Download T RM T CM

Common Clonal Origin of Resident Memory and
Central Memory T Cells after Immune
Responses in Skin
7th Drug Hypersensitivity Meeting
Thomas S. Kupper, MD
Thomas B. Fitzpatrick Professor
Harvard Medical School
Chair, Departments of Dermatology
Brigham and Women’s Hospital
Dana Farber Cancer Institute
Disclosure
In relation to this presentation, I declare the following, real or
perceived conflicts of interest:
- Scientific Advisory Board, Adaptive Biotechnologies
- Scientific Founder, TRmRx (no equity)
A conflict of interest is any situation in which a speaker or immediate family members have interests, and those may cause a conflict
with the current presentation. Conflicts of interest do not preclude the delivery of the talk, but should be explicitly declared. These
may include financial interests (eg. owning stocks of a related company, having received honoraria, consultancy fees), research
interests (research support by grants or otherwise), organisational interests and gifts.
Memory T Cell Tissue Migratory Polarization:
Development of T cell tissue tropism
Lymph
node
homing
L-selectin
Central memory (TCM)
blood
CCR7
Skin Homing (re-circulating)
Skin
Naïve
T cell
CLA
CCR4
Skin Resident TRM
VLA-1
Skin LN
Lung Homing (recirculating)
Lung LN
Lung
Lung Resident TRM
Gut LN
Effector T Cell (TEff)
Gut Homing (recirculating)
Gut
Gut Resident TRM
47
CCR9
Skin is one of several “Barrier Tissues”
Self-renewing epithelia
Unique Microbiomes
Sites of pathogen entry
Sites of environmentally
induced carcinomas
Skin resident T cells
2.0 x 1010 T cells in skin
20 Billion T Cells
1.1 x1010 T cells in blood
Clark, Kupper et al., Journal Immunology,
2006
Skin resident T cells
Skin tropic CLA+ effector memory T cells
• 98% are in normal appearing skin
•
2% are in blood
Different from:
• T cells in human blood
• Exclusively memory
• Potent effector function
Clark, Kupper et al., Journal Immunology, 2006
High throughput sequencing of
human TCR genes
Random insertion of
nucleotides
Every naive  T cell has a unique Tcr
V CDR3 sequence
Skin T cells are diverse
Deep sequencing of TCR V CDR3 genes from healthy human blood and
skin (split into dermis and epidermis)
R. Clark, T. Kupper
Do other Barrier Tissues host TRM?
Self-renewing epithelia
Unique Microbiomes
Sites of pathogen entry
Sites of environmentally
induced carcinomas
Lung
Skin
Gut
T cell diversity in human lung
R. Clark, T Kupper
T cell diversity in human gut
R. Clark, T. Kupper
Top 10 skin clones
Blood
Gut
Lung
Skin
R. Clark, T. Kupper
Top 10 gut clones
Blood
Gut
Lung
Skin
R. Clark, T. Kupper
Top 10 blood clones
Blood
Gut
Lung
Skin
R. Clark, T. Kupper
Barrier Tissues
Self-renewing epithelia
Unique microbiomes
Sites of pathogen entry
Sites of environmentally
induced carcinomas
Contain diverse
populations of TRM
Skin
TRM
Gut TRM
Lung
TRM
Barrier Tissues (TRM)
Immune Memory : Tissue TRM , lymph node TCM
CLA/
E selectin
ligand
TCM
L selectin
CCR7
Skin TRM
?
Lymph Nodes (TCM)
TEff
Lung TRM
47
Gut TRM
Memory T cells
enter tissue and
take up
residence as TRM
Understanding human T cell biology using
mouse models
Vaccinia virus (VACV)
skin infection model
CD8 TCM and TRM
Lisa Liu, MD, PhD
Xiaodong Jiang, MD, PhD
Skin Immunization
Model
Olivier Gaide, MD
TCR high
throughput
sequencing for TCM
and TRM
When Do Activated T Cells Commit
to Tissue Tropism?
Experimental plan using VACV
Cervical LN
(skin)
Mediastinal LN
(lung)
Skin infection
Pulmonary infection
GI infection
Mesenteric LN
(GI)
Sorting activated CD8+ T cells in LN by cell division
using CFSE
Principal Component Analysis of the Transcriptional Profiling
Data Shows Unique Patterns for Skin, Lung, and Gut
Imprinting as early as P1
Each T cell population generated in a barrier tissue lymph node has
Results
Venn
diagramof
analysis
of genes
mostly
differentially
expressed
in
a subset
unique
genes;
Skin
and
Lung
are
mostly
overlapping
+
activated CD8 T cells (P5 vs Naïve, cutoff 4 fold change)
CD8 Skin TRM Cells: 60 days post
VACV infection
Day 60 after VACV: LN TCM and Skin TRM are generated
TCM
Distinct
transcriptional
profiles (7x)
TRM
TCM
TRM
Tian and Kupper, unpublished
Do TRM re-circulate at steady state?
TCM in lymphoid tissues re-circulate freely
between parabiotic partners
Mice joined for four weeks
Non immunized
Parabiotic partner
Immunized
+OT-1 cells
Jiang and Kupper, Nature 2012
Skin resident TRM do not re-circulate between
parabiotic partners
Non immunized
parabiotic partner
Immunized
parabiotic partner
Jiang and Kupper, Nature 2012
Are skin resident TRM protective in the
absence of antibody and TCM?
Rechallenge
Vaccinia
FTY720
Circulating
TCM
B cell deficient mouse:
Is the mouse protected?
Liu and Kupper, Nature Medicine 2010
Skin TRM
Skin TRM protect against re-infection in the
absence of TCM and antibody
10,000-fold
difference
TRM+TCM
Only skin TRM
Liu and Kupper, Nature Medicine, 2010
Are TRM better than TCM in providing protective
antiviral immunity in skin?
No transgenic
T cells,
B cell
deficient mice
TRM are superior to TCM at mediating rapid protective
antiviral immunity in skin
Day 6 (- or + FTY 720)
**
10 7
**
10 6
10 5
-
+
10 4
10 3
-
-
+
+
10 2
-
+
10 1
10 0
10 -1
FT
Y7
20
20
no
r.p
no
Naïve
Parabiont
TCM
only
ar
a.
+
TY
7
a.
-F
r.p
ar
ra
.
+F
TY
72
0
72
0
m
u.
pa
TRM + TCM
im
im
m
u.
TRM + TCM
Immunized
Parabiont
TY
20
pa
ra
.-F
+F
T
Y7
20
m
u.
TY
7
72
0
m
u.
-F
im
l+
FT
Y
ct
Immunized
Control
im
Naïve
Control
72
0
TY
l-F
ct
viral DNA copies / g DNA
10 8
Jiang and Kupper, Nature 2012
Jiang and Kupper, Nature 2012
TRM are more effective than TCM in mediating
protective antiviral immunity in skin
Day 14 (- or + FTY 720)
10 7
10 6
10 5
10 4
-
+
10 3
10 2
10 1
-
-
+
-
+
+
10 0
72
0
FT
Y
TCM only
a.
+
a.
-F
ar
r.p
no
no
r.p
ar
ra
.
pa
m
u.
72
0
TY
TY
+F
-F
ra
.
im
72
0
TRM + TCM
TY
Y
FT
m
u.
pa
im
m
u.
+
im
-F
TY
TRM + TCM
72
0
72
0
72
0
72
0
m
u.
im
l+
FT
Y
Naive
ct
TY
72
0
l-F
N/A
N/A
10 -1
ct
viral DNA copies / g DNA
10 8
JiangJiang
and Kupper
2012
and, Nature
Kupper,
Nature 2012
TRM are much more effective than TCM in mediating
protective antiviral immunity in skin
Day 26 (- or + FTY 720)
10 7
10 6
-
10 5
10 4
-
+
+
10 3
10 2
10 1
-
-
+
+
10 0
20
72
0
Y7
ar
a.
+
FT
TY
r.p
r.p
a
ra
.-F
+F
no
TCM only
no
im
m
u.
pa
ra
.
ra
.
pa
72
0
TY
TY
TRM + TCM
-F
TY
+F
m
u.
im
72
0
72
0
72
0
im
m
u.
-F
m
u.
im
ct
TRM + TCM
TY
Y7
20
Naive
l+
FT
72
0
TY
l-F
N/A
N/A
10 -1
ct
viral DNA copies / g DNA
10 8
Jiang
Kupper,
Jiang andand
Kupper,
Nature 2012 Nature, 2012
What determines whether an individual
naïve T cell becomes a TCM ,TEM, or TRM
TCM
TCM
TEM
TRM
Tian and Kupper, unpublished
What determines whether a naïve T cell
gives rise to a TRM?
L-selectin
CCR7
Central memory (TCM)
blood
CLA
CCR4
TCR affinity?
Random
Stochastic?
Naïve
T cell
Lymph
node
homing
Skin TRM
Skin LN
Lung LN
Gut LN
Effector T Cell
(TEff)
Tissue
Resident
Memory
T Cell
(TRM)
VLA-1
Lung TRM
47
CCR9
Gut TRM
TRM vs TCM cell fate decision
Affinity model
Stochastic model
Low affinity
TCM
TCM
APC
High affinity
APC
TRM/EM
TRM/EM
Context model
APC
Single Responder model
TCM
TCM
APC
APC
TRM/EM
TRM
Skin immunization with three distinct antigens:
protein, hapten, and virus
Day -8
Day -7
Day 0= immuniz.
Day 35
+OTI iv
OVA+CT
OVA
DNFB
DNFB
MVA ss
MVA
Tail skin /
Left inguinal LN
Tail skin, Ear skin /
Cervical LN, right inguinal LN
Extraction of DNA from LN and Skin at different time points for
high throughput sequencing of the TCR V CDR3
Epicutaneous Ova + CT generates skin TRM
and LN TCM with identical TCR V
TCR percentage (specific/total)
T cell concordance at two skin sites
10 -
Tail 6 wks post OVA+CT to ear
Clone frequency (%)
Ear 6 wks post OVA to ear
OVA + CT
CASSRANYEQYF
Clone Frequency (%)
10
1-
1
C ASSPDKYYAEQFF
0.1 -
C ASGGGGSYEQYF
0.1
C ASSTNSDYTF
C ASSDTRGQETLYF
0.01 -
C ASSLERLGLGDQDTQYF
0.01
LN
Pre-
Gaide and Kupper, Nat Med, 2015
Tail
C ASSRANYEQYF
Ear
Drain.
LN
Tail
Dist. LN
Postimmunization
Comparably sized shared clones in skin (TRM)
and LN (TCM) after OVA immunization
j
Clone presence (color coded)
Average Clone Size
Expanded Ear/Tail
17
Also in
distant LN
10
Also in
draining LN
13
Gaide and Kupper, Nat Med, 2015
TRM
TCM
DNFB Contact Hypersensitivity generates TCRidentical TRM and TCM
TCR percentage (specific/total)
Ear 6 wks post DNFB to ear
10
Clone Frequency (%)
DNFB
T cell concordance at two skin sites
1
CTCSAQGHTLYF
CASSLGTGAETLYF
0.1
CTCSAAGNTEVFF
CASSQDGVNQDTQYF
CASSRQYNSLYF
0.01
LN
Tail 6 wks post DNFB to ear
Gaide and Kupper, Nat Med, 2015
Tail
Ear
CASSISANSDYTF
Drain
LN
.
Pre
Tail
Dist. LN
Post immunization
Comparably sized shared clones in skin (TRM)
and LN (TCM) after DNFB contact dermatitis
Clone presence (color coded)
Average Clone Size
Expanded Ear/Tail
18
Also in
distant LN
23
Also in
draining LN
Gaide and Kupper, Nat Med, 2015
10
TRM
TCM
Blood Contamination Does Not Account for Skin
T Cell Clones
1000
100
Blood
Skin: Tail
Inguinal LN
10
Skin: R ear
R Cervical
A6_1_B24h
A6_1_Inguinal
Skin:
1
1
2
3
A6_1_RightEar
4
5
Gaide and Kupper, Nat Med, 2015
6
7
8
A6_1_LeftEar
9
10
LN
L ear
L Cervical LN
Resident Memory T Cell (TRM)Polarization:
non-stochastic, independent of TCR affinity
Lymph
node
homing
L-selectin
CCR7
Central memory (TCM)
blood
CLA
CCR4
Skin TRM
Naïve
T cell
Common
TM precursor
Skin LN
Lung LN
Gut LN
Effector T Cell
(TEff)
Tissue
Resident
Memory
T Cell
(TRM)
VLA-1
Lung TRM
47
CCR9
Gut TRM
Is Contact Hypersensitivity Mediated by TRM or TCM ?
Day 0, 1, 7
Day 35
DNFB
sensitization.
naive
Parabiotic
surgery
4 weeks
Surgical separation
2 weeks
Challenge
(DNFB)
Naive
Sens.
Parab.
sens.
Parab.
naive
4 weeks
2nd DNFB
challenge
TRM mediate rapid CHS while TCM mediate a
delayed and limited response
Naive
Sens.
Parabiotic naive (TCM only)
Parabiotic sens.
Ear Swelling Response
45
40
35
30
25
20
89
90
91
92
93
94
95
Days post sensitization
96
97
98
99
Gaide and Kupper, Nature Medicine, 2015
TRM Contact Hypersensitivity is FTY 720 resistant, while
the TCM response is FTY 720 sensitive
Parab. sens.
Parab. sens. + FTY720
45
Parab. naive
40
Parab. naive + FTY720
35
30
25
20
89
90
91
92
93
94
95
Days post sensitization
96
97
98
99
Gaide and Kupper, Nature Medicine, 2015
HumanDPCP-associated
Contact Hypersensitivity
to Abundance
Diphencyprone
(DPCP)*
T Clonal
in Skin
RM
Dayday
3 3
DPCP
Day 14
Day 120
DPCP day 14
(Normalized TCR counts
/ 400ng tissue) DPCP 4 months
Patch test site
Placebo
Placebo
HE
1000
CD3 stain
100
10
CD3
0.1
D3 Placebo vs. D3 DPCP
3
1
5
2
4
Human Skin, patient A, Placebo skin
D3_Placebo
D120 DPCP vs. Day 14 DCPC
Hu Skin, 14 days after DPCP exp.
1
Hu Skin, patient A, 3 days after DPCP
DPCP = di-phenyl-cyclo-propenone
D3_DPCP
1
5
4
2
3
Human Skin, patient A, 4 month after DCPC exp.
D14_DPCP D120_DPCP
Gaide and Kupper, Nature Medicine, 2015
Barrier Tissues (TRM)
New View of Immune Memory
Staph aureus
Strep pneumonia
Rotavirus
Candida
Mycoplasma pneumonia
E.Coli
HSV
Reserve
Influenza
Rapid
Immunity:
Shigella
Protective
Can
Immunity
Augment
Against
TRM over
Infection
Time
Two compartments of immune
memory: peripheral tissues and
secondary lymphoid tissue
Lymph Nodes
(TCM)
TRM in Human Disease
T cell mediated drug reactions in barrier tissues (esp skin)
Chang and Kupper, Nature Medicine 2015
Acknowledgements
funded by National Institutes of Health
(NIAMS, NIAID, NCI)
Olivier
Gaide
Rachael Clark
Adaptive Biotech
H. Robins
R. Emerson
C. Desmarais
Rockefeller University
N. Gulati
J. Krueger
Xiaodong Jiang
Lisa Liu
Rahul Purwar
Changook Park