In vitro T cell expansion

Identification of vaccine-specific TCR-beta clonotypes by sequencing to track those clonotypes in memory CD4 T cell repertoires at day 0 pre-vaccination and day 60 post-vaccination with Engerix-B vaccine. Conclusion: We detected a significant rise in the frequency of unique vaccine-specific TCRβ sequences within memory CD4 T cell repertoire at day 60 post-vaccination compared to pre-vaccination. Notes: Peripheral blood mononuclear cells from day 60 were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with a pool of peptides spanning hepatitis B (HB) surface antigen (HBsAg) and selected single peptides (from epitope mapping). At day 7 of in vitro expansion, we sorted CFSElow CD4 T cells and extracted mRNA for quantitative assessment of vaccine-specific TCR? clonotypes by sequencing. Gates for CFSElow CD4 T cells were set using cells left unstimulated.