Date of Award:

5-1-1998

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Committee Chair(s)

LeGrande C. Ellis

Committee

LeGrande C. Ellis

Committee

Robert W. Sidwell

Committee

David B. Drown

Committee

Bill B. Barnett

Committee

M. Kevin Jackson

Committee

Anthony R. Torres

Abstract

Adoptive immunotherapy using in vitro expanded tumor-infiltrating lymphocytes (TIL) continues to be the subject of research and clinical trials for the treatment of cancer. However, the culture methods used to expand large numbers of TIL are limited and often ineffective. In this study using murine TIL, the level of in vitro expansion, expression of adhesion molecules and infiltration into tumors after infusion was evaluated. A comparison was made between lymphocyte culture fluid (LCF), a bovine plasma-based medium, and a bovine serum-based medium (SCM) as the culture media. Because plasma-based media lack the lymphocyte inhibitory factors associated with serum-based media, it was anticipated that LCF would be a superior growth medium to SCM. An animal model in which C3H/HEN mice were injected with RD-995 fibrosarcoma cells was used to provide tumors from which TIL were extracted. After 22 days in culture, TIL in SCM lost vigor and by day 35 all cultures had died. However, TIL cultured in LCF expanded significantly (p < 0.01) faster than TIL cultured in SCM from day 10 through 46 days of culture. The majority (> 90%) of the TIL cultured in both SCM and LCF expressed the CD4- CD8+ T cell phenotype during long-term culture. The ability to lyse specific tumor cells was higher (p < 0.01) for TIL cultured in LCF as compared to SCM. TIL cultured in LCF maintained a higher level expression of the cell-surface adhesion molecules very late antigen-1 (VLA-1), hyaluronic acid cellular adhesion molecule (HCAM), and lymphocyte function antigen-1 (LFA-1) than TIL cultured in SCM. When in vitro-expanded TIL were infused into tumor-infected mice, TIL cultured in LCF were found to infiltrate tumors to a greater degree than TIL cultured in SCM. An infusion of 5 X 108 TIL from LCF cultures into tumor-infected mice was slightly (p < 0.05) better at reducing tumor size than an infusion of the same number of TIL from SCM cultures. These studies collectively demonstrate the benefits of LCF as a culture medium for murine TIL. They provide the impetus for further studies in the application of LCF for the culture of human lymphocytes and the development of a human plasma-based lymphocyte culture medium.

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