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International Congress of Immunology 2016

Abstract Book

T-bet/Blimp1 double-deficiency resulted in the derepression of

several molecules usually associated with IL-17 producing CD8

+

T (Tc17) cells and severe immune pathology. Overall, our data

reveal two major pathways of effector differentiation governed

by the availability of Blimp-1 and T-bet and suggest a model

for cytokine-induced transcriptional changes that combine

quantitatively and qualitatively to promote robust effector CD8

+

T-cell differentiation.

757

The role of SATB1 in virus-specific CD8

+

T cell mediated

immunity

Nuessing, S., Russ, B.E., Grant, E.J., Kedzierska, K., Turner, S.J.

University of Melbourne, Peter Doherty Institute for Infection and

Immunity, Microbiology and Immunology, Melbourne, Australia

CD8

+

T cells are a critical component of the immune response

to viral infection such as the influenza virus. Upon virus

recognition immunologically

naïve

T cells undergo a program

of proliferation and functional differentiation resulting in a large

pool of

effector

cells secreting cytotoxic molecules and pro-

inflammatory cytokines required for viral clearance. Importantly,

a long-lived pool of virus-specific

memory

T cells survives after

resolution of the infection and responds more readily following

re-infection, allowing faster viral clearance and providing the

basis of protective T cell-mediated immunity.

Changes to the structure of DNA-protein complexes, within

respondingT cells underpin the differences in gene transcription

that confer the functional and phenotypic differences between

naïve, effector, and memory T cells. SATB1 is a master regulator

of chromatin structure, and as such, a key regulator of

transcription. We have shown that

Satb1

is expressed in naïve

T cells, down-regulated as naïve cells differentiate to effector

and then memory, in both mice and human. Additionally,

characterization of mice baring a dysfunctional

Satb1

gene

revealed a depletion of T cells baring the CD44

hi

signature,

a gene up-regulated after T cell activation. On this basis, we

hypothesize that SATB1 regulates CD8

+

T cell differentiation. To

address this hypothesis, we use next generation sequencing

methods to characterize the genome-wide distribution of

SATB1 binding at each stage of T cell differentiation. Combining

these data with transcriptomic analyses of the wild-type and

Satb1

mutant mice will provide insights into the role of SATB1 in

mediating virus-specific CD8

+

T cell differentiation.

1665

Transcriptional networks that establish B cell identity

Ikawa, T.

1

, Miyai, T.

1

, Kawamoto, H.

2

1

RIKENCenter for IntegrativeMedical Sciences, Yokohama, Japan,

2

KyotoUniversity, Institute for Frontier Medical Sciences, Kyoto, Japan

B lymphocytes are generated from hematopoietic stem

cells (HSCs) through a successive series of lineage restriction

processes. Although many transcription factors (TFs) and

epigenetic regulators have been implicated in controlling the B

cell fate specification, exact mechanisms remain elusive.

We have recently established a culture system that can

examine gene regulatory networks of HSCs differentiating

into B cell lineage (Ikawa et al. 2015). We overexpressed Id3

protein fused with ERT2 (Estrogen receptor) protein whose

nuclear translocation is induced by 4-hydroxytamoxifen (4-

OHT) in hematopoietic progenitors and cultured them in B cell

differentiation condition. B cell development of Id3-transduced

cells was blocked at an early developmental stage and the

cells grew enormously with maintaining multipotency in the

presence of 4-OHT. Virtually all cells became CD19+ B cells by

simply withdrawing 4-OHT within 7 days. We then performed

RNA-Seq analysis at multiple time points in this system.

The expressions of “master” TFs such as Ebf1 and Pax5 were

dramatically upregulated after 48hrs of induction. Notably,

the sequential upregulation of TFs before the onset of the key

TF program was found. The earliest responding TFs such as

Egr1 and Klf4 had a peak at 0.5-2hrs followed by a continuous

stream of TFs within 24hrs. Knockdown of these genes led to

the defected B cell generation both in vivo and in vitro. Taken

together, our findings identified a genome-wide view of the

dynamic transcriptional waves in B cell fate determination.

1387

Hierarchical regulation of enhancer establishment and gene

expression by transcription factors during mononuclear

phagocyte development

Kurotaki, D.

1

, Nakabayashi, J.

2

, Nishiyama, A.

1

, Sasaki, H.

1

, Kaneko,

N.

1

, Ozato, K.

3

, Suzuki, Y.

4

, Tamura, T.

1

1

Yokohama City University Graduate School of Medicine,

Department of Immunology, Yokohama, Japan,

2

Yokohama City

University, Advanced Medical Research Center, Yokohama, Japan,

3

NICHD, NIH, Bethesda, United States,

4

Graduate School of Frontier

Sciences, University of Tokyo, Department of Computational

Biology, Chiba, Japan

Monocytes and dendritic cells (DCs) are mononuclear

phagocytes essential for innate and adaptive immunity.

These phagocytes develop from hematopoietic stem cells

via intermediate progenitors, such as granulocyte-monocyte

progenitors (GMPs), monocyte-DC progenitors (MDPs), and

common monocyte progenitors (cMoPs) or common DC

progenitors (CDPs). However, the molecular mechanism

underlying their differentiation potential remains incompletely

understood. Recent studies suggest that promoter-distal

enhancers are key for cell fate decision. In this study, we

performed

chromatin

immunoprecipitation-sequencing

(ChIP-seq) analysis of enhancer-related histone modifications

(H3K4me1 and H3K27ac) in GMPs, MDPs, cMoPs, CDPs,

monocytes, DCs, and neutrophils. We found that monocyte- and

DC-specific enhancers were gradually established at progenitor

stages prior to gene expression. DNA motif analysis implicated

that these enhancers were regulated by combinations of

lineage-determining transcription factors such as PU.1, RUNX,

C/EBP, and IRF. Interestingly, the motifs specifically enriched

in the mononuclear phagocyte lineage were the IRF binding

motifs. Among IRFs, IRF8 is known to be highly expressed

from the MDP stage and necessary for MDP-to-CDP and

cMoP-to-monocyte transitions. Somewhat unexpectedly,

however, global gene expression patterns were comparable