Download Cells Deaminase, and Ig Class Switch in Human B Factor E2A

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Aging Down-Regulates the Transcription
Factor E2A, Activation-Induced Cytidine
Deaminase, and Ig Class Switch in Human B
Cells
Daniela Frasca, Ana Marie Landin, Suzanne C. Lechner,
John G. Ryan, Robert Schwartz, Richard L. Riley and
Bonnie B. Blomberg
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2008 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2008; 180:5283-5290; ;
doi: 10.4049/jimmunol.180.8.5283
http://www.jimmunol.org/content/180/8/5283
The Journal of Immunology
Aging Down-Regulates the Transcription Factor E2A,
Activation-Induced Cytidine Deaminase, and Ig Class Switch
in Human B Cells1
Daniela Frasca,*§ Ana Marie Landin,* Suzanne C. Lechner,† John G. Ryan,‡ Robert Schwartz,‡
Richard L. Riley,* and Bonnie B. Blomberg2*
A
ging affects the humoral immune response both quantitatively and qualitatively with reduced Ab specificity,
affinity, and isotype switch (1–3). The changes in the
humoral immune response during aging significantly contribute to
the increased susceptibility of the elderly to infectious diseases and
reduce the protective effects of vaccination. The decreased ability
of aged individuals to produce high affinity protective Ab responses against infectious agents or experimental Ags likely also
results from additional defects in required T cell signaling to B
cells (4 – 6). However, although B cell function may suffer from
lack of a good T cell help in aging, intrinsic changes in B cells
occur and have a significant impact on Ab production and mutation
(3, 7–9).
During an immune response, B cells can switch the expression
of surface Ig from IgM to IgG, IgE, or IgA. This DNA recombination takes place between two switch (S)3 regions, one located
*Department of Microbiology and Immunology, †Department of Psychiatry, and ‡Department of Family Medicine, University of Miami Miller School of Medicine, Miami, FL 33101; and §Graduate School of Cell Biology and Development, University
of Rome La Sapienza, Rome, Italy
Received for publication September 13, 2007. Accepted for publication February
15, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AG17618,
AG23717, AG28586 (to B.B.B.), AG025256, and AI064591 (to R.L.R.).
2
Address correspondence and reprint requests to Dr. Bonnie B. Blomberg, Department of Microbiology and Immunology, University of Miami, Miller School of Medicine, P.O. Box 016960 (R-138), Miami, FL 33101. E-mail address: bblomber@
med.miami.edu
3
Abbreviations used in this paper: S, switch; AID, activation-induced cytidine deaminase; CSR, class switch recombination; CTs, circle transcripts; GC, germinal center;
HIGM, hyper IgM; Ig, immunoglobulin; mRNA, messenger RNA; PBMC, peripheral
www.jimmunol.org
immediately upstream (5⬘) of the ␮ CH (donor site) and one 5⬘ of
one of the other CH regions (␥, ␧, or ␣) (acceptor site) to produce
IgG, IgE, or IgA. The class switch recombination (CSR) requires
chromatin opening of S regions, recognition and cleavage of the
target DNA by an endonuclease, and repair and ligation of the
cleaved ends (10 –15). CSR is extremely important for the humoral
immune response, because it generates Abs of the same specificity
but with different effector functions. In vitro, human naive B cells
can be activated to undergo CSR with anti-CD40 or CD40L, together with cytokine signals which can direct CSR to distinct
classes. The critical role of CD40-CD40L interaction for CSR is
provided by the discovery that mutations in either the CD40 or
CD40L (CD154) gene cause the hyper IgM (HIGM) syndrome,
HIGM3 or HIGM1, respectively, characterized by very low levels
of serum IgG, IgA, and IgE, with normal or elevated IgM, associated with defective germinal center (GC) formation (16).
Human B cell percentages and numbers have been shown to
decrease significantly with age (17, 18). It has also been shown
that the percentage of IgM memory B cells, which are responsible
for the response to Streptococcus pneumoniae infection, is significantly decreased whereas that of naive B cells is increased in old
individuals (19). The reduction in the IgM cells has been suggested
to cause reduced specific Ab titers in elderly individuals vaccinated against pneumococcal polysaccharides. Others have
shown that the percentage of CD27⫹ memory B cells increase
with age but this increase is not significant (20). Also in the
human tonsil, naive B cells have been shown to increase with
blood mononuclear cells; SHM, somatic hypermutation; sq, semiquantitative; q,
quantitative.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
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Elderly humans have compromised humoral and cellular immune responses, which lead to reduced protection to infectious agents
and to vaccines. Currently, available vaccines suboptimally protect the elderly population. The capacity to class switch the Ig H
chain is critical to the effectiveness of humoral immune responses in mice and humans. We have previously shown in mice that
the E2A-encoded transcription factor E47, which regulates many B cell functions, is down-regulated in old splenic B cells. This
leads to a reduction in the activation-induced cytidine deaminase (AID), which is known to induce class switch recombination and
Ig somatic hypermutation. The old activated murine B cells also have less AID and less switched Abs. We have extended our study
here to investigate whether aging also affects Ab production and E47 and AID expression in B cells isolated from the peripheral
blood of human subjects (18 – 86 years). Our results obtained with activated CD19ⴙ B cells show that the expression of E47, AID,
and Ig␥1 circle transcripts progressively decrease with age. We also show an age-related decline in the percentage of switch
memory B cells (IgGⴙ/IgAⴙ), an increase in that of naive B cells (IgGⴚ/IgAⴚ/CD27ⴚ) for most individuals, and no decrease
in that of IgM memory cells in peripheral blood, consistent with our data on the decrease seen in class switch recombination
in vitro. Our results provide a possible molecular mechanism for a B cell intrinsic defect in the humoral immune response
with aging and suggest avenues for improvement of vaccine response in elderly humans. The Journal of Immunology, 2008,
180: 5283–5290.
5284
Materials and Methods
Subjects
Experiments were conducted using PBMC isolated from healthy volunteers
of different ages (18 – 86 years) after appropriate signed consent, as well as
initial experiments with spleen cells from cadaveric donors (20 –56 years),
obtained from the Department of Surgery, Division of Transplantation of
the University of Miami Miller School of Medicine. Cadaveric spleen cells
were used to optimize the conditions for cell preparation and culture (data
not shown). The individuals participating in the study were screened for
diseases known to alter the immune response or for consumption of medications that could alter the immune response. In particular, the following
categories were excluded: established diagnosis of diabetes; one or more of
the following comorbid conditions including malignancy (patients without
a recurrence in the last 5 yrs have been allowed), Congestive Heart Failure,
Cardiovascular Disease (unstable, ⬍6 mo), Chronic Renal Failure, significantly impaired renal or hepatic function, autoimmune diseases, infectious
disease, recent (⬍3 mo) trauma or surgery, pregnancy, or documented
current substance and/or alcohol abuse. The psychological status of all
participants was within normal limits. Participants were 46 healthy subjects (mean age ⫽ 51.3; SD ⫽ 19.22). There were 25 females and 21
males in this sample. Two were Asian, 14 Black, and 30 White (of
which 13 identified themselves as Hispanic Whites). We have achieved
highly significant differences in young and aged samples (even before
reaching the number of 46 individuals). Of course we plan on extending
this study but show that the total amount of data (and the number of
different variables analyzed in our individuals) in the current paper is
very significant as is. t test analyses showed that there were no differences between male and female participants on any of the parameters
measured in this study (all values of p ⬎ .60). Similarly, no differences
were noted between participants of different ethnicities/races in
ANOVA analyses (all values of p ⬎ .20).
B cell enrichment
PBMC were collected by density gradient centrifugation on LSM Lymphocyte Separation Medium (ICN Biomedicals). Cells were then washed
three times with medium (RPMI 1640). B cells were isolated from the
PBMC as follows. Briefly, cells were washed three times with medium
(RPMI 1640) and incubated for 20 min at 4°C with 20 ␮l/107 cells of
anti-CD19 Microbeads (Miltenyi Biotech), according to the MiniMacs protocol (Miltenyi Biotech). Cells were then purified using magnetic columns.
At the end of the purification procedure, cells were found to be almost
exclusively (⬎85%) CD19⫹ by cytofluorimetric analysis (described below,
Flow Cytometry). Comparable numbers of CD19⫹ cells were obtained
after negative selection. After the isolation procedure was ended, cells were
maintained in serum-free medium for 1 h at 4°C to minimize potential
effects of anti-CD19 Abs on B cell activation and our parameters are measured 24 h or later.
Cell sorting
To evaluate which cell subset was responsible for class switching in the
whole CD19⫹ B cell population, PBMC were sorted into memory
(CD19⫹CD27⫹) or naive (CD19⫹CD27⫺) B cells (see description of Abs
below, Flow Cytometry). Cells were sorted on a FACS Aria (BD Immunocytometry Systems). Cell preparations were typically ⬎98% pure.
B cell culture
B cells were cultured in complete medium (RPMI 1640, supplemented with
10% FCS, 10 ␮g/ml gentamicin, and 2 ⫻ 10⫺5 M 2-ME and 2 mM Lglutamine). Cells (1 ⫻ 106/ml complete medium) were stimulated in 24well culture plates with purified anti-human CD40 Abs (BD Pharmingen
555587; 1 ␮g/ml), alone or together with recombinant human IL-4 (Biosource PHC0044; 10 ng/ml), for 1–7 days.
At the end of this time, cells were harvested, RNA extracted (for semiquantitative (sq) and quantitative (q)RT-PCR) and supernatants collected
(for ELISA). In four of the old (⬎65 years) subjects, total PBMC were
stimulated because of extremely low numbers of B cells. Only B cells were
stimulated by this procedure.
Memory or naive B cells were stimulated (104 cells/200 ␮l) in round-bottom 96-well plates with anti-CD40/IL-4, alone or together with F(ab⬘)2 of goat
anti-human Ig (2 ␮g/ml; Jackson ImmunoResearch Laboratories no.109-006006) for 1–7 days.
Flow cytometry
After magnetic enrichment, B cells (3–5 ⫻ 105/tube) were stained with
allophycocyanin-conjugated anti-CD19 (BD Pharmingen 555415) (all volumes are 20 ␮l) for 20 min at 4°C. To detect naive B cells (IgG⫺/IgA⫺/
CD27⫺), IgM memory B cells (IgG⫺/IgA⫺/CD27⫹), and switch memory
B cells (IgG⫹/IgA⫹), CD19⫹ cells (3–5 ⫻ 105/tube) were stained with
PE-conjugated anti-CD27 (1/40 diluted; BD Pharmingen 555441), biotin-SP-conjugated ChromPure human IgG (Jackson ImmunoResearch
Laboratories no.009-060-003), and 20 ␮l biotin-SP-conjugated
ChromPure human IgA (Jackson ImmunoResearch Laboratories
no.009-060-011). After washings, biotin-conjugated Abs were revealed
with allophycocyanin-conjugated streptavidin (1/40 diluted; BD Pharmingen 554067). Samples of 3–5 ⫻ 105 cells were analyzed on a LSR
flow cytometer (BD Biosciences) using logarithmic amplification. For
four-color analysis, controls were included in every experiment to determine background fluorescence.
ELISA
Human IgGs were titrated in the collected supernatants and sera by ELISA,
according to a sandwich protocol. Briefly, wells of microtiter plates (12565-136; Nunc) were coated (overnight, 4°C) with 100 ␮l of a purified
Goat anti-Human IgG, F(ab)2 capture Ab (Jackson Immunoresearch
Labs no.109-006-006) at a concentration of 2 ␮g/ml in 1⫻ PBS. Reactions were blocked by adding 200 ␮l of 1⫻ PBS containing 10% FCS
(blocking buffer) to each well for 30 min at room temperature. Wells
were then washed thoroughly with 1⫻ PBS containing 0.05% Tween 20
(washing buffer). Serial dilutions of the culture supernatants or of the
IgG standard (Human IgG whole molecule; Jackson ImmunoResearch
Laboratories no.009-000-003), at a concentration of 1120 ng/ml in
blocking buffer, were added to the wells (100 ␮l/well) and incubated
overnight at 4°C. Wells were washed thoroughly with washing buffer
before receiving 100 ␮l/well of the detecting Ab (anti-human IgG-HRP;
BD Biosciences no.555788), at a 1/5000 dilution in blocking buffer.
After 1-h incubation at room temperature, wells were washed and given
100 ␮l of the substrate solution (TMB chromogen; Biosource no.SB01).
Wells were incubated 15–20 min at room temperature to allow reactions
to develop. Well contents were measured for absorbance at 405 nm.
RNA extraction, reverse transcription, sqPCR, and qPCR
The mRNA was isolated from 0.5 ⫻ 104-105 B cells using the ␮MACS
mRNA isolation kit (Miltenyi Biotech), according to the manufacturer’s
protocol, eluted into 75 ␮l preheated elution buffer, and stored at ⫺80°C
until use. RT-PCR was performed in a Mastercycler Eppendorf machine.
Briefly, 10 ␮l of mRNA were used as template for cDNA synthesis in the
RT reaction.
The sqPCR was used to determine ␥1 circle transcripts (CTs). PCR
conditions for ␥1 CTs (608 bp) were: 1 min denaturation at 94°C, amplification of cDNA for 30 cycles, each cycle programmed for denaturation at
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age (21). In mice, the number of peripheral B cells is not decreased with age; this discrepancy with humans likely reflects a
different mechanism in mice to maintain B cell numbers, possibly by increased survival (22). So far, no one has studied
age-related changes in naive and memory B cells in mice, likely
because good memory B cell markers as is the CD27 in humans
were not available.
We have previously shown in mice (8, 9) that the E2A-encoded
transcription factor E47 is down-regulated in old splenic B cells,
leading to a reduction in activation-induced cytidine deaminase
(AID) and in CSR. This intrinsic defect in old splenic B cells
generates reduced amounts of switched Abs. We have extended
our study to investigate whether aging also affects Ab production
and both E47 and AID expression in B cells isolated from the
peripheral blood of human subjects (18 – 86 years). Results herein
show that the expression of E47, AID, and CSR progressively
decrease with age. We also show an age-related decline in the
percentage and numbers of switch memory B cells, no change in
the percentage but a decline in the numbers of IgM memory B
cells, and an increase in the percentage but not in the numbers of
naive B cells, consistent with our data on the decrease seen in CSR
in vitro. In addition, we suggest that the subsets of B cells able to
class switch in culture are the naive and the IgM memory population. Our results give possible molecular mechanisms for an intrinsic decrease in B cell function and the humoral immune response in aging.
Ig CLASS SWITCH IN AGING HUMAN B CELLS
The Journal of Immunology
5285
94°C for 1 min, annealing at 60°C for 1 min, and elongation at 72°C for 1
min, followed by a final extension phase of 3 min at 72°C. Primers were:
I␥1 forward, GGG CTT CCA AGC CAA CAG GGC AGG ACA; and C␮
reverse, GTT GCC GTT GGG GTG CTG GAC. Conditions and primers
for GAPDH have been published previously (8, 9). The PCR products were
separated on 1.5% agarose gels. Gels were photographed using the AlphaImager Enhanced Resolution Gel Documentation and Analysis System
(Alpha Innotech) and images were quantitated using the AlphaEaseFC 32bit software.
During the course of these studies, we switched over from sqPCR to
qPCR for the evaluation of E47 and AID, with results being consistent with
both techniques. To perform qPCR, 2 ␮l of cDNAs were added to 8 ␮l of
2⫻ SYBR Green PCR Master mix (Applied Biosystems no.4367659), 1 ␮l
of 5 ␮M forward primer, 1 ␮l of 5 ␮M reverse primer, and deionized water
in a final volume of 20 ␮l. Reactions were conducted in MicroAmp 96-well
plates (Applied Biosystems no.N8010560). Calculations were made with
ABI software. Briefly, we determined the cycle number at which transcripts reached a significant threshold (Ct) for E47, AID, and GAPDH as
control. A value of the target gene (E47 or AID), relative to GAPDH, was
calculated and expressed as ⌬Ct. Samples are expressed as percentages of
the highest ⌬Ct value, taken as 100. Primers for PCR amplification were:
E47 forward, GCG AGG GCA CCC ACT TC; E47 reverse, GGT CCC
AGG AAT GTG GAT GA; AID forward, CAC CGC GCG CCT CTA C;
AID reverse, CCT TCC CAG GCT TTG AAA GTT; GAPDH forward,
CCA GGT TGT CTC CTC CGA CTT; and GAPDH reverse, CCA GCG
TCG AAG GTG GAA. Primers for E47 were designed with the Applied
Biosystems 7300 software and were selected according to the best penalty
score. A set of primers for AID (AID forward, GCC ACG TGG AAT TGC
TCT TC; and AID reverse, AGC GGC CAG GGT CTA GGT) was initially
designed with the Applied Biosystems 7300 software and selected according to the criteria of having the best penalty score. However, these primers
were not optimal in amplifying small amounts of AID such as those found
in B cells from elderly people. Therefore, we chose primers for AID (23)
but used different conditions, presented here and also checked these for a
low penalty score. Conditions for both E47 and AID were: 50°C for 2 min,
55°C for 10 min, 95°C for 15 s (40 cycles), and 60°C for 1 min. Because
the primers for CTs gave a high penalty score, we measured CTs by sqPCR
only. We have fewer samples analyzed for the qPCR because they were set
up after the study began.
Statistical analyses
Before conducting the main analyses, we examined the data for normality of the distribution and outliers. Four outliers were observed but
there was no discernible pattern to these outliers. Outliers were scores
with ⬎2 SDs above and below the mean. For these four variables, the
score was changed to the mean score to ensure a normal distribution for
conducting the parametric statistical analyses. Linear correlations between age and the following immune parameters were calculated: B cell
percentages and numbers, and B cell subset percentages and numbers,
CTs, AID, E47, and IgG in culture supernatants and sera. Because the
graphical representations of these relationships had a nonlinear appearance, we also examined nonlinear (i.e., quadratic) relationships between
age and immune parameters. That is, a nonlinear relationship might
postulate that middle aged are higher than expected by linear curve.
Values are shown in each figure legend.
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FIGURE 1. Age-related changes in B cell percentages and numbers. B cells isolated from the PB of subjects of different ages using CD19 magnetic beads, were
analyzed for the percentage of cells expressing membrane CD19 (A, top) and the absolute number of CD19⫹
cells calculated (A, bottom). The percentages and numbers of naive B cells (IgG⫺/IgA⫺/CD27⫺), IgM memory B cells (IgG⫺/IgA⫺/CD27⫹), and switch memory B
cells (IgG⫹/IgA⫹) are shown in B, C, and D, respectively. Pearson’s r values for the linear curves expressing the correlation between age and immune variables
and the significance of these analyses are the following:
B cell percentages: r ⫽ ⫺0.64, p ⫽ 0.001; B cell numbers: r ⫽ ⫺0.68, p ⫽ 0.001; Naive B cell percentages
r ⫽ 0.54, p ⫽ 0.002; Naive B cell numbers: r ⫽ 0.19,
p ⫽ 0.31; IgM memory B cell percentages: r ⫽ ⫺0.23,
p ⫽ 0.220; IgM memory B cell numbers: r ⫽ 0.67, p ⫽
0.001; Switch memory B cell percentages: r ⫽ ⫺0.48,
p ⫽ 0.003; and Switch memory B cell numbers; r ⫽
0.726, p ⫽ 0.001. Naive B and IgM memory B cell
percentages showed significant quadratic relations to
age. Quadratic, nonlinear coefficient ␤s were, respectively: ⫺2.519 (t value ⫽ ⫺2.561, p ⫽ 0.016) and 2.64
(t value ⫽ 2.279, p ⫽ 0.03). Significant differences between young and old are indicated by ⴱ, p ⬍ 0.05; and
ⴱⴱ, p ⬍ 0.01. Solid line refers to linear regression and
dashed line refers to quadratic regression.
5286
Ig CLASS SWITCH IN AGING HUMAN B CELLS
Results
Switch memory B cells decrease whereas the percentage of
naive B cells increase with age
Aging down-regulates CSR in activated PB-derived human
B cells
B cells were stimulated in vitro with anti-CD40/IL-4. CSR was measured by ␥1 CTs. Briefly, CSR generates an extra-chromosomal reciprocal switch DNA recombination product, which includes the intronic H chain promoter 5⬘ of the targeted CH gene, the DNA segment
between S␮ and the targeted S region, and C␮ and is referred to as the
CT (26). Results in Fig. 2A show that these transcripts are reduced in
B cells from elderly individuals. We have evidence that in the CD19⫹
B cell population only IgM memory B cells switch in response to
anti-CD40/IL-4, as we show below (see Both memory and naive B
cells show intrinsic defects in class switch with age), and as expected
from other’s results (27). Naive B cells require additionally a BCR
stimulus (anti-␮) to be stimulated. We have also looked at germline
transcripts and found that they are present in B cells from young and
elderly individuals at a similar extent (not shown), similar to that as
we have seen in the murine system (9). Thus, our results indicating
less CSR products suggest that the defect in aged B cells occurs at the
CSR event and is not due to problems with accessibility, nor with the
cytokine signaling pathway that leads to ␥1 accessibility, as we have
shown in murine B cells (9).
We also measured IgG production by ELISA of culture supernatants after 7 days stimulation with anti-CD40/IL-4. Results in Fig. 2B
(top) show that IgG levels in culture supernatants decrease with age
and, as expected, parallel the data shown in Fig. 2A. Conversely,
when we measured IgG levels in serum, we found they increased with
age (Fig. 2B, bottom), as others have shown consistently (17, 28).
FIGURE 2. Aging decreases class switch in activated PB-derived human B cells. A, B cells (106 cells/ml), isolated from the PB of subjects of
different ages using CD19 magnetic beads (see Materials and Methods),
were cultured with anti-CD40 (1 ␮g/ml) and IL-4 (10 ng/ml) for 6 –7 days.
At the end of this time, cells were harvested, RNA extracted, and sqRTPCR performed to evaluate CTs and GAPDH transcripts. Undiluted and
1/4 diluted RT-PCR from five representative subjects are shown (top). The
graph (bottom) shows the densitometric analyses (arbitrary units) of CTs,
normalized to GAPDH, from 46 subjects of different ages. Numbers shown
for each sample are percentages of the highest value (randomly chosen),
taken as 100. Pearson’s r values for the linear curves expressing the correlation between age and immune variables and the significance of this
analysis are r ⫽ ⫺0.84, p ⫽ 0.001. B, Top, B cells were cultured as
described in A. IgG levels in culture supernatants were determined by
ELISA as described in Materials and Methods. Pearson’s r values expressing the correlation between age and immune variables and the significance
of this analysis are: r ⫽ ⫺0.75, p ⫽ 0.001. B, Bottom, IgG levels in the
serum were determined by ELISA as described in Materials and Methods.
Pearson’s r values expressing the correlation between age and immune
variables and the significance of this analysis are: r ⫽ 0.572, p ⫽ 0.001.
Solid line refers to linear regression and dashed line refers to quadratic
regression.
Aging down-regulates AID in activated PB-derived human
B cells
Because the reduction in CSR in old B cells could be due to reduced levels of AID, as already shown in murine B cells (3, 9), we
investigated the levels of AID transcripts in antiCD40/IL-4-stimulated CD19⫹ B cells from individuals of different
ages. Preliminary results (not shown) indicated that stimulation
with anti-CD40/IL-4, and to a lesser extent with IL-4, induced AID
mRNA expression in PB-derived B cells from both young and
elderly subjects. The level of AID expression was lower in old as
compared with young B cells stimulated not only with anti-CD40/
IL-4 (Fig. 3, top) but also with IL-4 (data not shown). Human B
cells require the activation of both CD40 and IL-4 signaling pathways to undergo optimal AID expression and likely Ig class switch
(29). Fig. 3, top shows that aging decreases AID mRNA expression in anti-CD40/IL-4-stimulated CD19⫹ B cells from subjects of
different ages, as determined by qPCR.
Aging down-regulates the E2A-encoded transcription factor
E47 in activated PB-derived human B cells
To gain insight into the mechanisms underlying AID regulation,
we analyzed the levels of expression of the E2A-encoded transcription factor E47 in PB-derived B cells. Preliminary results (not
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We have analyzed the composition of the peripheral B cell pool in
46 individuals of different ages (18 – 86 years), i.e., the percentages
and the absolute numbers of total CD19⫹ B cells, as well as the
percentages and absolute numbers of naive (IgG⫺/IgA⫺/CD27⫺),
IgM memory (IgG⫺/IgA⫺/CD27⫹), or switch memory (IgG⫹/
IgA⫹) B cells. We decided to look at IgG⫹/IgA⫹ cells as switch
memory cells because, in addition to the IgG⫹/IgA⫹/CD27⫹ population, a fraction of IgG⫹/IgA⫹/CD27⫺ B cells also expresses
mutated IgV region genes, increased expression of the costimulatory molecules CD80 and CD86, and higher in vitro Ig secretion as
compared with naive B cells, thus showing the features of a memory B cell population (24). Our results show that both the percentages and the numbers of total CD19⫹ B cells decrease with age
(Fig. 1A), consistent with data previously published (19, 25). The
percentage of naive B cells increases with age (Fig. 1B, top) at
least for most of the individuals tested (such that the linear curve
accounted for greater amounts of variance than the quadratic function, though both were statistically significant). Due to the decrease in
number and percentage of CD19⫹ cells, the number of naive B cells
is not significantly different in young and old (Fig. 1B, bottom). The
percentage of IgM memory B cells are not statistically different between young and old (Fig. 1C, top, linear curve) but show an increase
in old age compared with the middle aged group with the quadratic
curve. The absolute number of IgM memory B cells is decreased with
age (Fig. 1C, bottom). Switch memory B cells decrease in both percentage (Fig. 1D, top) and number (Fig. 1D, bottom) with age. The
significant decrease in switch memory B cells and the increase in the
percentage of naive and IgM memory B cells (from middle to old age)
suggest an intrinsic defect in the ability of old B cells to undergo CSR.
Therefore, we wanted to investigate whether CSR was decreased in
CD19⫹ B cells stimulated in vitro with anti-CD40/IL-4, a stimulus
leading to IgG production.
The Journal of Immunology
shown) determined optimal dose response and kinetics and indicated that stimulation with anti-CD40/IL-4, and to a lesser extent
with anti-CD40, induced E47 mRNA expression in PB-derived B
cells from both young and elderly subjects. IL-4 alone was unable
to induce E47 mRNA expression. Fig. 3, middle, shows that aging
decreases E47 mRNA expression in anti-CD40/IL-4-stimulated
CD19⫹ B cells from subjects of different ages, as determined by
qPCR. Results in Figs. 3 show that E47 and AID progressively and
similarly decrease with age. The significant correlation between
E47 and AID is shown in Fig. 3, bottom. Results show that as E47
increased, AID also increased (r ⫽ 0.80, significant at the 0.01
FIGURE 4. Both memory and naive B cells are impaired in their ability to undergo in vitro class switch. Sorted memory (CD19⫹CD27⫹) or
naive (CD19⫹CD27⫺) B cells were stimulated (104 cells/200 ␮l) with
anti-CD40/IL-4, alone or together with F(ab⬘)2 of goat anti-human Ig,
for 5 days (at later time points, viability in culture significantly dropped
off). The graph shows the amounts of AID mRNA (qPCR) normalized
to GAPDH, expressed as ⌬Cts (see Materials and Methods), from four
pairs of young (white columns) and old (black columns) subjects. For
each pair, results are expressed as percentages of the anti-CD40/IL-4stimulated memory B cells from young subjects, taken as 100. Ages of
young no.1, 2, 3, and 4 were 20, 21, 24, and 23 and those of old no.1,
2, 3, and 4 were 72, 82, 74, and 71. These subjects are not represented
in Fig. 3. The differences between anti-CD40/IL-4-stimulated memory
B cells from young and old subjects (columns 1 vs 5) are p ⬍ 0.05 for
the four pairs, and between anti-CD40/IL-4/IgM-stimulated memory B
cells from young and old subjects (columns 2 vs 6) are p ⬍ 0.01 (pair
no.1 and no.3), p ⬍ 0.05 (pair no.4), and not significant (pair no.2). The
differences between anti-CD40/IL-4/IgM-stimulated naive B cells from
young and old subjects (columns 4 vs 8) are p ⬍ 0.05 for the four pairs.
level, 2-tailed). Higher E47 was also associated with higher CTs
(r ⫽ 0.65, significant at the 0.01 level, 2-tailed, not shown). Likewise, there was a positive significant association between AID and
CTs where greater AID was associated with greater CTs (r ⫽ 0.79,
significant at the 0.01 level, 2-tailed, not shown).
Both memory and naive B cells show intrinsic defects in class
switch with age
We have so far shown that CD19⫹ B cells lose their ability to
undergo in vitro class switch with age and we have shown that
there is an intrinsic defect in the expression of E47 and AID. The
data shown herein do not clarify whether there is an intrinsic defect
in the subsets of memory B cells or whether the decrease we saw
in class switch depends on the age-related reduction in the numbers of memory B cells. To clarify this point, we sorted naive and
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FIGURE 3. Aging decreases AID mRNA expression in activated PBderived human B cells. Top, B cells (106 cells/ml), isolated from the PB of
subjects of different ages using CD19 magnetic beads were cultured with
anti-CD40 (1 ␮g/ml) and IL-4 (10 ng/ml) for 6 –7 days. At the end of this
time, cells were harvested, RNA extracted, and qRT-PCR performed to
evaluate AID and GAPDH transcripts. The graph shows the amounts of
AID mRNA, normalized to GAPDH, from 34 subjects of different ages
[these are 34 of the 46 evaluated by sqPCR (not shown)]. Values for seven
subjects between the ages of 67 and 79 were undetectable (at the x-axis).
Numbers are percentages of the highest value (randomly chosen), taken as
100. Pearson’s r values for the linear curves expressing the correlation
between age and immune variables and the significance of these analyses
are: r ⫽ ⫺0.78, p ⫽ 0.001. AID mRNA showed significant quadratic
relations to age. Quadratic, nonlinear coefficient ␤s were the same for qRTPCR. Middle, B cells (106 cells/ml) were cultured as above but for 1 day
(the optimal time point for E47 mRNA expression). The graph shows the
amount of E47 mRNA, normalized to GAPDH, from 30 subjects of different ages [these are 30 of the 46 evaluated by sqPCR (not shown)].
Pearson’s r values are: r ⫽ ⫺0.75, p ⫽ 0.001. E47 mRNA expression also
showed significant quadratic relations to age. Quadratic, nonlinear coefficient ␤s were: ⫺1.866 (t value ⫽ ⫺2.745, p ⫽ 0.009). Bottom, sqRT-PCR
data for E47 and AID were positively correlated. Correlation is significant
at the 0.01 level (2-tailed). Solid line refers to linear regression and dashed
line refers to quadratic regression.
5287
5288
Ig CLASS SWITCH IN AGING HUMAN B CELLS
Table I. Percentages of naIve, IgM memory, and switch memory
B cells in the subjects of Fig. 4a
Young
Old
Naive
IgM Memory
Switch Memory
17,21,20,15
64,39,73,69
46,44,52,48
29,31,18,26
37,35,28,36
7,30,10,5
a
The order from left to right 1– 4 corresponds to the number of the pair in the
figure. Phenotype (% cells) of old No. 2 is “young-like” (see text).
FIGURE 5. Class switch in subsets of B cells. B cells from three young
subjects were sorted into IgM memory, switch memory, or naive B cell
subsets and stimulated (104 cells/200 ␮l) with anti-CD40/IL-4, together
with F(ab⬘)2 of goat anti-human Ig, for 5 days. Columns show the amounts
of AID mRNA (qPCR) normalized to GAPDH. Results (mean ⫾ SE) are
expressed as percentages of the IgM memory B cells, taken as 100.
Parameters
Young Mean ⫾ SE
Old Mean ⫾ SE
p Value
B cell %
B cell numbers
Naive B cell %
Naive B cell
numbers
IgM memory B
cell %
IgM memory B
cell numbers
Switch memory B
cell %
Switch memory B
cell numbers
sqRT-PCR CTs
IgG in culture sups
qRT-PCR AID
qRT-PCR E47
14.30 ⫾ 1.12
337.58 ⫾ 18.90
30.93 ⫾ 3.20
103.14 ⫾ 15.86
6.68 ⫾ 0.69
153.48 ⫾ 20.66
48.72 ⫾ 4.75
57.18 ⫾ 8.10
0.001
0.001
0.01
0.019
36.51 ⫾ 5.70
32.62 ⫾ 4.76
0.61
118.87 ⫾ 19.43
41.20 ⫾ 12.89
0.004
28.80 ⫾ 3.31
18.65 ⫾ 2.18
0.02
104.17 ⫾ 14.36
23.79 ⫾ 5.26
0.001
82.63 ⫾ 4.83
44.67 ⫾ 4.41
79.90 ⫾ 4.66
84.51 ⫾ 2.72
28.91 ⫾ 4.03
11.23 ⫾ 1.07
22.77 ⫾ 3.14
51.31 ⫾ 5.00
0.01
0.01
0.01
0.01
a
Young: 18 –30 years (12 subjects). Old: ⬎65 years (15 subjects). Independent
samples t test were calculated.
Significant differences in B cell functions were observed
between young (18 –30) and old (⬎65) subjects
The younger age group (individuals from 18 to 30 years) differed
from the older age group (aged greater than 65 years) when analyzed by independent samples t test (Table II). The groups differed
on every variable with the exception of numbers of naive B cells
and IgM memory percentage, which is consistent with the findings
above (Fig. 1, B and C).
Discussion
In the present paper, we show both quantitative and qualitative
changes occurring in human B cells with aging. In particular, we
confirm data from other groups showing that B cell percentages
and numbers decrease with age (17–19, 25), whereas the percentage of naive B cells increase with age (19, 25). Moreover, we show
for the first time that the subset of switch memory B cells decreases with age. Because of the decreased numbers of total B cells
with age, the absolute number of switch memory B cells is extremely low in elderly subjects, whereas that of naive B cells is
maintained. We also show herein, as others have reported (19), that
IgM memory B cells are reduced in numbers, not in percentage.
Therefore, the depletion in both switch memory and IgM memory
B cells seems to be a feature of aging human B cells.
The significant decrease in the number of naive T cells and the
shrinkage in their TCR repertoire reported in the elderly (30) can
both account for the reduced stimulation of memory B cells in
older ages in vivo, but likely not for intrinsic B cell differences we
see here.
The decrease in switch memory B cells, the increase in the
percentage of naive B cells, and the absence of a decrease in the
percentage of IgM memory B cells (or an actual increase in
some individuals in IgM memory B cells, at least compared
with middle age, see quadratic curve, Fig. 1C) suggest an intrinsic defect in the ability of old B cells to undergo CSR. We
have indeed shown in the present study that CSR induced in cultures of whole PB-derived B cells by anti-CD40/IL-4 is significantly impaired by aging. In more detail, our preliminary data on
sorted subsets of B cells have shown that the defect we observed
in the ability of CD19⫹ cells to class switch upon in vitro stimulation with anti-CD40/IL-4 is not simply depending on a reduction
in the numbers of memory B cells (IgM memory/switch) but by an
intrinsic defect in AID in both naive and (IgM) memory B cells.
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memory B cells which were then stimulated in vitro by anti-CD40/
IL-4, a stimulus mainly for memory B cells. Sorted cells were also
stimulated with anti-CD40/IL-4 and F(ab⬘)2 of anti-human IgM,
used as surrogate Ag, because naive B cells require the activation
of the BCR signal transduction to undergo class switch (27). Results in Fig. 4 show that memory B cells express AID in response
to anti-CD40/IL-4, independently of the presence of anti-IgM. We
conclude that the subset of memory B cells able to class switch in
culture is likely the IgM memory population, although here we
only looked at AID, and not also CT. In the experiments shown in
Fig. 4, naive B cells do not express AID in response to anti-CD40/
IL-4 and up-regulate AID only in the presence of BCR triggering
by anti-IgM. Even in the best conditions of stimulation (antiCD40/IL-4 ⫹ anti-IgM), the response of naive B cells was lower
than that of memory B cells. Therefore, both IgM memory and
naive B cells are negatively affected by aging. The old subject in
pair no.2 is clearly exceptional from the data in Fig. 4 as well as
Fig. 3. We can conclude from these preliminary data on sorted
subsets of B cells that the defect we observed in the ability of
CD19⫹ cells to class switch upon in vitro stimulation with antiCD40/IL-4 does not simply depend on a reduction in the numbers
of memory B cells but is an intrinsic defect of the switched cells
(IgM memory in previous experiments and naı̈ve ⫹ IgM memory
here). The percentages of naive, IgM memory and switch memory
B cells in the subjects of Fig. 4 are shown in Table I.
Both switch memory and IgM memory B cells have been shown
to proliferate and differentiate into plasma cells in response to antiCD40/IL-4 (27), but this differentiation should not to be expected
to increase AID at least in the switch memory cell population. We
have shown this to be true in a preliminary series of experiments,
where we sorted naive, IgM, and switch memory B cells from
young subjects and found that in the three subjects we tested naive
and IgM memory B cells had comparable levels of AID transcripts,
whereas switch memory B cells had 4-fold less as compared with
the other subsets (Fig. 5). Thus, in the unsorted populations AID
expression to anti-CD40/IL-4 is predominantly measuring the IgM
memory population.
Table II. Age group differences for immune parametersa
The Journal of Immunology
Acknowledgments
We are grateful to the people who participated as subjects in this study. We
thank Dr. J. Mathew from the Departments of Surgery, Division of Transplantation and Microbiology/Immunology of the University of Miami
Miller School of Medicine for the human spleen samples, Emil Jackson and
the nurses of the Family Medicine Department, Jim Phillips and the Sylvester Comprehensive Cancer Center Flow Cytometry Core Resource, and
Michelle Perez for secretarial assistance.
Disclosures
The authors have no financial conflict of interest.
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Ig CLASS SWITCH IN AGING HUMAN B CELLS