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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,


(encoding T-bet) and


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


CD62L and



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


Next-generation detection of antigen-responsive

T cells using DNA barcode-labeled peptide-major

histocompatibility complex I multimers

Bentzen, A.K.


, Marquard, A.M.


, Lyngaa, R.


, Saini, S.K.


, Andersen,



, Donia, M.


, Svane, I.M.


, thor Straten, P.


, Szallasi, Z.



Jakobsen, S.N.


, Eklund, A.C.


, Hadrup, S.R.



Section for Immunology and Vaccinology, Technical University of

Denmark, Frederiksberg, Denmark,


Center for Biological Sequence

Analysis, Technical University of Denmark, Lyngby, Denmark,


Center for Cancer Immune Therapy, Copenhagen University

Hospital, Herlev, Denmark,


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


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-


T cells and will advance our understanding of immune

recognition from model antigens to genome-wide immune

assessments on a personalized basis.


Multiplexed spatially resolved protein detection from FFPE

samples for oncology and immunotherapy

Rhodes, M.


, Warren, S.


, Jung, J.


, Merritt, C.


, Webster, P.



Dunaway, D.


, Mills, G.


, Tumeh, P.


, Beechem, J.



Nanostring Technologies, Seattle, United States,


MD Anderson

Cancer Center, Houston, United States,


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



of protein expression and localization on both tumor and

infiltrating immune cells and facilitates the development of

novel biomarkers.


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