of islet antigenspecific Tregs Bluelab meeting 1 0914 Daqi The role of Treg in T1D Pathogenesis of T1D Autoreactive T cells are responsible for the onset of T1D ID: 930412
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Slide1
Specificity and repertoire of islet antigen-specific Tregs
Bluelab
meeting
1
0/9/14
Daqi
Slide2The role of Treg in T1DPathogenesis of T1DAuto-reactive T cells are responsible for the onset of T1D (Peterson and Haskins. Diabetes. 1996)
Impaired
Treg in T1D
Loss of suppression function of Treg accelerates T1D and other autoimmune diseases
(
Brusko
T.
Immun
reviews.
2008)
Compromised IL-2 signaling:
pSTAT5
(Long SA. 2010 Diabetes)
Plasticity: IFNγ producing
Treg，methylated
TSDR
(
McClymont
SA et al. JI.2001)
Other autoimmune diseases: RA, SLE, MS, IBD…
Slide3Ongoing and reported Treg Clinic Trial
Slide4To improve Treg cell therapy: Antigen-specific TregsAdvantages of Antigen-specific Tregs:More Efficient: Although Tregs can function in a bystander way, Ag-specific Tregs suppress more efficiently than polyclonal/non Ag specific Tregs.
Less
potential risk: Not
global
suppression
Tang, et al. 2004
JEM
Masteller, et al. 2005 JI
25x10
6
NOD splenocytesNOD rag ko or NOD TCRa KO miceopen circle: +2x106 expanded BDC2.5 Treg
2x10
6
expanded Treg
pre diabetic CD28-/- NOD mice
Slide5Current approaches to identify Ag+
suppressor
cells
Suppressor
Antigens
Assay
other
notes
Tr
1/Th2
IL-10
IL-4
GAD65 (4.13, 164) (Reijonen, H., R. 2004
Diabetes
)
(
Reijonen
, H., R.
200
6
Diabetes
)
(
Gebe
JA, 2009. JI)
ProINS
(73-90)
(
Durinovic-Belló
A.
2
006
PNAS)
IA
-2
(Thomas
A.
2004
JCI)
(Tree
TI.
2010
Diabetes)
INS
-B
(11-30) (Tree
TI.
2010
Diabetes)
E
lispot
HLA
class
II
-peptide
Tet
in
vitro
APC-
T
assay
I
mmunization
HLA-DR4
mouse
iTreg
Foxp
3
IL-
10
TGF-
β
GAD
65
(Long
SA.
2009
EJI)
IGRP
(Long
SA.
2009
EJI)
HLA
class
II
-peptide
Tet
Ag
+
Tconv
+
irradiated
APC
Ag
+
iTreg
(14 day
stimulation
)
IL
-10,
TGF
-
β
dependent
suppression
Treg
(
in
vivo)
Foxp
3
IL-10
cancer
Ags
:
colorectal carcinoma
(
Bonertz
A,
2009.
JCI)
breast
cancer/
mammaglobin
(mam34–48) (
schmidt
HH, 2013.
OncoImmunology
)
Elispot
suppression
assay (Ag
+
Teff
)
HLA
class
II-peptide
Tet
IL
-10
Elispot
sub
-
cloning
+
suppression
assay
(Ag+
better
suppression
)
Slide6Islet antigen-specific Tregs in humanHuman Foxp3+ Treg specific
for
islet
antigens
have
not been
identified although the importance
role of
these Tregs
in T1D;low frequencyanergic status of TregThe
frequency and function of
the antigen specific Foxp3 nTregs in T1D have not been
analyzed
in
T1D
compared
to
healthy
subjects
;
The
tTregs
may represent
different regulatory
TCR
repertoire
than
converted
iTreg
:
non-regulatory and regulatory T cells: 10-20% overlap;
(Hsieh CS, NI. 2006)
Tissue
-
resident
Tregs
have
enriched
TCR
Tissue
-
resident
Treg(VAT,
muscle)
has
little
overlap
with
Tconv
(either
tissue
or
lymphoid
organ)
(
Feuerer
M,
2009. Nat Med;
Burzyn
D.
2013
Cell)
Compared
to
the
iTreg
strategy,
naturally
occurring
Foxp3
Tregs
are
lineage/function
stable
Slide7Hypothesis and GoalHypothesis: The TCR diversity, quantity and function of islet-Ag specific Tregs in T1D are impaired compared to healthy individuals. Aim 1: Screen and identify islet Ag-specific Treg cells in healthy individuals and T1D patientsAim 2: Compare
islet Ag-specific Tregs of healthy individuals and T1D patients: frequency,
TCR
repertoire
,
suppression
Goal
Identify the islet-Ag specific Tregs
and compare the Ag-specific Tregs in healthy individuals and T1D patients.In the long run: More efficient Treg cell therapy with less off target effects
Slide8Human regulatory T cell libraryMethod:
(…or pancreas and LN from
nPOD
)
.
+[3H] Thymidine
cpm
readout
3 day culture
16hr 3H pulse
Ag+Treg
: phenotype, function in ctrl
v.s
. T1D
14-day expansion
CD3CD28 beads+IL2
Network for Pancreatic Organ Donors with Diabetes (
nPOD
)
Slide9Advantage: highlight rare population
96
cells:
polyclonal
95x1000cpm+1x5000cpm
≈
96x1000cpm
polyclonal
expansion:
anti-CD3+anti-CD28
95
well:
1000cpm
the
antigen-specific
well:5000cpm
---Why
we
want
to
use
Sallusto’s
method:
sub-culuture
polyclonal
expansion
Ag-driven
proliferation
screening
Slide10put the drawing in the white board
in
Slide11Islets antigens that elicit auto-reactive T cell responses (Human)Roberto Mallone. et al., Clinical and Developmental Immunology. 2011MyLinh Dang., et al. JI 2011
Peter A.
Gottlieb. et al., Journal of Autoimmunity. 2014
+
+
Slide12Autoantigen distribution: CD4 T cell epitopes in autoimmune diabetes in humans
T. P. Di
Lorenzo
,M
.
Peakman
and B. O.
Roep. Clinical and
Experimental Immunology. 2007
Slide13Candidate antigensZnT8
GAD
65
ProINS
PreProINS
IGRP
Antigens
:
whole
protein
InsulinIA-2Proinsulin
Slide14Insulin-specific Treg in healthy individuals
2000/well
n=31
n=25
Ag: Insulin
CTRL: irradiated autologous monocytes w/o Ag
INS: irradiated autologous monocytes pulsed with insulin
4
0/
10
6
16/10
6
Slide15Ag-specific Treg response: readoutCPM (INS-CTRL)=CPM (monocyte w/ INS) – CPM (monocyte w/o Ag pulsing)
responses > μ+2σ(p<0.05)
Slide16INS-specific Treg in T1D subject, PBMCCTSA0022T1DHLA
:
DR4
DQ8
detectable c-peptide
n=96
1000 cells/well
Slide17Insulin-specific Treg in T1D
responses > μ+2σ(p<0.05)
donor#3, H
donor#5, T1D
no c-peptide
n=96
2000 cells/well
5/
10
6
5/
10
6
donor#7, H
donor#6, T1Ddetectable c-peptiden=484000 cells/well0/106
1
0/
10
6
cpm
(INS-CTRL)
Slide18Antigen recallthe antigen-specific cultures identified from primary antigen screening
assay
were
recalled with different dose of corresponding antigens, including insulin,
proinsulin
and IA-2
stimulation
index
P
rimarySecondary
Slide19Antigen recall: insulin
samples
CTRL
INS
-
1
st
(primary)
INS
-2nd (recall)1st
-roundCTRL
CTRLINS
2nd-roundCTRLINSINS
Donor: Healthy,
DRB1 0401n=49, 2000 cells/well initially
Slide20TCR
enrichment
after
antigen
encountering
TCR-beta, adaptive TCR sequencing
Slide21ZnT8-specific Treg
ctrl
ZnT8
#1
#20
#30, negative ctrl
Donor
:
Healthy,
DRB1
0401n=49, 2000 cells/well initially
sort Tregpolyclonal expansionof Treg
library
Ag
screening
assay:
Ag
-driving
proliferation
CFSE
Slide22CaseID: 6178 Donor Type: No diabetes AutoAb (RIA): Negative Age (years): 24.5 Diabetes Duration (years): Gender: Female Ethnicity: Caucasian C-peptide (
ng
/ml): 4.55
HbA1c: 5
BMI (chart): 27.5
ClinicalHistory
:
Cause of Death: Anoxia
Histopathology : Ins+/Gluc+ normal islets. Low Ki67. No infiltrates. HiRes HLA: A*02:01 , 24:02
DRB1*04:01 , 15:01 DQA1*01:02 , 03:01DQB1*03:01 , 06:02HLA_Transplant: A*02/24, B*27/44, DR*04/15, DQ*06/03 Data Group: nPOD
nPOD
sample, #6178
: healthy subject
Slide23nPOD#6178, PLN Treg library
：
screening
for
INS+Treg
INS
#9
#53
ZnT
8
stimulation
index: ratio of (CFSE_diluted[Ag+]%/CFSE_diluted[ctrl]%)colored dot: SI>2xSD+Mean
(p<0.05)
A
g:
INS
4000 Treg cells/well, n=96
INS---
Ctrl---
pZnT8---
Ctrl---
#47
Insulin
screening
ZnT
8
screening
Slide24CaseID: 6266 Donor Type: T1D AutoAb (RIA): GADA+ IA-2A+ mIAA+ ZnT8A+ Age (years): 30
Diabetes Duration (years): 23
Gender: Male
Ethnicity: Caucasian
C-peptide (
ng
/ml): <0.05
HbA1c: 13 BMI (chart): 27.1 ClinicalHistory: Cause of Death: Anoxia Histopathology : Ins-/
Gluc+ islets present in reduced numbers, atrophic. Moderate acinar atrophy and fatty replacement with moderate interlobular fat. Chronic pancreatitis- multifocal, mild. HiRes HLA: A*01:01,03:01
DRB1*03:01,04:04 DQA1*03:01,05:01 DQB1*02:01,03:02
HLA_Transplant: A*01/03 B*08/61 DR*04/17 DQ*02/08 Data Group: nPOD
nPOD sample：#6266, T1D
Slide25nPOD#6266, PLN
Treg
library
：
screening
for
INS+ and ZnT8+ Treg
stimulation index: ratio of
(CFSE_diluted[Ag+]%/
CFSE_diluted[ctrl]%)colored
dot: SI>2xSD+Mean (p<0.05)Ag: INS,
ZnT8 peptide mix4000 Treg cells/well, n=96
#93
#
95
#27
INS---
Ctrl---
pZnT8---
Ctrl---
Insulin
screening
ZnT
8
screening
INS
ZnT
8
Slide26Enhance Treg proliferation upon antigen stimulationOptimize APCmdDC (human GM-CSF+IL-4)HdAc
inhibitor
HdAc
inhibitor I:
entinostat
(ms-275)
Slide27mdDC as APC in antigen screening assay
ratio: 10.4/10
6
HLA
-DR
CD
86
isotype
HLA
-DR
isotype
CD
86
Slide28HdAc inhibitor: EntinostatClass I Histone Deacetylase Inhibitor Entinostat (MS-275)Reduction of
Foxp3 (mouse, in vivo, in vitro)
Mechanism
:
STAT3
acetylation
Shen. L 2012
PlosOne
Suppression
Foxp
3
expression
Slide29Class I inhibition, not class II inhibition suppresses Foxp3 expression in TregsClass I inhibitor
Class II
inhibitor
Pan
-inhibitor
Shen
L
.
2012
PlosOne
Slide30Entinostat enhanced Treg proliferation, while reduced Foxp3 expression
Slide31Entinostat(ms-275) treatment during Treg library polyclonal
expansion
ms-275 treatment during polyclonal expansion
Antigen
:
insulin
sort
Treg
polyclonal
expansionof
Treg libraryAg
screening assay:Ag-driving proliferationw/o
ms-275
with
ms-275
Slide32Entinostat (ms-275) treatment during antigen screening assay
ms275 treatment
only
during antigen
priming
mean
=623
mean
=1464
Antigen
: ZnT8 peptide
mixsort
Tregpolyclonal expansionof Treg libraryAg screening
assay:
Ag
-driving
proliferation
w
/o
ms-275
with
ms-275
Slide33Plans to optimize the assaystudy more
on
the
kinetics
of
ms-275 to reduce
foxp3combine mdDC+ms-275 treatment
Slide34Summaryinsulin-specific Tregrespond to antigen recallTCR-b
enrichment
ZnT
8-specific
Treg
Treg polyclonal expansion enhanced by
HdAC inhibitor I/ms-275Insulin and ZnT8 specific Treg candidates (
nPOD PLN)were identified and ready for deep sequencing
Slide35Future stepsIslet antigen-specific Treg TCR specificity and repertoireScreen Treg library: islet antigens
deep sequencing to uncover the enriched TCR specificity
single cell level deep-sequencing to identify enriched TCR and related phenotypes (Periphery
v.s
. PLN/Inflamed tissue)
Optimization of Ag-Treg screening assays
HdAC
class I inhibitor
sBcartificial APC: K562-DR4-CD86
Slide36Treg TCR repertoirenon-regulatory and regulatory T cells: 10-20% overlap; (Hsieh CS, NI. 2006)Tissue-resident Tregs
have
enriched
TCR
Tissue-resident Treg(VAT,
muscle) has little overlap with
Tconv (either
tissue or
lymphoid organ) (Feuerer M,
2009. Nat Med; Burzyn D. 2013 Cell)
Slide37Islet antigen-specific repertoireIslet antigen-specific repertoire:(Aim2)Healthy v.s
. T1D subject
tTreg, pTreg, Tconv
PBMC/Spleen, pancreatic LN, pancreas (
nPOD
sample)
Slide38Identification of human tTreg vs pTregHelios+,Helios-: but
not
surface
TIGIT
,
CD226: surface markers
data from Todd
Brusko
38%
H
elios+91% Helios+
TIGIT
CD
226
pTreg
t
Treg
Slide39AcknowledgementJeff BluestoneAmy PutmanAngela LaresFredric Van GoolEle Trotta
Shen
Dong
Hilary Thomas
Weihong
Liu
Michel
DuPage
Armando VillataCaroline Raffi
Mike LeeVinh NguyenGaurav ChopraAndy Xia (summer student)
Everyone in Bluelab!
Marc Martínez-
LlordellaTodd BruskoBart Roep (IA-2) Leiden University Medical
CenterBIOMM Techology (Proinsulin)
Slide40To enhance Ag-specific proliferation: Antigen recall
+10 IU/ml huIL2, 3 days
+Autologous monocytes pulsed with insulin
(Ag recall)
+300 IU/ml huIL2, 4 days
responses > μ+2σ(p<0.05)
Treg
Teff