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Department of Microbiology and Immunology, University of Miami Medical School, Miami, FL 33136
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Th2 cells promote humoral immunity and
secrete IL-4, IL-5, and IL-10. Both Th1 and Th2 function are required
for comprehensive immunity. However, the inappropriate skewing of a
response to either the Th1 or Th2 lineage can result in pathological
conditions such as autoimmunity or the failure to clear infectious
agents. Thus, achieving and maintaining the appropriate balance between
Th1 and Th2 function are critical for protective and nonpathological
immune responses. This is especially important for the neonatal period
of life when tolerance to self-Ags as well as reactivity to a host of
novel non-self-Ags must be established. Neonatal animals are considered to be at high risk for infectious agents which have relatively minor effects in adults. In many cases, vigorous Th1-mediated responses appear to play a critical role in protecting the adult animals. Therefore, it had been widely thought that neonates were deficient in Th1 lineage development in vivo. Despite this expectation, it has recently been shown that neonates are fully competent to develop Ag-specific Th1 responses in situ (reviewed in Ref. 4). These mature Th1 responses were elicited with the use of strong Th1-promoting agents, including DNA vaccines (5, 6, 7, 8, 9, 10), strong Th1-promoting adjuvants (11), CpG-containing oligonucleotides (12), or IL-12 (13).
Although Th1 responses can be elicited from neonates, Th2-biased responses often emerge. In the early 1990s, Streilein and his colleague (14) first discovered a Th2 bias of neonatal allogeneic responses. Subsequently, Siegrist and his colleague (7) observed Th2 skewing in neonatal mice immunized with a variety of Ags precipitated with aluminum, the only adjuvant approved for pediatric use in humans (12). Bona and colleagues (9, 10) reported Th2 responses in neonates immunized with live or attenuated virus while comparably immunized adults developed Th1 responses. Using protein Ag (keyhole limpet hemocyanin (KLH)3) in PBS, we have also found that animals initially immunized as neonates show Th2-dominant memory responses (15). Finally, Zaghouani and colleagues (16, 17, 18) have described Th2-polarized responses in the lymph nodes of adult animals originally tolerized as neonates to peptide Ag. Thus, under many conditions, neonates appear to be biased to Th2 lineage function.
To understand how neonates arrive at Th2-dominant memory responses, we have examined all phases of the immune response, from naive cells to memory effectors. First, we have tested the influence of primary Th1/Th2 function on subsequent memory responses. We had previously found that naive neonatal lymph nodes develop mature, mixed Th1/Th2 primary effectors (15, 19) but that the splenic primary effectors are exclusively Th2-like (19). The possibility that the exclusive Th2 primary responses in the spleen account for Th2-biased memory responses was addressed. Experiments using splenectomized neonates showed no significant differences in the Th2-skewed memory responses in the lymph nodes. Therefore, the Th2-biased primary responses in the neonatal spleen are not required to generate Th2-dominant memory in neonates. Second, we have compared the duration of the primary effector phase in neonates and adults. These experiments showed that although primary Th2 function in adult lymphoid organs disappears rapidly, high-level primary Th2 function is retained in both the spleen and lymph nodes of neonates for weeks. Third, we have analyzed the kinetics of the generation of memory effector cells in neonates and adults. These studies revealed that animals initially immunized as neonates are deficient in the capacity to generate Th1 memory effector function. Together, these data demonstrate that neonates are biased to Th2 function at all stages of the immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice, originally obtained from Charles River Breeding
Laboratories (Wilmington, MA), were bred and housed under barrier
conditions in the Division of Veterinary Resources at the University of
Miami Medical School. Periodic screening showed the colony to be free
of commonly occurring infectious agents. Females from timed matings
were monitored closely from days 19–21 of gestation and the date of
delivery was recorded. Birth day was called day 0. Neonatal animals
were defined as 
1-day old.
Splenectomy
Neonatal mice 1-day old were anesthetized by burial up to the
neck in wet ice for 
2–3 min. Adult mice were anesthetized by
injection of a mixture of xylazine (Cetus, Rockville Center, NY) and
ketaset (Fort Dodge Laboratories, Fort Dodge, IA) i.p. at,
respectively, 0.2 and 1 mg/10 g body weight. Animals were placed on
their stomachs and a small incision was made on the right back side,
just below the rib cage. Spleens were pinched off with forceps and the
wounds were closed with silk braided sutures (Ethicon, attached 5-0
needle) for neonates or with surgical staples for adults.
Immunization
Unless otherwise indicated in the text, 1-day-old neonatal or
adult (6–8 wk old) mice were immunized with, respectively, 10 or 100
µg KLH (Calbiochem, San Diego, CA). Mice >1 day but <6 wk old were
weighed and immunized with 5 µg/g KLH. In most cases, a solution of
KLH in PBS was used for immunization. For the experiment shown in Fig. 6
, KLH precipitated with aluminum potassium sulfate (alum) was used.
For Alum immunization, a 1:1 (v/v) mixture of 1 mg/ml KLH and a 10%
(w/v) solution of aluminum potassium sulfate dodecahydrate was
prepared. A sufficient volume (20% of the final volume) of 1.0 N
NaOH was added to achieve a pH of 6.5. The mixture was allowed to stand
for 30 min at room temperature and then was diluted with an excess of
PBS and centrifuged for 5 min (1000 rpm). The pellet was resuspended
with PBS to achieve a final ratio of 0.5 mg aluminum potassium
sulfate/10 µg KLH.
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Preparation of total spleen and lymph node cells
Pools of tissues from 
2 adults or 6 newborn animals were
used for the cell preparations. Total spleen cell suspensions were
prepared (15) and RBC were removed by incubation in
hypotonic lysis buffer (0.15 M NH4Cl, 0.001 M
KHCO3, and 0.1 mM EDTA). Mesenteric, inguinal,
axillary, brachial, and cervical lymph nodes were pooled and used for
total lymph node cell suspensions (15).
Preparation of CD4+ cells
Enriched (95–98% CD4+) CD4+ cells were positively selected (MS+ columns) using the Miltenyi Biotec (Bergisch-Gladbach, Germany) MACS system, precisely per the manufacturer’s directions.
Preparation of adult splenic APC
Total spleen cells from naive adult animals were treated with anti-Thy-1 (mAb 42–21) plus complement, followed by treatment with 50 µg/ml mitomycin C, as described earlier (20, 21).
Culture conditions for ELISAs
For cytokine production by total lymph node or spleen cells,
5 x 105 cells were plated in 200 µl of
culture medium and stimulated with 50 µg/ml KLH. Culture medium
consisted of RPMI 1640 (Life Technologies, Grand Island, NY) containing
1 mM sodium pyruvate (Life Technologies), 2 mM L-glutamine
(Life Technologies), 5 x 10-2 mM 2-ME
(Life Technologies), 1% penicillin-streptomycin (Life Technologies),
and 10% heat-inactivated (56°C, 30 min) FCS (HyClone, Logan, UT).
For cytokine production by CD4+ T cells, 2
x 105 CD4+ (prepared as
described above) were coplated in 200 µl of culture medium with
4 x 105 adult splenic APC (as above) and
stimulated with 50 µg/ml KLH. Culture supernatants were harvested
72 h later and IFN- and IL-4 content were assessed using
mouse-specific cytokine ELISA kits (Endogen, Woburn, MA), precisely
according to the manufacturer’s directions.
Culture conditions for enzyme-linked immunospot (ELISPOT) assays
To activate cells for the ELISPOT assays, lymph node or spleen
cells from immunized mice were cultured at 5 x
105 cells/200 µl of culture medium containing
50 µg/ml KLH. At 36–48 h later, the cells were harvested and
processed for ELISPOT, as previously described in detail
(19). Briefly, Nunc Maxisorp plates (Nunc, Naperville, IL)
were coated by overnight incubation at room temperature with 100 µl
of a 5-µg/ml solution of anti-mouse IL-4 (PharMingen, San Diego,
CA) or anti-mouse IFN- mAb (PharMingen). The plates were washed
and the wells were then blocked with 100 µl of culture medium for
1 h at room temperature. Different dilutions of the harvested
cells (above) were added to the wells and the plates were incubated for
20 h at 37°C in an atmosphere of 5% CO2.
The plates were then washed and 100 µl of biotinylated anti-IL-4
or anti-IFN- mAb (PharMingen) was added to each well. Following
a 90-min incubation at room temperature and additional washes, 100 µl
of 0.2 µg/ml streptavidin-alkaline phosphatase (Jackson
ImmunoResearch, West Grove, PA) was added to each well. The wells were
incubated for 60 min at room temperature, washed, and 100 µl of a 1:4
mixture (v/v) of 3% melted low EEO type 1 agarose (Sigma, St. Louis,
MO) and 2.3 mM 5-bromo-4-chloro-3-indolyl phosphate (Sigma) in AMP
buffer was added to each well. AMP buffer was prepared by mixing 75 mg
MgCl2 hexahydrate, 50 µl Triton X-405, 500 mg
NaN3, and 47.9 ml 2-amino-2-methyl-1-propanol
(Sigma) in 350 ml of H2O. The mixture was brought
to pH 10.25 with HCl and the final volume was then adjusted to 500 ml
with H2O. The developed spots were counted with
the aid of a dissecting scope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We had previously found that the nature of the primary Th1/Th2
response in neonates is highly dependent on the site of initial Ag
exposure. In the lymph nodes, neonates develop primary, mixed Th1/Th2
effector function that is indistinguishable from that found in adults
(15, 19). In striking contrast, there is the exclusive
development of primary Th2 responses in the neonatal spleen
(19). Since neonatal memory responses become Th2 skewed
under similar immunization conditions, the question that arises is, are
the Th2-dominant memory responses of neonates due to the preferential
development of primary splenic Th2 function? To address this question,
we wished to separate the lymph node from the splenic responses in
situ. In particular, we wanted to eliminate the primary neonatal
splenic responses to test whether memory responses in the lymph nodes
remained Th2 biased or became more Th1 dominant, as in adult animals
(15). For this purpose, 1-day-old or adult BALB/c mice
were splenectomized. Two to 4 h later, the neonatal and adult
animals were immunized both s.c. and i.p. with, respectively, 10 and
100 µg of KLH in PBS. Nonsplenectomized age-matched control animals
were similarly immunized in parallel. Two weeks later, the animals were
reimmunized with KLH in PBS; adults were reimmunized with 100 µg and
the 2-wk-old animals were weighed and immunized with 5 µg/g KLH.
Following an additional 2 wk, the animals were sacrificed and
CD4+ lymph node cells were cultured with adult
splenic APC and 50 µg/ml KLH. Seventy-two hours later, supernatants
were harvested and tested for IFN- and IL-4 content using
cytokine-specific ELISA kits. As shown in Fig. 1, there was little difference in the
cytokine secretion patterns in the splenectomized vs the control
animals for either neonatal or adult animals; animals initially
immunized as neonates still produced less IFN- and more IL-4 than
did adults. This Th2 bias observed in vitro appeared to reflect the in
vivo availability of cytokines since secondary serum antihapten Ab
responses were skewed to the Th2-associated IgG1 isotype in
splenectomized mice (data not shown), as previously seen in
nonsplenectomized neonates (15). Therefore, even in the
absence of the neonatal spleen, Th2-skewed memory responses ensue. From
these experiments, we conclude that the exclusive Th2 primary responses
in the neonatal spleen are not required for the development of
Th2-dominant memory in neonates.
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The splenectomy experiments indicated that the generation of
dominant Th2 memory in neonates was likely regulated by an event(s)
downstream of primary effector development. We next decided to compare
the primary effector phase in neonates and adults. In one from an
elegant series of in vivo studies, Swain and colleagues
(22) earlier described the kinetics of Th1/Th2 primary
effector development in adult lymph nodes. Following a single
immunization with KLH, both Th1 and Th2 primary effectors developed
rapidly, peaking in cytokine secretion activity 5–7 days after
immunization. The production of both IFN- and IL-4 declined rapidly
thereafter and was evident only at low levels by 2 wk after
immunization. To examine similarly the primary effector phase in our
system, neonates and adults were immunized with KLH in PBS and cytokine
production by lymph node cells restimulated with KLH in culture was
analyzed 1 and 2 wk after immunization (Fig. 2). Between 1 and 2 wk after
immunization, IFN- secretion declined modestly in both neonatal and
adult lymph node cell cultures. Thus, neonatal and adult Th1 effector
function showed similar changes with time following immunization. A
different picture emerged when IL-4 production was examined. As was
previously observed by Swain and colleagues (22), IL-4
production by adult lymph node cells was virtually undetectable by 2 wk
after immunization. In contrast, neonatal lymph node cells continued to
produce high levels of IL-4, as late as 2 wk following a single
immunization. Thus, unlike adults, neonates appear to retain
substantial Th2 primary effector function in the lymph nodes for at
least 2 wk after immunization.
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). Moderate changes in IFN-
production were observed among 1, 2, and 5 wk after immunization in
neonatal and adult lymph nodes and in adult spleen. Neonatal spleens
showed a large increase in IFN- production between 1 and 5 wk,
likely due to the greatly increased numbers of T cells accumulating
over the intervening period of development (Ref. 23 ; B.
Adkins, unpublished observation). Nonetheless, the corresponding
lymphoid organs in neonates and adults showed similar levels of IFN-
production at the end of the 5-wk period. As previously seen (Fig. 2
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IL-4 secretion was undetectable as early as 2 wk after immunization in
adult lymph nodes, and a similar drastic reduction was seen in the
adult spleen. In neonatal lymph nodes, IL-4 production decreased 5- to
10-fold by 5 wk after immunization but was nonetheless readily
detectable. As observed for IFN- production, IL-4 secretion in the
neonatal spleen increased markedly by 5 wk after immunization. Thus,
although Th1 function is similar in neonates and adults late following
primary immunization, Th2 effector function shows marked differences;
in adults Th2 function disappears rapidly while neonates show prolonged
high Th2 activity.
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). Minor differences in IFN-
production were observed between total and CD4+
cells in the lymph nodes of both neonates and adults. IFN-
production was greatly enhanced among CD4+ cells
in the neonatal spleen, most likely because there are still low
frequencies of CD4+ cells present at 2 wk of life
among total splenocytes (23). The prolonged Th2 effector
function seen among total neonatal lymph node and spleen cells was even
more dramatic among the CD4+ fraction.
CD4+ cells from neonates made from 5-fold
(spleen) to >500-fold (lymph node) more IL-4 than did adult
CD4+ cells. Therefore, the retention of the
capacity for high-level primary Th2 function in neonates is not
dependent on the presence of neonatal APC in culture.
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The dissimilarities in the duration of primary Th2 activity in
neonates and adults might result from differential Ag clearance. For
example, if neonates clear Ag less efficiently than do adults, the
persistence of Ag may result in the continual recruitment of new Th2
primary effectors. Similarly, if adults efficiently eliminate Ag, new
Th2 cells could not continue to be enlisted. We have tested this idea
using three approaches. First, we have immunized neonates with 10-fold
less KLH to reduce the amount of Ag that is present late during the
primary response phase. We have also immunized adults with 10-fold more
KLH to provide the potential opportunity for late-phase recruitment of
new Th2 cells. Immunizing neonates with 10-fold less or adults with
10-fold more KLH resulted in minor differences in IFN- production 1
or 2 wk later, in either the lymph node or spleen (Fig. 5). A similar major decline in IL-4
production was observed in both the lymph nodes and spleens of adults
immunized with either the standard amount, 100 µg, of KLH or with
10-fold more. Immunization of neonates with 10-fold less KLH did not
reduce the levels of IL-4 secreted at 2 wk and, indeed, resulted in a
major increase from 1 to 2 wk, especially in the lymph nodes. Thus, it
seems unlikely that large differences in effective Ag dose in vivo
account for the retention of prolonged Th2 function in neonates and the
corresponding loss of this function in adults.
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). As we had seen for immunization
with KLH in PBS, neonates and adults showed similar patterns of IFN-
production. In the lymph nodes, IFN- decreased modestly between 1
and 2 wk, whereas in the spleen, IFN- increased modestly.
Contrasting patterns were observed between neonates and adults for IL-4
production. Between 1 and 2 wk of immunization in adults, IL-4
production decreased 5-fold. On the other hand, IL-4 production
increased in both the lymph nodes and spleens of neonates. Therefore,
the use of adjuvant does not obviously prevent the decline in primary
Th2 function in adults nor does it interfere with the prolonged
retention of this activity in neonates.
The rates of migration of newly generated cells from the thymus to
peripheral organs appear to be similar in neonates and adults
(24). However, the relative proportions of recent thymic
emigrants, compared with resident cells, are much higher in neonates
than in adults. Thus, in the third approach, we tested the possibility
that, in neonates, prolonged Th2 primary function resulted from the
continued recruitment of new thymic emigrants into the response. Two
groups of 1-day-old neonates were immunized with KLH. One week later,
animals in one group were thymectomized while animals in the other
group were kept as unmanipulated controls. One additional wk later
(i.e., 2 wk after the initial immunization), CD4+
lymph node and spleen cells were restimulated with KLH in the presence
of adult splenic APC and supernatants were harvested for ELISA (Fig. 7). IFN- production was increased 2-
to 4-fold in the lymph nodes and spleens of thymectomized, as compared
with control, neonates. Similarly, IL-4 production increased 4 fold
in the lymph nodes of thymectomized vs control neonates while IL-4
production was similar in the spleens of the two groups. Therefore,
removal of the thymus 1 wk after primary immunization did not result in
a reduction, and actually resulted in an increase in the lymph nodes,
in prolonged Th2 primary function.
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The large differences in IL-4 secretion seen in bulk cultures of
adult and neonatal cells at late times following primary immunization
could result in one of at least two possible ways. First, neonatal
tissues may contain many more IL-4-secreting cells compared with adult
tissues. Second, neonatal and adult Th2 cells may be present at similar
frequencies but neonatal cells may be capable of greater IL-4
production per cell. To distinguish between these possibilities, the
frequencies of IFN-- and IL-4-producing cells were measured by
ELISPOT analyses 1 and 5 wk after immunization (Fig. 8). In the lymph nodes, neonates
developed 2- to 3-fold more of both IFN-- and IL-4-secreting cells
than did adults 1 wk after immunization. As we have previously reported
(19), the neonatal spleen contained very few
IFN--secreting cells but mature levels of IL-4-producing cells 1 wk
after immunization. With the exception of the large increase in the
neonatal spleen, the frequencies of IFN--producing cells showed
modest changes from 1 to 5 wk after immunization in both neonates and
adults. The frequencies of IL-4-producing cells declined in the lymph
nodes of both neonates and adults between 1 and 5 wk of immunization.
Surprisingly, the fold decline was not very different between the two,
a decrease of 2- to 3-fold for neonates and 3- to 4-fold for adults
between 1 and 5 wk. In the spleen, neonates showed a modest increase in
the frequency of IL-4-secreting cells while the frequency declined in
adult spleens. However, the decline was modest, with close to 1000
IL-4-secreting cells/106 total cells still
remaining in adult spleens 5 wk following immunization. Since total
IL-4 production in bulk cultures is low to undetectable by this time
point in both adult lymph node and spleens, primary adult Th2 cells may
lose robust function while neonatal Th2 cells efficiently retain
it.
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The prolongation of primary Th2 effector function in neonates
could have a significant impact on the generation of memory effectors.
Bradley et al. (25) have shown that IL-4 can drive the
preferential development of adult Th2 memory effector cells.
Accordingly, the increased IL-4 produced by long-lived Th2 primary
effector function in neonates may serve to skew the development of
memory effectors to the Th2 lineage. To compare memory effector
development in neonatal and adult animals, the mice were immunized with
KLH in PBS and then reimmunized 5 wk later. Th1/Th2 cytokine production
was then assessed at different intervals following reimmunization (Fig. 9). The development of IL-4-secreting
memory effector function in adults was very similar to that previously
described by Bradley et al. (26). In both lymph nodes and
spleen, IL-4 production appeared rapidly, peaking 3 days after
reimmunization. Neonates showed similar rapid increases in IL-4
production although maximal production was observed as early as day 2
after reimmunization. There was also a rapid increase in IFN-
production in adult lymph node and spleen. In striking contrast, there
was no increase in IFN- production at any time point analyzed in
either the neonatal lymph nodes or spleen. Similar results were
obtained using CD4+ cell populations (data not
shown). Thus, in addition to the prolongation of primary Th2 function
in neonates, there appears to be the selective impairment in the
development of Th1 memory effector function.
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). Two groups each of
neonates and adults were immunized with KLH. Four weeks later, one
group of each was reimmunized with KLH; the other group of each was not
reimmunized. Three days later, lymph node and spleen cells from all
four groups were restimulated with KLH and subsequently processed for
ELISPOT, as described in Materials and Methods. For both
adults and neonates, the frequencies of cells secreting IL-4 increased
10-fold following reimmunization. The frequency of IFN--secreting
cells also increased 2-fold in adult lymph nodes and spleens. As we
had expected, there was no increase in the frequency of
IFN--secreting neonatal spleen cells following reimmunization.
However, the frequency of IFN- secretors did increase in neonatal
lymph nodes and, surprisingly, the increase was at least as great as
that seen in adult lymph nodes. Thus, in the lymph nodes, neonates
developed Th1 memory effectors in frequencies similar to those that
developed in adult lymph nodes. Since total IFN- secretion as
assessed by ELISA did not increase (Fig. 9), these results imply that
neonatal lymph node Th1 memory effectors may produce less IFN- per
cell than do adult Th1 memory effectors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4. In our system,
adult animals also show somewhat prolonged IFN- production. What
sets neonates apart from adults is the extended period of time (5 wk)
over which high-level IL-4 production remains evident. Why this occurs
is currently unclear. In the adult, it is known that shortly after the
peak of a primary response, at 5–7 days, the vast majority of
Ag-specific cells either leave the lymph nodes or die in situ (reviewed
in Refs. 28, 29). This may not occur in neonates, i.e.,
neonatal Th2 cells may either fail to exit the nodes or fail to undergo
apoptosis. If this were the case, the frequencies of IL-4-secreting
cells in neonates would be expected to be much higher than in adults
late following primary immunization. However, as late as 5 wk after a
single immunization, the frequencies of IL-4-secreting cells in adults
were only 3-fold lower than the frequencies in the corresponding
neonatal tissues. This sharply contrasts with total IL-4 production
which, at this late time after primary immunization, is 100 times
lower in adult than in neonatal tissues. One important question that
arises is, is the extended Th2 function in neonates due to the
retention of robust primary function or to the continual recruitment of
new effectors by residual Ag? We have tried to address this possibility
by decreasing the amount of Ag used to immunize neonates. The rationale
is that this approach may decrease the amount of Ag present at later
times to below the threshold level for the activation of naive cells;
the result would be greatly reduced Th2 function. However, immunizing
neonates with 10-fold less Ag did not alter the late-phase retention of
Th2 primary function. Moreover, thymectomy at 1 wk of life to eliminate
any ongoing recruitment of new thymic emigrants into the response did
not diminish the prolonged primary Th2 activity in neonates. This
finding argues that the extended primary Th2 function in neonates is
largely not due to the persistent activation of new naive T cells.
Together, these data suggest that prolonged primary Th2 function in
neonates is achieved, at least in part, by the retention of the
capacity for high-level IL-4 production by individual Th2 cells.
Animals initially immunized as neonates mounted robust Th2 memory
effector responses. However, as assessed by total IFN- levels
measured by ELISA, there was essentially no development of Th1 memory
effector function. In the spleen, this appears to be mainly due to the
failure to develop Th1 memory effectors since there was no increase in
the frequency of IFN- secretors following reimmunization. In
contrast, IFN--secreting memory effectors did develop in neonatal
lymph nodes and their frequencies were similar to those developing in
adult lymph nodes. However, since the levels of IFN- produced in
bulk cultures by neonatal lymph node cells were 5-fold less than
those produced by adult lymph node cells, neonatal lymph node Th1
memory cells may secrete less IFN- per cell as compared with adult
Th1 memory cells. Thus, animals initially immunized as neonates show
regionally distinct patterns of attenuation of Th1 memory responses: in
the spleen, Th1 memory effectors fail to develop; in the lymph nodes,
these cells develop but show limited function. Regionally discrete
responses have been previously reported for neonates. First, we
demonstrated that neonatal primary effector responses are mature, mixed
Th1/Th2-like in the lymph nodes but exclusively Th2-like in the spleens
(19). Second, Zaghouani and colleagues
(16, 17, 18) have reported different regional responses in
adult animals originally tolerized as neonates to peptide Ag. In their
case, Ag-specific responses in the lymph nodes were Th2-polarized while
Ag-specific splenic T cells were anergized. Together, these results
indicate that the spleen and lymph nodes of neonates are functionally
distinct sites and the influence of the different environments can
persist well into adult life.
One interesting observation is that the exclusive primary Th2 function in the neonatal spleen does not appear to be required to develop Th2-biased memory responses in the lymph nodes. Experiments using splenectomized neonates showed no major changes in Th1/Th2 cytokines produced in a memory response in neonatal lymph nodes. These results indicate that intrinsic properties of the lymph node T cells and/or the neonatal lymph node environment are sufficient to generate dominant Th2 memory responses. One straightforward way to test this idea is to separate the T cells from the environment by transfers of neonatal T cells to adoptive adult hosts and vice versa. Determining the relative contributions of T cell intrinsic properties and environmental influences is a major goal of the laboratory. Although primary splenic responses may not have a major effect on later memory responses, the clear Th2 bias in neonates is likely to be important during the neonatal period. Based on the regional responses in neonates, we have developed a working hypothesis to explain the differential responses: during early neonatal life, two opposing but necessary processes are occurring at frequencies that are almost certainly higher than in adult life. First, the vast majority of T cells are newly generated and are encountering many peripheral Ags not present in the thymus. Tolerance to these self-Ags must first be established during the neonatal period. Second, the neonate has no immunological experience and must, therefore, establish productive responses against a wealth of newly encountered non-self-Ags. We propose that, in the neonate, the spleen is the primary site of tolerance induction to self-Ags in the periphery whereas the lymph nodes are the site of immune responsiveness to foreign Ags. There are a number of specific predictions from this hypothesis, such as neonatal splenectomy should result in an increased incidence of early onset autoimmune disease. Using this and related approaches, we are currently trying to determine the relative roles of splenic vs lymph node responses in neonates.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Becky Adkins, Department of Microbiology and Immunology, R-138, University of Miami Medical School, P.O. Box 016960, Miami, FL 33136.
3 Abbreviations used in this paper: KLH, keyhole limpet hemocyanin; alum, aluminum potassium sulfate; ELISPOT, enzyme-linked immunospot.
Received for publication August 15, 2000. Accepted for publication October 24, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated splenic anergy. J. Exp. Med. 188:2007., and IL-12 regulate the development of subsets of memory effector helper T cells in vitro. J. Immunol. 155:1713.[Abstract]
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I. Malhotra, P. Mungai, E. Muchiri, J. Ouma, S. Sharma, J. W. Kazura, and C. L. King Distinct Th1- and Th2-Type Prenatal Cytokine Responses to Plasmodium falciparum Erythrocyte Invasion Ligands Infect. Immun., June 1, 2005; 73(6): 3462 - 3470. [Abstract] [Full Text] [PDF]
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 B. Gunnlaugsdottir, S. M. Maggadottir, and B. R. Ludviksson Anti-CD28-induced co-stimulation and TCR avidity regulates the differential effect of TGF-{beta}1 on CD4+ and CD8+ naive human T-cells Int. Immunol., January 1, 2005; 17(1): 35 - 44. [Abstract] [Full Text] [PDF]
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 S. Chang-Rodriguez, R. Ecker, G. Stingl, and A. Elbe-Burger Autocrine IL-10 partially prevents differentiation of neonatal dendritic epidermal leukocytes into Langerhans cells J. Leukoc. Biol., September 1, 2004; 76(3): 657 - 666. [Abstract] [Full Text] [PDF]
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B. Adkins Peripheral CD4+ Lymphocytes Derived from Fetal versus Adult Thymic Precursors Differ Phenotypically and Functionally J. Immunol., November 15, 2003; 171(10): 5157 - 5164. [Abstract] [Full Text] [PDF]
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 M. Obuchi, M. Fernandez, and G. N. Barber Development of Recombinant Vesicular Stomatitis Viruses That Exploit Defects in Host Defense To Augment Specific Oncolytic Activity J. Virol., August 15, 2003; 77(16): 8843 - 8856. [Abstract] [Full Text] [PDF]
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J. W. Upham, P. T. Lee, B. J. Holt, T. Heaton, S. L. Prescott, M. J. Sharp, P. D. Sly, and P. G. Holt Development of Interleukin-12-Producing Capacity throughout Childhood Infect. Immun., December 1, 2002; 70(12): 6583 - 6588. [Abstract] [Full Text] [PDF]
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 F. J. Culley, J. Pollott, and P. J.M. Openshaw Age at First Viral Infection Determines the Pattern of T Cell-mediated Disease during Reinfection in Adulthood J. Exp. Med., November 18, 2002; 196(10): 1381 - 1386. [Abstract] [Full Text] [PDF]
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H. J. Ezelle, D. Markovic, and G. N. Barber Generation of Hepatitis C Virus-Like Particles by Use of a Recombinant Vesicular Stomatitis Virus Vector J. Virol., October 25, 2002; 76(23): 12325 - 12334. [Abstract] [Full Text] [PDF]
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, Control; 
, splenectomized.
