Transcript Slide 1

The role of hyaluronan and
hyaladherins in T cell
proliferation
Mark Mummert, Ph.D.
UNT Health Science Center
Seminar Outline
I.
Hyaluronan overview
II.
Hyaluronan mediated T cell
proliferation
III. CD44 regulated T cell proliferation
Biochemical Properties of Hyaluronan (HA)
Unbranched glycosaminoglycan
Repeating N-acetyl-glucosamine
and glucuronic acid subunits
Molecular weight at least >300kDa
HA Synthesis
HA Degradation
HAS1 HMW-HA polymer
Hyal-1
Tetra- and hexasaccharides
HAS2 HMW-HA polymer
Hyal-2
HMW fragments
HAS3 Intermediate HA
Hyal-3 Unknown
Hyal-4 Unknown
HA Synthase
UDP-GlcA
UDP-GlcNAc
Nucleus
Functions of HA
Tissue hydration
Structural integrity
Lubrication
Cell activation
Cell trafficking
Cell proliferation
WF/GM/OE-HABP
LYVE-1
CD54
CD38
BEHAB
Inter-a-trypsin inhibitor related proteins
LEC HA receptor
Neurocan
Hyaluronectin
Versican
Aggrecan
Link Protein
RHAMM
Hyaluronan
CD44
WF/GM/OE-HABP
LYVE-1
CD54
CD38
BEHAB
Inter-a-trypsin inhibitor related proteins
LEC HA receptor
Neurocan
Hyaluronectin
Versican
Aggrecan
Link Protein
RHAMM
Hyaluronan
Collagens
Fibronectin
Chondroitin sulfates
Heparin
Heparin sulfate
Serglycines
Osteopontin
CD44
Available HA Probes
Hyaluronan Binding
Protein (HABP)
Comments
Enzymatic digest of bovine nasal
cartilage
Variation in quality of different batches
Expensive
HA Receptor-Fc Proteins
Produced recombinantly
Not available commercially
Synthesis is typically low
Specificity is questionable
Frequency of Sequences from Phage
Clones
Peptide Designation
Frequency
1
13/19
2
2/19
3
1/19
4
2/19
5
1/19
*
Metabolic Labeling
With 35S
B16-F10
*
*
*
HA-coated Plate
* *
Lyse Cells
*
* *
*
Wash Plate
β-counter
*
**
Background
No Inhibitor
RP: SATPASAPYPLA
**
Pep-1: GAHWQFNLATVR
Scrambled: RNHALVGWQFTA
Scrambled: AGNHLATVFWQR
Scrambled: QVNLAHTWARFG
Scrambled: WRHGFALTAVNQ
0
5
10
15 20 25
% Bound
30
35
40
Background
No Inhibitor
Pep-1 (WT)
RP
Pep-1 (G A)
Pep-1 (H A)
Pep-1 (W A)
Pep-1 (Q A)
Pep-1 (F
A)
Pep-1 (N A)
Pep-1 (L
A)
Pep-1 (T
A)
Pep-1 (V A)
Pep-1 (R A)
**
**
**
**
**
0
5
10 15 20 25 30 35
% Bound
Functional roles of HA in DC-dependent Ag
restricted T cell proliferation
Diana I. Mummert
Dale Edelbaum
Francis Hui
Hiroyuki Matsue
Akira Takashima
Dendritic cells (DC)
Derived from bone marrow precursors and are found in
lymphoid and non-lymphoid tissues. They have a dendritic
morphology and are potent stimulators of T cells.
T cells
A subset of lymphocytes defined by their development in the
thymus and by heterodimeric receptors (α:β or γ:δ) associated
with the CD3 complex (CD3γ,δ, ε, and dimeric ζ chains).
Adhesive Molecules and Antigen Presentation
T cells
TCR
LFA-1
ICAM-3
DC
Ag/MHC II
ICAM-1
DC-SIGN
CD44 Overview
Glycoprotein
Expressed by leukocytes
Major receptor for HA
Leukocyte trafficking
Signaling co-receptor
CD44/ HA Molecular Interaction and T cells
Stimulated T cells express functional CD44
isoforms
Exogenous HA enhances mitogen induced T cell
proliferation
Anti-CD44 blocks HA augmented proliferation
Figure 3. CD44 in Signaling
A. There is little evidence for direct CD44 signaling upon ligation with hyaluronan.
B. CD44 can function as a co-receptor, physically linked to other classical signaling receptors.
C. CD44 can serve as a docking protein for pericellular proteins, such as MMPs or, for cytoplasmic proteins
such as kinases, Smad1, actin-binding proteins and other adapter proteins, which can subsequently activate
signal pathways (pink arrows).
D. A two-step proteolytic cleavage (red lightening bolts) releases the CD44 intracellular domain (ICD), which
exhibits nuclear translocation and functions in transcriptional activation.
HA Inhibitor
+
Murine T Cells
Human T Cells
**
**
XS106
None
**
None
+ Anti-CD44
Not Tested
**
+ Control Ig
+
**
Not Tested
Pep-1
**
**
+
**
RP
0
4
8
12
16 0
5
10
15
% Bound
20 0
20
40
60
HAS1
HAS2
HAS3
Hyal-1
Hyal-2
Hyal-3
b-Actin
T Cells, (+)Con A
T Cells, (-)Con A
BM-DC
XS106 DC
XS52 DC
XS52 DC
100
101
BM-DC
102
103
104 100
101
FL2-Height
102
101
103
104 100
101
FL2-Height
Splenic T cells (-ConA)
100
Splenic DC
102
FL2-Height
103
104
102
103
FL2-Height
Splenic T cells (+ ConA)
100
101
102
FL2-Height
103
104
104
In Vitro Antigen Presentation Assays
3H-thymidine
Bone Marrow DC
DO11.10 T cells
Ova Peptide
uptake
CFSE fluorescence profile
Cytokine Production
Impact of Pep-1 on Ag specific T cell proliferation
Proliferation (cpm x 104)
25
20
RP
15
*
**
10
Pep-1
****
5
0
0
5
DC number/well (x103)
10
N
C
Monomer
C
C
N
N
PEG
N
C
Tetramer
N
C
Multivalent Pep-1 enhances T cell inhibition
**
**
% Inhibition
100
PEG-Pep-1
60
**
**
Pep-1
20
0
0
100
200
Pep-1 (mM)
300
Proliferation (cpm x 104)
25
20
15
PEG-RP
**
10
**
**
5
PEG-Pep-1
0
0
2
4
6
8
10
DC number/well (x103)
T cell proliferation: CFSE staining
(-) OVA
PBS
(+) OVA
PBS
PEG-Pep-1 PEG-RP
Inhibition of T cell proliferation by bPG
DC T cells OVA Inhibitor
+
+
+
+
+
+
+
+
+
+
+
bPG
+
+
+ denat. bPG
**
0
1
2
3
4
5
Proliferation (cpm x 105)
6
100
**
**
Cell Adhesion
**
% Inhibition
80
**
60
40
Antigen Presentation
20
**
0
0
20
40
mAb (mg/ml)
60
**
Impact of PEG-Pep-1 on T cell cytokine secretion
IL-2
DC T cells OVA Inhibitor
+
+
+
+
+
+
+
+
+
+ PEG-Pep-1
PEG-RP
+
+
+
IFNg
**
**
0
1.0
2.0 0
0.5
1.0
Cytokine concentration (ng/ml)
Summary
DC and T cells express mRNAs
for HA synthases and
hyaluronidases
DC and activated T cells express
HA polymers on their surfaces
DC induced T cell activation is
blocked by two HA inhibitors
Concept #1
CD44
Exogenous HA
Proliferation
Concept #2
HA Synthesis
CD44
Proliferation
Anti-CD3 Anti-CD28 Inhibitor
-
-
-
+
-
-
-
+
-
+
+
-
+
+
Pep-1
+
+
RP
**
0.0
0.5
1.0
1.5
Proliferation (cpm x 105)
Impact of endogenous HA on T cell
proliferation
Christie Mahaffey
4-Methylumbelliferone (4-MU)
Inhibitor of HA synthesis
Natural product found in plants
Celery
Parsley
Manna Ash
German Chamomile
ConA
4-MU
-
-
+
-
+
+
**
0
200
400
600
800 1000 1200 1400
3H-glucosamine
uptake
Impact of 4-MU on ConA stimulated T cells
7000
100
80
5000
% viability
3H-thymidine
uptake
6000
4000
3000
**
60
40
2000
20
**
1000
**
0
0
0
20
40
60
80 100
4-MU,μg/ mL
0
20
40
60
80 100
T cell proliferation by different stimuli
14000
PMA / ionomycin
Allogeneic Spleen Cells
30000
3H-thymidine
uptake
12000
25000
10000
20000
**
8000
15000
**
6000
**
10000
4000
**
2000
**
**
5000
0
0
0
20
40
60
80
100
4-MU,μg/ mL
0
20
40
60
80
100
Impact of 4-MU on CD69 expression
19%
67%
71%
Impact of 4-MU on cytokine secretion
ConA 4-MU
-
-
+
-
+
+
IFNγ
IL-2
**
**
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
A450
ConA 4-MU IL-2
+
+
-
+
-
+
+
+
+
0
2
4
3H-thymidine
6
uptake
8
HA Synthesis
Concept #3
CD44
IL-2 Secretion
Proliferation
IL-2R
Adhesion of ConA stimulated T cells to HA
ConA
ConA + 4-MU
No HA
Control IgG
Anti-CD44
*
0
2
**
4
6
8
10 12
0
2
% Adhesion
4
6
8
10 12
Impact of anti-CD44 on IL-2 secretion
Control IgG
Anti-CD44
No ConA
0.0
0.2
0.4
A450
0.6
0.8
HA Synthesis
Concept #4
?
IL-2 Secretion
Proliferation
IL-2R
SUMMARY
Pep-1 blocked T cell proliferation and IL-2 secretion
4-MU inhibited HA synthesis by T cells
4-MU inhibited T cell proliferation in a dose dependent
fashion but did not globally impair T cell activation
4-MU inhibited the secretion of IL-2 and IFNγ
Addition of IL-2 to T cells treated with ConA and 4-MU
reversed the block in proliferation
Blocking the HA binding module of CD44 did not
recapitulate the effects of 4-MU on IL-2 production or T
cell proliferation
CONCLUSION
Endogenously synthesized HA plays a role in IL-2
mediated T cell proliferation. Furthermore, HA
inhibitors (e.g., Pep-1) or HA synthase inhibitors
(e.g., 4-MU) may have utility in regulating T cell
responses.
CD44 and the Proliferation of Jurkat T cells
Li-Shu Zhang
He-Wen Ma
Weilan Zuo
Henry Greyner
Immortalized line of T cells originally derived from the
peripheral blood of a 14 year old boy with T cell leukemia.
they are CD44 negative.
Expression of CD44 in stably transfected Jurkat cells
RT-PCR
Flow Cytometry
Vector
CD44
Impact of CD44 on Jurkat cell morphology
Cell density-dependent proliferation of Jurkat cells
25,000 / well
5,000 / well
1,000 / well
Up- and down-regulated genes (2-fold cut-off)
Accession
No.
Symbol
Fold up- or
downregulation
Gene Name
Description
Down-regulation:
NM_001964
EGR1
-6.81
AT225/G0S30
Early growth response 1
NM_021784
FOXA2
-4.17
HNF3B/TCF3B
Forkhead box A2
NM_002982
CCL2
-3.40
GDCF-2/GDCF-2
HC11
Chemokine (C-C motif) ligand 2
NM_020182
TMEPAI
-2.85
PMEPA1/STAG1
Transmembrane, prostate androgen induced
RNA
NM_002228
JUN
-2.61
AP1/c-Jun
Jun oncogene
NM_000639
FASLG
-2.41
APT1LG1/CD178
Fas ligand (TNF superfamily, member 6)
NM_003202
TCF7
-2.21
TCF-1
Transcription factor 7 (T-cell specific, HMGbox)
NM_000589
IL4
-2.15
BSF1/IL-4
Interleukin 4
NM_000584
IL8
15.98
3-10C/AMCF-I
Interleukin 8
NM_004591
CCL20
10.94
CKb4/LARC
Chemokine (C-C motif) ligand 20
NM_000450
SELE
5.75
CD62E/ELAM
Selectin E (endothelial adhesion molecule 1)
NM_000598
IGFBP3
5.14
BP-53/IBP3
Insulin-like growth factor binding protein 3
NM_015869
PPARG
5.07
NR1C3/PPARG1
Peroxisome proliferator-activated receptor
gamma
NM_000230
LEP
3.68
OB/OBS
Leptin
NM_000418
IL4R
3.45
CD124/IL4RA
Interleukin 4 receptor
NM_005551
KLK2
3.21
KLK2A2/hK2
Kallikrein-related peptidase 2
NM_002416
CXCL9
2.62
CMK/Humig
Chemokine (C-X-C motif) ligand 9
Up-regulation:
EGR-1 (Early Growth Response Gene-1)
Transcription Factor
Nuclear Phosphoprotein
Zinc Finger Motif
Binds GCGGGGGCG promoter sequences
Transiently expressed in lymphocytes
EGR-1 transcript levels in Jurkat cells
EGR-1 protein expression in Jurkat cells
Pharmacological Inhibitors to Study Cell Proliferation
SB239063: Inhibits p38 activation
UO126: Inhibits ERK 1/2 activation
Wortmannin: Inhibits Akt activation
LY294002: Inhibits Akt activation
Impact of inhibitors on cell proliferation and EGR-1 expression
Akt is hypophosphorylated in CD44 expressing Jurkat cells
Impact of myristolated Akt on Jurkat cell proliferation
Vector
CD44
CD44 + Akt
0
2000
4000
6000
3H-Thymidine
8000 10000
Impact of EGR-1 siRNA on Jurkat cell proliferation
CD44 EGR-1 siRNA
+
-
-
+
0
20
40
60
% Inhibition
80
100
SUMMARY
CD44 knock in inhibited Jurkat cell proliferation
EGR-1 expression was significantly reduced in CD44
knock in Jurkat cells
EGR-1 expression was correlated with Akt activation
EGR-1 siRNA could partially block Jurkat cell proliferation
CONCLUSION
CD44 inactivates Akt and subsequent EGR-1 expression