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The role of hyaluronan and hyaladherins in T cell
proliferation
Mark Mummert, Ph.D.UNT Health Science Center
I. Hyaluronan overview
II. Hyaluronan mediated T cell proliferation
III. CD44 regulated T cell proliferation
Seminar Outline
Biochemical Properties of Hyaluronan (HA)
Unbranched glycosaminoglycan
Repeating N-acetyl-glucosamineand glucuronic acid subunits
Molecular weight at least >300kDa
HA DegradationHAS1 HMW-HA polymer
HAS2 HMW-HA polymer
HAS3 Intermediate HA
Hyal-1 Tetra- and hexasaccharides
Hyal-2 HMW fragments
Hyal-3 Unknown
Hyal-4 Unknown
HA Synthesis
UDP-GlcA
UDP-GlcNAc
HA Synthase
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--trypsin inhibitor related proteins
LEC HA receptor
Neurocan
Hyaluronectin
Versican
Aggrecan
Link Protein
RHAMM
CD44Hyaluronan
WF/GM/OE-HABP
LYVE-1
CD54
CD38
BEHAB
Inter--trypsin inhibitor related proteins
LEC HA receptor
Neurocan
Hyaluronectin
Versican
Aggrecan
Link Protein
RHAMM
CD44HyaluronanCollagens
Fibronectin
Chondroitin sulfates
Heparin
Heparin sulfate
Serglycines
Osteopontin
Hyaluronan BindingProtein (HABP)
HA Receptor-Fc Proteins
Enzymatic digest of bovine nasal cartilage
Variation in quality of different batches
Expensive
Produced recombinantly
Not available commercially
Synthesis is typically low
Specificity is questionable
Available HA Probes Comments
Frequency of Sequences from Phage Clones
Peptide Designation
1
2
3
4
5
Frequency
13/19
2/19
1/19
2/19
1/19
* *
*
*
*
*** **
* β-counter
Metabolic LabelingWith 35S
HA-coated Plate Wash Plate
Lyse Cells
B16-F10
% Bound
0 5 10 15 20 25 30 35 40
Scrambled: WRHGFALTAVNQ
Scrambled: QVNLAHTWARFG
Scrambled: AGNHLATVFWQR
Scrambled: RNHALVGWQFTA
Pep-1: GAHWQFNLATVR
RP: S A T P A S APYPL A
No Inhibitor
Background
**
**
% Bound0 5 10 15 20 25 30 35
Pep-1 (V A)Pep-1 (T A)Pep-1 (L A)
Pep-1 (F A)Pep-1 (Q A)Pep-1 (W A)
Pep-1 (G A)RP
No InhibitorBackground
Pep-1 (WT)
Pep-1 (N A)
Pep-1 (H A)
Pep-1 (R A)
**
**
**
**
**
Functional roles of HA in DC-dependent Agrestricted T cell proliferation
Diana I. MummertDale Edelbaum
Francis HuiHiroyuki MatsueAkira Takashima
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.
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).
Dendritic cells (DC)
T cells
T cellsTCRLFA-1ICAM-3
DCAg/MHC IIICAM-1DC-SIGN
Adhesive Molecules and Antigen Presentation
CD44 Overview
Glycoprotein
Expressed by leukocytes
Major receptor for HA
Leukocyte trafficking
Signaling co-receptor
Stimulated T cells express functional CD44 isoforms
Exogenous HA enhances mitogen induced T cell proliferation
Anti-CD44 blocks HA augmented proliferation
CD44/ HA Molecular Interaction and T cells
Figure 3. CD44 in SignalingA. 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.
XS106
0 20 40 60
Inhibitor
None
None
Anti-CD44
Control Ig
Pep-1
RP
HA
-
+
+
+
+
+
0 4 8 12 16
Murine T Cells
% Bound
Human T Cells
0 5 10 15 20
Not Tested
Not Tested
**
** **
**
**
****
**
HAS3
HAS2
HAS1
Hyal-1
Hyal-2
Hyal-3
-Actin
XS
52
DC
XS
106
DC
BM
-DC
T C
ells
, (+
)Co
n A
T C
ells
, (-)
Co
n A
100 101 102 103 104
FL2-Height
XS52 DC
Splenic T cells (+ ConA)
100 101 102 103 104
FL2-Height
Splenic T cells (-ConA)
100 101 102 103 104
FL2-Height
Splenic DC
100 101 102 103 104
FL2-Height
BM-DC
100 101 102 103 104
FL2-Height
In Vitro Antigen Presentation Assays
Bone Marrow DCDO11.10 T cellsOva Peptide
Cytokine Production
3H-thymidine uptake
CFSE fluorescence profile
DC number/well (x103)
Pro
life
rati
on
(cp
m x
104
)
0 5 100
5
10
15
20
25
****
**
*
Pep-1
RP
Impact of Pep-1 on Ag specific T cell proliferation
C
C
C
C
NN
N N
N C Monomer
TetramerPEG
Pep-1 (M)
% I
nh
ibit
ion
0 100 200 3000
20
60
100**
**
****
PEG-Pep-1
Pep-1
Multivalent Pep-1 enhances T cell inhibition
Prol
ifera
tion
(cpm
x 1
04 )
DC number/well (x103)
0 2 4 6 8 10
0
5
10
15
20
25
**
**
**
PEG-RP
PEG-Pep-1
PBS PBS PEG-Pep-1 PEG-RP
(+) OVA(-) OVA
T cell proliferation: CFSE staining
DC
+
-
-
-
+
+
+
T cells
-
+
-
+
+
+
+
OVA
-
-
+
+
+
+
+
Inhibitor
-
-
-
-
-
bPG
denat. bPG
Proliferation (cpm x 105)0 1 2 3 4 5 6
**
Inhibition of T cell proliferation by bPG
% I
nh
ibit
ion
mAb (g/ml)0 20 40 60
0
20
40
60
80
100
**
** ****
**
**
Cell Adhesion
Antigen Presentation
IL-2
0 1.0 2.0 0 0.5 1.0
IFN
Cytokine concentration (ng/ml)
DC+--+++
T cells-+++++
OVA--++++
Inhibitor----
PEG-Pep-1
PEG-RP
** **
Impact of PEG-Pep-1 on T cell cytokine secretion
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
Exogenous HACD44
Proliferation
Concept #1
CD44
HA Synthesis
Proliferation
Concept #2
0.0 0.5 1.0 1.5
Proliferation (cpm x 105)
Anti-CD3
-
+
-
+
+
+
Anti-CD28
-
-
+
+
+
+
Inhibitor
-
-
-
-
Pep-1Pep-1
RPRP
Impact of endogenous HA on T cell proliferation
Christie Mahaffey
Inhibitor of HA synthesis
Natural product found in plantsCeleryParsleyManna AshGerman Chamomile
4-Methylumbelliferone (4-MU)
ConA 4-MU - -
+ -
+ + **
3H-glucosamine uptake
0 200 400 600 800 1000 1200 1400
0 20 40 60 80 1000
20
40
60
80
100
0 20 40 60 80 1000
1000
2000
3000
4000
5000
6000
7000
3 H-t
hym
idin
e u
pta
ke
% v
iab
ilit
y
4-MU,μg/ mL
**
****
Impact of 4-MU on ConA stimulated T cells
T cell proliferation by different stimuli
0 20 40 60 80 1000
5000
10000
15000
20000
25000
30000
0 20 40 60 80 1000
2000
4000
6000
8000
10000
12000
14000
4-MU,μg/ mL
3 H-t
hym
idin
e u
pta
ke
**
****
******
PMA / ionomycin Allogeneic Spleen Cells
Impact of 4-MU on CD69 expression
19% 67% 71%
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
A450
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70
Impact of 4-MU on cytokine secretion
-
- -
+
++
ConA 4-MU
** **
IL-2 IFNγ
0 2 4 6 8
-
+ -
+
++
ConA 4-MU
+
+
+
IL-2
3H-thymidine uptake
HA Synthesis
Proliferation
IL-2 Secretion
CD44
Concept #3
IL-2R
No HA
Control IgG
Anti-CD44
ConA ConA + 4-MU
* **
Adhesion of ConA stimulated T cells to HA
% Adhesion
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Control IgG
Anti-CD44
No ConA
Impact of anti-CD44 on IL-2 secretion
A450
0.0 0.2 0.4 0.6 0.8
HA Synthesis
Proliferation
IL-2 Secretion
??
Concept #4
IL-2R
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
SUMMARY
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.
CONCLUSION
CD44 and the Proliferation of Jurkat T cells
Li-Shu ZhangHe-Wen MaWeilan 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.
SymbolFold up- or down-regulation
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, HMG-box)
NM_000589 IL4 -2.15 BSF1/IL-4 Interleukin 4
Up-regulation:
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
Transcription FactorNuclear PhosphoproteinZinc Finger MotifBinds GCGGGGGCG promoter sequencesTransiently expressed in lymphocytes
EGR-1 (Early Growth Response Gene-1)
EGR-1 transcript levels in Jurkat cells
EGR-1 protein expression in Jurkat cells
SB239063: Inhibits p38 activation
UO126: Inhibits ERK 1/2 activation
Wortmannin: Inhibits Akt activation
LY294002: Inhibits Akt activation
Pharmacological Inhibitors to Study Cell Proliferation
Impact of inhibitors on cell proliferation and EGR-1 expression
Akt is hypophosphorylated in CD44 expressing Jurkat cells
3H-Thymidine
0 2000 4000 6000 8000 10000
Vector
CD44
CD44 + Akt
Impact of myristolated Akt on Jurkat cell proliferation
% Inhibition
0 20 40 60 80 100
-
- +
+
CD44 EGR-1 siRNA
Impact of EGR-1 siRNA on Jurkat cell proliferation
CD44 knock in inhibited Jurkat cell proliferation
EGR-1 expression was significantly reduced in CD44knock in Jurkat cells
EGR-1 expression was correlated with Akt activation
EGR-1 siRNA could partially block Jurkat cell proliferation
SUMMARY
CD44 inactivates Akt and subsequent EGR-1 expression
CONCLUSION