Date of Award

12-2010

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Microbiology

Advisor

Wei, Yanzhang

Committee Member

Yu , Xianzhong

Committee Member

Tzeng , Jeremy T. R.

Abstract

Immune cell tumor infiltration represents one of the mechanisms that the immune system responds to tumor cells. The tumor infiltrated immune cells include lymphocytes (TIL), dendritic cells (DCs), macrophages, and natural killer (NK) cells. Although TILs have been extensively studied in order to develop adoptive transfer based immunotherapies, how to effectively isolate, culture, and in vitro boosting the killer cells from TILs remains a challenge. Meanwhile, OK-432, a heat and penicillin-treated lyophilized preparation of the low-virulence strain of Streptococcus pyogenes, has been shown to exhibit immunomodulatory activities and potential antitumor therapeutic function, including the activation of DCs, neutrophils, macrophages, lymphocytes, and NK cells as well as the induction of multiple cytokines production, such as IL-1, IL-2, IL-6, TNF-a, IFN-y, and IL-12. This research aims to establish an in vitro culture system, in which highly cytotoxic and tumor cells specific CTLs can be expanded, using OK-432, IL-2 and other cells and/or cytokines. TILs were extracted from experimental mouse melanoma B16F0 tumors grown on mice using two different methods. The isolated TILs were then cultured in vitro in four different culture conditions: 1) low dose IL-2, 2) low dose IL-2 + OK-432, 3) low dose IL-2 + DCs, and 4) low dose IL-2 + OK-432 + DCs. The cell count, phenotype, and cytotoxicity of these cultured TILs were evaluated using techniques including fluorescent activated cell sorting (FACS) and cytotoxicity assay. The results demonstrated that the additions of OK-432 and DCs into the culture system increased the cell growth rate and the percentages of CD8+ cells in some culture conditions. More importantly, the addition of OK-432 and DCs into the culture system significantly increased the cytotoxicity of 14-day cultured TILs from 18% (low dose IL-2 alone), to 44% (low dose IL-2 + OK-432), 40% (low dose IL-2 + DC), or 73% (low dose IL-2 + OK-432 + DC). Therefore, this research provides an improved in vitro TIL culture system that effectively enhances the cytotoxicity of the killer cells.

Included in

Microbiology Commons

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