Role of regulatory T cells in in vitro human culture systems


BACKGROUND: Regulatory T cells (Tregs) are an essential subset of T cells that despite over 10 years of research have yet to be fully characterized. These suppressive immune cells, derived from the thymus, express high levels of interleukin-2 receptor alpha-chain (CD25). Tregs are needed to maintain self-tolerance and to control responses to non-self-antigen. The mechanism of Treg repression is unknown. The direct cell-to-cell contact through binding of cell surface molecules as well as secretion of suppressive cytokines has been shown to suppress the proliferation of Thl and Th2 cells against auto, allo and foreign antigens. The role of Tregs in regulating B cell response is also uncertain. The objective of this study is to determine how the removal of T regulatory cells can increase B cell responses in vitro. METHOD: This study focuses on the effect Tregs have on the generation of an antigen specific immune response in vitro. T cells with and without Tregs were co-cultured with monocyte derived dendrtic cells. The antigen specific activation was determined by analyzing cytokine production using intracellular cytokine staining, a flow based assay. Next, the effects of Tregs on both recall and naive B cell responses was analyzed using a co-culture of B cells, CD4 T cells with and without Tregs and monocyte-derived dendritic cells. Analysis of lymphoproliferation, activation, and antibody production was analyzed by using flow cytometry, Elispot and ELISA assays. RESULTS: An antigen specific response against gp120 was generated in naive T cell culture. Tregs were shown to inhibit antigen specific cytokine production in CD4 T cell culture to de novo antigens. The activation in the absence of Tregs was superior to the addition of exogenous factors of IL-2 and IL-7 with a third less non-specific background activation. When analyzed in a TT recall B cell assay, however, the removal of Tregs proved to have an inhibitory effect on antigen secreting cells detected by Elispot. This inhibition appeared at both a 1: 1 and 1 :4 T to B cell ratios though was slightly decreased at the 1 :4 ratio. The same was true for na1ve B cell assay showing a decrease in the generation ofMSPl-42 IgM antigen secreting cells. ELISA assays also confirmed the results showing a nearly 2.5 fold decrease in the amount of MSP 1-42 specific IgM Ab in the L TE cell culture supernatant. Conclusion: While the removal of T regulatory cells is beneficial for the activation of na1ve T cells, the removal of Tregs seems to be inhibitory to B cell activation in the LTE. This inhibitory effect maybe due to T cells becoming too stimulatory before culture with B cells. Studies involving a wider range of T to B cell ratios and culture times may be beneficial to determine if the depletion of Tregs would benefit this culture method.


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Thesis Completion





Khaled, Annette


Bachelor of Science (B.S.)


Burnett College of Biomedical Sciences

Degree Program

Molecular Biology and Microbiology


Biomedical Sciences -- Dissertations, Academic;Dissertations, Academic -- Biomedical Sciences







Access Status

Open Access

Length of Campus-only Access


Document Type

Honors in the Major Thesis

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