Abstracts (complete list) - Wissenschaft Online

Sonja Schmucker, Mario Assenmacher, Anne Richter
Monocytes and myeloid dendritic cells are both able to induce
primary activation of naïve MART-1-specific CD8 + T cells in
vitro
Adoptive T cell therapy for cancer and viral infections in immuno-compromised patients
is hampered by the lack of reliable protocols for primary activation and expansion of
naïve tumor or virus-specific T cells.
In this study we examined the capacity of monocytes and myeloid dendritic cells (MDC)
to in vitro prime naïve MART-1-specific CD8 + T cells.
Therefore we magnetically isolated CD14 + monocytes, CD1c + myeloid dendritic cells
(MDC), and naïve CD8 + CD25- CD45RO- CD45RA + CD62L + T cells from peripheral blood
mononuclear cells (PBMC) of healthy HLA-A2-positive donors. Naïve T cells were
stimulated with either autologous MART-126-35 peptide-pulsed CD14 + monocytes or
CD1c + MDC in the presence of CD28 antibodies, IL-7 and IL-15. For further expansion
IL-2 was added subsequently from day three on. At day eight to ten the frequency, the
degree of expansion, and the expression of CD45RA, CD45RO and CD62L of HLA-A2/
MART-1-Tetramer + CD8 + T cells was determined. Additionally, production of IFN-? by
the expanded T cells was analysed after restimulation with MART-1-pulsed monocytes.
In PBMC of healthy donors naïve HLA-A2/MART-1-Tetramer + CD8 + T cells are
detectable at frequencies of around 0.07% of the CD8 + T cell population. Eight to ten
days after in vitro primary activation the frequencies of HLA-A2/MART-1-Tetramer + CD8
+ T cells increased to 0.3% - 5.7%, irrespective of whether monocytes or MDC were
used as antigen presenting cells (APC). We found a 90-fold expansion of MART-1specific
T cells, which showed a CD45RA + CD45RO-/+ CD62L- effector/effector memory
phenotype. Restimulation of the activated and expanded CD8 + T cells with MART-126-35 peptide- but not irrelevant peptide-pulsed monocytes led to IFN-? production in up to
70% of the HLA-A2/MART-1-Tetramer + CD8 + T cells, confirming antigen specificity and
indicating functionality of the cells.
In conclusion we established an in vitro protocol for priming and expansion of MART-1specific
CD8 + T cells based on the use of peptide-pulsed monocytes or MDC as APC