Abstracts (complete list) - Wissenschaft Online

Rebekka Geiger, Antonio Lanzavecchia, Federica Sallusto
THE “T CELL AMPLIFICATION METHOD” AS A NEW TOOL TO
ANALYZE THE REPERTOIRE OF HUMAN T CELLS
By using T cell receptor (TCR) gene amplification and subsequent sequencing it was
shown that circulating human T cells contain 2.5x107 distinct TCRs in the naïve pool
and 2x105 TCRs in the memory pool. These molecular approaches, while showing TCR
heterogeneity, cannot establish a direct relationship between TCR sequence and antigenspecificity.
We are developing a new method to analyze the antigen-specific repertoire
of human T cells. Highly pure naïve CD4+ T cells were sorted from peripheral blood
mononuclear cells (PBMC’s) of several healthy donors on the basis of CD45RA and CCR7
expression and stimulated polyclonally in limiting dilution (1,000 cells/well) by PHA,
irradiated feeder cells and IL-2. For comparison, memory T cells (CD45RA-) were also
isolated from the same donors and stimulated in limiting dilution (400 cells/wells) under
the same conditions. After a ~1000-5000-fold amplification, each line was stimulated
with autologous monocytes and a panel of naïve and recall antigens. Proliferation was
measured using 3H-Thymidine incorporation as read-out. Using this method we
assessed frequencies of T cells specific for different antigens, e.g. KLH, Anthrax PA, TT,
DerpI and PPD. The data obtained so far indicate that frequencies of antigen-specific T
cells in the naïve pool are much higher than previously thought, whereas the
frequencies of antigen-specific T cells in the memory pool vary with the antigenic
experience of the individual.