functions by fractions of<i>Alchornea cordifolia</i>(Euphorbiaceae) leaf extract

The linoleic acid derivative DCP-LA selectively activates PKC-ɛ, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.

Effects of genistein and daidzein on the cell growth, cell cycle, and differentiation of human and murine melanoma cells

Genistein and daidzein are two major isoflavonoids in dietary soybean that have inhibition effect on the cell growth of different tumor cell lines. We previously reported the anti-tumor activities of genistein and daidzein in human co1on tumor (HCT) cells and their different ability to enhance the activation of murine lymphocytes. In the present study, the effect of genistein and daidzein on the cell growth, cell cycle progression, and differentiation of murine K1735M2 and human WM451 cel1s was investigated. It was found that genistein could inhibit the cell growth of two metastatic melanoma cell lines, murine Kl735M2 and human WM45l in a dose-dependent manner. Flow cytometry showed that genistein could cause arrest of both Kl735M2 and WM45l at G 2/M phase, while daidzein increased the cell numbers at S phase, decreased the cell numbers at G 1 phase. Detection of melanin and morphological observation showed that genistein can induce Kl735M2 and WM45l to produce dendrite-like structure and produce more melanin by 80%. In contrast, daidzein only retarded the growth of K1735M2 and did not induce differentiation in either K1735M2 or WM451. These results suggest that genistein and daidzein in soybean can inhibit certain malignant phenotype of melanoma via different mechanisms and be potential medical candidates for melanoma cancer therapy.

Cloning of Murine LAG-3 by Magnetic Bead Bound Homologous Probes and PCR (GENE-CAPTURE PCR)

In recent years PCR-based gene cloning strategies have found wide application in molecular biology, due to the power, speed, and relative simplicity of the PCR methodology. We have set up a novel PCR cloning strategy to isolate homologous genes, which is based on the capture of the cDNA sequence(s) of interest with a biotinylated probe and streptavidin-coupled magnetic beads followed by PCR amplification of the selected molecules. This method does not require sequence information on 5′ and 3′ regions of the cDNA of interest and permits gene isolation to be sensitive, fast, simple, and specific even when the conventional screening procedures give rise to high backgrounds. By using this technique, which we propose to call gene-capture PCR (GC-PCR) cloning, we were able to isolate the full-length murine lymphocyte activation gene 3 (LAG-3) cDNA from total RNA of activated thymocytes. The GC-PCR technique represents a powerful tool for easy isolation not only of homologous genes from related species, but also of genes sharing conserved regions of suitable length, gene variants, and gene encoding proteins where only limited knowledge of the amino acid sequence exists.

Activation of murine lymphocytes by lipopolysaccharide incorporated in fusogenic, reconstituted influenza virus envelopes (virosomes).

We have studied the in vitro activation of murine lymphocytes with LPS incorporated in the membranes of both phospholipid vesicles (liposomes) and vesicles composed of fusogenic, reconstituted influenza virus envelopes (virosomes). The incorporation of Salmonella minnesota rough-LPS in liposomes reduced the potency of LPS to stimulate splenocyte proliferation and cell surface kappa-light chain expression on 70 Z/3 pre-B cells by over 100-fold. Salmonella minnesota rough-LPS inserted into virosomes was at least 10-fold more potent than free LPS, both when prebound virosomes were allowed to be taken up by the cells at neutral pH and when the virosomes were fused into the plasma membrane by low pH treatment. Inactivation of the virosomes by low pH pretreatment reduced the potency of the virosomal LPS to the level of liposome-incorporated LPS. The association of the various LPS forms with the cells was quantitated using radio-iodinated LPS. Correcting for uptake, virosomal LPS remained 2- to 10-fold more potent than free LPS in stimulating B lymphocytes and at least 100-fold more active than liposomal LPS or fusion-inactivated virosomes. After low pH-induced fusion with the plasma membrane, the majority (80%) of the prebound virosomes had fused with the cells, compared with about 8% after neutral uptake. From these results we conclude that LPS inserted into the plasma or endosomal membranes efficiently activates murine lymphocytes. The fusion data suggest that the incorporation into endosomal membranes might be a more effective stimulus.

Microvascular expression of mala-2 correlates withIn vivo lymphocyte trafficking and is preferentially enhanced in tumors by tumor necrosis factor-α and interleukin-1α

The role of endothelial cell adhesion molecule expression in the immune response to tumors is unknown. We have investigated the expression of murine lymphocyte activation antigen (MALA-2), the murine equivalent of intercellular adhesion molecule-1 (ICAM-1), in blood vessels of normal murine tissues and in melanoma tumors and evaluated the relationship between MALA-2 expression and lymphocyte trafficking in vivo. C3H/HeN mice were injected both i.p. and s.c. with a clone of K-1735 syngeneic melanoma cells. Day 11 tumor-bearing mice were killed and vascular expression of MALA-2 was quantified using immunohistochemistry. MALA-2 expression was high in lung, liver and spleen and low in lymph node, small bowel, muscle and tumor. Systemic administration of either recombinant tumor necrosis factor alpha (rTNF alpha) or recombinant interleukin-1 alpha (rIL-1 alpha) over 2 days prior to organ harvest resulted in an increase in the number of tumor vessels expressing MALA-2, with no change in MALA-2 expression in other tissues. In vivo lymphocyte trafficking was evaluated using cultured, activated splenocytes radiolabeled with 111In. 111In-labeled splenocyte distribution correlated closely with MALA-2 expression, with high localization to spleen, liver and lung and poor localization to lymph node, small bowel, muscle and tumor. Systemic administration of rTNF alpha, but not rIL-1 alpha, resulted in a significant increase in 111In-labeled splenocyte distribution to tumor, but neither rTNF alpha nor IL-1 alpha altered 111In-labeled splenocyte distribution to normal organs. Our data demonstrate the in vivo pattern of vascular MALA-2 expression in normal murine tissues and tumors and suggest that the expression of MALA-2 can be preferentially enhanced in tumors by systemic administration of cytokines. Lymphocyte distribution in vivo correlates closely with the pattern of MALA-2 expression, and these data support the conclusion that MALA-2 plays an important role in the regulation of lymphocyte trafficking.

In vitro potentiation of lymphocyte activation by dehydroepiandrosterone, androstenediol, and androstenetriol.

Dehydroepiandrosterone (5-androstene-3 beta-ol-17-one, DHEA) has been shown to protect mice from lethal viral and bacterial infections. However, recently, androstenediol (5-androstene-3 beta-17 beta-diol, AED) and androstenetriol (5-androstene-3 beta-7 beta-triol, AET), metabolites of DHEA, have each shown to be more potent endocrine regulators of the immune response. In contrast to glucocorticosteroids, in vivo, these steroid hormones up-regulated the cellular immune response of the host to limit virus-mediated pathology. These experiments first examined the in vitro influences of DHEA, and AED, or AET on mitogen-stimulated activation of murine lymphocytes. From physiologic to pharmacologic doses, DHEA suppressed proliferation of mixed splenocyte cultures activated with Con A or LPS by 20 to 70% whereas AED had little influence on the response. In sharp contrast, AET potentiated the response with both mitogens to 50 to 70% above control. Analogous to these observations was the regulation of IL-2 and IL-3 secretion from Con A-activated lymphocytes by each of these hormones which again was depressed analyzed. The suppressive effects of hydrocortisone on Con A-induced lymphocyte proliferation and cytokine production were strongly counteracted by coculture with AET. DHEA did not counteract hydrocortisone activity whereas AED showed moderate antiglucocorticoid function.

Combined activation of murine lymphocytes with staphylococcal enterotoxin and interleukin-2 results in additive cytotoxic activity.

This report demonstrates that in vitro activation of murine spleen cells with interleukin-2 (IL-2) or the bacterial superantigen staphylococcal enterotoxin A (SEA) results in different patterns of activation and function of cytotoxic cells. Lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity (ADCC) are mainly mediated by IL-2 activated natural killer (NK) cells. SEA is the most powerful T cell mitogen known so far and retargets cytotoxic T lymphocytes (CTL) to tumors expressing major histocompatibility complex (MHC) class II in staphylococcal-enterotoxin-dependent cellular cytotoxicity (SDCC). Culture of mouse spleen cells with SEA led to expansion and activation of T cells, which demonstrated strong SDCC activity and some NK-like cytotoxicity after 5 days in culture. Cell sorting revealed that both CD8+ and CD4+ T cells mediated SDCC but the former were more effective. Phenotypic analysis showed that SEA preferentially stimulated and expanded T cells expressing T cell receptor V beta 11, in particular CD8+ T cells. Combined activation with SEA and IL-2 resulted in simultaneous induction of T and NK cell cytotoxicity. Moreover, IL-2 had additive effects on SEA-induced SDCC. Combined treatment with SEA and IL-2 might therefore be an approach to induce maximal cytotoxicity against tumors and to recruit both T and NK cells in tumor therapy.

Combined activation of murine lymphocytes with staphylococcalenterotoxin and interleukin-2 results in additive cytotoxic activity

Combined activation of murine lymphocytes with staphylococcalenterotoxin and interleukin-2 results in additive cytotoxic activity

Activation of Murine Lymphocytes by Exogenous Phosphatidylethanolamine- and Phosphatidylcholine-Specific Phospholipase C

Enzymes of the protein kinase C (PKC) family have a decisive role in the activation process of T cells. Under physiological conditions. PKC undergoes activation by diacylglycerol (DAG) which is transiently produced following activation of an antigen receptor-coupled phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and hydrolysis of PIP2. In vitro activation of distinct PKC isoenzymes is differentially regulated by various synthetic DAGs and membrane phospholipids. We tested whether exogenous PLC obtained from Bacillus cereus can affect PKC-dependent functions in mouse lymphocytes. The results indicated that B. cereus PLC, which preferentially hydrolyzes phosphatidylethanolamine (PE) and phosphatidylcholine (PC), induces the expression of functional cell surface IL2 receptors and that, in the presence of exogenous IL2, it leads to cell proliferation. The PE/PC-PLC reversed the inhibitory effect of PC on enzymatic activity of T cell-derived PKC in vitro. Furthermore, it augmented the activity of PKC over the background level suggesting that the PC-derived DAG may function as a positive regulator of T cell-specific PKC isoenzymes.

Lysis of Murine Macrophages by Lymphokine‐Activated Killer‐Like Cells Induced by BCG in vitro

The lymphokine-activated killer (LAK)-like activity was found to be induced in mouse splenocytes cultured together with Bacillus Calmette-Guérin (BCG). The killer cells induced by BCG were capable of killing both NK-sensitive (YAC-1, P388D1) and NK-resistant (P815) tumor cells. As an important finding, they also lysed syngeneic macrophages (M phi). The anti-M phi killer activity appeared on day 2, and reached a peak on day 5 of culture. Phenotype analysis of the killer cells by depletion techniques using monoclonal antibody (mAb) and complement indicated that the majority of these anti-M phi killer cells were Thy-1+ and asialo GM1+. This M phi cytolysis could be inhibited by the addition of cold M phi, YAC-1 tumor cells, and P815 tumor cells, suggesting that the same population of the effector cells recognize M phi and tumor cells. The addition of anti-MHC class I, anti-MHC class II, anti-L3T4, or anti-Ly-2 mAb directly to assay cultures did not affect anti-M phi cytolysis, suggesting that the MHC molecules are not involved in the cytolysis of M phi by the BCG-induced killer cells. The addition of anti-LFA-1 mAb partially inhibited the cytotoxicity, suggesting importance of the contact between targets and effectors in the cytolysis. Our present data suggest that activation of murine lymphocytes with BCG induces LAK-like cells capable of killing a wide variety of tumor cells as well as M phi and this anti-M phi cytolysis is mediated by nonspecific killer cells.

Induction of tumor cytotoxic immune cells using a protein from the bitter melon ( Momordica charantia)

The fruit and seeds of the bitter melon ( Momordica charantia) have been reported to have anti-leukemic and antiviral activities. This anti-leukemic and antiviral action was associated with an activation of murine lymphocytes. A partially purified protein factor from the bitter melon caused an infiltration and activation of peritoneal exudate cells in C57B1/6J, C3H/HeJ, and C3H/HeN mice. When the extract was injected twice a week at 8 μg of protein per ip injection for 0–4 weeks, the peritoneal exudate cells from the treated mice were cytotoxic in a long-term (18-hr) 51Cr-release assay against a range of labeled targets; L1210, P388, and MOLT-4 tumor cells. Cytotoxicity was also observed against YAC-1 targets in a short-term (4-hr) assay. Fractionation of the cytotoxic immune cells implicated a nonadherent cell population which was capable of killing an NK-sensitive cell line in a 4-hr 51Cr-release assay. Unit gravity sedimentation studies indicated that the cytotoxicity was due to either a neutrophil or a large lymphocyte. Antibody depletion experiments using antibody to asialo GM1, an NK cell-specific antibody, depleted cytotoxicity observed in nonadherent, Ficoll/Hypaque-separated PEC. This suggests that at least part of the anti-leukemic activity of the bitter melon extract is due to the activation of NK cells in the host mouse.

MALA‐2, mouse homologue of human adhesion molecule ICAM‐1 (CD54)

In humans, lymphocyte adhesion to cells is mediated by the protein heterodimer CD11a/CD18 (Leu-CAMa, LFA-1) and its ligand CD54 (ICAM-1). Although the murine CD11a/CD18 is well characterized, the mouse homologue of human ICAM-1 has not been identified. In the present study a rat monoclonal antibody to the murine lymphocyte activation antigen MALA-2 was found to inhibit in a dose-dependent manner the phorbol ester-enhanced aggregation of mouse lymphoblasts, an adhesion-specific assay, and hence to define an adhesion molecule. By immunofluorescence flow cytometry the antigen expression was low on spleen cells but it largely increased after stimulation with mitogens. The antigen was expressed by some, but not all, lymphoid cell lines, and myelomonocytic and mastocytoma cells were also positive. In frozen tissue sections MALA-2 was mainly detected on germinal center B cells, dendritic cells, macrophages and vascular endothelium, including high endothelial venules. Cell surface labeling followed by immunoprecipitation and gel electrophoresis indicated that the antigen is a sialoglycoprotein which has a relative molecular mass of 95 kDa and displays a faster electrophoretic mobility under non-reducing conditions. The function, cellular distribution and molecular properties of MALA-2 are indistinguishable from those of human ICAM-1.

Increased Assembly of Clathrin Occurs in Response to Mitogenic Activation of Murine Lymphocytes

The unassembled (soluble) and assembled (particulate) pools of clathrin in murine lymphocytes have been separated by centrifugation, and specifically quantified by immunoblotting of cellular extracts with an anticlathrin heavy chain monoclonal antibody. In resting spleen lymphocytes only 25-30% of the total cellular clathrin was found to be present in an assembled form. Upon activation of lymphocytes with B or T cell mitogens (lipopolysaccharide or concanavalin A), the levels of assembled clathrin increased to 60% of the total. These changes in the levels of assembled clathrin were not due to an increase in total cellular clathrin concentration following lymphocyte activation, but rather to changes in the steady state ratio of assembled to unassembled clathrin. The increase in assembled clathrin preceded the expression of transferrin receptors, as measured by the cell surface binding of an antitransferrin receptor monoclonal antibody, and maximal DNA synthesis, indicating that clathrin assembly occurs early after lymphocyte activation and precedes cell division. Immunofluorescence analysis of activated lymphocytes with an anti-clathrin heavy chain monoclonal antibody revealed a punctuate staining pattern characteristic of coated pits and vesicles. Activated B lymphocytes displayed particularly prominent staining in the perinuclear region compared to T cells, suggesting that clathrin assembly may be important for B cell functions such as immunoglobulin synthesis or secretion. These results suggest that in lymphocytes, clathrin assembly is a dynamic process that is triggered by mitogenic stimuli.