!
104!
3.3.2 Plasmablasts in extrafollicular responses were IgM
+

Our data, thus far, indicated that extrafollicular responses in apoE
-/-

mice may be responsible for the generation of total IgM
+
and also oxLDL-
specific IgM
+
plasma cells. Thus, we performed immunofluorescence staining
to evaluate whether IgM
+
plasmablasts were generated from extrafollicular
responses. Our result showed that IgM
+
plasmablasts were indeed colocalizing
with CD11c
hi
DCs at the bridging channel of the follicles (Figure 21A).
Extending our findings, we observed these IgM
+
plasmablasts that colocalized
with CD11c
hi
DCs were proliferating as they incorporated thymidine analog,
EdU in a 12hr pulsed chase experiment (Figure 21B).
Because of the lack of tools to evaluate if these IgM


!
107!Figure 21. IgM
+
plasmablasts were generated in extrafollicular responses
in the spleen of apoE
-/-
mice.
(A) Representative image of IgM
+
plasmablasts colocalizing with CD11c
+

3.3.3 Summary
Collectively, we provide direct evidence that IgM
+
plasmablasts were
generated through the extrafollicular response pathway in the spleen of apoE
-/-

mice. The increased humoral IgM responses in spleen of apoE
-/-
mice were not
due to defective antibodies class-switching from the GC reactions. Also, we
showed that extrafollicular responses, but not GC reactions, were elicited in
WT mice when we immunized WT mice with oxLDL.

!
109!
3.4 Evaluation of molecular cues to direct extrafollicular responses in the
spleen of apoE
-/-
mice
Activated B cells that migrate to the bridging channel in extrafollicular
responses requires the expression of chemokine receptor, EBI2 (Gatto et al.,
2009). Until recently, the natural ligand for EBI2 was identified to be 7α, 25-
OHC (Hannedouche et al., 2011; Liu et al., 2011). The formation of 7α, 25-
OHC is by the stepwise actions of two enzymes, CH25H and CYP7B1
(Figure 2). This oxysterol could be further metabolized into 4-cholesten-7α,
25-ol-3-one by HSD3B7 (Figure 2). The deficiency of any of the three
enzymes is associated with decreased antigen-specific plasma cell numbers
(Hannedouche et al., 2011; Yi et al., 2012). Therefore, we investigated if the
robust splenic extrafollicular responses seen in apoE

3.4.2 Increased oxysterol in the spleen of apoE
-/-
mice
To confirm our hypothesis, we collaborated with Dr. Andreas Sailor
from Novartis (Basel, Switzerland) to measure the amount of oxysterol in the
spleen of apoE
-/-
mice compared to WT mice using high performance liquid
chromatography mass spectrometry (HPLC-MS). Indeed, our data analysis
showed higher amount of 25-OHC and 7α, 25-OHC oxysterol in the spleen of
apoE
-/-
mice compared to WT mice (Figure 22E & 22F). Therefore, our data
indicates the possibility that the robust splenic extrafollicular responses in
apoE
-/-
mice may be supported, at least in part, by increased bioavailability of
7α, 25-OHC.

!
111!

Figure 22. Elevated 7α, 25-OHC oxysterol in the spleen of apoE
-/-
mice.
(A-D) Quantitative mRNA transcript expression of (A) EBI2, (B) CH25H, (C)
CYP7B1 and (D) HSD3B7, relative to HPRT1 (mean ± SEM, n = 8). Data
were pooled from two independent experiments. (E-F) Quantitative oxysterol
measurement of (E) 25-OHC and (F) 7α, 25-OHC (n=5) by LC-MS using D6-
7α, 25-OHC as reference. *, P < 0.05

B1a and CD19
+
CD5
-
B1b cells. Since the amount of oxLDL-specific IgM
autoantibodies in circulation were elevated in apoE
-/-
mice and B1a cells are
implicated in the production of oxidation epitope-specific antibodies in
atherosclerosis (Chou et al., 2009), we examined if B1a cell population
increased in apoE
-/-
mice. These B1a cells had also been described to retain
CD5
+
marker before losing expression 5 days after LPS stimulation (Yang et
al., 2007). Therefore, it allows a window of opportunity to investigate B1a
cells differentiating into IgM
+
CD138
+
ASCs (Yang et al., 2007).
3.5.1 B1a cells were not expanded in PEC of apoE
-/-
mice
Our flow cytometry analysis demonstrated that there were no
differences in relative percentage and number in B1a cell population in PEC
of apoE
-/-
mice compared to WT mice (Figure 23A & 23B). Furthermore,

-/-
mice.
Our preliminary analysis of CD19
+
CD5
+
B1a cell population showed
that apoE
-/-
mice had higher relative percentage of B1a in the spleen (Figure
24A). However, we did not detect an increase in relative cell number of B1a
population in the spleen of apoE
-/-
mice (Figure 24B). Therefore, these sets of
observations suggest increased frequency of splenic B1a cell population was
due to changes in frequency of other lymphocyte sub-populations instead of
indications that there was B1a cell population expansion in apoE
-/-
mice.
Next, we examined if there were more B1a cells differentiating into
IgM
+
ASCs in apoE
-/-
mice. Preliminarily, we observed a non-statistical
significant increase in relative percentage but a statistical significant increase
in relative number of extracellular IgM
+
CD138
+

+
CD138
+
CD19
+
CD5
+
died rapidly to account for the decreased
population. We favoured the latter possibility that these IgM
+
plasmablasts
migrate to the bone marrow for long-term maintenance that we will explore in
the subsequent sections.

!
116!Figure 23. No difference in B1a cell population in peritoneal cavity of
apoE
-/-
mice.
(A-B) Comparative flow cytometry analysis of relative (A) percentage and (B)
number of CD19
+
CD5
+
B1a cells (mean ± SEM, n = 9). Data were pooled
from three independent experiments. (C-D) Comparative flow cytometry
analysis of relative percentage and number of extracellular

IgM
+
CD138
+
CD19
+
CD5
+
B1a cells in the spleen (mean ± SEM, n=6). Data
were pooled from two independent experiments. (E-F) Comparative flow
cytometry analysis of relative (E) percentage and (F) number of intracellular
IgM
+
CD138
+
CD19
+
CD5
+
B1a cells in the spleen (mean ± SEM, n=9). Data
were pooled from three independent experiments. * P < 0.05; ** P < 0.01

!
118!
3.5.3 Summary
Collectively, our data suggests that PEC B1a cells were not affected in
the apoE
-/-
mice. In addition, our preliminary data suggests that although B1a
cells did not expand in the spleen of apoE

of B cells could be due to ASCs existing in the old apoE
-/-
mice.
3.6.1 Adoptive transfer of splenic CD138
+
ASCs into apoE
-/-
mice
To investigate this, we carried out a pilot experiment in which sorted
CD138
+
splenic ASCs from apoE
-/-
or WT mice were adoptively transferred
via i.v. route into 10 weeks old apoE
-/-
recipient mice. Due to cell number
limitation ( < 1.0 x 10
6
cells) after sorting from six spleens from donor mice,
only one recipient mouse per group was used. Recipient mice were sacrificed
at 28 weeks old and aorta analysis for lesion was carried out. We performed
Oil Red-O staining to visualize lipid distribution throughout the aortic tree of
the mice. As reported, aortic arches of the aorta are lesion prone sites and most
affected in disease severity (Figure 25A). Our observation of the whole mount
Oil Red-O stained aortas suggested that less amount of lipids were
accumulated in the aortic arch region of apoE
-/-
ASCs recipient mouse (Figure
25A). To confirm, we sectioned the aortic arch and performed
Figure 25. No differences in lesion size of apoE
-/-
mice after adoptive
transfer of ASCs.
(A) Representative whole mount images of aorta from apoE
-/-
mice (n=2), WT
mice (n=3), apoE
-/-
mice recipient for apoE
-/-
ASCs (n=1) and apoE
-/-
mice
recipient for WT ASCs (n=1). (B) Representative immunofluorescence image
of aorta staining to reveal lesion size. Scale bar represents 200µm. (C)
Quantification of lesion size in aorta of apoE
-/-
mice (n=2), WT mice (n=3),
apoE
-/-
mice recipient for apoE
-/-
ASCs (n=1) and apoE
-/-
mice recipient for
WT ASCs (n=1) (mean ± SEM).
!

the bone marrow, we reasoned that the decrease in intracellular IgM
+
CD138
+

B1a plasma cells population in the spleen may be explained by their
subsequent migration into the bone marrow of apoE
-/-
mice. To support our
hypothesis, recent reports showed that IgM
+
plasma cells could be detected in
the bone marrow after immunization (Bortnick et al., 2012; Foote et al., 2012;
Racine et al., 2011).
3.7.1 Accumulation of IgM
+
long-lived plasma cells in the bone marrow of
apoE
-/-
mice
Because B cells also express CD138 in almost all stages of B cell
development in the bone marrow of adult mice (Tung et al., 2006), we
included the non-proliferative state of plasma cells as an additional parameter
when examining plasma cells in the bone marrow. To begin our investigation
into ASCs in the bone marrow of apoE
-/-
mice, we maintained apoE
-/-
mice
with thymidine analog, BrdU in drinking water for one month. The

showed that IgM
+
plasma cells were increased in relative percentage and
number in bone marrow of apoE
-/-
mice (Figure 26C & 26D). However, our
ELISpot analysis revealed that there were no changes in total IgM ASCs in
bone marrow of apoE
-/-
mice (Figure 26E). This suggests that while
frequency of IgM
+
ASCs in bone marrow of apoE
-/-
mice was similar to WT
mice, the changes occurred in the IgM
+
plasma cells compartment. More
importantly, in our ELISpot analysis, we were able to detect statistically
significant increased frequency of oxLDL-specific IgM ASCs in the bone
marrow of apoE
-/-
mice (Figure 26F).

!
126!

Figure 26. Increased IgM
+
long-lived plasma cells in bone marrow of

pooled from three independent experiments. * P < 0.05; ** P < 0.01

!
127!
3.7.2 Summary
Collectively, our data established an accumulation of plasma cells in
the bone marrow of apoE
-/-
mice. Because we maintained the mice on BrdU in
drinking water for one month, the result is also indicative that these plasma
cells accumulated in the bone marrow of apoE
-/-
mice were long-lived.
Subsequent analysis revealed that the accumulated plasma cells in the bone
marrow of apoE
-/-
mice were of IgM isotype, accompanied by observation of
increased oxLDL-specific IgM ASCs.

!
128!
3.8 Evaluation of IgM
+
plasmablasts migration from the spleen to bone
marrow of apoE
-/-
mice

IgM autoantibodies was also similar, if not non-statistical significantly higher
than that of sham-operated apoE
-/-
mice (Figure 27B) in agreement with
previous report that increased MDA-LDL IgM autoantibodies in Sx apoE
-/-

mice was associated with increased atherosclerotic lesion size in the aorta
compared to sham-operated mice (Caligiuri et al., 2002). This implied that the
humoral responses in Sx apoE
-/-
mice were not affected by the lack of spleen.