Galectin-9 helps govern human B cell adhesion to and migration through human vascular endothelium

1. Galectin-9 helps govern human
B cell adhesion to and migration
through human vascular
endothelium
Lee Seng Lau
2nd year PhD Student in Biomedical Sciences
Mentor: Dr. Charles Dimitroff
1

2. Biorender.com
Glycans and immune cell function 2
What are glycans ?
 Heterogeneous carbohydrates “Sugars”
 Present on cell surface proteins and secreted proteins
 Key regulators during immune response
 Bind to proteins know as lectin
Suila, H. (2014). Glycobiological insights in characterization and targeting of umbilical cord blood

3. Galectins 3
 Lectins that bind to β-linked galactose sugar
 Bound to proteins by either N-linked or O-
linked glycosylation
 Promote pro- or anti-inflammatory responses
Varki, A. (1970, January 1). Essentials of Glycobiology. Retrieved from
https://www.ncbi.nlm.nih.gov/books/NBK1908/
Galectins
affect B cell
Physiology
Nature Reviews Immunology ISSN 1474-1741

4. Sugars modify B cell Biology 4
 B cell development is accompanied by progressive remodeling of its surface glycan
 Gal-9 suppresses B cell activation by binding Cd45 decreasing calcium signaling mediated
by Lyn-CD22 SHIP-1 dependent mechanism
Giovannone N, atFront Immunol. 2018 Dec 14;9:2857. doi: 10.3389/fimmu.2018.02857. PMID: 30619255; PMCID: PMC6302748.

5. Biorender.com
5
Produce antibodies
that trap invading
viruses or bacteria in
large clumps.
Antibody
production
Production
of cytokines
Activate innate
immune cells to
kill pathogens
Question:
How do B cells protect us
from pathogens?
Answer:
B cells
Disruption of B-
cell activation
leads to
autoimmune
diseases

6. 6
Typical route for leukocytes to gain access to tissues
FEBS Letters, Volume: 585, Issue: 20, Pages: 3148-3158, First published: 05 August 2011, DOI: (10.1016/j.febslet.2011.07.039)
However not much is known on how B cells
migrate to the peripheral Lymph node.

7. 7
Hypothesis
Gal-9 influences circulating B cell homing to
peripheral Lymph Nodes (LN) by modulating B cell
and endothelial cell interaction.

8. 8
Experimental Approaches
 Localization
o Flowcytometry
o Immunofluorescence staining
 Migration
o Static Assay
o Adhesion Assay
 Mechanism
o Immunoblotting
o RT-qPCR
Question 1: Is Gal-9 found in Lymph node tissue?

9. Gal-9 co-localizes with vascular structures in human LN and tonsil
9
IHC staining of tonsil tissues
with anti-Gal-9 (brown)
Strong Gal-9 staining in
naïve B cells of the mantle as
well as in high endothelial
venules (HEV) (black arrows).
Hematoxylin showed high Gal-
9 expression on Endothelial
Cells of Human endothelial
venules (HEVs white arrows)
and spatial localization with
parenchymal and circulating B
cells undergoing diapedesis.

10. 10
N
a
iv
e
C
D
4
+
T
N
a
iv
e
C
D
8
+
T
T
C
M
C
D
4
+
T
C
M
C
D
8
+
T
E
M
C
D
4
+
T
E
M
C
D
8
+
N
a
iv
e
B
M
e
m
o
r y
B
Ig
M
M
e
m
o
r y
B
0
5
1 0
1 5
2 0
2 5
H
E
C
A
-
4
5
2
E
x
p
r
e
s
s
i
o
n
(
%
)
sLeX/sLeA
N
a
i
v
e
C
D
4
+
T
N
a
i
v
e
C
D
8
+
T
T
C
M
C
D
4
+
T
C
M
C
D
8
+
T
E
M
C
D
4
+
T
E
M
C
D
8
+
N
a
i
v
e
B
M
e
m
o
r
y
B
I
g
M
M
e
m
o
r
y
B
0
2 5
5 0
7 5
1 0 0
C
D
6
2
L
E
x
p
r
e
s
s
i
o
n
(
%
)
L-selectin
Human circulating B cells express low levels of sialyl LewisX/A and L-selectin
Key homing molecules sLeX/sLeA and
L- Selecting are not highly expressed
indicating that another molecule is
driving homing of B- cells
Biorender.com byCharlotte Butterworth

11. 11
d
Naïve
B cells
IgM Memory
B cells
IgD
Human CD19+ B cells
Gal-9
+
anti-
Gal-9
Count
Human CD19+ B cells
Naïve
IgM Mem
Mem
f
Gal-9 Ligand
IgD
+ Lac
Human CD19+ B cells
Count
Gal-9 Ligand
Gal-9
+ Lac
+
anti-
Gal-9
Naïve
IgM Mem
Mem
Naïve
B cells
IgM Memory
B cells
CD27
CD27
Human circulating B cells express high levels of Gal-9 ligand expression
PBMC were stained B
cell subsets and
found Gal-9 ligand
expressed at high
levels when treated
with Gal-9
Memory
Memory

12. IFN-treated human vascular EC express a high level of Gal-9
12
B
Untreated HUVEC
Anti-Gal-9
Anti-Gal-9 + Lac
Isotype control
Counts
Gal-9
Anti-Gal-9
Anti-Gal-9 + Lac
Isotype control
IFN-ß/γ-treated HUVEC
Counts
Gal-9
A
C D
HUVEC were incubated
with IFN-β and/or IFN-γ or
TNF-α and analyzed with
anti-Gal-9
Surface Gal-9 E-Selectin

13. 13
Anti-Gal-1
Lac + Anti-Gal-1
Isotype control
IFN-ß/γ-treated HUVEC
Gal-1
Counts
Anti-Gal-3
Lac + Anti-Gal-3
Isotype control
IFN-ß/γ-treated HUVEC
Gal-3
Counts
Gal-1 and Gal-3 were not express on
HUVECs treated IFN-β and/or IFN-γ
IFN-treated human vascular EC did not affect Gal-1 and Gal-3

14. 14
Experimental Approaches
 Localization
o Flowcytometry
o Immunofluorescence staining
 Migration
o Static Assay
o Adhesion Assay
 Mechanism
o Immunoblotting
o RT-qPCR
Question 2: Does Gal-9 affect B cell homing?

15. 15
B
aseline
G
al-9
G
al-9
+
Lactose
G
al-1
G
al-3
IFN
IFN
+
Lactose
0
1
2
3
Adherent
B
cells
(Fold
Change)
✱✱
✱✱✱
*
✱✱✱
Human circulating B cells adhere to human vascular EC dependent on Gal-9
Under physiological migration shear flow assay B cells were
treated with Gal-9 (with or without 50mM lactose), cells were
infused over into the chamber, shear stress was increased in 0.1
dynes/cm2 every 30 sec from 0.2 – 2 dynes/cm2.

16. Gal-9 slows human B cell trans-endothelial migration (TEM)
16
Fold
Change
(
SEM)
C
o
n
t
r
o
l
G
a
l
-
9
G
a
l
-
9
+
L
a
c
t
o
s
e
0.0
0.5
1.0
1.5
TEM of Human Circulating B cells
Quantification
Migrating
B cell
Gal-9
B cells
HUVECs

17. 17
Experimental Approaches
 Localization
o Flowcytometry
o Immunofluorescence staining
 Migration
o Static Assay
o Adhesion Assay
 Mechanism
o Immunoblotting
o RT-qPCR
Question 3: What is the role of Gal-9 in migration?

18. 18
Total FAK
pFAK
(Tyr397)
β-Actin
150kD
100kD
150kD
100kD
50kD
35kD
LFA1-Kinase mediated B cell migration
Giannoni E, at. Biol Chem. 2003 Sep 19;278(38):36763-76. doi: 10.1074/jbc.M302686200. Epub
2003 Jun 18. PMID: 12815062.

19. 19
 SLAMF7 leads to B cell activation or inactivation
depending on downstream targets
 B cell homotypic adhesion is aided by SLAMF7
No effect on Key
migratory
molecule RHOA
which is required
for cell migration
Gal-9 treatment upregulates immunomodulators and
adhesion molecule SLAMF7

20. 20
SLAMF7 is increase in naïve cell when
co-cultured with Gal-9
SLAMF7 protein expression is increased in naïve B cell
IFN treatment increase Gal-9
production by HUVECs

21. 21
Suppression of
EAT2- primary
downstream target
Gal-9 treatment decreases EAT2 in naïve B cells

22. 22
Gal-9 treatment suppresses EAT2
Human tonsillar naïve B cell
Human circulating naïve B cell

23. 23
Human circulating B cell
Gal-9 treatment suppresses EAT2 on circulating B Cell

24. 24
Gal-9 immunoregulation

25. 25
 High endothelial and post-capillary venules are rich in galectin (Gal)-9.
 Circulating naïve B cells express low levels/ absence of homing molecules L-
selecting and sLeX/A.
 There is robust Gal-9 binding (Gal-9 ligand) in all circulating B cell subsets.
 Gal-9 mediates human B cell - vascular endothelial cell adhesion while reducing
trans-endothelial migration.
 SLAMF7 protein is induce by Gal-9-binding in the absence of BCR-activation
 Activation of SLAMF7 is counteracted by EAT-2
 Gal-9 drives phosphorylation of pro-survival signaling factor, ERK
Conclusion

26. 26
Future Directions
 Are gene expression levels of ICAM1 and VCAM1
altered with Gal-9 expression? – Key adhesion
molecules
 Is SLAMF7 a Gal-9 ligand?
 How can levels of Gal-9 and its ligands be applied to
boost or blunt Ab- responses during vaccination
and autoimmunity?
Remaining Questions
Consequences of this observation in Lupus and Myeloma, where Gal-9 levels are
elevated in patients

27. Acknowledgement 27
Dr. Charles J Dimitroff
Dr. Asmi Chakraborty
Caleb Staudinger
Norhan Mohammed
Angela Bernasconi
Kimberly Justin
Mariana Perez
Liettel Ortega
Joseph Souchak
Jordan Carroll
Dr. Dimitroff’s Lab
Department Translational Medicine
Herbert Wertheim College of Medicine
Florida International University
McKnight Doctoral Fellowships