Abstract Book 2010 - CIMT Annual Meeting

027 Low | Immune monitoring
Culturing PBMC in the presence of antigen results in a measurable
expansion of memory T cells
Lindsey Low, Ann Mander, Kathy Tier, Christian Ottensmeier
Cancer Research UK Clinical Centre, Cancer Sciences Division, University of Southampton
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Following an in vivo encounter with an antigen,
there is a primary expansion of effector CD8 T cells,
followed by a contraction in which 90 – 95% of
effector cells die. A small population of memory
cells remain which can persist long term, and will
expand to produce effector cells if the antigen is encountered
again. Culturing of PBMC in the presence
of antigen is believed to stimulate this expansion,
producing antigen-specific effector cells which can
be detected using standard immunological assays.
The protocol was developed using cryporeserved
PBMC from HLA A2 positive heathy donors, cultured
in the presence of IL-2 with the viral peptide
antigens, cytomegalovirus (CMV), CMV pp65 493-
499, (NLVPMVAVT); influenza A, Matrix 1 58-66,
(GILCFVFTL); Epstein Barr virus (EBV), BMLFI
259-217 (GLCTLVAML), and measles, non-structural
C protein 84-92 (KLWESPQEI). Variables optimized
included cell concentration, peptide concentration,
well size, time spent in culture, feeding
regimen, medium and use of serum. The resulting
protocol was shown to give an optimum yield of antigen-specific
effector cells at the end of the culture
process. Cultured cells were used in immunological
assays to determine phenotype and fuctionality.
FACS analysis of pre- and post-culture cells has been
used to show a measureable post-culture increase
in the population of effector cells. Use of cultured
cells in standard IFNγ ELISpots shows a noticeable
amplification of antigen-specific response rates
when compared to non-cultured cells for both the
test viral antigens and for low frequency tumour
associated antigens in clinical trial usage.
This indirect measurement of the memory cell population
provides a sensitive and reliable method to
monitor the long term effectiveness of immunotherapeutic
vaccination.