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Murine Neonatal Lymphocytes Show Rapid
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J Immunol 2003; 170:4548-4556; ;
doi: 10.4049/jimmunol.170.9.4548
http://www.jimmunol.org/content/170/9/4548
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References
Becky Adkins, Tonya Williamson, Patricia Guevara and
Yurong Bu
The Journal of Immunology
Murine Neonatal Lymphocytes Show Rapid Early Cell Cycle
Entry and Cell Division1
Becky Adkins,2 Tonya Williamson, Patricia Guevara, and Yurong Bu
mmune responses during neonatal life in the mouse are typically weak in magnitude and poorly protective. This period
of life is characterized by first exposures to a vast array of
potentially pathogenic microorganisms and environmental Ags.
Because strong immunological reactions can have deleterious effects, mature responses to many of these Ags simultaneously
would probably be detrimental to neonates. Overly strong responses in newborns appear to be prevented by several mechanisms. First, the relatively low number of immune cells present in
neonates almost certainly contributes to dampened responses when
neonates receive the same doses of agents as do adults (1–3). Second, the cells participating in an immune response may be developmentally immature. One striking example of immature function
is seen in the APC compartment. Numerous reports have demonstrated that neonatal APC are deficient in both number and function (4 –7). Third, neonatal immune responses may be deviant.
This phenomenon is well characterized for murine neonatal T cell
responses. In particular, exposure to Ag during the neonatal period
often results in Th2-dominant responses in later life (reviewed in
Refs. 8 –10). Because Th2 responses are anti-inflammatory in nature, this deviance undoubtedly contributes to the relatively poor
capacity of neonates to mount protective cellular immune responses. Last, unlike in adult animals, T cells in the peripheral
I
organs of neonates are almost exclusively naive. The absence of
memory cells in the neonate would be expected to result in responses that are both slower to develop and lower in magnitude.
Although it may be important to limit immune responses in
early life, it is almost certainly equally important to allow sufficient
responsiveness to achieve protection from disease and death.
Given the restrictions to responsiveness described in the paragraph
above, how can this be achieved? In this study, we describe a
phenomenon that may partially compensate for the limitations in
immune cell function in neonates. Following CD3/TCR-dependent
or -independent activation, greater proportions of neonatal T cells
entered the cell cycle more rapidly than did adult T cells. This
rapid cell cycle entry occurred both in vitro and in vivo, during
homeostatic proliferation in lymphopenic hosts. Experiments in
which exogenous cytokines or anti-cytokine mAb were added indicated that the differences between neonatal and adult early cycling were not dependent on differences in early cytokine production. In addition to T cells, greater proportions of neonatal,
compared with adult, B cells also showed more rapid cycling following lineage-specific activation. Thus, populations of lymphocytes present in early life have the capacity to enter the cell cycle
more quickly and hence may be able to mobilize a more rapid
response from an otherwise completely naive population.
Department of Microbiology and Immunology, University of Miami Medical School,
Miami, FL 33136
Materials and Methods
Received for publication September 10, 2002. Accepted for publication March
3, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant RO1 AI44923-02.
2
Address correspondence and reprint requests to Dr. Becky Adkins, Department of
Microbiology and Immunology, R-138, 1600 NW 10th Avenue, Rosenstiel Medical
Science Building Room 3152A, University of Miami Medical School, Miami, FL
33136. E-mail address: [email protected]
Copyright © 2003 by The American Association of Immunologists, Inc.
Mice
BALB/c mice, originally obtained from Charles River Laboratories (Wilmington, MA), OVA-specific TCR transgenic DO11.10 mice (The Jackson
Laboratory, Bar Harbor, ME), and C57BL/6 mice (The Jackson Laboratory) 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.
0022-1767/03/$02.00
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Neonatal animals are highly susceptible to infectious agents. At least part of this susceptibility is due to the virtual absence of
immunological memory in newborns. One of the hallmarks of memory is the rapidity of the response. We show in this study that
neonates may make up for their lack of memory, at least in part, by the rapid entry of large proportions of naive lymphocytes into
the cell cycle. Following activation, greater percentages of both CD4ⴙ and CD8ⴙ neonatal, as compared with adult, lymph node
cells showed early cell cycle entry; this was assessed by propidium iodide staining, CFSE labeling profiles, [3H]thymidine uptake,
and up-regulation of early activation markers. This rapid cycle entry was observed following polyclonal activation with anti-CD3
or with PMA and ionomycin and in both C57BL/6 and BALB/c mice. Stimulation with specific peptide also elicited more rapid
proliferative responses from neonatal vs adult TCR transgenic CD4ⴙ cells. In addition, more rapid cycle entry was observed in
vivo, in lymphopenic RAG2ⴚ/ⴚ hosts. For both CD4ⴙ and CD8ⴙ cells, this phenomenon was observed out to 3 wk of life, although
the differences between neonatal and adult cells became smaller with increasing time postbirth. These properties of peripheral
neonatal T cells appeared to be inherited from their thymic precursors, because CD4ⴙ8ⴚ single-positive cells in the neonatal
thymus also showed more rapid cycle entry, compared with their counterparts in the adult thymus. Interestingly, rapid early
cycling was also observed among activated neonatal B cells, compared with adult B cells. Thus, early cell cycle entry by large
proportions of cells may allow the naive lymphocyte population to efficiently mobilize responses against the broad range of
pathogens first encountered in neonatal life. The Journal of Immunology, 2003, 170: 4548 – 4556.
The Journal of Immunology
RAG2⫺/⫺ mice were purchased from Taconic (Germantown, NY) and
similarly bred and housed under barrier conditions.
Cell preparation
Lymph node preparations. Pools of tissues from ⱖ10 neonatal (day 7) or
ⱖ2 adult (6–8 wk old) mice were used for the cell preparations. Mesenteric, inguinal, axillary, brachial, and cervical lymph nodes were collected
to prepare total lymph node cell suspensions (11).
Enriched CD4⫹8⫺ thymocyte preparations. Thymus cell suspensions
were incubated with anti-CD8 microbeads (Miltenyi Biotec, Auburn, CA),
and CD8⫹ cells were depleted on negative-selection columns, following
the manufacturer’s suggested protocol. The resultant population contained
⬍1% CD8⫹ cells and ⬃50% CD4⫹8⫺ and 50% CD4⫺8⫺ cells.
CFSE labeling
CFSE was purchased from Molecular Probes (Eugene, OR). Total lymph
node cells were labeled with CFSE according to the manufacturer’s instructions, with the concentrations of cells and CFSE in the labeling reaction adjusted to 5.0 ⫻ 106/ml and 0.5 ␮M, respectively.
Cell culture conditions
cells were suspended in HBSS containing 50 ␮g/ml PI (Sigma-Aldrich)
and 50 ␮g/ml RNase A (Boehringer Mannheim, Indianapolis, IN) and incubated for 1 h at room temperature. Debris and doublets were eliminated
from the analyses using pulse width/area discrimination.
Staining with Hoechst and pyronin Y
Cells (1 ⫻ 106) were stained with fluorescein-conjugated anti-CD4 mAb
and then fixed and permeabilized in 200 ␮l of Cytofix/Cytoperm (BD
PharMingen) for 45 min at 4°C. Cells were washed once with Perm/Wash
(BD PharMingen) and resuspended in 1.0 ml 5 ␮g/ml Hoechst 33342 (Molecular Probes) in PBS containing 0.1% glucose. The cells were incubated
at 37°C for 45 min, and pyronin Y (in PBS, 0.1% glucose) was added to
a final concentration of 5 ␮g/ml. After an additional 45 min at 37°C, samples were analyzed on a BD Biosciences (San Jose, CA) LSR equipped
with a 488 nm laser line and an HeCd 325 nm UV laser.
Results
Neonatal CD4⫹ and CD8⫹ cell populations divide more rapidly
than adult cells in response to CD3/TCR-dependent or
-independent stimulation
We previously observed that freshly isolated neonatal T cells, unlike adult T cells, produced large quantities of IL-4 relatively early
(within 48 h) following activation with anti-CD3 mAb (13). For
adult T cells, IL-4 production generally begins after ⱖ3 days and
a number of rounds of proliferation (17–19). Therefore, we wondered whether the early appearance of IL-4 in neonatal cultures
Cell activators
Peripheral T cell activation. 145-2C11 mAb, anti-murine CD3-⑀ (12),
ascites fluid was used at a 1/2000 dilution, unless indicated otherwise. In
some cases, cultures were supplemented with the indicated concentrations
of rIL-2 or rIL-4 (R&D Systems, Minneapolis, MN) or with neutralizing
anti-IL-4 mAb (11B11; BD PharMingen, San Diego, CA). PMA (SigmaAldrich, St. Louis, MO) was used at 12 nM in combination with ionomycin
(Calbiochem, La Jolla, CA) at 0.15 ␮M for adult cells or 0.30 ␮M for
neonatal cells (13). OVA peptide 323–339 (14) was synthesized by the
Protein Analysis Core Facility (Department of Biochemistry and Molecular
Biology, University of Miami Medical School) and used at a final concentration of 0.1 ␮g/ml, unless otherwise indicated.
Thymocyte activation. Enriched CD4⫹8⫺ thymocytes were cultured in
96-well culture dishes precoated with 10 ␮g/well of purified 145-2C11
mAb (11), in the absence or presence of anti-CD28 mAb (BD
PharMingen).
B cell activation. LPS (Sigma-Aldrich) was added to a final concentration
of 50 ␮g/ml. Anti-CD40 mAb was a generous gift of Dr. S. MarshallClarke (University of Liverpool, Liverpool, U.K.) and was used at 5 ␮g/ml
together with 50–200 U/ml murine rIL-4 (Genzyme, Cambridge, MA).
[3H]Thymidine incorporation
Triplicate cultures of lymph node cells were activated with different concentrations of soluble anti-CD3 mAb (145-2C11), as described in Cell
activators. At 3, 24, or 48 h of culture, 1 ␮Ci of [3H]thymidine (Amersham
Pharmacia Biotech, Piscataway, NJ) was added per 96-well culture. Cells
were incubated at 37°C for an additional 20 h, and the cultures were harvested using a PHD harvester (Cambridge Technology, Watertown, MA).
Ab staining
For Ab staining, anti-CD4, anti-CD8, anti-B220, anti-CD25, and antiCD69 were purchased from BD PharMingen and used as previously described (15).
Propidium iodide (PI)3 staining
Cells were first stained with fluorescein-conjugated anti-CD4, anti-CD8, or
anti-B220 mAb and then processed for PI staining as described previously
in detail (16). Briefly, cells were suspended in HBSS containing 50% FCS,
fixed by the dropwise addition of ice-cold 70% ethanol, to a final concentration of 50%, and held on ice for at least 1 h. After extensive washing, the
3
Abbreviations used in this paper: PI, propidium iodide; PLC, phospholipase C.
FIGURE 1. Both neonatal CD4⫹ and CD8⫹ cell populations show more
rapid division than their adult counterparts in response to anti-CD3 stimulation. Total lymph node cells from 7-day-old or adult BALB/c mice were
labeled with CFSE and cultured in the absence (unactivated) or presence
(activated) of anti-CD3 mAb. Twenty-four or 42 h later, the cells were
harvested and stained with anti-CD4 or anti-CD8 mAb. Shown are the
CFSE profiles of gated CD4⫹ cells or CD8⫹ cells. Unactivated cells from
the 24 h time point only are shown, although the profiles were very similar
for unactivated cells at 42 h. One experiment typical of five independent
analyses for CD4⫹ cells and three individual experiments for CD8⫹ cells
is shown.
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Total lymph node cells (5 ⫻ 105) were cultured in 200 ␮l of medium in
96-well culture dishes in the presence or absence of various activators.
Culture medium consisted of RPMI 1640 (Life Technologies, Grand Island, NY) containing 1 mM sodium pyruvate (Life Technologies), 2 mM
⫺2
L-glutamine (Life Technologies), 5 ⫻ 10
mM 2-ME (Life Technologies), 1% penicillin-streptomycin (Life Technologies), and 10% heat-inactivated (56°C; 30 min) FCS (HyClone, Logan, UT). For cytokine assays,
culture supernatants were harvested at 24 h, and ␥-IFN and IL-4 content
were assessed using mouse-specific cytokine ELISA kits (Pierce Endogen,
Rockford, IL).
4549
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FIGURE 2. More rapid division by neonatal CD4⫹ or CD8⫹ cells is
seen with TCR-independent stimulation, in different strains of mice, in
response to specific peptide, and in vivo in lymphopenic hosts. Left panel,
Total lymph node cells from day 7 or adult BALB/c mice were labeled with
CFSE and stimulated with PMA and ionomycin for 42 h. The CFSE profiles of gated CD4⫹ cells are shown. Second panel from left, Total lymph
node cells from day 7 or adult C57BL/6 mice were labeled with CFSE and
stimulated with anti-CD3 mAb for 44 h. The CFSE profiles of gated CD4⫹
cells are presented. Third panel from left, Total lymph node cells from day
7 or adult TCR transgenic DO11.10 mice were CFSE labeled and stimulated with 0.1 ␮g/ml OVA peptide for 41 h. The CFSE staining of gated
CD4⫹clonotype⫹ cells is shown. Right panel, Total lymph node cells from
day 7 or adult BALB/c mice were CFSE labeled, and 5 ⫻ 106 cells were
injected i.v. into RAG2⫺/⫺ hosts. Forty-eight hours later, spleen and lymph
node cells were pooled and stained with anti-CD8 and anti-TCR␣␤ mAb.
The CFSE profiles of gated CD8⫹TCR␣␤⫹ cells are shown. Each experiment was performed at least twice.
showed more rapid division in vivo, upon transfer to lymphopenic
RAG2⫺/⫺ hosts (Fig. 2, right column). Together, these results indicate that faster kinetics of proliferation is a universal phenomenon among neonatal T cell populations—it occurs both in vivo and in
vitro, it occurs in several strains of mice, and it occurs whether stimulation is through the CD3/TCR complex or is TCR independent.
Faster kinetics of division are due to early cell cycle entry by
greater proportions of neonatal T cells
In the CFSE experiments described in the previous section, division peaks were not detectable among either neonatal or adult cultures until ⱖ40 h. The cell population present at this time is the
cumulative product of the ongoing processes of cell survival, cell
proliferation, and cell death. For example, there may be relatively
more death among undivided cells within the neonatal population.
If so, the proportion of cells in the divided CFSE peaks would be
increased proportionally, even if the amount of division was similar in the neonatal and adult cultures. Thus, to minimize the effects
of longer term culturing, neonatal and adult cells were analyzed
earlier, using PI staining. This method allowed us to monitor two
things: 1) the percentage of cells in the different phases of the cell
cycle and 2) the presence of apoptotic or sub-G0 cells. Neonatal or
adult BALB/c lymph node cells were activated with soluble antiCD3, and 21 h later, the cells were stained with anti-CD4 mAb,
fixed and permeabilized, and stained with PI. Among freshly isolated cells, a small but consistently greater (⬃2-fold) percentage of
neonatal CD4⫹ cells were in the S plus G2/M stages of the cell
cycle, compared with adult cells (Fig. 3, left panel). At 21 h of
activation, nearly 50% of neonatal CD4⫹ cells had entered the cell
cycle, compared with ⬃20% of adult CD4⫹ cells (Fig. 3, right
panel). This cell cycle entry was clearly dependent on activation,
because similarly cultured but unactivated cells showed low levels
of cycling (Fig. 3, middle panel). These experiments also demonstrated similar modest proportions (⬍10%) of apoptotic (sub-G0)
cells among the activated neonatal or adult CD4⫹ populations at
this early culture time, and although the proportions increased
FIGURE 3. Earlier division of neonatal CD4⫹ cells is preceded by more
rapid cell cycle entry. Total lymph node cells from day 7 or adult BALB/c
mice were cultured for 21 h in the presence (activated) or absence (unactivated) of anti-CD3. The cells were harvested and stained with anti-CD4
mAb and PI. Freshly isolated lymph node cells (fresh) from day 7 or adult
animals were stained in parallel. The PI stainings of gated CD4⫹ cells are
shown. The percentages of cells in the S plus G/M stages of the cell cycle
are indicated. One experiment typical of five separate analyses is shown.
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was due to accelerated proliferation by neonatal T cells. To test
this idea, total lymph node cells from 7-day-old neonatal or adult
BALB/c mice were CFSE labeled and stimulated with soluble antiCD3 mAb. Parallel cultures were prepared in the presence of medium only (unactivated). Twenty-four and 42 h after the initiation
of culture, the cells were stained with anti-CD4 or anti-CD8 mAb
and analyzed on the flow cytometer (Fig. 1). At 24 h of activation,
there was no obvious division by either neonatal or adult cells. By
42 h of activation, both cell types showed clear division peaks. As
has been recently reported for Ag-driven stimulation (20), adult
CD4⫹ cells showed more limited proliferation than adult CD8⫹
cells. This relative phenomenon was also seen for neonatal CD4⫹
and CD8⫹ cells. However, among both the CD4⫹ and CD8⫹ populations, neonatal cells had clearly undergone more extensive division than the corresponding adult cells. This more rapid division
did not require neonatal APC, because similar results were obtained using purified neonatal or adult CD4⫹ cells and adult APC
(data not shown).
We next sought to determine more about the conditions that
elicit rapid division by neonatal T cells. We first found that CD4⫹
cells from the same strain of mice (BALB/c) also proliferated more
quickly than adult CD4⫹ cells when stimulation bypassed the
TCR, using PMA plus ionomycin (Fig. 2, left column). The phenomenon of more rapid division was not limited to the BALB/c
strain of mice because CD4⫹ cells from C57BL/6 neonates also
demonstrated more rapid proliferation in response to anti-CD3
stimulation (Fig. 2, second column from the left). Third, direct
stimulation through the Ag receptor using OVA peptide elicited
more rapid proliferation by neonatal DO11.10 TCR transgenic
CD4⫹ cells (Fig. 2, second column from the right). Last, the phenomenon was not limited to in vitro activation, because CD4⫹
cells (data not shown) and CD8⫹ cells from BALB/c neonates also
RAPID CELL CYCLE ENTRY BY NEONATAL LYMPHOCYTES
The Journal of Immunology
FIGURE 4. Slower cycle entry by adult CD4⫹ cells is not due to limiting Ag concentration. Total lymph node cells from day 7 or adult
DO11.10 TCR transgenic mice were stimulated with different concentrations of OVA peptide. Twenty-four hours later, the cells were harvested
and stained with anti-CD4 mAb and PI. The PI profiles of gated CD4⫹ cells
are shown. The percentages of cells in the sub-G0/apoptotic region and in
the S plus G2/M cell cycle stages are indicated.
FIGURE 5. Neonatal T cells show higher levels of DNA synthesis early
but, by 48 h of culture, show lower levels than do adult T cells. Total lymph
node cells from BALB/c mice were activated with the indicated concentrations of anti-CD3 mAb. Following 3, 24, or 48 h of culture, [3H]thymidine was added, and the cultures were incubated for an additional 20 h.
One experiment typical of two independent setups is shown.
results were obtained by adding [3H]TdR 48 h after activation and
allowing incorporation to occur over the next 18 –20 h. In this
study, we have reported events occurring within the initial 48 h of
activation. Therefore, the simplest reconciliation of the two results
may be the differences in times of analysis. To test this idea, neonatal and adult lymph node cells were activated with different concentrations of soluble anti-CD3 mAb, and [3H]thymidine was
added 3, 24, and 48 h following culture initiation. The cells were
then harvested after an additional 20 h of culture (Fig. 5). As predicted from the experiments with PI, neonatal T cells incorporated
substantially more (ⱕ5-fold) [3H]thymidine between 3 and 23 h of
culture. Between 24 and 44 h of culture, [3H]thymidine uptake by
neonatal T cells was approximately twice that of adult T cells.
However, by 48 h of culture, adult T cells showed greater [3H]thymidine incorporation. Strikingly, while the radioactivity incorporated by adult T cells was still ⱖ100-fold higher than the levels
seen in the absence of activation, activated neonatal T cells incorporated ⱕ2-fold as much [3H]thymidine as the corresponding unactivated cultures. Together, these data indicate that the faster cycle entry of neonatal T cell populations is followed by their earlier
cessation of proliferation.
Stimulation of T cells is accompanied by the up-regulation of
activation markers. As an independent measure of entry into the
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later, neonatal and adult CD4⫹ cells also showed similar percentages of apoptotic cells at 48 h (data not shown) (16). Similar results were obtained when the CD8⫹ populations were compared,
with 47– 68% of activated neonatal CD8⫹ cells in cycle compared
with 12–16% of adult CD8⫹ cells in S plus G2/M at 21 h of culture
(data not shown). These experiments argue strongly that a greater
proportion of activated neonatal, compared with adult, T cells enter the cell cycle early.
In the experiments described thus far, neonatal and adult T cells
were activated with concentrations of reagents previously determined to be optimal for cytokine secretion or for later proliferation
assessed by [3H]TdR incorporation (13). Nonetheless, it seemed
possible that these conditions were not optimal for the early cycle
entry of adult T cells, thus leading to the apparent discrepancies in
kinetics between neonatal and adult cells. To investigate this issue,
neonatal and adult total lymph node cells from DO11.10 TCR
transgenic mice were stimulated with a 1000-fold range of specific
peptide concentration. Twenty-four hours later, the cells were
stained with anti-CD4 mAb, fixed and permeabilized, and stained
with PI (Fig. 4). In the absence of stimulation (no peptide), both
neonatal and adult CD4⫹ cells showed substantial proportions of
cells in the sub-G0/apoptotic regions and minimal proportions of
cells in S plus G2/M. This contrasts with BALB/c mice in which
⬍10% of the cells are apoptotic in the absence of stimulation (data
not shown) and may be a reflection of the relative heterogeneity of
the TCR repertoire in normal and transgenic mice. With increasing
peptide concentration, the percentages of apoptotic cells declined
while the proportions in S plus G2/M increased, for both neonatal
and adult cells. For all concentrations of peptide, a greater proportion of neonatal than adult T cells were in the S plus G2/M stages
of the cell cycle. Neonatal CD4⫹ cells showed relatively large
increases in the percentages of cells in S plus G2/M out to 50
␮g/ml peptide. However, there was minimal further increase for
adult cells between 5 and 50 ␮g/ml, indicated that optimal activation conditions for adult cells were obtained by a concentration of
5 ␮g/ml peptide. Thus, under conditions clearly optimal for the
early proliferation of adult T cells, greater proportions of neonatal
vs adult CD4⫹ cells showed early cell cycle entry.
Earlier studies from our laboratory (13) found that activated
murine neonatal T cells are highly impaired, compared with adult
T cells, in [3H]TdR incorporation. How can we reconcile these
earlier findings to the data presented in this study showing faster
early cell cycle kinetics among neonatal T cells? The previous
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RAPID CELL CYCLE ENTRY BY NEONATAL LYMPHOCYTES
CD4⫹ and CD8⫹ cells also showed rapid up-regulation of CD25
expression (Fig. 6, A and B, bottom panels). The rapid up-regulation of these markers was also observed following anti-CD3 stimulation of TCR transgenic DO11.10 neonatal vs adult CD4⫹ cells
(data not shown). These data, together with those obtained with the
PI experiments, clearly demonstrate that proportionally more neonatal T cells enter the cell cycle rapidly, compared with adult cells.
Rapid cell cycle entry also occurs among neonatal CD4⫹8⫺
thymocytes and begins to disappear among peripheral T cells by
3 wk of life
cell cycle, activated neonatal and adult CD4⫹ or CD8⫹ cells were
examined for the expression of the activation markers CD69 and
CD25. Lymph node cells from BALB/c neonatal or adult mice
were activated with soluble anti-CD3, and at various time intervals, the cells were stained with anti-CD4 or anti-CD8 and antiCD69 or anti-CD25 mAb. Within 2 h of activation, ⬃70% of neonatal CD4⫹ cells showed CD69 expression, as opposed to ⬃50%
of adult CD4⫹ cells (Fig. 6A, top panel). CD8⫹ cells showed similar relative patterns of CD69 expression (Fig. 6B, top panel). Although somewhat more delayed, greater proportions of neonatal
Table I. More rapid cell cycle entry also occurs among neonatal CD4⫹8⫺ thymocytes
Cellsa
Anti-CD3
Anti-CD28
% Apoptoticb
% G0/G1b
% Sb
% G2/Mb
% S ⫹ G2/Mb
Day 1
⫺
⫹
⫹
⫺
⫹
⫹
⫺
⫹
⫹
⫺
⫺
⫹
⫺
⫺
⫹
⫺
⫺
⫹
25.0
23.0
16.5
34.4
21.2
15.4
16.8
20.0
11.2
68.5
54.1
49.5
60.0
57.5
55.4
81.1
70.9
77.7
3.1
15.4
23.6
2.4
12.8
19.2
1.1
6.3
7.8
3.5
7.7
10.8
3.3
8.7
10.3
1.1
2.9
3.5
6.6
23.1
34.4
5.7
21.5
29.5
2.2
9.2
11.3
Day 3
Adult
a
Enriched CD4⫹8⫺ thymocytes from day 1, day 3, or adult BALB/c mice were cultured in medium alone (⫺/⫺) or activated
with plate-bound anti-CD3 with or without soluble anti-CD28. Twenty-four hours later, cells were stained with anti-CD4 mAb
and PI.
b
Percentage among gated CD4⫹ cells.
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FIGURE 6. More rapid up-regulation of early activation markers by
neonatal, compared with adult, CD4⫹ and CD8⫹ cells. Total lymph node
cells from BALB/c mice were activated with anti-CD3 mAb. Following 20
min, 2 h, 6 h, and 24 h of culture, cells were harvested and stained with
anti-CD4 or anti-CD8 mAb in combination with anti-CD69 or anti-CD25
mAb. The percentages of gated CD4⫹ (A) or CD8⫹ (B) cells staining with
the anti-CD69 or anti-CD25 mAb are indicated on the y-axis. This experiment was repeated once more for both CD4⫹ and CD8⫹ cells in BALB/c
mice and for CD4⫹ cells in DO11.10 mice with comparable results.
Newborn mice contain greatly reduced (50- to 100-fold) numbers
of peripheral lymphocytes (2). As a result, neonates can be considered lymphopenic relative to adults. T cells isolated from the
secondary lymphoid organs of neonates may be primed by peripheral homeostatic signals to undergo rapid cycle entry. Alternatively, the rapid cycling of peripheral T cells in the neonate may be
due to their derivation from the neonatal vs the adult thymus. To
distinguish between these possibilities, we compared cycling
among neonatal vs adult thymocytes. To exclude the major population of immature thymocytes (CD4⫹8⫹ double-positive cells)
from the analyses, thymocytes from day 1, day 3, and adult animals were enriched for CD4⫹8⫺ cells (see Materials and Methods). The enriched populations were activated for 24 h with plastic-immobilized anti-CD3 mAb with or without anti-CD28 mAb;
control unactivated cultures were plated in medium only in nonmAb-coated wells. The cells were then stained with anti-CD4
mAb, fixed, permeabilized, and stained with PI (Table I). The proportions of apoptotic CD4⫹8⫺ cells was higher, especially among
unactivated cells, than generally observed for peripheral CD4⫹8⫺
cells (see, for example, Fig. 3). However, in common with peripheral T cells, neonatal CD4⫹8⫺ thymocytes showed somewhat
more (⬃3-fold) spontaneous proliferation, compared with adult
thymocytes, in the absence of activation. Moreover, when activated with anti-CD3 or with anti-CD3 plus anti-CD28, greater proportions of neonatal, compared with adult, CD4⫹8⫺ thymocytes
entered the cycle (percentage of S plus G2/M). Therefore, the rapid
cell cycle entry of peripheral T cells in the neonate appears to be
inherited from their immediate precursors in the thymus.
In these experiments, we defined neonatal mice as 7 days old.
An immediate question that arises is how long postbirth this phenomenon continues to occur. To address this question, BALB/c
mice 1, 2, and 3 wk postbirth were compared with adult mice.
Total lymph node cells were activated with anti-CD3 mAb. Twenty-four hours later, the cells were stained with anti-CD4 or antiCD8 mAb, fixed and permeabilized, and stained with PI (Table II).
The Journal of Immunology
4553
Table II. Rapid cell cycle entry by peripheral neonatal T cells begins
to disappear by 3 wk postbirth
% S ⫹ G2/M
Table III. Relative entry into cell cycle by adult and neonatal CD4⫹
cells is not dependent on early cytokine levels
Cellsa
Agea
CD4⫹
CD8⫹
1 wk
2 wk
3 wk
Adult
52.7
45.4
35.2
19.4
66.2
59.0
60.5
46.6
a
Total lymph node cells from BALB/c mice of the indicated ages were activated
with anti-CD3. Twenty-four hours later, the cells were stained with PI and anti-CD4
or anti-CD8.
Differences in early cell cycle entry are not due to differences in
early cytokine levels or to differences in the cell cycle status of
unactivated neonatal and adult T cells
When neonatal lymph node cells are activated with anti-CD3, i.e.,
the mode of activation used for most of the cycling experiments,
we found that they produced high levels of both Th1 and Th2
cytokines as early as 24 h of activation (Fig. 7). This contrasts with
adult total lymph node cell cultures that contained markedly less of
the Th1 cytokine ␥-IFN and no detectable IL-4 (Th2 cytokine) at
this early time point. Thus, one possible explanation for the faster
cycling kinetics of neonatal T cells is that the high levels of cytokine present early act to drive cell cycle entry. This possibility
was tested in two ways. First, adult cells were activated in the
presence of exogenous IL-2. The prediction in this study is that if
adults are cycling more slowly simply because of limiting amounts
of early cytokine, the exogenous IL-2 should increase the speed of
cycle entry to resemble neonatal T cell cycling. However, over a
broad range of rIL-2 concentrations, there was no change in the
percentage of adult CD4⫹ cells entering the cycle (Table III). Similar negative results were obtained in the presence of exogenous
rIL-4 (data not shown). In the second approach, neonatal cells
FIGURE 7. Neonatal T cells produce high levels of both Th1 and Th2
cytokines earlier than do adult T cells. Total lymph node cells from day 7
or adult DO11.10 mice were activated with soluble anti-CD3 mAb. Twenty-four hours later, supernatants were harvested, and the IFN-␥ and IL-4
content was determined by specific ELISA. One experiment typical of
three is shown.
1 wk neonate
Anti-IL-4 (␮g/ml)
% S ⫹ G2/M
⫺
1.0
5.0
25.0
50.0
⫺
⫺
⫺
1.0
5.0
25.0
50.0
⫺
⫺
⫺
⫺
⫺
⫺
⫺
20
100
⫺
⫺
⫺
⫺
⫺
20
100
13.1
11.6
11.6
15.6
10.5
9.4
9.0
42.1
42.0
44.5
46.3
41.9
43.3
43.2
a
Total lymph node cells from 1 wk neonatal or adult BALB/c mice were activated
with anti-CD3 for 21 h and stained with PI plus anti-CD4. Values are taken from the
percentages of S plus G2/M events among the gated CD4⫹ populations.
were activated in the presence of neutralizing anti-IL-4 mAb. The
rationale here is that early cycle entry by neonatal T cells may be
promoted by their high IL-4 production, something completely
lacking in the early adult cultures. In this case, elimination of the
IL-4 signal should slow down cycle entry to resemble adult T cell
cycling. Again, however, the presence of anti-IL-4 in the cultures
had no effect on the proportions of cycling neonatal CD4⫹ cells
(Table III).
Another possibility is that the rapid cycle entry of neonatal T
cells may be because they are actually ahead of adult T cells before
activation. That is, adult T cells taken from unprimed animals are
largely in the quiescent G0 phase of the cell cycle. If freshly isolated neonatal T cells were already in the G1 phase of the cell
cycle, their progression into the S phase would appear to be more
rapid. To test this idea, we took advantage of a staining method
that allows the discrimination between the G0 and G1 stages (21).
During cell cycle progression, there is a constant increase of cellular RNA, largely because of the increased production of rRNA
(22, 23). Simultaneous staining of viable cells with the RNA-specific fluorochrome pyronin Y and the DNA-specific fluorochrome
Hoechst can be used to determine cell cycle stages and, particularly, differentially mark cells in G0 vs G1 (21). Total lymph node
cells from day 7 or adult BALB/c mice were isolated and stained
with anti-CD4 mAb followed by fixation and permeabilization and
FIGURE 8. Similar percentages of cells in G0 among freshly isolated
neonatal or adult CD4⫹ cells. Total lymph node cells from day 7 or adult
BALB/c mice were stained with anti-CD4 mAb, followed by Hoechst and
pyronin Y staining. Control cells stained in parallel consisted of 24-h antiCD3 activated adult lymph node cells. The Hoechst and pyronin staining
profiles of gated CD4⫹ cells are shown. One example of three separate
experiments is shown.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Compared with adult cells, elevated percentages of both CD4⫹ and
CD8⫹ cells from animals up to 3 wk old showed early cell cycle
entry. Nonetheless, the proportions of cells entering the cycle early
declined between 1 and 3 wk of life, especially for CD4⫹ cells.
These results demonstrate that rapid responses to activation stimuli
are gradually lost over the first few weeks of life.
Adult
rIL-2 (ng/ml)
4554
RAPID CELL CYCLE ENTRY BY NEONATAL LYMPHOCYTES
Discussion
staining with pyronin Y and Hoechst (Fig. 8). To allow definition
of cells in various stages of the cycle, lymph node cells activated
48 h earlier with anti-CD3 mAb were stained in parallel (Fig. 8,
left panel). Using the activated cells as a standard, a region defining cells in G1 was drawn. A small percentage of freshly isolated
adult CD4⫹ cells appeared within this G1 region. Neonatal CD4⫹
cells showed a small but consistent increase (⬃1.5-fold) over adult
CD4⫹ cells in the percentage of cells in G1. These findings are
consistent with the PI results presented in Figure 3, indicating that
⬃1.5–2 times as many CD4⫹ cells in neonates, compared with
adults, appear to be spontaneously cycling. Nonetheless, the percentage in cycle is low, and it seems unlikely that spontaneous
cycling contributes substantially to the large proportion of neonatal
CD4⫹ cells in the S plus G2/M stages of the cell cycle after only
24 h of activation (see Fig. 3).
Like neonatal T cells, greater proportions of neonatal than adult
B cells show early cell cycle entry
The rapid responses of neonatal T cells to activation signals may
be a quality unique to T lineage cells. Alternatively, rapid cycle
entry may be a feature common to all lymphoid cells of this developmental age. To distinguish between these possibilities, lymph
node cells from neonatal or adult BALB/c mice were stimulated
with LPS or with anti-CD40 mAb plus rIL-4 for 24 h. The cells
were then stained with anti-B220 mAb, to detect B cells, fixed and
permeabilized, and stained with PI. Unlike the T cell populations,
freshly isolated and unactivated cultured B cells from both neonates and adults showed a considerable percentage (10 –20%) of
cells in S plus G2/M (Fig. 9, first and second panels from the left).
Following 24 of activation with either agent, there was essentially
no increase in cycling among adult B cells (Fig. 9, third and fourth
panels from the left). In contrast, the percentage of cycling cells in
the neonatal B cell population increased at least 2-fold in response
to either LPS or anti-CD40 plus rIL-4 stimulation. Similar results
were obtained when cells were stimulated with anti-␮ mAb (data
not shown). Thus, both the B and T cell populations present in
early life respond to lineage-specific activation with rapid cell cycle entry.
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FIGURE 9. Proportionally more neonatal B cells also enter the cycle
faster than adult B cells in response to in vitro activation. Total lymph node
cells from day 7 or adult BALB/c mice were activated for 24 h with LPS
or with anti-CD40 mAb in the presence of IL-4. Parallel cultures contained
no activators (unactivated). The cultured cells, together with freshly isolated day 7 or adult lymph node cells, were then stained with anti-B220
mAb followed by PI. The PI staining profiles of B220⫹ cells are shown and
the percentage of these cells that are in the S plus G2/M phases of the cell
cycle are indicated. This experiment was performed three times.
We have shown in this study that greater proportions of neonatal
lymphocytes, including CD4⫹ and CD8⫹ T cells and B cells, respond to lineage-specific activation by entering the cell cycle earlier than their adult counterparts. This is likely to be a physiologically significant event, because 1) it occurs in vitro in response to
direct TCR␣␤ activation, 2) it occurs in vivo in response to the
peptides and/or signals stimulating homeostatic proliferation (reviewed in Refs. 24 and 25), and 3) it is seen in several strains of
mice. At least in part, early cell cycle entry may compensate for
the lack of immunological memory in neonates, allowing the rapid
mobilization of immune effector mechanisms.
The major finding that led to these experiments was that neonatal T cells, unlike adult T cells, make high levels of IL-4 within
48 h of initial activation (13). For adult T cells, Reiner and colleagues (17) made the original observation that IL-4 production
occurs at increased frequencies in cells that have undergone at
least three cell divisions. We reasoned that neonatal T cells may
make high levels of IL-4 early because they divide more rapidly
than adult cells. While proportionally more neonatal than adult T
cells entered the cell cycle early, no cell division was detected
before ⬃36 h of activation (data not shown; see Figs. 1 and 2). In
these experiments, we found that large quantities of IL-4 were
secreted by neonatal T cells within 24 h of activation (Fig. 7).
Thus, neonatal T cells produce high levels of IL-4 before undergoing a single round of cell division. In this regard, neonatal T
cells resemble the relatively small proportion of adult cells that can
acquire IL-4-secreting potential in the absence of proliferation (26,
27). However, it is important to point out that ⱖ37 h of culture
time was required before IL-4 production by undivided adult cells
was apparent. Thus, whether the cells proliferate or not, some extended time period may be required to remodel the IL-4 locus (17,
28 –31) to allow efficient gene expression. Because neonatal T
cells produce IL-4 so rapidly, in a division-independent fashion, it
is tempting to speculate that the IL-4 locus is relatively more accessible, e.g., less methylated, in neonates compared with adults.
Experiments to test this intriguing possibility are currently
underway.
It is generally believed that most fetal, and possibly neonatal,
cells proliferate faster than adult cells. However, direct comparisons of cell cycle entry by fetal/neonatal and adult cells are generally lacking. What is clear is that fetal/neonatal cells appear to
cycle spontaneously at a higher rate than their adult counterparts.
For example, a higher percentage of freshly isolated fetal CD34⫹
hemopoietic stem cells appear to be in cycle compared with the
adult CD34⫹ population (32, 33). This spontaneous proliferation is
not limited to hemopoietic cells. In first-passage cultures, a greater
proportion of newborn than adult lung fibroblasts spontaneously
enter the S phase of the cell cycle at any time point (34). Smooth
muscle cells from neonatal rats, unlike those from adult rats, appear to be competent to proliferate in the absence of exogenous
mitogens (35). Among T cell populations, cord blood cells, but not
naive adult cells, proliferate in response to IL-7, in the absence of
stimulation through the TCR (36, 37). Indeed, in our system, although spontaneous proliferation is not high, there is a reproducibly greater (⬃2-fold) percentage of freshly isolated neonatal
lymph node T cells that appear to be spontaneously in cycle (see
Fig. 3, left panel). In addition, a recent report (38) describes significantly greater spontaneous proliferation of neonatal splenic T
cells in situ. However, the observations reported in this study are
unique in that rapid early entry into the cell cycle is observed
following lineage-specific activation of fully differentiated cells.
The Journal of Immunology
Acknowledgments
We are grateful to Dr. Kathy Welsh for critical evaluation of the manuscript and stimulating scientific discussion and to Jim Phillips for outstanding technical assistance with flow cytometry.
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The demonstration that greater proportions of neonatal B cells as
well as T cells enter the cycle faster than their adult counterparts
indicates that this phenomenon is common to all lymphocytes of
this developmental stage. It will be interesting to determine
whether other neonatal blood cells, like macrophages and dendritic
cells, also share this property.
What are the characteristics of the neonatal lymphocyte population that lead to rapid early cell cycle progression? For T cells,
one possibility we entertained was that a greater proportion of
neonatal T cells were already in G1 and, thus, entered the S phase
of the cycle faster. However, low proportions of both freshly isolated neonatal and adult T cells were found to be in G1 (see Fig. 8).
Because neonatal T cells made high levels of cytokine more rapidly than adult T cells, we then postulated that these high early
levels of cytokine drove proportionally more neonatal T cells into
cycle early. This also did not appear to be the case because 1)
adding saturating amounts of exogenous cytokine did not result in
faster cell cycle progression by adult T cells and 2) neutralizing
anti-IL-4 mAb did not slow down neonatal cell cycle progression
(see Table III). Yet another possibility may be found in the relative
compositions of neonatal and adult populations. For example,
Turka and colleagues (39) have shown that up to 40% of adult T
cells fail to progress through the cell cycle, despite showing evidence of an activated state. Due to developmental immaturity, neonatal lymphocytes may lack this population, resulting in a greater
proportion of cells responding to activation with early proliferation. Thus, it will be important to measure the absolute number of
precursors among the neonatal and adult populations that respond
to activation with proliferation (39).
The activation of T cells results in signaling cascades that ultimately lead to cellular proliferation and the acquisition of effector
function. Lineage-specific molecular signals are activated, often by
phosphorylation, and these signals, in turn, interface with cell cycle machinery common to all cells. The more rapid cycling of
neonatal T cells might result from the dysregulation of one or more
of these signals. For example, the initial entry of all cells into cycle
is controlled by a combination of the up-regulation of positive
signals and the down-regulation of negative signals. p27kip is expressed in resting cells of all types and must be degraded to allow
cycle entry (reviewed in Refs. 40 and 41). It is conceivable that
resting neonatal T cells underexpress p27kip or that p27kip is expressed at normal levels but degraded more rapidly upon activation. T lineage-specific signals may also be affected in neonates.
For example, phospholipase C (PLC)-␥1 is a central signaling molecule in T cells. Phosphorylation of PLC-␥1 on multiple tyrosine
residues is required for its activation (reviewed in Ref. 42). It is
conceivable that these phosphorylation events occur more rapidly
or efficiently in neonatal T cells or even that PLC-␥1 may be partially tyrosine phosphorylated in resting neonatal T cells. Clearly,
there are numerous candidate signals in the regulation of the relative cell cycle entry of neonatal and adult T cells. We are currently beginning experiments to distinguish among the many
possibilities.
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