Abstracts for the 25th Annual Scientific Meeting of the International ...

J Immunother Volume 33, Number 8, October 2010
Abstracts
human cancer patients, and (2) an inability to safely and effectively
label human T cells and NK cells.
Cell tracking of perfluorocarbon-labeled cells by 19F magnetic
resonance imaging (MRI) provides a highly-specific signal to
quantitatively assess in vivo migration and persistence. Previously,
19F MRI has been used to observe and measure the accumulation
of antigen-specific murine T cells to anatomic sites in vivo. A novel,
self-delivering, perfluoropolyether (PFPE) emulsion designed for
optimal MRI detection was tested for the ability to safely and
effectively label human primary, in vitro-expanded T and NK
lymphocytes.
Activated human T lymphocytes and NK cells and were effectively
labeled with PFPE, as measured by NMR spectroscopy, with no
evidence of apoptosis or cell death. A dual-mode fluorescent PFPE
probe revealed dose-dependent labeling by flow cytometry, and
localization of the tracer within vesicles in the cytoplasm using
confocal microscopy. The ability of labeled T cells to produce IFNg
and proliferate in response to TCR stimulation indicated no
impairment in these effector functions. Similarly, labeled NK cells
exhibited equal IFN-g secretion and cytotoxicity against target cells
as their unlabeled counterparts.
These results indicate that both T cells and NK cells can be labeled
with PFPE tracer agents for MRI without alterations in functional
properties. The clinical translation of this PFPE tracer for 19F MRI
may enable the quantification of effective homing and persistence in
the tumor microenvironment in cancer patients, significantly
advancing the effectiveness of adoptive immunotherapies.
INNATE/ADAPTIVE IMMUNE
INTERPLAY IN CANCER
Monocytes Enhance Natural Killer Cell Cytokine Production
in Response to Antibody Coated Tumor Cells in the Presence
of IL-12
Neela S. Bhave*, Robin Parihar*w, Adrian Lewis*, Volodymyr
Karpa*, Cassandra Skinner*, William E. Carson*. *The Ohio State
University, Columbus; w Cleveland Clinic Children’s Hospital, Cleveland,
OH.
Our group has shown in vitro, in murine tumor models and in
phase I clinical trials that co-stimulation of NK cells via the
interleukin-12 receptor (IL-12R) and the FcgRIIIa activates the
extracellular signal-regulated kinase (ERK) signaling pathway,
which in turn promotes the secretion of interferon-gamma (IFN-g)
thereby promoting potent anti-tumor effects. We hypothesized that
NK cell cytokine secretion would be significantly enhanced
following simultaneous stimulation of the NKG2D receptor and
that monocytes could serve as a source of NKG2D ligands. Costimulation
of purified NK cells with trastuzumab coated HER2+
SKBR3 breast cancer cells and IL-12 (10 ng/mL) resulted in
synergistic production of IFN-g (>20,000 pg/mL) as compared to
the single conditions (4 fold) as compared to un-supplemented NK cells
exposed to Ab and IL-12. A dose-response effect was observed with
increasing numbers of added monocytes. Pre-treatment of monocytes
with LPS and/or IFN-g led to increased expression of
NKG2D ligands (MICA and MICB) and increased the ability of
monocytes to act as co-stimulators of NK cell cytokine secretion.
The stimulatory effects of MICA/B+ monocytes could be
duplicated by the use of a MICA over-expressing cell line (C1R-
MICA) but not the parental MICA-negative cell line (2-3.2 fold
increase). Incubation of C1R MICA cells with a MICA neutralizing
antibody prior to co-culture with NK cells led to a significant
reduction in IFN-g secretion. The stimulatory effects of monocytes
were also observed in whole peripheral blood mononuclear cells
(PBMC) in that depletion of monocytes from PBMC markedly
inhibited the production of IFN-g by the NK cell compartment
whereas supplementation of PBMC with additional monocytes led
to dose-dependent increases in cytokine production. These data
suggest that stimulation of NK2D by monocyte ligands can
enhance the NK cell cytokine response to Ab-coated targets.
Enhancement of NK monocyte interactions could increase the
efficacy of Ab-based anti-cancer therapies.
Peripheral Blood Lymphocytes Induce Survival and Autophagy
in Human Renal and Bladder Carcinoma Cell Lines
William Buchser, Thomas Laskow, Tara Loux, Pawel Kalinski,
Per Basse, Herbert Zeh, Michael Lotze. Surgical Oncology,
University of Pittsburgh, Pittsburgh, PA and UPCI, University of
Pittsburgh, Pittsburgh, PA.
Objectives/Background: Macroautophagy is an important physiologic
process in stressed cells as well as being critical for antigen
processing and cross-presentation within Class II MHC molecules.
Many tumors expressing stress receptors such as MICA/MICB are
susceptible to natural killer (NK) cell induced apoptosis, interacting
with NKG2D on the cell surface.
Methodology: We first confirmed that human primary NK cells
could kill epithelial cancers cell lines-renal RCC4, T-24 bladder
carcinomas and others. NK cells co-cultured with RCC4 renal
cancer cells killed their targets at high ‘‘effector to target ratios’’
(E:T, 100:1) following 16 hours in the presence of 500 IU/mL IL-2.
Results: NK cells not only kill targets, but may enhance
programmed autophagy/cell survival in epithelial tumors. Interestingly,
we have observed enhanced autophagy in the spared target
cells in ‘‘high NK-kill’’ conditions. Both the fraction and the
number of autophagic cells increased (10% to 64%, 4 to 12 cells per
field), comparing no PBLs to 100:1 E:T. The effect was robust
(ANOVA P