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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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- or IL-4-producing cells, adults developed mixed Th1/Th2
responses in both organs. Neonatal lymph nodes contained mature
frequencies of IFN-- and IL-4-producing cells. In striking contrast,
while mature frequencies of Th2 cells developed in neonatal spleens,
virtually no IFN--secreting cells were detected. Exclusive Th2
function was observed in both BALB/c and C57BL/6 neonates, strains in
which the Th2 and Th1 lineages, respectively, are favored in adults.
Although Th1 effectors were virtually undetectable, the addition of
rIL-12 boosted the frequency of IFN--secreting cells to adult
levels. Therefore, Th1 effectors apparently developed in situ, but Th1
effector function either was not promoted or was inhibited upon
subsequent exposure to the Ag in culture. Together, these results
indicate that the quality of a primary Th response in neonates is
strongly dependent on the site of initial Ag exposure; responses
initiated in the lymph nodes are mixed Th1/Th2, whereas responses
occurring in the spleen are heavily Th2 biased. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
mediates delayed-type hypersensitivity and protection against
intracellular pathogens. The Th2 subset produces IL-4 and IL-5 and is
important in humoral responses. Immune responses inappropriately skewed
toward Th1 or Th2 function can exacerbate infectious diseases, allergic
reactions, and autoimmunity. Thus, generating and maintaining the
appropriate Th1/Th2 balance is critical for effective, nonpathological
immune function. Since the landmark studies of Medawar and colleagues (4) in the 1950s, it had been generally held that the poor T cell-mediated responses of neonates are due to immaturity in the T cell compartment. Indeed, a great deal of in vitro data, including a number of publications from our laboratory (5, 6, 7), led to the hypothesis that neonatal mice would be unable to mount adult-level Th1 responses in situ. However, it is now clear that neonates are capable of developing mature Th1 function in vivo. Strong Th1-inducing adjuvants (8), reduced loads of infectious agents (9), and some DNA vaccines (10, 11, 12, 13) have all been shown to elicit adult-level Th1 responses in neonates. We also reported that in response to a single immunization with protein Ag, neonates produced a mixed primary Th1/Th2 lymph node response quantitatively indistinguishable from that made by adults (14). Thus, under selected conditions, neonatal Th1 function can develop at adult levels in situ.
Our studies (14) showing that T cells from 
1-day-old
mice develop mature primary Th1 function are in seeming conflict with
reports by Siegrist and colleagues (15). They immunized
7-day-old neonatal mice with a variety of vaccine Ags and examined
responses 2–3 wk later. Their results showed a complete failure of Th1
and exclusive Th2 development. Two major differences between these
systems were in the ages of the neonates and the organs examined. To
resolve this apparent discrepancy, we have now compared the frequencies
of primary, Ag-specific Th1 vs Th2 effectors generated in the lymph
nodes vs spleens of 1-day-old animals. The lymph nodes of neonates
contained mature frequencies of Th1 and Th2 primary effector cells. In
striking contrast, spleens from the same animals contained mature
levels of Th2 effectors but essentially no Th1 effectors. Thus, in the
mouse the quality of Th responses mounted in vivo in early life is
strongly influenced by whether the responses initiate in the spleen or
in the lymph nodes.
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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 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. C57BL/6 mice were purchased from The
Jackson Laboratory (Bar Harbor, ME). Females from timed matings were
monitored closely from days 19 to 21 of gestation, and the date of
delivery was recorded. Birth day was called day 0. Neonatal animals
were defined as 24 h old.
Immunization
Adult (6–8 wk old) and newborn mice were immunized with 5
µg/g keyhole limpet hemacyanin
(KLH;3 Calbiochem, San
Diego, CA). In most cases, a solution of KLH in PBS was used for
immunization. Where indicated, KLH was delivered in the vehicles CFA or
aluminum potassium sulfate (ALUM). For the CFA, an emulsion was
prepared using a 1/1 (v/v) mixture of CFA (Sigma, St. Louis, MO) and
KLH. For ALUM, 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 1 µg of ALUM/20 µg of KLH. Each mouse
was injected in three sites i.p. and s.c. between the shoulder blades
and at the base of the tail. Adults received 100 µl/site, and
neonates received 10 µl/site.
Preparation of total spleen and lymph node cells
Pools of tissues from two or more adults or six or more newborn animals were used for the cell preparations. Total spleen cell suspensions were prepared (14), 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 (14).
Complement-mediated depletion of CD4+ or CD8+ cells
Spleen cells (4 x 107/ml) were incubated at 4°C for 45 min in HBSS supplemented with 1% calf serum and 10 mM HEPES buffer, pH 7.0, containing a 1/20 dilution of anti-CD8, HO2.2 (16) or anti-CD4, RL172.4 (17) ascites. Following the incubation, the cells were diluted with HBSS supplemented with 1% calf serum and 10 mM HEPES buffer, pH 7.0, and centrifuged through an underlayer of calf serum. The cells (4 x 107/ml) were then incubated for 45 min at 38°C in a 1/5 dilution of Cedar Lane Low Tox M Rabbit Complement (Accurate Chemicals, Westbury, NY) in medium 199 (Sigma), at pH 6.8–72. The cells were then washed, and the entire procedure was repeated with volume adjustments made for cell losses.
In vitro culture conditions
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 with or
without 50 µg/ml of KLH or, for the experiment shown in Table I
, 100 µg/ml of chicken egg albumin
(OVA; Calbiochem). 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). Where indicated, cultures
also included 0.1 ng/ml rIL-12 (a gift from Dr. Maurice K. Gately,
Hoffmann-La Roche, Nutley, NJ), 5 µg/ml neutralizing goat
anti-mouse IL-10 Ig (R&D Systems, Minneapolis, MN), or 25 µg/ml
neutralizing anti-IL-4 mAb, 11B11 (18), purified from
ascites fluid using protein G chromatography (19).
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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, clone 11b11 (PharMingen, San Diego,
CA), or anti-mouse IFN- mAb, clone R4-6A2 (PharMingen). The
plates were washed with PBS containing 0.1% Tween 20 (Fisher,
Fairlawn, NJ; PBS/0.1% Tween) for 5 min and then washed twice with
PBS. The wells were blocked with 100 µl of culture medium for 1
h at room temperature. The cells (above) were harvested 36–48 h after
the initiation of culture and placed in the precoated, blocked wells in
quadruplicate using five different cell concentrations in 2.5-fold
dilutions (from 2.5 x 105 to 6.4 x
103 cells/well). The plates were incubated for
20 h at 37°C in an atmosphere of 5% CO2.
The plates were then washed three times with PBS, followed by three
washes with PBS/0.1% Tween. To each well, 100 µl of biotinylated
anti-IL-4 (PharMingen clone BVD6-24G2) or anti-IFN- mAb
(PharMingen clone XMG1.2) was added, and the plates were incubated at
room temperature for 90 min. Following four washes with PBS/0.1%
Tween, 100 µl of 0.2 µg/ml of streptavidin-alkaline phosphatase
(Jackson ImmunoResearch Laboratories, West Grove, PA) was added to each
well. The wells were incubated for 60 min at room temperature and then
washed three times with PBS/0.1% Tween followed by three washes with
PBS. A 1/4 mixture (v/v) of 3% melted low EEO type 1 agarose (Sigma)
and 2.3 mM 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma) in AMP
buffer was added at 100 µl/well, taking care to minimize bubbles. AMP
buffer was prepared by mixing 75 mg of MgCl2
hexahydrate, 50 µl of Triton X-405, 500 mg of
NaN3, and 47.9 ml of 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. The quadruplicate wells yielding 
20
and 150 spots were used to calculate the average ± SD, which
were then normalized to the frequencies per 106
total cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In previous studies (14) we examined the development
of primary, Ag-specific Th1/Th2 responses in situ in neonatal lymph
nodes. Th1 or Th2 effector function was assessed in ELISA assays by
measuring the production of IFN- or IL-4, respectively, in response
to restimulation with the Ag in culture. Those experiments showed that
neonatal lymph nodes develop mature Th1 and Th2 effector function in
vivo. To determine whether the neonatal spleen was also capable of
mounting mature Th responses, we similarly analyzed cytokine production
by neonatal spleen cells. ELISA measurements, however, failed to reveal
detectable cytokine production by neonatal splenocytes (data not
shown), perhaps because there are few T cells present in the spleen in
the first week of life (20). Therefore, we adopted the
ELISPOT assay, which measures the frequencies of cytokine-producing
cells and which, in our experience, is more sensitive than the ELISA
assay.
Neonatal and adult BALB/c mice were immunized with KLH in PBS. Six to
eight days later spleen cell suspensions were prepared, and ELISPOT
analyses were performed as described in Materials and
Methods. Ag-specific cells secreting IFN- (Th1) or IL-4 (Th2)
were readily detected among adult splenocytes (Fig. 1). Th2 effectors also developed in the
neonatal spleen and at frequencies comparable to those found in the
adult spleen. In striking contrast, few Th1 effectors were detected in
the neonatal spleen.
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production by
neonatal lymph node T cells using the ELISA (14), we
wanted to directly compare the frequencies of IFN-- and
IL-4-producing cells developing in the spleens and lymph nodes of the
same neonatal animals. Neonatal and adult BALB/c mice were immunized
with KLH in PBS, and 6–8 days later the frequencies of IFN-- and
IL-4-producing cells in both spleen and lymph nodes were examined (Fig. 2). Th1 and Th2 effectors developed in
the lymph nodes of both neonates and adults. The frequencies of both
types of effectors were 2- to 3-fold higher in neonatal than in adult
lymph nodes. As we had previously seen, while the neonatal spleen
produced mature levels of Th2 effectors, few Th1 effectors were
detected. Thus, while adult animals developed mixed Th1/Th2 responses
in both the lymph nodes and spleens, the responses of neonates were
highly dependent on the microenvironment; in the lymph nodes mature
Th1/Th2 responses developed, while in the spleen exclusive Th2 effector
responses were observed.
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- and
IL-4-secreting cells were measured by ELISPOT (Fig. 3). Adults of the C57BL/6 strain, like
BALB/c mice, developed both Th1 and Th2 primary splenic effectors. Also
similar to BALB/c mice, C57BL/6 neonates developed exclusively Th2
primary effectors. In fact, the development was even more striking,
because, while roughly equivalent numbers of Th2 effectors developed in
BALB/c neonates and adults, up to 5-fold more Th2 effectors were
present in neonatal compared with adult C57BL/6 spleens. Therefore, the
exclusive development of primary Th2 effectors in the neonatal spleen
is not confined to one strain of mouse and is evident even in mice in
which the Th1 lineage is often favored.
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The cellular composition of the neonatal spleen is quite different
from that of the adult spleen. For example, T cells are undetectable by
flow cytometric methods at birth and represent <5% of the total cells
at 1 wk of life (20) (B. Adkins, unpublished observation).
This contrasts with adult spleens, which, in BALB/c mice, contain
30–40% T cells. The B cell compartment in neonatal spleens is also
different; unlike in adult spleens, the neonatal spleen is enriched in
nonconventional CD5 B cells (21). Lastly, the neonatal
spleen contains relatively high proportions of mast cells (22, 23). Because of these differences, it was very important to
establish the specificity of IL-4 production by neonatal splenocytes in
this assay system. This was investigated in several ways. First, as
presented in Figs. 1
and 2, the frequencies of IL-4-producing cells in
the spleens of unimmunized, age-matched neonates was at least 5-fold
lower than the specific frequency observed in immunized neonates. Thus,
the detection of IL-4 in this assay is unlikely to be due to
spontaneous IL-4 production by nonprimed T cells (or non-T cells).
Second, when an isotype-matched Ab control for the
anti-IL-4-detecting mAb was used, no spots developed (data not
shown), indicating that the Ab system used was indeed specific for
IL-4. Third, BALB/c neonates and adults were immunized with KLH, and
their spleen cells were restimulated in culture with either KLH or OVA
(Table I). The frequency of IL-4-secreting cells was 4- to 10-fold
higher when restimulation occurred with KLH vs OVA. This provides
evidence that the production of IL-4 is Ag specific, as would be
expected if primed T cells were generating the response. Lastly,
CD4+ or CD8+ cells were
eliminated from the cell suspensions using complement-mediated lysis
(Fig. 4). Anti-CD4 mAb plus complement
treatment resulted in a severe depletion of IL-4-producing cells from
adult spleens. The same treatment did not completely remove
IL-4-secreting cells from the neonatal spleen, but did reduce the
frequency 10-fold. Together, these results indicate that 1) we are
specifically detecting IL-4 in this system; and 2) the IL-4 production
is mediated by Ag-specific CD4+ cells.
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Poor Th1 development in the neonatal spleen could arise in several
ways. One possibility is that the APC population in the neonatal spleen
might be unable to efficiently promote Th1 function. Results from in
vitro studies indicate that neonatal splenic APC are poor in promoting
functions mediated by Th1 cells (24, 25, 26, 27, 28). In the
experiments reported above, PBS was used as the vehicle to deliver the
Ag. In the absence of adjuvant, the capacity of neonatal APC to
costimulate Th1 responses may have been minimal. To achieve optimal APC
function in vivo, we next tested the capacity of neonatal spleens to
develop Th1 effectors when the Ag was introduced in adjuvants. Neonatal
and adult BALB/c mice were immunized with KLH in PBS, in CFA, or in
ALUM precipitates, and the frequencies of IFN-- and IL-4-producing
cells were assessed by ELISPOT 1 wk later (Fig. 5). The neonatal spleen appeared to be
able to respond to the effects of adjuvant; modest, but significant
(p < 0.001), increases in IL-4-producing cells
were observed in response to CFA. However, neither CFA nor ALUM
immunization increased the frequency of IFN--producing cells in the
neonatal spleen. Thus, Th1 development and/or function is not promoted
in the neonatal spleen, even in the presence of strong adjuvants.
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production in vitro by spleen cells from KLH-immunized
neonates. The pattern of cytokine-producing cells observed under these
conditions was indistinguishable from that seen with total neonatal
splenocytes, i.e., very low frequencies of IFN--secreting and mature
frequencies of IL-4-secreting cells (data not shown). One possible
interpretation of this result is that neonatal T cells cannot respond
to the Th1-promoting activity of adult APC. However, naive splenic APC
are relatively poor promoters of Th1 function. Therefore, we decided to
use IL-12, an APC-derived cytokine with powerful Th1-promoting activity
(reviewed in Ref. 29). Neonatal and adult BALB/c mice were
immunized with KLH in PBS; 7 days later, splenocytes were cultured with
or without KLH and with or without rIL-12 for ELISPOT assays (Fig. 6). As previously observed, neonatal
cultures stimulated with KLH alone contained few IFN--secreting
cells. The presence of IL-12 alone resulted in a small increase in this
number. In striking contrast, in those cultures containing both KLH and
rIL-12, the frequency of IFN--secreting cells among neonatal
splenocytes was increased to mature, adult levels. In these experiments
the culture period was brief (48 h). Nonetheless, it was possible
that the IFN--secreting cells developed de novo from unprimed cells
within this time period. However, this seems unlikely, because the same
cells generated similar low frequencies of IFN--secreting cells when
cultured with IL-12 plus an irrelevant Ag (OVA) or with IL-12 alone
(data not shown). Therefore, this result suggests that Ag-specific Th1
effectors did develop within the neonatal spleen in situ. However, they
may have been unable to execute effector function in vitro due to
limiting levels of IL-12.
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). The frequencies of IL-4-secreting
cells were similar among neonatal or adult splenocytes alone and in the
mixtures. As we previously observed, Th1 effectors were virtually
absent from the neonatal spleen, but were present at levels of
>1000/106 cells in the adult spleen. In the
mixtures, the frequency of IFN--producing cells was significantly
(p < 0.001) lower than the predicted average
of values in neonatal and adult cells. Values significantly lower than
the predicted average were also observed when primed adult spleen cells
were mixed with spleen cells from naive neonatal animals (data not
shown). Thus, these experiments are consistent with the idea that
neonatal spleens do not efficiently promote, and may inhibit, the in
vitro development of Th1 effector function by primed T cells.
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). The presence of anti-IL-4 mAb,
even in quantities 5-fold greater than that required for
neutralization in bioassays (5), had no effect on
IFN--secreting cells. Anti-IL-10 treatment did result in a modest
increase in the frequency of IFN--producing cells in mixes of
neonatal and adult splenocytes and among adult splenocytes alone.
However, there was no increase in the frequency of IFN--secreting
cells in the cultures of neonatal splenocytes alone. Thus, IL-4 or
IL-10 alone does not appear to account for the depression of Th1
function in the neonatal spleen.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting cells were essentially
undetectable. This severe Th2-skewing was observed in both Th2-prone
BALB/c mice and Th1-biased C57BL/6 mice. Although the neonatal spleen
contains few T cells, the IL-4 production detected in the ELISPOT
appeared to be mounted by T cells, because 1) the response was Ag
specific; and 2) elimination of CD4+ cells also
eliminated IL-4-producing cells. Cell-mixing experiments and
experiments with supplemental additions indicate that poor Th1
development and/or function in the neonatal spleen may arise from a
combination of insufficient Th1-promoting agents and inhibition of the
Th1 lineage.
A number of in vitro studies (24, 25, 26, 27, 28) have indicated that
neonatal spleens have poor APC function. Although Th1 cytokine
production was not measured directly, the in vitro assays employed
generally relied on Th1 function. Therefore, it is perhaps not
surprising that the neonatal spleen poorly promoted Th1 effector
function in vivo. Our initial interpretation of these data was that
even in the presence of adjuvants, neonatal APC function is
insufficient to promote Th1 lineage development. Probably the single
most important cytokine in directing Th1 development is IL-12 (reviewed
in Ref. 29). IL-12 also acts to enhance IFN- production
by committed Th1 effector cells (29). To our surprise, the
addition of IL-12 boosted the frequency of IFN--producing neonatal
spleen cells >1000-fold, to the level found in mature adult spleens.
This indicates that Th1 lineage cells do indeed develop in neonatal
spleens in situ, but they are apparently unable to become effectors in
culture unless exogenous Th1 promotion, in this case excess IL-12, is
provided.
Mixing experiments provided a potential explanation for the inability
of neonatal Th1 cells to perform an effector function. When neonatal
and adult splenocytes were cocultured, the frequency of
IFN--secreting cells was decreased to a level significantly below
the theoretical value predicted from the average of the two individual
values. There are at least two possible interpretations of these
studies. First, in 1/1 coculture mixes, both adult T cells and APC are
decreased 2-fold. This may result in the dilution of mature APC
activity below a threshold required for optimal Th1 effector function.
Second, neonatal splenocytes may inhibit the development of Th1
effector activity by primed adult T cells. We favor the latter
possibility, since there is an extensive literature indicating that
neonatal splenocytes suppress Th-mediated functions by adult T cells
(26, 27, 28, 34). Two well-characterized inhibitors of the Th1
lineage are IL-4 and IL-10. Both these cytokines inhibit IFN-
production by committed Th1 cells, presumably by down-regulating APC
IL-12 production (30, 31, 32, 33). However, experiments using
neutralizing Abs showed that neither IL-4 nor IL-10 alone is
responsible for the putative inhibition of Th1 function. This is
perhaps not surprising, because the mechanism of neonatal splenic
suppression appears to be complicated, involving several distinct
soluble factors and several cell types, including monocytes, mast
cells, and T-lineage cells (26, 27, 28, 34). The identities of
the signals and cell types operating in our system to suppress Th1
function are currently under investigation in the laboratory.
In a number of experimental settings, memory responses in mice
initially immunized as neonates are Th2 skewed (reviewed in Ref.
35). This phenomenon occurs in both spleen and lymph nodes
(14). The observation that the primary splenic responses
of 1-day-old neonates are exclusively Th2 provides an explanation for
Th2-dominant memory in the spleens, i.e., the vast majority of cells
responding in the first exposure to Ag are Th2, and thus the majority
of cells converting to memory cells will probably be Th2. However,
another explanation is required for the lymph nodes, because neonatal
lymph node T cells mount mature mixed Th1/Th2 primary responses (Ref.
14 and this manuscript). How the transition from mixed
Th1/Th2 to Th2-dominant memory in lymph nodes occurs is unknown, but
the possibilities under consideration include 1) that unique
recirculation patterns of splenic memory Th2 cells in neonates lead to
Th2 dominant memory in the lymph nodes; and 2) that neonatal and adult
Th1 or Th2 lymph node cells have different stabilities in
vivo.
It is generally agreed that the mouse is immunologically less mature than humans at birth, and thus the newborn mouse may be more accurately compared with human fetuses. In human newborns Ag-specific Th responses that apparently developed in utero have been described (reviewed in Ref. 35). In some cases Th1-like responses prevail, and in other cases primarily Th2 activity is observed. These reports have demonstrated that human fetuses, like newborn mice, are competent to develop mature Th1 function in situ. However, they do not reveal why Th1 responses developed in some cases and Th2 responses in others. Based on our work in the mouse, it is tempting to speculate that regional differences also exist in humans in early ontogeny. Perhaps Th1 responses arise when exposure occurs in the fetal lymph nodes, but Th2 responses arise in the spleen. If this phenomenon also occurs in humans, this would suggest that there are important evolutionary implications for the regional control of Th responses in early development.
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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 33101. E-mail address:
3 Abbreviations used in this paper: KLH, keyhole limpet hemocyanin; ALUM, aluminum potassium sulfate; ELISPOT, enzyme-linked immunospot.
Received for publication August 17, 1999. Accepted for publication December 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by murine peripheral T lymphocytes. J. Immunol. 151:6117.
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B. Adkins, Y. Bu, and P. Guevara The Generation of Th Memory in Neonates Versus Adults: Prolonged Primary Th2 Effector Function and Impaired Development of Th1 Memory Effector Function in Murine Neonates J. Immunol., January 15, 2001; 166(2): 918 - 925. [Abstract] [Full Text] [PDF]
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