Development of Limiting Dilution Analysis as a Method for Analyzing Tumor Infiltrating Lymphocyte Trafficking and Survival in Vivo and in Vitro
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Authors
Mali, Vikram
Issue Date
1991
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
Retroviral mediated transduction and expression of a bacterial gene for
Neomycin resistance (Neo R) labels T cells with a unique gene sequence and
allows them to grow in the Neomycin analog (G418. Recently, this approach
has been utilized to identify cell survival and trafficking in both animal and
human systems. In this study we examined whether the selectable growth
advantage of T cells transduced with the Neo R gene could be exploited in
Limiting Dilution Analysis (LDA) to quantitatively assess the frequency of
labeled cells present in a population. A dose of G418 (400 µg/ml active
concentration) was established which prevented growth of nontransduced
cells. Both normal spleen cells and cultured T cells were found to be adequate
feeder cells and the addition of mitogens to the LDA did not increase the
cloning frequency of gene modified cells directly out of culture. The observed
frequency of clones growing in LDA was a good approximation to the number
of transduced cells plated. Twenty million Neo R T cells were adoptively
transferred i.v. into mice, and the mice received 30,000 U IL-2 i.p. After 48
hours, the mice were sacrificed and their organs were processed. Limiting
dilution analysis of the organ preparations has demonstrated recovery of the
adoptively transferred TIL (as these cells grew in G418). Recovery of these
cells was documented at frequencies ranging from 1/200 to >1/5000. Cells
from control animals or recipients of nontransduced T cells fail to grow in
LDA with G418. Therefore, the combination of LDA and Neo R T cells may
provide an additional approach to quantitate trafficking of gene modified T
cells and might be utilized to analyze qualitative differences of the isolated
cells.
Description
vi, 47 p.
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