The goals of this experiment are to induce activation of T-cell by Brios statin one ion mycin, as well as differentiation of T-cells by gamma chain cytokines begin with the isolation of tumor sensitized T-cells from spleen and lymph nodes. Then activate with brios statin one and ion mycin two intracellular signal mimetics that increase protein kinase C activity and intracellular calcium respectively. Culture in the presence of IL seven IL 15, and IL two for the differentiation and expansion of tumor reactive T cells.
The powerful combination of results from the interferon gamma EISA and the fact cytotoxicity assay can shed light on tumor reactivity of the expanded T cells and offer potential insights to adoptive immunotherapy. This method of exvivo expansion of tumor reactive T cells can help answer key questions in tumor immunology questions such as what is the role of common gamma imaging cytokines in the differentiation of tumor reactive T cells. And what is the role of each T-cell phenotype such as effector cells, effector memory cells, and central memory cells in generating long-term memory response against cancer.
The insulation of lymph nodes and spleen and the septic handling of the lymphocytes are difficult procedures to implement but can be learned rapidly. By observation. The FVBN 2 0 2 transgenic female mice express an inactivated rat new transgene under the regulation of MMTV promoter and as a result, developed spontaneous mammary carcinoma between four and 10 months of age.
In this protocol, these spontaneous tumor bearing mice are used as donors of tumor reactive T cells. To begin isolate the tumor draining lymph nodes and spleens from tumor bearing FVBN 2 0 2 transgenic mice. Now prepare a single cell suspension in ice cold RPMI 1640 supplemented with 10%FB sce A culture at 10 to the six cells per milliliter in complete medium containing 15%FBS with one micromolar cin five nano molar brios statin one and 80 units per milliliter.
IL two for 16 hours with our PMI medium equilibrated at 37 degrees Celsius, wash the cells three times, then culture at 10 to the six cells per milliliter in complete medium with 10 nanograms per milliliter, each of IL seven and IL 15 for 24 hours. In order to further activate the cells pulse with an addition of 40 units per milliliter IL two for 24 hours. Now split the cells and culture them with 10 nanograms per milliliter, each of IL seven and IL 15 for four more days if necessary, change the medium and split the cells every two days.
Harvest the expanded primary T cells by centrifugation, enumerate the cells by light microscopy and calculate the fold expansion of T cells. Now prepare the cells for flow cytometry to determine the proportion of CD eight positive and CD four positive T cells. First block non-specific binding of antibodies to FC receptors by incubating the cells with anti CD 16, CD 32 antibody for 20 minutes on ice.
Then wash the cells twice with two milliliters of ice cold PBS supplemented with 1%sodium azide stain the cells by incubating with Fitz CD four and PE CD eight antibodies for 20 minutes on ice. Wash twice with two milliliters of ice cold PBS supplemented with 1%FBS and 0.1%sodium azide. Finally, fix the cells with 1%paraform aldehyde and analyze the samples on a Beckman Coulter FC 500 using summit version 4.3 software in six well culture plates, pipette T cells with mammary tumor cells in a 10 to one effector to target ratio and three milliliters per well.
Complete medium containing 20 units per milliliter of IL two incubate at 37 degrees Celsius and 5%carbon dioxide for 48 hours. Perform three color antibody staining for new, followed by PE anti-US IgG and Xin five fite and propidium iodide according to the manufacturer's protocol gate facts on new positive tumor cells and analyze viability of the tumor cells with an Xin five pi. An activation of T cells with BI for 16 hours results in killing of naive T cells that are not sensitized with the tumor in vivo.
After the bi selectivity of tumor reactive T cells, they expand up to 2.8 fold within a six day culture with the gamma chain cytokines, both CD eight positive and CD four positive T cells are equally expanded with the gamma chain cytokines. After a six day ex vivo culture, the ex vivo expanded T cells show high responsiveness against the tumors that donor mice were sensitized to as evaluated by the production of interferon gamma. In the presence of new positive mouse mammary carcinoma or MMC tumor cells, the ex vivo expanded T cells can induce apoptosis in the new positive MMC tumor cells, such that viability of the tumor cells drops from 92%to 61%within 48 hours.
While attempting this procedure, you should remember to work under aseptic conditions. After watching this video, you should have a good understanding of how to select and expand tumor active T-cells for adaptive T-cell therapy of cancers.
