J Appl Physiol 97: 491– 498, 2004;
10.1152/japplphysiol.01404.2003.
Influence of age and physical activity on the primary in vivo antibody
and T cell-mediated responses in men
Taro P. Smith, Sarah L. Kennedy, and Monika Fleshner
Department of Integrative Physiology, University of Colorado at Boulder, Boulder, Colorado 80302
Submitted 31 December 2003; accepted in final form 31 March 2004
characterized by a progressive
decrease in responsiveness to exogenous antigens (16, 25).
This dysregulation in immune function is problematic for
several reasons. A decline in antigen-specific adaptive immunity is a risk factor for increased incidence of infectious disease
(12, 15). In addition, older persons are often inadequately
protected from diseases because of ineffective responses to
vaccination (13), a fact that is in part due to an impairment of
the immunoglobulin (Ig) response (30). Both the Ig response
(28) and the delayed-type hypersensitivity (DTH) response
(10) to antigenic challenge are altered in older human populations, although the precise nature and cellular mechanisms of
these alterations remain unknown (3).
Regular moderate exercise training (cardiovascular training,
such as walking, cycling, etc.) may offset some of the immune
function abnormalities found in healthy older populations (27,
33, 35). A majority of the previous work testing the effect of
physical activity on immune function is limited because primarily in vitro measures of immunity were utilized. In vitro
assessment of immune function can be problematic because it
is difficult to link circulating blood lymphocyte response to
overall immune system function (45), and changes in in vitro
immune function, when cells are removed from the organism
and placed in cultures, often do not predict in vivo responses,
perhaps because interactions with neural and hormonal factors
that are important in modulating optimal immune responses are
not present. Measurement of the antibody response to vaccination and DTH response to a skin challenge provide in vivo
methods to measure B cell and T cell function, respectively.
A few studies have tested the effect of physical activity on
in vivo assessment of immune function. For example, Kohut et
al. (22) reported an improved anti-influenza antibody response
to influenza vaccination in older human populations of higher
trained status. Bruunsgaard et al. (1) did not find any changes
in antibody production to tetanus and pneumococcal vaccines
after a race event in triathletes compared with controls. Rall et
al. (32) measured DTH responses in the elderly after a 12-wk
resistance training intervention; however, they did not find any
changes in response to training compared with sedentary controls. One limitation of the previous work is that the antigenic
challenge tested was not novel to the person and thus stimulated a secondary or tertiary immune response.
To better understand potential mechanisms of how physical
activity may improve immunity, it is best to challenge subjects
with a novel antigen. After antigen presentation, the B cell first
produces the IgM isotype. The B cell then undergoes antibody
class switching via T cell help to produce the IgG isotype. IgG
consists of four subclasses in humans (IgG1, IgG2, IgG3, and
IgG4). Each subclass differs in its function and cytokine
requirement. IgG1, for example, is better for opsonization,
whereas IgG2 is better for antigen neutralization (18). In
humans, Th1 cells produce IL-2 and IFN-␥, which induces B
cells to produce opsonizing antibody such as IgG1 and IgG3,
whereas Th2 cells produce IL-4 and IL-10 that induce B cells
to produce neutralizing antibodies such as IgG2 and IgG4 (18).
If there is an improvement in IgM production with physical
activity, then perhaps this is due to improved antigen presenting cell and B cell interaction. If there is a change in the
response of a particular IgG subclass, then this could be
reflective of changes in Th1 or Th2 cytokine production.
CD4⫹ T cell function and numbers decline with age (25).
Moreover, the ability to form a novel T cell response is also
compromised because of an increase in memory subset and a
decrease in naive T cell populations over time. The generation
of a DTH response after sensitization with a novel antigen
provides an in vivo measure for memory T cell function of the
CD4⫹ subset. Memory T cells will migrate to the spot of an
Address for reprint requests and other correspondence: M. Fleshner, Dept. of
Integrative Physiology, Campus Box 354, Univ. of Colorado at Boulder,
Boulder, CO 80309 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
immune function; vaccination; human
THE AGING IMMUNE SYSTEM IS
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Smith, Taro P., Sarah L. Kennedy, and Monika Fleshner. Influence of age and physical activity on the primary in vivo antibody and T
cell-mediated responses in men. J Appl Physiol 97: 491– 498, 2004;
10.1152/japplphysiol.01404.2003.—The aging immune system is characterized by the progressive decline in the antibody and T cellmediated responses to antigen. Little is known, however, about the
benefits of exercise in aging on the generation of a primary immune
response to antigen and the subsequent antibody and memory T
cell-mediated response. Most in vivo immune research to date has
utilized vaccines or recall antigens to elicit an immune response.
Therefore, the purpose of this experiment was to examine the association of aging and physical activity on the primary antibody and T
cell response to the novel protein antigen keyhole-limpet hemocyanin
(KLH). Forty-six physically active and sedentary, young (20 –35 yr)
and older (60 –79 yr) men were recruited. Subjects were intramuscularly immunized with 100 ␮g of KLH, and blood samples were
collected at days 0, 7, 14, 21, and 28. Samples were measured for
anti-KLH IgM, IgG, IgG1, and IgG2 by ELISA. On day 21 after
intramuscular KLH administration, subjects received an intradermal
injection with 1 ␮g of KLH of inflammation recorded at 24, 48, 72,
96, and 120 h to assess anti-KLH delayed-type hypersensitivity
response. There was a significant reduction in all anti-KLH measures
with aging except for anti-KLH IgG2. The physically active older
group had significantly higher anti-KLH IgM, IgG, IgG1, and delayed-type hypersensitivity responses, but not IgG2 compared with
the sedentary older group. In conclusion, regular physical activity in
older men is associated with a more robust immune response to novel
antigenic challenge.
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EXERCISE, AGING, AND IMMUNITY
METHODS
Subjects
The experimental protocol was approved by the Human Research
Committee at the University of Colorado-Boulder. Voluntary informed consent forms outlining the purpose and risks of the study
were collected from all subjects. A total of 19 healthy men (n ⫽ 8) and
women (n ⫽ 11) aged 20 –35 participated in testing the KLH specificity (experiment 1). A total of 46 healthy men participated in the
physical activity aging study (experiment 2): 10 young (aged 20 –35)
and 10 older (aged 65–79) sedentary and 12 young and 14 older
physically active men. The sedentary men had performed no regular
exercise for ⬎2 yr. The endurance-trained men had performed regular
aerobic physical activity at least three times per week for ⬎2 yr. All
subjects were healthy and free of overt disease as assessed by physical
examination, medical history, resting blood pressure, and fasting
blood chemistries (e.g., complete blood count with differential, lipid
panel, etc.). All older subjects were evaluated for coronary artery
disease via an exercise stress test with electrocardiograms and exercise blood pressures. Subjects were excluded if they were smokers,
were on medications, or had high alcohol intake. Furthermore, because KLH is a derivative of sea mollusks, subjects with shellfish
allergies were excluded from the study.
J Appl Physiol • VOL
Treadmill Exercise Test
All experiment 2 subjects participated in a graded treadmill exercise test with a modified Balke protocol. Maximal oxygen consumption was measured via computer-assisted open-circuit spirometry.
Heart rate, blood pressure, and rating of perceived exertion were
measured at each workload.
Administration of KLH
KLH is a widely used protein isolate that has been used extensively
in humans (4 – 6, 31, 37, 38). KLH (Pierce Biotechnology, Rockford,
IL) was conjugated to alum adjuvant (Pierce Biotechnology) according to manufacturer instructions. Briefly, 100 ␮g of KLH were
adsorbed to 900 ␮g of alum in a ratio as previously published (36) and
injected intramuscularly in the deltoid via a 11/2-in. 23-gauge needle.
In experiment 1, a subset of subjects (n ⫽ 7; 3 men and 4 women)
were immunized, with the remaining serving as negative controls (n ⫽
12; 5 men and 7 women). All subjects in experiment 2 (n ⫽ 46) were
immunized.
Blood Sampling
Ten milliliters of blood were collected via intravenous forearm
draws at days 0 (baseline) and 21 for experiment 1 and at days 0, 7,
14, 21, and 28 for experiment 2 after KLH injection. Blood was
allowed to clot and then was spun at 3,000 rpm for 10 min, and serum
was separated into 1-ml aliquots and frozen at ⫺20°C for later
analysis of antibody.
Anti-KLH Antibody ELISA
Experiment 1 serum concentrations of anti-KLH IgM and IgG were
measured at days 0 and 21 via ELISA. Experiment 2 anti-KLH IgM
was measured at days 0, 7, 14, and 21, whereas anti-KLH IgG IgG1,
and IgG2 were measured at days 0, 7, 14, 21, and 28 via ELISA.
Ninety-six-well microtiter plates (Nunc, Rochester, NY) were coated
with 1 ␮g of KLH in 100 ␮l of carbonate coating buffer per well and
incubated for 2 h at 37°C then overnight at 4°C. Plates were washed
four times with PBS-Tween 20 buffer and blocked with 1% dry skim
milk in PBS. Plates were washed an additional four times, and 100 ␮l
of sera were added at the appropriate dilutions (1:100 for IgM, 1:200
for IgG, 1:2 for IgG1, and 1:1 for IgG2). Plates were incubated for 45
min and washed six more times. One hundred microliters of horseradish peroxidase-conjugated anti-human IgM (lot no. 080, DAKO,
Carpinteria, CA), IgG (DAKO lot no. 128), IgG1 (lot no. 20167766,
Zymed, South San Francisco, CA), and IgG2 (Zymed lot no.
20264084) secondary antibody were added at a concentration of 1:500
in 1% dry skim milk in PBS-Tween to the appropriate plates and
incubated for 45 min. Plates were washed six more times, and 100 ␮l
of 2,2-azino-di(3-ethylbenzthiazoline)sulfonic acid (ABTS)⫹H2O2 in
citrate buffer (Zymed ABTS substrate kit for horseradish peroxidase)
were added to all wells. Plates were incubated for 15–30 min and read
at an optical density (OD) of 405 nm. Anti-KLH antibody is presented
as absolute OD for experiment 1 to allow for the inspection of the
levels of nonspecific binding in sera of preimmunized subjects.
Anti-KLH antibody was calculated for each subject as a subtraction of
preimmunization (day 0) OD and then as a proportion of its plate
positive to account for plate-to-plate variability (7).
Anti-KLH DTH Response
A DTH intradermal challenge (1 ␮g KLH in 10 ␮l sterile H2O on
the volar aspect of the arm) was administered 3 wk after KLH
immunization via a 3/4-in. 29-gauge needle. Timing of the intradermal KLH challenge was determined from anti-KLH DTH experiments
as previously published (6, 36). The diameter (mm) of the DTH
response was measured 24, 48, 72, 96, and 120 h postchallenge.
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intradermal antigenic skin challenge after sensitization with the
same antigen. The T cell then produces IFN-␥ to recruit
macrophages to the location (18), subsequently producing local
inflammation that peaks at 48 h postchallenge and slowly
resolves over the next 120 h. Improvement in this response
with physical activity in the aged would suggest that there is
either a restoration in the number of naive T cells, an increase
in T cell function, or an increase in macrophage recruitment.
This study proposes the use of two in vivo measures of
acquired immune function in response to a novel antigen for
studying the effect of aging and the potential benefits of regular
moderate exercise in younger and older populations. The
immune responses assessed are the in vivo generation of the
primary Ig and DTH responses after antigenic challenge with
keyhole limpet hemocyanin (KLH), a benign T cell-dependent
protein. KLH has been previously used in humans to measure
immune responses to stress (37, 38), as a vaccine conjugate
(23, 31), and for bladder cancer therapy (19, 20). Reduction in
the level of KLH antigen-specific antibody is suggestive of
impairments in antigen presenting cell–T cell–B cell function,
whereas the DTH response offers an in vivo measure of the
ability of T cells to proliferate and migrate to antigen. Two
experiments were performed. The first study verified that KLH
immunization in humans would stimulate detectable and antigen-specific antibody and DTH responses. This was accomplished by comparing pre- and postimmunization anti-KLH
IgM and IgG values, as well as comparing anti-KLH DTH
responses between KLH-immunized and nonimmunized subjects. The second study tested whether there was 1) an ageassociated decline in the primary antibody to a novel antigen;
2) an age-associated decline in memory T cell-mediated responses to a skin challenge (DTH) with the antigen; 3) an
effect of regular physical activity on these antigen-specific
responses; 4) an association of age and physical activity in
antigen-specific antibody isotype (IgM and IgG) and subclass
(IgG1 and IgG2); and 5) differences in specific antibody
response that can be accounted for by total circulating IgM,
IgG1, IgG2, IgG3, and IgG4 in aging and physical activity.
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EXERCISE, AGING, AND IMMUNITY
Serum samples were assayed according to manufacturer instructions
for total circulating IgG1, IgG2, IgG3, and IgG4 at baseline and day
21 postimmunization. Total IgG was determined by summing the total
quantity of IgG subclass (IgG1–IgG4) at both preimmunization and
day 21.
Statistical Analysis
Data were analyzed by 2 ⫻ 2 ANOVA (age ⫻ trained status) with
repeated measures as indicated. When significant differences were
found, Fisher’s protected least significant difference (PLSD) post hoc
analyses were utilized to determine group differences. All data are
expressed as means ⫾ SE. Statistical significance was determined at
P ⬍ 0.05.
RESULTS
Experiment 1: Specificity of the KLH Response
Measurement of Total IgM
Total IgM was measured in experiment 2 at preimmunization and
day 21 postimmunization via ELISA according to manufacturer instruction (Total Human IgM Assay, Alerchek, Portland, ME).
Measurement of Total IgG, IgG1, IgG2, IgG3, and IgG4
Zymed Laboratories total IgG subclass ELISA kits were used for
the determination of total IgG and IgG subclass in experiment 2.
Experiment 2: Aging and Physical Activity
Subject characteristics. Select subject characteristics for
experiment 2 are presented in Table 1. The mean age difference
between the young and older groups was 40 yr. Body mass was
not related to age or body composition and was not different
between the two older groups; however, physically active older
subjects had lower body mass index than sedentary older
subjects (P ⬍ 0.05). Lymphocyte counts were significantly
higher in the physically active older subjects compared with
their sedentary counterparts (P ⬍ 0.05) but were still within
normal clinical ranges.
Table 1. Selected subject characteristics
Sedentary
Physically Active
Variable
Young (n ⫽ 10)
Older (n ⫽ 10)
Young (n ⫽ 12)
Older (n ⫽ 14)
Age, yr
Body mass, kg
Body fat, %
Body mass index, kg/m2
Systolic BP, mmHg
Diastolic BP, mmHg
V̇O2 max, ml䡠kg⫺1䡠min⫺1
WBC, K/␮l
Neutrophils, K/␮l
Lymphocytes, K/␮l
Monocytes, K/␮l
Basophils, K/␮l
27.6⫾1.2
86.0⫾5.6
20.4⫾2.0
27.8⫾1.1
117⫾3
78⫾3
42.8⫾2.5
5.6⫾0.5
3.1⫾0.4
1.8⫾0.1
0.52⫾0.05
0.07⫾0.01
72.1⫾1.2*
82.6⫾3.0
27.5⫾1.6*
27.2⫾0.9
124⫾4
76⫾2
24.9⫾0.8*
6.2⫾0.5
3.8⫾0.5
1.5⫾0.1
0.61⫾0.05
0.05⫾0.01
25.3⫾1.0
73.9⫾5.0
10.1⫾1.1†
22.8⫾0.7†
118⫾4
80⫾3
57.8⫾0.8†
5.6⫾0.3
3.2⫾0.3
1.8⫾0.1
0.49⫾0.04
0.06⫾0.01
69.4⫾0.9*
75.0⫾3.2
26.3⫾1.6*
24.9⫾0.5*†
128⫾4
79⫾3
34.3⫾1.1*†
6.9⫾0.5*
3.9⫾0.4
2.2⫾0.3†
0.53⫾0.04
0.09⫾0.01*†
Values are means ⫾ SE. BP, blood pressure; V̇O2
†P ⬍ 0.05 vs. age-matched sedentary.
max
, maximal O2 uptake; WBC, white blood cells; K, 1,000. *P ⬍ 0.05 vs. young of same trained status;
J Appl Physiol • VOL
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Fig. 1. Experiment 1 [keyhole limpet hemocyanin (KLH) specificity] subjects
were immunized with KLH and had blood samples collected for determination
of anti-KLH IgM and IgG. Both IgG and IgM were elevated 21 days post-KLH
immunization in our test subjects (A). Intradermal injection of KLH into the
forearm resulted in a delayed-type hypersensitivity (DTH) response in subjects
who were immunized with KLH 21 days before challenge vs. nonimmunized
subjects (B). *P ⬍ 0.05.
Both the anti-KLH IgM and IgG titers are elevated at day 21
postimmunization (Fig. 1A, P ⬍ 0.05). This demonstrates that
our ELISA does detect KLH-specific Ig and that KLH immunization is effective at inducing the primary antibody response.
Nonspecific reactivity or cross-reactivity in preimmunized subjects resulted in an OD of 0.1– 0.3; however, this was acceptable given that the overall OD range was 0 –3.0. Intradermal
KLH challenge resulted in a DTH reaction as measured by
inflammation diameter (mm) that peaked at 48 h and persisted
up to 120 h in the KLH-immunized subjects; however, nonimmunized subjects had no resultant inflammation, indicating
that the DTH reaction only occurs in previously immunized
subjects and is specific to memory for KLH (Fig. 1B).
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EXERCISE, AGING, AND IMMUNITY
Fig. 2. Serum concentrations of anti-KLH IgM (A) and IgG (B) were assessed
in young and older physically active and sedentary men after KLH immunization. Physically active men had a robust antibody response that was similar
to that of the younger groups and significantly greater than the older sedentary
group.
Effect of age and physical activity status on the anti-KLH
serum antibody response. There was a main effect of age as
indicated by a reduction anti-KLH IgM [F(1,42) ⫽ 16.73, P ⬍
0.001; Fig. 2A] and IgG [F(1,42) ⫽ 19.99, P ⬍ 0.0001; Fig.
2B] antibody response over time in the older subjects. There
was a main effect of physical activity in anti-KLH IgG
[F(1,42) ⫽ 4.74, P ⬍ 0.05] indicated by an increase over time
that is likely due to a greater response in older physically active
subjects and not that of younger subjects. There was only a
trend toward an increase in anti-KLH IgM [F(1,42) ⫽ 3.10;
P ⫽ 0.0857] in the physically active subjects. Post hoc analysis
revealed a reliably higher anti-KLH IgM (Fisher’s PLSD, P ⬍
0.05) response by day 21 in the older physically active compared with the older sedentary group that was not significantly
different than the young sedentary subjects (Fisher’s PLSD,
P ⫽ 0.3038). The older physically active group had an elevated
anti-KLH IgG concentration compared with the older sedentary group at both day 21 (Fisher’s PLSD, P ⬍ 0.05) and day
28 (Fisher’s PLSD, P ⬍ 0.05), which was not different on day
21 from the young sedentary (Fisher’s PLSD, P ⫽ 0.5572) and
the young physically active groups (Fisher’s PLSD, P ⫽
0.2951).
J Appl Physiol • VOL
Fig. 3. Serum concentrations of the anti-KLH IgG subclasses IgG1 (A) and
IgG2 (B) were assessed in young and older physically active and sedentary
men after KLH immunization. Serum changes of anti-KLH IgG in the
physically active older men could be largely accounted for by changes in the
IgG1 (A) subtype and not the IgG2 (B) subtype.
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Only anti-KLH IgG1 demonstrated an effect of training
[F(1,42) ⫽ 14.24, P ⬍ 0.001; Fig. 3A] and a reliable age ⫻
trained status [F(1,42) ⫽ 5.26, P ⬍ 0.05] interaction. Post hoc
analysis revealed that older trained subjects had elevated antiKLH IgG1 response at days 14 (Fisher’s PLSD, P ⬍ 0.0001),
21 (Fisher’s PLSD, P ⬍ 0.01), and 28 (Fisher’s PLSD, P ⬍
0.001) compared with their sedentary counterparts. In addition,
the anti-KLH IgG1 responses of the physically active older
subjects were not statistically different at days 21 (P ⫽ 0.93)
and 28 (P ⫽ 0.207) compared with the young physically active
controls. This effect was isotype specific such that anti-KLH
IgG2 responses were not affected by age (P ⫽ 0.513) or
physical activity (P ⫽ 0.144) (Fig. 3B).
Effect of age and physical activity on the anti-KLH DTH
response. All subject groups mounted a DTH response to the
intradermal challenge over time [F(5,190) ⫽ 38.601, P ⬍
0.0001; Fig. 4]. Statistical analysis revealed a main effect of
training [F(1,38) ⫽ 4.922, P ⬍ 0.05] over time and a time ⫻
trained status interaction [F(5,190) ⫽ 2.699, P ⬍ 0.05]. AntiKLH DTH response was higher in both physical activity age
groups, as indicated by a lack of main effect of aging
[F(1,38) ⫽ 0.2306, P ⫽ 0.6339] or age ⫻ trained status
interaction [F(1,38) ⫽ 1.000, P ⫽ 0.3236]. Post hoc analysis
EXERCISE, AGING, AND IMMUNITY
revealed that the sedentary older subjects had age-related
reduction in the anti-KLH DTH response at 48 h compared
with the young physically active subjects (Fisher’s PLSD, P ⬍
0.05). DTH response was significantly elevated in older physically active subjects compared with their sedentary counterparts (Fisher’s, P ⬍ 0.05) and was similar to that of the
younger controls (P ⫽ 0.6237). However, 120 h after the
intradermal injection the older physically active subjects had
not fully resolved their inflammation, which was significantly
elevated compared with young sedentary (Fisher’s PLSD ⫽
2.832, P ⬍ 0.05) and the young physically active (Fisher’s
PLSD ⫽ 3.75, P ⬍ 0.001) subjects.
Effect of age and physical activity on total IgM, IgG, IgG1,
IgG2, IgG3, and IgG4. There were no differences in total
circulating IgM, IgG, IgG1, IgG2, IgG3, and IgG4 antibodies
between the subject groups. Furthermore, there were no differences in total antibody concentrations 21 days postimmunization between groups (P ⬎ 0.05). These data support the idea
that significant changes in a specific immune response may
occur in the absence of changes in total circulating Ig.
DISCUSSION
This is the first study to clearly demonstrate by use of a
novel in vivo antigenic challenge that physical activity is
associated with elevated generation of a primary antigenspecific T cell-dependent antibody and DTH responses in aging
humans. The primary observations of this study are the following: 1) there is an age-related decline in the primary
antibody response to the novel antigen KLH; 2) there is an
age-related decline in the memory T cell response to KLH; 3)
older physically active subjects have an improved antibody and
DTH response compared with older sedentary subjects that is
equal to that of younger subjects; 4) the changes in anti-KLH
IgG production are primarily of the IgG1 subclass; and 5) the
detected changes in antibody are reflective of improvements in
antigen-driven responses because total antigen nonspecific Ig is
not affected.
Previous studies examining vaccine efficacy in young vs.
older subjects have demonstrated declines in anti-influenza
(13) and no changes in anti-tetanus (2) antibody response. It is
J Appl Physiol • VOL
important to account for previous exposure to antigen when
utilizing vaccines to compare in vivo immune responses between subject groups, because there is a high likelihood of
previous encounter and resultant immunological memory. On
the basis of our specificity results, the KLH response is antigen
specific and is only present in immunized individuals. As such,
KLH challenge is a useful tool in determining the effects of
aging and trained status on the in vivo immune response in
humans as well as animals. In fact, it has been recently used to
assess the extent of immune dysfunction in persons with
diabetes (40).
CD4⫹ T cell function has been reported to decline with
aging, and that decline contributes to the subsequent reduction
in B cell Ig production (46). The decrease in T cell function is
characterized by a failure to proliferate in response to antigen,
possibly because of a reduction in IL-2 production (24, 29, 39).
Previously, Shinkai et al. (34) suggested that trained older
people have improved T cell function secondary to improved in
vitro IL-2, IFN-␥, and IL-4 production. This would be consistent with our data, because our trained older subjects had
improved antigen-specific IgM and IgG responses to a T
cell-dependent antigen, suggesting that the physical activity
may restore the anti-KLH antibody response by increasing T
cell help.
Similar to the influenza vaccination study by Powers (30),
our data demonstrated a reduction in specific IgG1 response in
the elderly individuals. In the present study, there was a change
only in the IgG1 antibody response and not in the IgG2
response. Perhaps there were no changes in the anti-KLH IgG2
responses between groups because protein-derived antigens
such as KLH drive primarily IgG1 and IgG3 antibody responses in humans (47). Alternately, the differential anti-KLH
IgG1 and IgG2 responses may be reflective of age-selective
declines in helper T cell function. Th1 cytokines stimulate
IgG1 and IgG3 production, whereas Th2 cytokines promote
IgG2 and IgG4 production from B cells in humans (18). Thus
the selective effect of aging and training on IgG1 may reflect
a greater sensitivity of Th1 cells to the effects of age and
activity. Although we were not able to directly measure the
changes in anti-KLH IgG3 or IgG4 in our study, we did
measure total nonspecific IgG3 and IgG4; however, these
results indicated that no subject group differences in these
antibody subtypes were present.
Anti-KLH DTH responses were higher in the physically
active older group compared with the sedentary older group.
This indicates that the in vivo measure of T cell-mediated
immune response is associated with higher physical activity
status. The DTH response is driven by memory CD4⫹ T cells
that release IL-2 and IFN-␥ to signal macrophages to an area
after an encounter with the recall antigen. There is also a shift
in the subset of T cells from naive (CD45RA⫹) to memory
(CD45RO⫹) with aging, resulting in a compromised ability to
deal with new antigens (3, 9, 14). Because there appears to be
a restoration in the in vitro T-cell cytokine production in
trained elderly (35), these results provide in vivo evidence that
physical activity is associated with higher T cell-mediated
responses. Furthermore, the DTH measure could have an
important clinical implication, because DTH anergy is a predictor of mortality in the elderly (42) and is a determinant of
infectious disease risk (10).
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Fig. 4. Anti-KLH DTH was assessed 21 days post-KLH immunization, and
resultant inflammation diameter was measured at 24, 48, 72, 96, and 120 h.
Peak DTH response was at 48 h, when the older physically active groups had
an improved DTH response that was similar to the young groups and significantly elevated compared with the sedentary older group.
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EXERCISE, AGING, AND IMMUNITY
Changes in B cell repertoire with age also contribute to the
decline in antigen-specific responses (8). There is a shift in the
population of B cells from B2 to B1 cells (43, 44). B1 cells
contribute to the increase in autoantibodies with age. Furthermore, there is a higher occurrence of autoantibody (e.g.,
␣-dsDNA) in older individuals compared with younger controls (17). Thus, in the case of aging, the problem is not a
reduction in the total concentration of antibody but rather a
decrease in functional antibody. The results from the present
study support this idea. As shown in Fig. 5, aging was not
associated with a decline in total Ig, rather only a decline in the
generation of new antibody in response to KLH. Importantly,
our study demonstrated a higher antigen-specific antibody
production in the physically active older group compared with
the sedentary older group and demonstrated that this increase
was detected in antigen-specific antibody independent of any
changes in total antibody. In addition, changes in total IgM,
IgG, IgG1, IgG2, IgG3, and IgG4 over time could not account
for changes in the anti-KLH IgG response. Older subjects did
not have reduced total antibody, and physical activity did not
increase total antibody. Assessment of total antibody is not
sensitive enough to detect either aging or physical activity.
Thus further questioning the validity of measuring total Ig as a
means of examining the effects of an intervention on immune
Downloaded from http://jap.physiology.org/ by 10.220.33.3 on June 17, 2017
Fig. 5. Total serum concentrations of IgM (A),
IgG (B), IgG1 (C), IgG2 (D), IgG3 (E), and IgG4
(F) were measured on day of KLH immunization
and 21 days postimmunization. The changes in
total antibody over time could not account for
the subject group difference in the antigen-specific KLH response as there were no total increases in any Ig isotype.
J Appl Physiol • VOL
97 • AUGUST 2004 •
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EXERCISE, AGING, AND IMMUNITY
ACKNOWLEDGMENTS
We thank all of the subjects who participated in this study as well as Dr.
Alison Smith for technical assistance.
GRANTS
This work was supported by National Institutes of Health awards AI48557
and 2 M01-RR-00051 from the General Clinical Research Center Program of
the National Center for Research Resources.
J Appl Physiol • VOL
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