Chapter 3 Results
3.1 FcγRIIB
232T/Tmice show persistent impaired affinity maturation over time
To investigate whether reduced inhibitory function of FcγRIIB could affect the
selection of GC B cells, we have generated FcγRIIB232T/T mice to test our concept. We
have recently shown that the FcγRIIB-I232T polymorphism, of which the 232th amino
acid was substituted from isoleucine to threonine, in mice results in impairment in the
negative selection of GC B cells during GC reaction (Jhou, et al., 2018). To evaluate the
long-term effect of FcγRIIB232T/T on affinity maturation, we immunized female
wild-type and FcγRIIB232T/T mice with 50 µg of NP-CGG admixed with an equal volume of alum adjuvant. Serum samples were collected on days 14, 28 and 35. Serum
levels of high-affinity NP-specific IgG and total NP-specific IgG were measured with
ELISA plates coated with NP7-BSA and NP30-BSA proteins, respectively. The affinity
maturation status was measured by dividing high-affinity NP-specific IgG by total
NP-specific IgG from ELISA readings. We found that on day 14, the serum levels of
NP-specific IgG of FcγRIIB232T/T mice showed significantly lower affinity maturation
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(P < 0.05, Figure 2) than those of wild-type mice. By day 35, the affinity maturation
status of serum NP-specific IgGs of wild-type mice was close to 1, indicative of nearly
total elimination of the low-affinity NP-specific GC B cells to produce mostly
high-affinity NP-specific IgGs. In contrast, FcγRIIB232T/T mice revealed continued
retention of low-affinity NP-specific IgGs and a slower affinity maturation kinetic
toward maturation to high-affinity NP-specific IgGs over time. These data from
FcγRIIB232T/T mice suggest that pharmacological down-regulation of FcγRIIB
expression might induce a similar Ab phenotype resulting from reduced inhibitory
activity of FcγRIIB.
3.2 Association of FcγRIIB and BCR with the lipid raft after coligation was impaired in FcγRIIB
232T/Tmutant mice
The I232T polymorphism is a single nucleotide polymorphism (SNP) in the
transmembrane domain of FcγRIIB proteins. Coligation of FcγRIIB with immune
complex results in apoptosis of the centrocytes. We hypothesized that the mouse
FcγRIIB-I232T would alter its stability in the association with lipid raft as observed in
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human counterpart. To investigate this, confocal microscopy was applied to visualize
the effect of FcγRIIB-I232T on BCR in the lipid microdomain at the cell surface. The
ganglioside GM1, a lipid raft marker that binds to the subunit B of cholera toxin, and
FcγRIIB were respectively labeled as green with FITC and red with Cy3-conjugated
Abs. As shown in Figure 3A, the wild-type B lymphocytes showed remarkable yellow
cap structure at 30 to 60 min, indicating that the FcγRIIB were stably coligated within
the lipid raft with BCR in response to whole anti-Ig crosslinking. In contrast, B
lymphocytes from FcγRIIB232T/T mice showed discrete FcγRIIB and lipid raft
co-localization. Metamorph analysis tool was applied to quantify the percentage of
FcγRIIB localized in the lipid raft. From 15 to 60 min, we found that a significantly
lower co-localization in isolated FcγRIIB-232T B cells in comparison with the
wild-type B cells in a time-dependent fashion (Figure 3B). Our findings FcγRIIB-232T
B cells are consistent with those observed in human peripheral mononuclear cells
isolated from FcγRIIB-232T carriers (Kono, et al., 2005).
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3.3 Nilotinib administration during GC reaction has a negative impact on affinity maturation
To assess whether it is applicable to manipulate affinity maturation with FcγRIIB
targeting agent, we examine the effects of nilotinib, a specific c-Abl inhibitor. As
reviewed in the introduction, c-Abl is the key downstream signal protein involved in the
apoptosis pathway induced by aggregation of FcγRIIB via non-cognate ICs. Mice were
immunized twice with NP-CGG 50 ug admix with equal volume of alum on day 1 and
day 28 (Figure 1).
After the second booster of NP-CGG, we administered the wild-type female mice
with nilotinib with the dose of 2 mg/kg/day daily from the sixth day (day 35) to the
ninth day (day 37), which was the most active time of GC reaction, clonal selection and
apoptosis of low-affinity GC B cells. Immunized mice were sacrificed on day 38, the
following day after last treatment of nilotinib. Sera and splenocytes were collected.
NP-specific IgG secreting PCs and IgG levels were detected and quantified with
ELISPOT and ELISA assays, respectively. Low-affinity NP-specific IgG secreting PCs
were measured with subtracting the NP7-specific IgG-secreting PC count from
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NP30-specific IgG-secreting PC count. In nilotinib-treated group, the low-affinity
NP-specific IgG secreting cell count was significantly higher (P < 0.05, Figure 4A).
Correspondingly, the affinity maturation status of the circulating NP-specific IgGs was
significantly reduced (P < 0.05, Figure 4B).
3.4 GW501516 increases respective total and low-affinity Ag-specific IgG PCs at the dose of 3 and 6 mg/kg/day during GC reaction
To observe the effects of GW501516 on the selection process of GC reaction, we
gave each wild-type female mouse with either 3 or 6 mg/kg/day on the sixth to the ninth
day after the secondary immunization when IgG ICs were abundantly present. The
choice of doses was based on a study of obese rhesus monkey, of which lipid-lowering
effect by GW501516 was dramatically observed at 3 mg/kg/day given for 1 month.
More importantly, the dosage of 3 mg/kg/day was safe and not reported to induce
carcinogenesis in mice in previous studies. Due to the short-term treatment of
GW501516 for 4 days, a higher dose of 6 mg/kg/day of GW501516 was also included
in our study to observe a potential dose-dependent effect. The control group was given
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an equal volume of diluted dimethyl sulfoxide (DMSO) in PBS, which is the vehicle of
GW501516. We performed ELISPOT assay to assess Ag-specific IgG secreting PCs in
spleen. We found that mice respectively given 3 and 6 mg/kg/day of GW501516 could
generate more NP30-specific IgG secreting PCs than those of control mice. Moreover,
the increase in the both groups of 3 and 6 mg/kg/day was statistically significant (P <
0.05, Figure 5B). The total Ag-specific IgG secreting cells show increment in each dose
but no dose dependent effect was observed. The number of NP7-specific IgG secreting
cells, considered as high affinity NP-specific IgG secreting cells, did not show a
significant change (Figure 5A). The low-affinity Ag-specific IgG secreting PC count,
defined as the numbers of NP30-specific IgG secreting PC numbers minus NP7-specific
IgG secreting PC numbers, increased at the dose of 3 and 6 mg/kg/day in a
dose-dependent fashion (P < 0.05, Figure 5C). The effect was apparent on 6 mg/kg/day
probably because of no significant increase and also mild decrease in some of the mice
in the numbers of high-affinity NP+ PCs.
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3.5 GW501516 increases the serum levels of total and low-affinity Ag-specific IgG at the dose of 3 and 6 mg/kg/day in wild-type mice
In order to assess whether the serum IgG level was associated with the numbers of
IgG secreting PCs, we performed indirect ELISA with either NP2-BSA (1 BSA
conjugated with 2 NPs) or NP30-BSA as the Ag coated onto 96-well plates. Serum
samples were diluted with PBS in a factor of 4 x 104. The relative NP Ag-specific IgG
concentrations expressed in arbitrary units per ml (AU/ml) were calculated from
semilog regression of the OD450 levels of standard serum. The average of total
antigen-specific (NP30-specific) IgGs increased approximately 2 folds in both 3 and 6
mg/kg/day groups but the results did not show a dose-dependent pattern (P < 0.05,
Figure 6B). The level of high-affinity IgGs (NP2-specific) did not show significant
differences (Figure 6A). We divided the high-affinity Ag-specific IgG level over total
Ag-specific IgGs as the antibody avidity index as a functional readout of affinity
maturation. The higher the ratio, the better the affinity maturation in terms of
high-affinity IgG production. The results showed a significant decrease in the affinity
maturation in 3 mg/kg/day but not in 6 mg/kg/day. As a result, the circulating IgG
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levels were highly relevant to the IgG secreting cell count in the spleen. Notably, the
interpretation of IgG levels is slightly distinct from antigen secreting cell count since the
affinity maturation terminates and the germinal centers diminished in the spleen after
the primary immunization. But the circulating IgGs continue to increase affinity till day
35 as shown in Figure 2. The average ratio was about 0.8 in 3 mg/kg/day (Figure 6C)
compared with 1 in control group, which indicating complete affinity maturation on
day10 after the second booster. The results were consistent with the prediction that
GW501516 can increase low-affinity antibody production and impair negative selection
during the GC reaction. On average, high-affinity antibody levels were not affected, but
the GW501516-treated mice presented a greater variation than the control group.
Namely, the GW501516 treatment has an ambiguous effect on high-affinity Abs.
3.6 GW501516 increases total Ag-specific IgM secreting PCs at the dose of 6 mg/kg/day during GC reaction
Our results had shown effects of GW501516 on the GC reaction and humoral
response of repeated immunization. Traditionally, IgM is not considered as the main
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component of affinity maturation since the centrocytes undergo class switch to IgG
secreting PCs. Especially after the secondary immunization, IgM has little role in the
humoral immunity. Circulating IgMs are produced from extrafollicular PCs that do not
participated in the GC reaction. To evaluate whether GW501516 had effects on
extrafollicular IgM secreting PCs, we performed ELISPOT assay to determine the
numbers of IgM secreting PCs in the spleen. GW501516 administration increased the
levels of NP30-specific (total NP-specific) IgM at the dose of 6 mg/kg/day (P < 0.05,
Figure 7B) but not 3 mg/kg/day. In the group of mice treated with 3 mg/kg/day, the
effect of GW501516 varies that some of the mice decreased but some increased in total
numbers of NP-specific PCs (Figure 7A). When the numbers of high-affinity IgM PCs
were analyzed, no significant difference was observed in each dose. However, the
numbers of low-affinity IgM NP+ PCs (total IgM PCs minus high-affinity IgM PCs)
increased significantly in mice treated with 6 mg/kg/day for 4 days (P < 0.05, Figure
7C). The avidity index was 0.5 in control group but the GW501516-treated group
display an average 0.6 and up to 0.8 in some mice.
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3.7 GW501516 increases the serum levels of high-affinity Ag-specific IgM at the dose of 3 and 6 mg/kg/day and total Ag-specific IgM at 6 mg/kg/day
To assess whether the result of Ag-specific IgM PCs is associated with serum
levels of Ag-specific IgMs, we performed ELISAs. The serum samples were diluted in a
factor of 2 x 104. The serum Ab levels were displayed in arbitrary units per ml.
Consistent with the findings in NP30-specific (total NP-specific) IgM secreting PCs, the
serum total Ag-specific IgM levels increased in mice treated with 6 mg/kg/day (P <0.05,
Figure 8B) but not in 3 mg/kg/day. On the other hand, the NP2-specific (high affinity
NP-specific) IgMs increased in both 3 and 6 mg/kg/day in a dose-dependent pattern,
which was not concordant with the results in IgM-secreting PCs (Figure 8B). The
antibody avidity index of the high-affinity Ag-specific IgMs over the total NP-specific
IgMs showed increased ratios in both groups of 3 and 6 mg/kg/day treatments owing to
the higher levels of high-affinity Ag-specific IgMs (Figure 8C). The result indicates
that the effect of GW501516 on FcγRIIB might not be the only factor to influence the
humoral immunity and affinity maturation. GW501516 might also affect the memory B
cell response, Ab secretion, and class switch of centrocytes and Tfh activity.
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Chapter 4 Discussion
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