Download A quantitative assessment of primary and secondary immune responses in cattle using the B cell ELISPOT assay

A quantitative assessment of primary and secondary immune
responses in cattle using the B cell ELISPOT assay
Eric A. Lefevre, B. Veronica Carr, Helen Prentice and Bryan Charleston
Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire, RG20 7NN, UK
Abstract
The B cell ELISPOT assay
We have developed a method allowing the identification
and quantification of Ag-specific plasma cells and memory
B cells in the blood of cattle. Using this model, we were
able to build a comprehensive picture of the appearance in
the blood stream of both these cell types within individual
calves immunised with ovalbumin. During the primary
response, we detected a burst of ovalbumin-specific
plasma cells at days 6 and 7 post-immunisation, which was
followed by the production of specific Ab, whereas a
gradual increase of memory B cells was only detected
from day 15. As expected, a boost immunisation performed
7 weeks later induced a quicker and stronger secondary
response. Indeed, a burst of plasma cells was detected in
the blood at days 3 and 4, which was followed by a strong
increase in Ab titres. Furthermore, a burst of memory B
cells, and not a gradual increase, was detected at days 5
and 6 post-boost immunisation. Importantly, we showed a
strong correlation between the anti-ovalbumin-specific IgG
titres and plasma cell numbers detected in the blood at the
peak response after secondary immunisation.
This assay allows, in the bovine model, the identification and quantification in the blood of Agspecific plasma cells and memory B cells, using ovalbumin as a model T-dependent Ag (fig 1).
As shown below, stimulation of quiescent, memory B cells within PBMC is necessary to
induce their differentiation into Ab-secreting cells (ASC) and subsequent detection by the
B cell ELISPOT assay.
Mixed cellulose ester
A.
B.
Blood sample from calf
membranes
Histopaque
Coating with Ag
(ovalbumin)
PBMC
Plasma cells:
No stimulation
required
ELISPOT assay
Memory B cells:
Need to be stimulated prior to the
ELISPOT assay in order to induce
the differentiation of these
quiescent B cells into ASC
Incubation overnight
with mixture of cells
Incubation with Sheep
anti-bovine IgG-HRP
Stimulation for 6 days
in complete medium containing
PWM+CD40 mAb+IL-2+IL-10
Spot
Incubation with
chromogen (AEC
substrate)
ELISPOT assay
Figure 1: Protocol and schematic representation of the B cell ELISPOT assay. (A) The B cell ELISPOT
assay was performed with freshly isolated and 6 day-stimulated PBMC for the detection of plasma cells
and memory B cells, respectively. (B) Schematic procedure of the detection of ASC by ELISPOT.
Results
ƒ Various reagents have been used for inducing the differentiation of human memory B cells, but these are not suitable when using bovine B cells. Here, we
show PWM + anti-bovine CD40 mAb+ rhIL-2 + rbIL-10 is a potent differentiation stimulus (fig 2A), which was used in all subsequent experiments aiming at
the detection of memory B cells. These activated PBMC were mostly T cell blasts, but numerous plasma cells (positively stained with an anti-bovine light
chain mAb) were also detected (fig 2B-D).
ƒ We determined the kinetics of the appearance of ovalbumin-specific IgG, plasma and memory B cells in the blood following an immunisation and boostimmunisation (fig 3): (i) Primary response: burst of ovalbumin-specific plasma cells detected at days 6 and 7 post-immunisation (pi), followed by the
production of specific Ab. Generation of memory B cells detected at a later time-point (starting between days 15 and 21pi and gradually increasing); (ii)
Quicker/stronger secondary response: burst of plasma cells detected at days 3 and 4 post-boost immunisation (pb), followed by a strong increase in Ab
titres. Burst of memory B cells detected at days 5 and 6 pb.
ƒ Subsequently, eight calves were immunised with various doses of ovalbumin (results detailed in fig 4A). No correlation was found between the antiovalbumin IgG titres and memory B cell numbers detected at the peak response during the secondary immune response (r value= -0.05, n=8, fig 4B).
However, we found a very strong correlation between the anti-ovalbumin IgG titres at day 17pb and plasma cell numbers at day 4pb during the secondary
response (r value= 0.95, n=8, fig 4C). A strong correlation (r value= 0.88, n=8) was also observed between the anti-ovalbumin IgG titres determined at a later
time point (5 months pb) and the plasma cell numbers detected in the blood at day 4 pb (data not shown).
45%
PWM
+IL-2
+IL-10
26%
PWM
+CD40
mAb
+IL-2
+IL-10
Counter-staining:
May Grunwald Giemsa
107
106
105
100000
4
10
10000
3
10
1000
101002
10101
1001
10000000
1000000
1000000
0
10
20
30
40
50
0
5
10
15
20
25
30
B: Plasma cells
D
7%
250
2000
200
1500
150
B
1000
100
50
500
0
0
0
10
20
30
40
50
0
5
10
15
20
25
30
CD8
γδ
IHC staining:
Anti-Light chain mAb
Figure 2: Differentiation of memory B cells into ASC.
(A) PBMC isolated from an ova-immunised calf were
cultured for 6 days in media alone or in the presence of
various combinations of stimuli. The number of ova-specific
ASC generated from 106 cultured PBMC (mean of
duplicates +/- SD) was determined for each culture
condition. (B-D) PBMC isolated from an ova-immunised calf
were stimulated for 6 days with PWM, anti-bovine CD40
mAb, rhIL-2 and rbIL-10. The % of CD4+, CD8+ and γδ+ T
cells within the resulting population was determined by flow
cytometry (B) and cytospin slides from stimulated PBMC
were stained with May-Grunwald-Giemsa (arrows indicate
cells with plasma cell morphology, C) or by
immunohistochemistry for the detection of intracellular Ig
(D). Original magnification: x40. Ova: ovalbumin; PWM:
Pokeweed mitogen; rhIL-2: recombinant human IL-2; rbIL10: recombinant bovine IL-10; ASC: Ab-secreting cells.
600
20000
15000
400
10000
200
5000
6
CD4
Induced-ASC number
C: Memory B cells
0
0
0
10
20
30
40
Days post-immunisation
(primary response)
50
0
5
10
15
20
25
30
Days post-boost immunisation
(secondary response)
Figure 3: Kinetics of Ab titres, plasma and
memory B cell numbers after immunisation and
boost-immunisation with ovalbumin. Four calves
were injected s.c. with 40mg ova per animal in
incomplete Freund’s adjuvant (primary response, left
panels), then boosted 49 days later with 10mg ova
per animal in incomplete Freund’s adjuvant
(secondary response, right panels). Blood samples
were taken at various time-points following these
injections and ova-specific IgG titres (Ab titre, A),
plasma cell (B) and memory B cell (C) numbers were
determined by ELISA and B cell ELISPOT.
Calf
number
Immunisation regime
Primary/Secondary
Peak Ab titre (log10)
(day 17pb)
Peak plasma cell
number (day 4pb)
Peak memory B cell
number (day 6pb)
#1
#2
40mg/10mg
40mg/10mg
5.28 ± 0.04
5.86 ± 0.02
70 ± 19
581 ± 44
2,021 ± 0
2,222 ± 226
#3
#4
10mg/10mg
10mg/10mg
5.96 ± 0.01
5.19 ± 0.01
728 ± 79
187 ± 7
382 ± 25
142 ± 14
#5
#6
1mg/10mg
1mg/10mg
5.44 ± 0.05
5.87 ± 0.04
229 ± 3
618 ± 33
352 ± 28
1,553 ± 21
#7
#8
0.1mg/10mg
0.1mg/10mg
6.13 ± 0.02
6.25 ± 0.04
1,601 ± 226
2,879 ± 433
485 ± 23
166 ± 11
C
r = -0.05
6.5
6.0
5.5
5.0
2.0
2.5
3.0
3.5
Log 10 ASC (memory B cells)
number per 106 PBMC (Day 6pb)
Log10 anti-ovalbumin IgG titre
(Day 17pb)
CD40
mAb
+IL-2
+IL-10
ASC number per 106 PBMC
Media
107
106
105
100000
4
10
10000
3
10
1000
101002
10101
1001
10000000
Log10 anti-ovalbumin IgG titre
(Day 17pb)
200
0
A
Ab titres
400
B
Mean fluorescence
intensity
A:
C
Anti-ovalbumin IgG titre
Ova-specific memory B cells
600
6 PBMC
pernumber
10 cultured
PBMC
ASC
per 10
ASC number
Induced-ASC
number
6 cultured
106 PBMC
per 10per
PBMC
A
r = 0.95
6.5
6.0
5.5
5.0
1.5
2.0
2.5
3.0
3.5
Log10 ASC (plasma cells)
number per 106 PBMC (Day 4pb)
Figure 4: Correlation of Log10 anti-ova IgG titres with
Log10 plasma cell numbers detected at the peak of the
secondary response. (A) Eight calves were injected s.c.
with 40, 10, 1 or 0.1mg ova per animal in incomplete
Freund’s adjuvant, and then boosted 51 days later with
10mg ova per animal in incomplete Freund’s adjuvant.
Blood samples were taken following the boost-injection at
the optimal time-points corresponding to the peak
response for ova-specific IgG titres (Ab titre, day 17pb),
plasma cells (day 4pb) and memory B cells (day 6pb).
Plasma cell and memory B cell numbers were expressed
as ASC number per 106 PBMC (mean of duplicates +/- SD).
(B & C) Pearson’s correlation coefficient (r) was determined
to examine the association between the Log10 anti-ova IgG
titres and the Log10 memory B cell (B) or plasma cell (C)
numbers detected. pb: post-boost immunisation.
Conclusion
Using the B cell ELISPOT assay, we precisely determined the kinetics of bovine plasma and memory B cells appearing in the blood in response to an
immunisation and a boost immunisation with ovalbumin. We also demonstrated a strong correlation between the anti-ovalbumin specific plasma cell
numbers at the peak of the secondary response and IgG titres detected subsequently in the blood.
The detection and quantification of plasma cells and memory B cells following an immunisation/vaccination strategy could constitute a very effective
means to predict, at an early time point, the magnitude and maintenance of the Ab response that will be generated afterwards. Such a method could
be valuable for determining the potency of new vaccines and immunisation protocols.