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Developing an In-Vivo Retroviral Expression System to Examine Downstream Signaling of T follicular helper cell and B cell Interactions within Germinal Centers Open Access

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Inducible Co-stimulator (ICOS) molecule is a member of the CD28 family of co-stimulatory molecules expressed on T cells. Previous studies demonstrate an important role for ICOS signaling in sustaining optimal numbers of T follicular helper (TFH) cells, a specialized T helper cell subset known for their role in B cell help and promoting germinal centers (GCs) in secondary lymphoid organs. B cells and the ICOS-ICOSL pathway have been shown to be required for TFH differentiation and more recently for their maintenance during the GC response. Although signaling events downstream of ICOS involving the Phosphoinositide-3 Kinase (PI3 Kinase) pathway have been shown to be required for TFH differentiation, the molecular events involved in the maintenance phase remain unknown. To investigate the downstream effects of B-TFH interactions and to dissect the specific events downstream of the ICOS-ICOSL pathway during the maintenance phase, an in-vivo retroviral expression system allowing forced expression of key signaling molecules was developed. The system works by selecting a specific donor mouse strain with trackable CD4+ T cells which are isolated, retrovirally transduced with a gene of interest, and transferred into recipient mice. The recipient mice are then immunized to promote TFH differentiation and a germinal center response. Here, I first optimized the conditions for retroviral transduction of CD4+ T cells with specific genes of interest, namely Bcl-2, myrAkt, and dnFOXO1 in donor cells for future investigation of signaling downstream of ICOS. Finally, by testing various different donor and recipient mouse strains and immunization techniques, I developed an in-vivo system which allows for optimal expansion of CD4+ T cells and TFH differentiation of donor CD4+ T cells. The methods established here can not only be used in the future to investigate signaling pathways such as ICOS in B cell mediated maintenance of TFH cells, but can also be used to overexpress and investigate other genes of interest and their relationship to reactions occurring within the germinal center.

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