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International Congress of Immunology 2016
Abstract Book
RNA-seq at each time point showed that distinct cohorts of
gene expression, including bivalently marked genes,
Tbx21
(encoding T-bet) and
Irf4,
are transcribed as early as 3hrs post-
activation. Importantly, inhibition of H3K27me3 demethylation
perturbed the immediate expression of these transcription
factors along with early activation markers
i.e.
CD62L and
CD69
.
Therefore, the timely resolution of chromatin bivalency
by H3K27me3 demethylation, significantly shapes early CTL
gene expression programs through the activation of these
transcription factors. These molecular signatures potentially
represents a novel regulatory step involved in the formation of
effector and memory CD8
+
T cells.
Emerging Technologies
1831
Next-generation detection of antigen-responsive
T cells using DNA barcode-labeled peptide-major
histocompatibility complex I multimers
Bentzen, A.K.
1
, Marquard, A.M.
2
, Lyngaa, R.
1
, Saini, S.K.
1
, Andersen,
S.R.
1
, Donia, M.
3
, Svane, I.M.
3
, thor Straten, P.
3
, Szallasi, Z.
2
,
Jakobsen, S.N.
4
, Eklund, A.C.
2
, Hadrup, S.R.
1
1
Section for Immunology and Vaccinology, Technical University of
Denmark, Frederiksberg, Denmark,
2
Center for Biological Sequence
Analysis, Technical University of Denmark, Lyngby, Denmark,
3
Center for Cancer Immune Therapy, Copenhagen University
Hospital, Herlev, Denmark,
4
Immudex, Copenhagen, Denmark
Identification of antigenic peptides recognized by T cells is
important for understanding and treating immune related
diseases. Current cytometry-based approaches are limited
to simultaneous screening of T cell reactivity towards 10-100
distinct peptide specificities, which poorly match the large
diversityofTcellrecognitioninhumans.Consequentlyithasbeen
impossible to comprehensively analyze T cell responsiveness in
cancer, infectious and autoimmune diseases. We present and
validate a novel technology that enables parallel detection
of numerous different peptide-MHC responsive T cells in a
single sample using >1000 different peptide-MHC multimers
labeled with individual DNA barcodes. After isolation of MHC
multimer binding T cells their recognition are revealed by
amplification and sequencing of the MHC multimer-associated
DNA barcodes. The relative frequency of the sequenced DNA
barcodes originating from a given peptide-MHC motif relates
to the size of the antigen-responsive T cell population. We have
demonstrated the use of large panels of >1000 DNA barcoded
MHC multimers for detection of rare
T cell populations of virus and cancer-restricted origin in
various tissues and compared with combinatorial encoding
of fluorescent-labeled MHC multimers. Finally, we have
demonstrated that this technology can be applied for multiplex
T cell detection both in limited biological samples, such as
uncultured tumor material, and for simultaneous assessment
of target recognition and functional capability of T cells. This
technology enables true high-throughput detection of antigen-
responsive
T cells and will advance our understanding of immune
recognition from model antigens to genome-wide immune
assessments on a personalized basis.
3509
Multiplexed spatially resolved protein detection from FFPE
samples for oncology and immunotherapy
Rhodes, M.
1
, Warren, S.
1
, Jung, J.
1
, Merritt, C.
1
, Webster, P.
1
,
Dunaway, D.
1
, Mills, G.
2
, Tumeh, P.
3
, Beechem, J.
1
1
Nanostring Technologies, Seattle, United States,
2
MD Anderson
Cancer Center, Houston, United States,
3
University of California, Los
Angeles, Los Angeles, United States
Traditional immunohistochemistry approaches to staining
proteins within FFPE tissue are suboptimal because they
only evaluate a few targets simultaneously, rely on signal
amplification, and require subjective assessment of staining
intensity. NanoString has developed a method for determining
abundance and localization of a large number of proteins
(potentially up to 800-plex) from FFPE samples using non-
amplified digital counting. Tissue sections are stained with
antibodies conjugated to UV-cleavable barcodes and proteins
are enumerated by counting UV-released barcodes with the
the nCounter® Analysis System. This allows the quantification
of proteins from either a whole slide (to facilitate biomarker
development in highly reproducible format) or from small
regions of interest within the tumor (to permit the discovery of
novel biology).
Here, we present two applications of the technology. First,
breast cancer samples are stained with a oncocktail of barcode
labeled antibodies including HER2, ER, PR, EGFR, histone, and
others. Regions of interest are selected and barcodes from the
cocktail of antibodies quantified. The robustness of the system
is demonstrated by digital counts which are proportional to
the illuminated area and strong concordance between visual
intensity of HER2 staining and the HER2 digital counts. In the
second example, a 30+ cocktail of antibodies recognizing
immunologically relevant targets (including cell lineage
markers and immune checkpoints) are used to stain tumors and
elucidate patterns of immune cell distribution and activation
states.
Thisnewtechnologyenableshighlymultiplexedcharacterization
of protein expression and localization on both tumor and
infiltrating immune cells and facilitates the development of
novel biomarkers.
2351
Proof of concept for MHC allelic replacement by CRISPR-
Cas9 assisted cassette exchange
Kelton, W., Waindok, A., Pogson, M., Parola, C., Reddy, S.
ETH Zürich, BSSE, Basel, Switzerland
Allogeneic cellular transplantation is widely employed to treat
various genetic diseases and hematological malignancies.
Locating suitable donors for these procedures is often
challenging, as MHC/HLA gene alleles need to be matched in
order to prevent transplant rejection. We report here a proof
of concept study for an ex-vivo CRISPR-Cas9-based approach
for the precise exchange of MHC alleles (~5kb) at the native
genomic locus. For initial evaluation, themethodwas performed