![Two days of in vivo administration of IL-7 enhances subsequent T cell responses independently of the disproportionate increase in CD8 T cells. C57BL/6 mice were injected i.p. twice a day with HBSS plus 0.1% NMS (HBSS; vehicle control) for 7 days or 10 g/injection of IL-7 for 2, 4, or 7 days. After cessation of treatment, single-cell suspensions were prepared from pooled peripheral lymph nodes from 6-15 mice/ group. A, Cells were cultured in medium alone or with anti-CD3 mAb and anti-CD28 mAb for 18 h. [ 3 H]Thymidine was added at the initiation of culture to assess the proliferative response. Each bar represents the mean cpm of three replicates SD. B, Cells were stained with anti-CD4 or anti-CD8 mAb. Cell surface expression of these phenotypic markers was determined by flow cytometric analysis. The percentage of positive cells bearing a particular marker was multiplied by the mean of the total number of lymph node leukocytes per mouse (six lymph nodes per mouse) to determine the number of cells within the subset.](https://www.researchgate.net/profile/Jeff-Subleski/publication/12124499/figure/fig2/AS:667825354063884@1536233380402/Two-days-of-in-vivo-administration-of-IL-7-enhances-subsequent-T-cell-responses_Q320.jpg)
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IL-7 Administration Alters the CD4:CD8 Ratio, Increases T
Cell Numbers, and Increases T Cell Function in the Absence of
Activation
1
Lisa A. Geiselhart,* Courtney A. Humphries,* Theresa A. Gregorio,
†
Sherry Mou,
†
Jeffrey Subleski,* and Kristin L. Komschlies
2†
IL-7 is vital for the development of the immune system and profoundly enhances the function of mature T cells. Chronic admin-
istration of IL-7 to mice markedly increases T cell numbers, especially CD8
ⴙ
T cells, and enhances T cell functional potential.
However, the mechanism by which these effects occur remains unclear. This report demonstrates that only 2 days of IL-7
treatment is needed for maximal enhancement of T cell function, as measured by proliferation, with a 6- to 12-fold increase in the
proportion of CD4
ⴙ
and CD8
ⴙ
T cells in cell cycle by 18 h of ex vivo stimulation. Moreover, a 2-day administration of IL-7 in vivo
increases basal proliferation by 4- and 14-fold in CD4
ⴙ
and CD8
ⴙ
T cells, respectively. These effects occur in the absence of
cytokine production, increases in most activation markers, and changes in memory markers. This enhanced basal proliferation is
the basis for the increase in T cell numbers in that IL-7 induces an additional 60% and 85% of resting CD4
ⴙ
and CD8
ⴙ
T cells,
respectively, to enter cell cycle in mice given IL-7 for 7 days. These results demonstrate that in vivo administration of IL-7
increases T cell numbers and functional potential via a homeostatic, nonactivating process. These findings may suggest a unique
clinical niche for IL-7 in that IL-7 therapy may increase T cell numbers and enhance responses to specific antigenic targets while
avoiding a general, nonspecific activation of the T cell population. The Journal of Immunology, 2001, 166: 3019–3027.
I
nterleukin 7 is a 25-kDa glycoprotein produced by thymic
and intestinal epithelial cells, bone marrow stromal elements
and keratinocytes and has been shown to be an essential
growth factor for B and T lineage cells (reviewed in Ref. 1). IL-7
also acts as a T cell costimulus and can enhance in vitro T cell
responses in an Ag-specific fashion when added simultaneously
with various stimuli (2–7). In vitro, IL-7 in the absence of any
other stimulus has been shown to induce proliferation of fresh T
cells in a dose-dependent fashion in some reports (3, 4, 8) but not
in others (5, 9). In addition, reports indicate that human T cells
may proliferate more robustly to IL-7 than mouse T cells, and
some results suggest that the proliferation induced by IL-7 is de-
pendent on the presence of APC (2–4, 8, 9). Although IL-7 does
not appear to switch T cells from CD45RA to CD45RO (10), up-
regulation of activation markers such as CD25, CD98, CD71,
CD11a, and CD40 ligand has been reported in vitro (3, 8, 11). In
addition, less mature T cells (such as those from human cord
blood) appear to proliferate more vigorously to IL-7 than do T
cells from adults (12).
In vivo administration of IL-7 results in the increase of B lin-
eage cell and T cell numbers with a preferential increase in CD8
⫹
T cells (13–15). Although this increase in T cell numbers appears
to be predominantly a thymic independent event (15), the mech-
anism by which the increase in T cell numbers occurs remains to
be determined. In addition, T cells from IL-7-treated mice generate
enhanced CTL and proliferative responses to subsequent ex vivo
stimulation. However, it has not been determined whether the in-
creased functional ability of T cells from IL-7-treated mice is due
to a direct alteration in the biological status of individual T cells or
whether it is a result of the alteration in the CD4:CD8 subset ratio
due to the disproportionate increase in CD8
⫹
T cells that simul-
taneously occurs.
The results presented in this report demonstrate that T cells from
IL-7-treated mice acquire enhanced functional capacity, as mea-
sured by proliferation, before the disproportionate increase in
CD8
⫹
T cells and resultant CD4:CD8 ratio alteration and involves
the enhanced activity of both CD4
⫹
and CD8
⫹
T cells. The en-
hanced functional capacity appears to be attributable to the ability
of IL-7 to increase the level of basal proliferation. Thus, T cells
from IL-7-treated mice already have the cell cycle machinery in
place to respond in an enhanced fashion to a subsequent stimula-
tion. This IL-7-induced proliferation appears to be nonactivating in
that these T cells are not producing cytokine. Moreover, adminis-
tration of IL-7 in vivo does not induce alterations in most activa-
tion and memory markers examined. This is in contrast to in vitro
models that have shown an up-regulation of several activation
molecules (3, 8, 11), thereby demonstrating that in this regard,
previously reported in vitro results are not representative of what
occurs in vivo. Finally, our results demonstrate that the increase in
T cell numbers after IL-7 administration is attributable at least in
part to the ability of IL-7 to induce additional T cells to enter cell
*Laboratory of Experimental Immunology, Division of Basic Sciences, and
†
Intra-
mural Research Support Program, Science Applications International Corp. Frederick,
National Cancer Institute-Frederick Cancer Research and Development Center, Fred-
erick, MD 21702
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.
Received for publication August 7, 2000. Accepted for publication December 18,
2000.
1
The content of this publication does not necessarily reflect the views or policies of
the Department of Health and Human Services, nor does mention of trade names,
commercial products, or organization imply endorsement by the U.S. Government.
This project has been funded in whole or in part with Federal funds from the National
Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000.
Animal care was provided in accordance with the procedures outlined in Guide for the
Care and Use of Laboratory Animals (National Institutes of Health Publication No.
86-23, 1985).
2
Address correspondence and reprint requests to Dr. Kristin L. Komschlies, Intra-
mural Research Support Program, Science Applications International Corp. Frederick,
National Cancer Institute-Frederick Cancer Research and Development Center, Build-
ing 560, Room 31-93, Frederick, MD 21702-1201. E-mail address: komschliesk@
mail.ncifcrf.gov
Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00
cycle. These results are important for the potential use of IL-7
clinically in that IL-7 increases T cell numbers and functional ca-
pacity via a nonactivating process. Thus, polyclonal activation of
T cells does not occur with IL-7 treatment in vivo and only T cells
with specificity to a particular Ag may respond with enhanced
vigor to such antigenic stimuli as unique tumor Ags or DNA vac-
cines encoding these Ags.
Materials and Methods
Mice
C57BL/6 and C57BL/6-CD45.1 mice were used at 2–3 mo of age and were
obtained from the Animal Production Area of the National Cancer Insti-
tute-Frederick Cancer Research and Development Center (Frederick, MD).
Breeding pairs of C57BL/6 (CD45.2)-IL-7R knockout mice were pur-
chased from The Jackson Laboratory (Bar Harbor, ME) and bred in our
animal facility to provide mice for this study. All mice were maintained
under specific pathogen-free conditions.
Treatment of mice
IL-7 (recombinant human IL-7) was generously provided by Sanofi Re-
cherche (Gentilly, France). The IL-7 had a biologic activity of 5.4 ⫻ 10
7
U/mg, as measured by the proliferation of a murine pre-B cell line (16); the
endotoxin levels were ⬍1.3 EU/mg of IL-7. Mice were injected i.p. twice
a day for a varying number of days with HBSS (without Ca
2⫹
,Mg
2⫹
,or
phenol red; BioWhittaker, Walkersville, MD) plus 0.1% normal mouse
serum (NMS)
3
as a vehicle control or with IL-7 at 10
g/injection diluted
in HBSS plus 0.1% NMS at 0.2 ml/injection. Mice were euthanized the day
after treatment completion, and their tissues were then analyzed.
Fluorescent dye labeling of cells and injection into recipient
mice
In one set of experiments a single-cell suspension of peripheral lymph node
(inguinal, axillary, and brachial) cells prepared as previously described
(15) from C57BL/6-CD45.1 mice were labeled with CFSE (Molecular
Probes, Eugene, OR) to monitor proliferation (17, 18). Cells were resus-
pended in PBS at 20 ⫻ 10
6
cells/ml. One milliliter of 200 nM CFSE (in
PBS) was added per 1 ml of cell suspension, followed by mixing and
incubation at room temperature for 15 min in the dark. After the incubation
period, 1 ml of FCS per 1 ml of CFSE-cell suspension was added to in-
activate the labeling reaction. Cells were washed once in PBS and counted.
CFSE-labeled cells were injected i.v. into C57BL/6-(CD45.2)-IL-7R
knockout mice. Twenty-four hours after injection of the cells, mice were
treated with HBSS plus 0.1% NMS or IL-7 as indicated above.
T cell stimulation assay
Peripheral lymph node cells were cultured as described previously (15) at
a concentration of 2 ⫻ 10
5
cells per well with medium alone or a 1:10
supernatant of anti-CD3 generated in vitro by the hybridoma clone 145/
2C11 (19) and 0.5
g/ml of anti-CD28 (BD PharMingen, San Diego, CA).
Cell cultures were pulsed with 0.5
Ci of [
3
H]thymidine (Amersham Life
Science, Piscataway, NJ) at the initiation of culture. After culture, the cells
were recovered by using a Harvester 96 (Tomtec, Hamden, CT). Prolifer-
ation was assessed in triplicate by measuring the amount of cellular incor-
poration of [
3
H]thymidine in cpm with a 1450 MicroBeta TRILUX liquid
scintillation and luminescence counter (Wallac, Turku, Finland). Cells
(2 ⫻ 10
6
/ml) also were stimulated in vitro with PMA (20 ng/ml) and
ionomycin (1
g/ml) or Con A (5
g/ml).
Surface phenotyping of cells
Single-cell suspensions of peripheral lymph nodes or spleens were pre-
pared in HBSS plus 0.5% BSA. RBC were lysed using ACK lysing buffer
(BioWhittaker). Cells were labeled with optimally titered Abs, and 10
4
cells were analyzed for the percentage of cells bearing a particular mark-
er(s) by using a FACScan flow cytometer affixed with a doublet discrim-
ination module (Becton Dickinson, Mountain View, CA) as described pre-
viously (20). Analysis was performed using CellQuest software (Becton
Dickinson). Subset analysis in Fig. 1 was performed using PE-conjugated
anti-CD4 mAb (Becton Dickinson) and biotin-conjugated anti-CD8 mAb
(Becton Dickinson) developed with streptavidin-RED670 (Life Technolo-
gies, Gaithersburg, MD). Surface expression of activation markers on T
cell subsets was determined by using FITC-conjugated mAb to CD25,
CD69, and CD71 (BD PharMingen) individually combined with PE-con-
jugated anti-CD4 mAb and biotin-conjugated anti-CD8 developed with
streptavidin-RED670. In addition, PE-conjugated mAb to CD137 (BD
PharMingen) was individually combined with FITC-conjugated anti-CD4
mAb (clone H129.19; Ref. 21; conjugated in our laboratory) or FITC-
conjugated anti-CD8 mAb (BD PharMingen) was used. Surface expression
of memory markers on T cell subsets were determined by using FITC-
conjugated mAb to CD44 and PE-conjugated mAb to CD62L (BD PharM-
ingen) combined with biotin-conjugated mAb to CD4 (BD PharMingen) or
biotin-conjugated mAb to CD8 developed with streptavidin-RED670.
Where required, cells from C57BL/6-CD45.1 mice (donor-origin) and
C57BL/6-CD45.2-IL-7R
⫺/⫺
mice (host-origin) were detected by using anti-
CD45.1 mAb (clone A-20-1.7; Ref. 22) or anti-CD45.2 mAb (clone
104.2.1; Ref. 22), respectively, developed with a PE-conjugated goat anti-
mouse IgG2a specific polyclonal antiserum (Southern Biotechnology As-
sociates, Birmingham, AL). In this system, CD4
⫹
and CD8
⫹
T cells were
detected by using biotin-conjugated anti-CD4 (clone H129.19, conjugated
in our laboratory) or anti-CD8 mAb developed with streptavidin-RED670.
Cell cycle analysis
Leukocytes were labeled with FITC-conjugated anti-CD4 or anti-CD8 (BD
PharMingen) to distinguish their cell surface phenotype as described
above. Surface-labeled cells were then permeabilized by resuspension in
saponin buffer (0.1% BSA, 0.01 M HEPES, 0.1% saponin in PBS) at a
concentration of 0.5 ⫻ 10
6
cells/ml followed by centrifugation at 1500 rpm
for 5 min at 4°C. The supernatant was decanted and the pellet was resus-
pended in 0.5 ml/10
6
cells of saponin buffer containing 200
g/ml of pro
-
pidium iodide (Sigma, St. Louis, MO) and 50
g/ml of RNase (Puregene,
Minneapolis, MN) followed by incubation for 15 min at 4°C. Labeled cells
were analyzed by using a FACScan flow cytometer affixed with a doublet
discrimination module to include only single cells in the cell cycle analysis.
Data were analyzed using CellQuest software.
Analysis of 5-bromo-2⬘-deoxyurindine (BrdU) incorporation by
lymph node cells
Leukocytes were cultured at 10
6
cells/ml in medium alone or anti-CD3 and
anti-CD28 as indicated above. For each culture condition, cells were cul-
tured either with or without 10
g/ml of BrdU (Sigma). After culture, cells
were washed in HBSS. Cell surface labeling was performed as above in
PBS without azide by using PE-conjugated anti-CD4 mAb and biotin-
conjugated anti-CD8 mAb developed with streptavidin-RED670 to distin-
guish T cell subsets at 10
6
cells per sample in a 96-well round-bottom plate.
After the final wash, the supernatant was removed and the pellets were
resuspended in 0.1 ml of PBS and transferred to 12 ⫻ 75-mm polystyrene
tubes containing 1 ml of PBS followed by centrifugation at 1500 rpm for
5 min at 4°C. The supernatant was removed and 0.5 ml of cold 0.15 M
NaCl was added per sample. While gently vortexing, 1.2 ml cold 95%
ethanol was added dropwise to each sample followed by a 30-min incu-
bation on ice. After incubation, 2 ml of PBS was added to the samples
followed by centrifugation at 1800 rpm for 5 min at 4°C. The supernatant
was removed and the cells were permeabilized by slowly vortexing and
adding 1 ml of PBS containing 1% paraformaldehyde (Sigma) and 0.01%
Tween 20 (Sigma). Cells were incubated for 30 min at room temperature
followed by centrifugation at 1800 rpm for 6 min at 4°C. Each pellet was
resuspended slowly while vortexing in 1 ml of DNase I (DNase I from
bovine pancreas at 50 Kunitz U/ml in 4.2 mM MgCl/0.15 M NaCl, pH 5;
Roche Molecular Biochemicals, Indianapolis, IN). Cells were incubated for
10 min at room temperature then washed in 2 ml PBS followed by cen-
trifugation at 1800 rpm for 6 min at 4°C. Twenty microliters of optimally
titered FITC-conjugated anti-BrdU mAb (BD PharMingen) diluted in PBS
was added; the cells were gently mixed, incubated for 30 min at room
temperature, and washed in 2 ml of PBS followed by centrifugation at 1800
rpm for 6 min at 4°C. Samples were resuspended in 0.2 ml of PBS and
placed on ice in the dark until analyzed by using a FACScan flow cytom-
eter. Cells cultured without BrdU were used as nonspecific binding controls
for the anti-BrdU mAb.
Immune complex kinase assay
Lymph node cells were washed twice in HBSS and disrupted in 1 ml of
lysis buffer (1% (v/v) Triton X-100, 50 mM NaCl, 10 mM Tris-HCl (pH
7.5), 5 mM EDTA, 30 mM sodium pyrophosphate, 5 mM NaF, 25 mM

-glycerolphosphate, 5 mM sodium orthovanadate, 0.1% p-nitrophe-
nylphosphate, 1 mM PMSF) at 4 ⫻ 10
7
cells/ml. Cell lysates were clarified
by centrifugation (5000 ⫻ g for 20 min at 4°C), and the protein concen-
tration was determined with the bicinchoninic acid protein detection kit
3
Abbreviations used in this paper: NMS, normal mouse serum; BrdU, 5-bromo-2⬘-
deoxyurindine; Rb, retinoblastoma.
3020 IL-7 INCREASES T CELL NUMBERS AND FUNCTION WITHOUT ACTIVATION
(Pierce, Rockford, IL). For Cdk2 kinase assay, 500
g of protein from
clarified cell lysates were incubated with 1.5
g of anti-Cdk2 (Santa Cruz
Biotechnology, Santa Cruz, CA); after 3 h, 50
l of a 1:1 slurry of protein
G-agarose was added and incubated for an additional 60 min. The immune
complexes were then washed three times with lysis buffer and two times in
a buffer of 10 mM HEPES, pH 7.2. The immune complexes were resus-
pended in 50
l of kinase buffer (10 mM HEPES, pH 7.2, 10 mM MgCl
2
,
10 mM MnCl
2
,50
Ci/ml [
␥
-
32
P]ATP, and 3
g of histone H1, a substrate
for cyclin-dependent kinases). The reaction was terminated after 20 min at
room temperature by the addition of 3⫻ SDS sample buffer, and the kinase
mixture was separated through a 10% polyacrylamide SDS gel and trans-
ferred to an Immobilon-P membrane (Millipore, Bedford, MA). Autora-
diography was performed to detect protein radiolabeled in the immune
complex kinase assay.
Immunoblotting
For detection of cyclin E and retinoblastoma (Rb) protein, lymph node cells
were solubilized in 1 ml of solubilization buffer (50 mM HEPES, pH 7.4;
15 mM EGTA; 137 mM NaCl; 15 mM MgCl
2
; 0.1% Triton X-100; 10 mM

-glycerophosphate, 1 mM Na
3
VO
4
; 1 mM PMSF; and 1
g/ml aprotinin/
leupeptin). Insoluble material was removed by centrifugation (5000 ⫻ g for
20 min at 4°C), and 20
g of total protein was resolved by 12% SDS-
PAGE, and transferred to an Immobilon-P membrane. The membrane was
blocked in TBST (20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween
20) containing 5% nonfat dry milk (6 h), washed twice, and then incubated
overnight with specific anti-Cdk2 antiserum, anti-cyclin E antiserum (Santa
Cruz Biotechnology), or anti-Rb mAb (clone G3-245; BD PharMingen).
After vigorous washing, blots were incubated first with a biotinylated sec-
ondary Ab (anti-rabbit or mouse, as appropriate), then with peroxidase-
conjugated streptavidin, and developed by ECL.
Results
Enhanced T cell response requires only 2 days of IL-7 treatment
in vivo
Previous studies have shown that chronic administration of IL-7 to
mice or expression of an IL-7 transgene results in an increase in T
cell numbers (13–15, 23, 24). Other studies revealed an ability of
IL-7 to prime mature T cells for polyclonal activation stimuli (e.g,
anti-CD3) (15). However, little is known about whether the ability
of IL-7 to prime T cells for activation is causally linked to the
induction of proliferation. To address this issue, mice were treated
twice a day with IL-7 (10
g/injection) for 2, 4, or 7 days. After
treatment, lymph node cells were stimulated in culture with anti-
CD3 and anti-CD28 for 18 h, and proliferation was monitored. The
results in Fig. 1A demonstrate that IL-7 administration for 2, 4, or
7 days resulted in increased proliferation by 5.0-, 5.1-, or 6.1-fold,
respectively, over that of vehicle control (HBSS plus 0.1% NMS).
Thus, IL-7 treatment induces peak priming for subsequent en-
hancement of proliferative response by 2 days. Moreover, as
shown in Fig. 1B, this enhanced response occurs independently of
the disproportionate increase in CD8
⫹
cells induced by IL-7 treat
-
ment after 4 days or more of IL-7 administration.
In a similar experiment with 2 days of IL-7 administration, cell
cycle analysis was performed to specifically identify the respond-
ing cell type(s) 18 h after the initiation of ex vivo stimulation with
anti-CD3 and anti-CD28. The results demonstrate that 1.96% and
3.60% of CD4
⫹
and CD8
⫹
T cells, respectively, were in S phase
or G
2
/M in the vehicle control-treated mice (Fig. 2
). However, in
lymph node cell cultures from mice treated with IL-7, 11.43% and
42.56% of CD4
⫹
and CD8
⫹
T cells, respectively, were in cycle.
This represents a 5.8-fold increase in CD4
⫹
T cells and an 11.8-
fold increase in CD8
⫹
T cells that were in cycle at 18 h of culture
with anti-CD3 and anti-CD28 when cells from IL-7-treated mice
were compared with controls. In addition, lymph node cells from
2-day HBSS- or IL-7-treated mice were cultured ex vivo for 24 h
with anti-CD3 and anti-CD28 in the presence of BrdU to deter-
mine the proportion of cells that entered cell cycle over the 24-h
period. The results in Fig. 3 (upper panels) demonstrate that the
total lymph node cells from IL-7-treated mice have a dramatic
increase in the proportion of cells entering cell cycle compared
with cells from control mice whose levels were only slightly above
background levels. More specifically, Fig. 3 (middle and lower
panels) shows that, after subtraction of background values, 1.4%
of CD4
⫹
T cells and 4.9% of CD8
⫹
T cells from vehicle control
mice had entered cell cycle. In contrast, 17.0% of CD4
⫹
T cells
and 41.4% of CD8
⫹
T cells from IL-7-treated mice had entered
cell cycle. Thus, pretreatment with IL-7 in vivo results in an ⬃12-
and 8-fold increase in the number of CD4
⫹
and CD8
⫹
T cells,
respectively, that enter cell cycle within the first 24 h of ex vivo
stimulation compared with cells from vehicle control mice.
IL-7 treatment in vivo results in an increase in the basal level of
T cell proliferation
One possible explanation for the accelerated proliferative capacity
of lymph node T cells from IL-7-treated mice is that IL-7 induces
T cells to move at least partially into cell cycle. The results in Fig.
1 support this possibility in that lymph node cells from mice
treated with IL-7 for 2, 4, or 7 days have a 7.5-, 9.0-, or 11.1-fold
FIGURE 1. Two days of in vivo administration of IL-7 enhances sub-
sequent T cell responses independently of the disproportionate increase in
CD8
⫹
T cells. C57BL/6 mice were injected i.p. twice a day with HBSS
plus 0.1% NMS (HBSS; vehicle control) for 7 days or 10
g/injection of
IL-7 for 2, 4, or 7 days. After cessation of treatment, single-cell suspen-
sions were prepared from pooled peripheral lymph nodes from 6–15 mice/
group. A, Cells were cultured in medium alone or with anti-CD3 mAb and
anti-CD28 mAb for 18 h. [
3
H]Thymidine was added at the initiation of
culture to assess the proliferative response. Each bar represents the mean
cpm of three replicates ⫾ SD. B, Cells were stained with anti-CD4 or
anti-CD8 mAb. Cell surface expression of these phenotypic markers was
determined by flow cytometric analysis. The percentage of positive cells
bearing a particular marker was multiplied by the mean of the total number
of lymph node leukocytes per mouse (six lymph nodes per mouse) to
determine the number of cells within the subset.
3021The Journal of Immunology
increase, respectively, in [
3
H]thymidine incorporation when cul
-
tured for 18 h in medium alone compared with HBSS control
lymph node cells.
Biochemical analysis of lymph node cells from mice treated
with IL-7 for 2 days revealed an increased level of Cdk2 kinase
activity as evidenced by the presence of phosphorylated histone
H1, a substrate of Cdk2 kinase; whereas HBSS-treated control
lymph node cells had no detectable level of Cdk2 kinase activity
(Fig. 4A, upper panel). This increase was not due to alterations in
the total amount of Cdk2 kinase, as the level of Cdk2 kinase was
equivalent in the two groups (Fig. 4A, lower panel). Moreover,
whole-cell lysates from lymph node cells from IL-7-treated mice
had elevated levels of cyclin E (Fig. 4B) and phosphorylated Rb
compared with cells from mice that had not received IL-7 (Fig.
4C). These results demonstrate that IL-7 administration can in-
crease activity/levels of these components critical for the move-
ment of cells from G
0
through G
1
and toward the S phase of cell
cycle.
To determine whether IL-7 had an effect on the basal prolifer-
ation levels of T cell subsets, cell cycle analysis was performed as
described above with lymph node cells from mice treated with
IL-7 or HBSS (vehicle control) for 2 days. The results in Fig. 5
demonstrate that in the vehicle control group, 0.88% of the CD4
⫹
T cells and 0.70% of the CD8
⫹
T cells were in either S phase or
G
2
/M. In contrast, in the IL-7-treated group, 3.77% of the CD4
⫹
T cells and 10.21% of the CD8
⫹
T cells were in either S phase or
G
2
/M. Thus, a 2-day administration of IL-7 in vivo induces a 4.3-
and 14.6-fold increase in CD4
⫹
and CD8
⫹
T cells, respectively,
that are in S/G
2
/M. Furthermore, in vivo IL-7 treatment results in
a differential response by T cell subsets in that 3-fold more CD8
⫹
T cells are in S/G
2
/M at a given time point compared with CD4
⫹
T cells. In contrast, cells from control-treated mice have an ap-
proximately equal proportion of CD4
⫹
and CD8
⫹
T cells in
S/G
2
/M. These observations are further supported when lymph
node cells from 2-day HBSS- or IL-7-treated mice were cultured
ex vivo for 24 h in the presence of BrdU. The results in Fig. 6
(upper panels) show that over the 24-h culture period there was no
detectable level of BrdU incorporated above background values in
total lymph node cells from control-treated mice. However, lymph
node cells from IL-7-treated mice had detectable levels of BrdU
incorporation. Detailed examination revealed that after subtraction
of background, 4.0% of the total CD4
⫹
T cells and 10.7% of the
total CD8
⫹
T cells from IL-7-treated mice entered cell cycle.
These results are similar to that of the cell cycle data and clearly
demonstrate that in vivo administration of IL-7 increases the basal
proliferation level in both T cell subsets, but preferentially in
CD8
⫹
T cells.
The enhanced basal proliferation induced by in vivo IL-7
administration does not result in a concomitant induction of
most activation markers, cytokine production, or alterations in
memory markers
To determine the effect of IL-7 on the activation and the naive/
memory status of T cells, lymph node cells from mice given a
2-day treatment of HBSS plus 0.1% NMS or IL-7 were analyzed
by using flow cytometry to determine the surface expression of
activation and memory markers on T cell subsets. The results in
Fig. 7 show that 2 days of IL-7 treatment in vivo (dark solid line)
does not change the cell surface expression of the activation mark-
ers CD25, CD69, and CD137 or the memory markers CD44 and
CD62L on CD4
⫹
or CD8
⫹
T cells compared with cells from
HBSS control-treated mice (dashed line). However, CD71 is up-
regulated but only on the CD8
⫹
T cells from IL-7-treated mice.
Although IL-7 induces the expression of CD71 on CD8
⫹
T cells,
the intensity of expression is less than that induced on stimulation
with PMA and ionomycin (light solid line). Based upon these
markers, IL-7 treatment in vivo does not have an overall activating
effect on T cells in that most of the activation markers remained
unchanged. Furthermore, the naive/memory status of T cells is not
altered with 2 days of IL-7 treatment. However, IL-7 has a limited
and differential effect on CD8
⫹
T cell activation compared with
CD4
⫹
T cells based upon the up-regulation of CD71.
To determine whether IL-7 treatment could induce another T cell
function in addition to proliferation, the ability of cells from IL-7-
treated mice to produce cytokine was examined. Lymph node cells
from mice treated with HBSS or IL-7 for 2 days were cultured for
FIGURE 2. IL-7 administration increases the propor-
tion of T cells that are in S/G
2
/M of the cell cycle at a
given time point in response to subsequent stimulation.
C57BL/6 mice were injected i.p. twice a day for 2 days
with HBSS plus 0.1% NMS (control) or IL-7 (10
g/
injection). Peripheral lymph node cells then were stim-
ulated in vitro with mAb to CD3 and CD28 for 18 h.
After culture, cells were surface-labeled with fluoro-
chrome-conjugated mAb to CD4 and CD8 to discrimi-
nate the two T cell subsets followed by treatment with
propidium iodide for detection of cell-cycle status by
using flow cytometric analysis. The histograms were
generated by gating on either the CD4
⫹
or CD8
⫹
T cell
subset and displaying the cell cycle status for that par-
ticular subset.
3022 IL-7 INCREASES T CELL NUMBERS AND FUNCTION WITHOUT ACTIVATION
24 h in medium. After culture, supernatants were assayed by ELISA
to determine whether the cytokines IL-2, IL-4, GM-CSF, or IFN-
␥
were produced. No detectable levels of any of the cytokines examined
were found in the supernatants from either group. In contrast, super-
natants from control cells stimulated with anti-CD3 and anti-CD28
contained considerable amounts of each of these cytokines (data not
shown). Thus, while in vivo administration of IL-7 increases basal
proliferation levels in T cells, this is not an activation-induced prolif-
eration as it is not associated with other hallmarks of cell activation
including cytokine production and up-regulation of the cell surface
markers typically associated with T cell activation.
Increase in T cell numbers induced by IL-7 administration in
vivo is due, at least in part, to proliferation of peripheral T cells
The ability of in vivo administration of IL-7 to induce increased
basal proliferation suggested that the IL-7-induced increase in T
cell numbers may occur via proliferation of peripheral T cells. To
test this hypothesis, lymph node cells from C57BL/6-CD45.1 con-
genic mice were labeled ex vivo with CFSE, a fluorescent dye that
binds irreversibly to cellular components. On division, CFSE is
distributed evenly between daughter cells and the mean fluores-
cence halves accordingly. CFSE labeled cells were injected i.v.
into C57BL/6-CD45.2-IL-7 receptor knockout mice. After allow-
ing the cells 24 h to home to lymphoid tissues, mice were injected
i.p. twice a day for 7 days with HBSS or IL-7. After treatment, the
splenocytes were examined by using flow cytometric analysis to
enumerate the number of donor-origin (CD45.1
⫹
) T cells and to
determine whether they had undergone proliferation. Fig. 8 dem-
onstrates that 71.5% of the donor-origin CD4
⫹
T cells from a
representative mouse treated with HBSS were in the nonprolifer-
ating portion (highest intensity of CFSE), whereas only 13.7% of
the donor-origin CD4
⫹
T cells from IL-7-treated mice had an
equivalent level of CFSE intensity. Similarly, 71.4% of the donor-
origin CD8
⫹
T cells from vehicle control-treated mice fell in this
range of CFSE intensity; in contrast, only 2.3% of the donor-origin
CD8
⫹
T cells from IL-7-treated mice fell into this range (Fig. 8).
FIGURE 3. IL-7 administration induces enhanced T cell proliferation in
response to subsequent stimulation. C57BL/6 mice were injected i.p. twice
a day for 2 days with HBSS plus 0.1% NMS (control) or IL-7 (10
g/
injection). Peripheral lymph node cells were stimulated in culture with
mAb to CD3 and CD28 for 24 h. At initiation of culture, BrdU was added
to determine the number of cells that entered cell cycle. After culture, cells
were surface-labeled with fluorochrome-conjugated mAb to CD4 and CD8,
then fixed, permeablized, and labeled intracellularly with a fluorochrome-
conjugated mAb to BrdU for flow cytometric analysis. The data repre-
sented in the plots are the profiles of the total leukocyte population. In the
upper panels, the solid line indicates the proportion of cells that incorpo-
rated BrdU and the dashed line indicates the nonspecific background bind-
ing of the anti-BrdU mAb that was generated using cells cultured under
similar conditions but in the absence of BrdU. The percentage of nonspe-
cific binding was ⬍0.3% per quadrant.
FIGURE 4. IL-7 treatment induces cell cycle proteins and kinase activ-
ity. C57BL/6 mice were injected i.p. twice a day for 2 days with HBSS plus
0.1% NMS or IL-7 (10
g/injection). Peripheral lymph node cells were
pooled by treatment group. A, Top, An immune complex kinase assay was
performed with Cdk2 immunoprecipitated (IP) from equivalent protein
amounts. Histone H1 was included as a phosphorylation substrate for
Cdk2. Samples were resolved with SDS-PAGE, transferred to an Immo-
bilon-P membrane, and radiolabeled proteins were detected with autora-
diography. Bottom, Immunoblotting (IB) with Cdk2-specific antiserum, il-
lustrates the total amount of Cdk2 protein (33-kDa doublet) in each sample.
B and C, Levels of cyclin E (50 kDa; B) or phosphorylated Rb protein
(105–110 kDa; C) were determined by using whole-cell lysates. An equiv-
alent amount of protein was loaded in each sample, and the samples were
resolved with SDS-PAGE and transferred to Immobilon-P membrane. Im-
munoblotting was performed with antiserum specific for cyclin E or Rb,
respectively. Hyperphosphorylated Rb (pRb) migrates more slowly than
the less phosphorylated forms lower in the blot. In C, the top band in all
lanes is attributable to nonspecific binding, and the control is actively pro-
liferating MOLT-4 cells.
3023The Journal of Immunology
Therefore, there is a 4- and 16.7-fold increase in the proportion of
CD4
⫹
and CD8
⫹
T cells, respectively, undergoing proliferation
compared with T cells from mice treated with HBSS. To determine
whether IL-7 induced additional cells to proliferate, the number of
cells that the fell into the nonproliferating vs proliferating catego-
ries were calculated in Fig. 9. The number of CD4
⫹
and CD8
⫹
T
cells that remained in a nonproliferation status was decreased by
59.7% and 84.6%, respectively, in the spleens from mice treated
with IL-7. Furthermore, these data demonstrate for the first time
that IL-7 acts directly on T cells (i.e., IL-7R
⫹/⫹
donor-origin T
cells) to achieve these effects as the recipient cells (IL-7R
⫺/⫺
)in
the microenvironment are genotypically unable to respond to IL-7.
In addition, the increase in donor-origin T cell numbers is not due
to differentiation of T cell precursors that are absent in lymph node
cell inoculum. Moreover, these data demonstrate that IL-7 admin-
istration preferentially induces CD8
⫹
T cells to proliferate. Thus,
IL-7 increases T cell numbers in vivo at least in part by directly
inducing additional nonprecursor peripheral T cells to undergo
proliferation.
Discussion
Although in vivo administration of IL-7 has been demonstrated to
enhance T cell functional capacity (15) and increase T cell num-
bers, particularly CD8
⫹
T cells (13–15), little is known regarding
the kinetics, responsible T cell subset(s), and mechanism(s) by
which these in vivo phenomena occur. To initiate investigation of
these issues, we speculated that the enhanced T cell functional
capacity, which we demonstrated in a previous report where IL-7
was administered in vivo for 7 days (15), may result from a change
in the composition of the T cell population from an approximately
equal proportion of CD4
⫹
and CD8
⫹
T cells in controls to a ma
-
jority of CD8
⫹
cells in the IL-7-treated group. In this report we
have demonstrated that the enhanced T cell functional capacity
induced by in vivo administration of IL-7 occurs after as little as
2 days of treatment with IL-7 (Fig. 1A) and before the alteration in
the CD4:CD8 ratio (Fig. 1B) and the increase in pre-B cell num-
bers (data not shown) that occurs after 4 or more days of IL-7
treatment. In addition, in vivo IL-7 administration enhances the
proliferative response to a subsequent ex vivo stimulus of the
CD4
⫹
and CD8
⫹
T cell subsets by ⬃6- and 12-fold, respectively
FIGURE 6. In vivo treatment with IL-7 induces T cell proliferation.
C57BL/6 mice were injected i.p. twice a day for 2 days with HBSS plus
0.1% NMS (control) or IL-7 (10
g/injection). Peripheral lymph node cells
were cultured for 24 h in medium alone with BrdU. After culture, cells
were surface-labeled with fluorochrome-conjugated mAb to CD4 and CD8,
then fixed, permeabilized, and labeled intracellularly with a fluorochrome-
conjugated mAb to BrdU for flow cytometric analysis. The data repre-
sented in the plots are the profiles of the total leukocyte population. In the
upper panels, the solid line indicates the proportion of cells that incorpo-
rated BrdU, and the dashed line indicates the nonspecific binding control as
described in Fig. 3. The percentage of nonspecific binding was ⬍0.5% per
quadrant.
FIGURE 5. IL- 7 administration increases the propor-
tion of T cells that are in cell cycle. C57BL/6 mice were
injected i.p. twice a day for 2 days with HBSS plus 0.1%
NMS (control) or IL-7 (10
g/injection). Peripheral
lymph node cells from these mice were surface-labeled
with fluorochrome-conjugated mAb to CD4 and CD8,
then treated with propidium iodide for detection of cell-
cycle status by using flow cytometric analysis. The his-
tograms were generated by gating on either the CD4
⫹
or
CD8
⫹
T cell subset and displaying the cell cycle status
for that particular subset.
3024 IL-7 INCREASES T CELL NUMBERS AND FUNCTION WITHOUT ACTIVATION
(Fig. 2). Furthermore, the ability of in vivo administration of IL-7
to enhance T cell function is not merely due to the reported co-
stimulatory effects of IL-7 (2–5) as a result of carry-over of IL-7
from the in vivo treatment into the ex vivo culture. This is evi-
denced by the fact that addition of IL-7 in culture failed to enhance
the proliferative response of lymph node cells from HBSS control-
treated mice stimulated with anti-CD3 and anti-CD28 by 24 h of
culture (data not shown). Thus, the enhancing effect of IL-7 on T
cell function, as measured by proliferation, occurs in both T cell
subsets and is independent of the disproportionate increase in
CD8
⫹
T cells. In terms of the clinical use of IL-7, these results
demonstrate that the functional enhancing properties of IL-7 may
be separated from the ability of IL-7 to increase cell numbers by
varying the length of treatment. Thus, only short periods of IL-7
treatment may be needed to enhance T cell function in patients.
In addition to the ability of IL-7 administration in vivo to en-
hance the proliferative response of T cells to a subsequent ex vivo
stimulus, results presented here demonstrate that IL-7 administra-
tion for as little as 2 days results in increased basal proliferation of
T cells. This increase in basal proliferation may be the basis for the
enhanced proliferative response to subsequent stimulation induced
FIGURE 9. In vivo administration of IL-7 acts directly to induce IL-
7R
⫹
donor T cells to enter cell cycle in IL-7R
⫺/⫺
recipient mice. The
results from Fig. 8 were analyzed to determine the number of donor-origin
T cells per spleen that were represented in the proliferating vs the nonpro-
liferating group based on CFSE intensity. This was calculated for individ-
ual spleens by multiplying the percentage of cells determined in Fig. 8 by
the total number of leukocytes per spleen. The values represent the mean
cell numbers ⫾ SD from three mice per group.
FIGURE 7. In vivo administration of IL-7 does not induce T cell acti-
vation markers or alter memory markers. C57BL/6 mice were injected i.p.
twice a day for 2 days with HBSS plus 0.1% NMS or IL-7 (10
g/injec-
tion). Peripheral lymph node cells were surface-labeled with fluorochrome-
conjugated mAb to CD4 and CD8 and mAb to the T cell activation markers
CD25, CD69, CD71, and CD137 and the memory markers CD44 and
CD62L. The histograms were generated by gating on either the CD4
⫹
or
CD8
⫹
T cell subset and displaying their expression of a particular activa
-
tion marker. The histogram of the control group is indicated by a dashed
line and that of the IL-7 group by a dark solid line. As a positive control
(light solid line) normal lymph node cells were activated in culture with
PMA and ionomycin or Con A, or anti-CD3 and anti-CD28, and labeled as
indicated above to demonstrate up-regulation of activation markers or al-
teration in memory markers.
FIGURE 8. In vivo administration of IL-7 increases the proportion of
donor T cells (CD45.1, IL-7R
⫹
) undergoing proliferation in IL-7R
⫺/⫺
re
-
cipient mice. Peripheral lymph node cells from C57BL/6-CD45.1 congenic
mice were labeled ex vivo with CFSE. C57BL/6-CD45.2
⫺
IL-7R
⫺/⫺
mice
were injected i.v. with 85 ⫻ 10
6
of the CFSE-labeled donor cells. These
donor cells contained 40.7% CD4
⫹
T cells and 37.8% CD8
⫹
T cells. After
allowing the injected cells 24 h to home to lymphoid tissues, mice were
injected i.p. twice a day for 7 days with HBSS plus 0.1% NMS (control)
or 10
g/injection of IL-7. Splenocytes were labeled with mAb specific to
CD45.1 and either CD4 or CD8 to distinguish donor-origin T cell subsets.
In addition, the cells were analyzed to determine the intensity of CFSE as
an indicator of proliferation. The histograms display the intensity of CFSE
of donor-origin CD4
⫹
T cells or CD8
⫹
T cells. This is a representative
profile of three mice per group. The histograms of the control group or the
IL-7-treated group are indicated by a dashed or solid line, respectively.
3025The Journal of Immunology
by IL-7 pretreatment in vivo. Indeed, the results presented here are
the first to demonstrate that IL-7 treatment in vivo induces Cdk2
kinase activity and Rb phosphorylation and increases cyclin E lev-
els; components involved in the G
1
/S transition. This complements
previous work by Itoh et al. (25) demonstrating that IL-7 increased
Cdk4 kinase activity in B cell precursors. Thus, IL-7 up-regulates
the cell cycle machinery, thereby allowing T cells from IL-7-
treated mice to undergo proliferation in an enhanced, accelerated
fashion after exposure to a subsequent stimulation.
The increased basal proliferation induced by IL-7 appears to be
more of a “homeostatic” proliferation rather than an “activation”
proliferation in that these T cells do not produce cytokine and they
do not express the markers CD25, CD69, CD71 (except for the
CD8
⫹
T cells), and CD137, which indicate activation. This is in
contrast to previous in vitro reports demonstrating that IL-7 in-
duces several activation markers, suggesting a state of activation
(3, 8, 11, 26). In addition, our results demonstrate that 2 days of
IL-7 treatment does not alter the expression of the activation/mem-
ory markers CD44 or CD62L (27–29) on either T cell subset,
suggesting that IL-7 does not activate naive cells or induce them to
become memory cells. Basal proliferation levels increase by day 2
in both T cell subsets, but occur disproportionately in the CD8
⫹
T
cells by a 3-fold greater amount, suggesting that IL-7 may have a
differential effect on CD8
⫹
T cells compared with CD4
⫹
T cells.
The up-regulation of CD71 only on the CD8
⫹
T cells and dispro
-
portionate increase in the number of CD8
⫹
T cells after 7 days of
IL-7 administration reported previously (15) support this hypoth-
esis. Although the disproportionate increase in the basal prolifer-
ation level of CD8
⫹
T cells could be attributable to the observation
that a higher percentage of CD8
⫹
T cells express the IL-7R com
-
pared with the CD4
⫹
T cell subset (30), this would not explain the
differential expression of CD71 on CD8
⫹
T cells compared with
the CD4
⫹
T cells with IL-7 treatment in vivo. Although these
results may not necessarily represent a role for IL-7 in normal
immune system homeostasis, it appears that exogenous adminis-
tration of IL-7 increases basal proliferation of T cells and enhances
functional capacity via a homeostatic mechanism. This may be
beneficial in the clinical setting in that IL-7 primes the T cells for
enhanced functional activity, but this potential is not realized un-
less the T cells are specifically activated. Thus, IL-7 therapy in
patients would avoid massive polyclonal activation of the T cell
population while concurrently enhancing responses to specific
stimuli such as peptides derived from tumor-associated Ags.
IL-7 administration has been shown previously to increase T
cell numbers in vivo (13–15). There are several possible ways that
this could be accomplished, including redistribution of T cells,
increased exportation from the thymus, expansion of peripheral T
cells by induction of proliferation often via activation, generation
of new T cells directly from precursors/progenitors via an extra-
thymic mechanism, and/or inhibition of apoptosis. It is unlikely
that the increase in T cell number is due to redistribution in that T
cell numbers are increased in all secondary lymphoid tissues ex-
amined (13–15). In addition, the increase in T cell numbers is not
attributable to increased exportation from the thymus, as demon-
strated by a previous report from our laboratory showing that T
cell numbers are increased to similar levels in both thymectomized
and normal euthymic mice given IL-7 in vivo (15). In this report,
we have examined the possibility that IL-7 increases T cell num-
bers via induction of proliferation. The data presented here dem-
onstrate that IL-7 increases basal proliferation in both T cell sub-
sets and disproportionately in CD8
⫹
T cells. Thus, we speculated
that the increase in T cell number and the alteration in the CD4:
CD8 T cell ratio that occurs with IL-7 administration in vivo could
be attributable to increased homeostatic proliferation of peripheral
T cells. Our results in Figs. 8 and 9 indicate that IL-7 does indeed
expand T cell numbers, at least in part, by inducing additional T
cells to undergo proliferation and not merely by increasing the
number of divisions of already proliferating cells. Because the host
mice in our studies had disrupted IL-7R, only the injected donor
cells were physically capable of responding to IL-7. Thus, our data
demonstrate that IL-7 acts directly on T cells to increase their
numbers and not through an indirect mechanism. Moreover, this
rules out the possibility that the increase in T cell numbers is
attributable to the generation of new cells from progenitors via a
thymic-independent mechanism. This is based on the fact that
host-origin progenitors are unable to respond to IL-7, as they have
no functional IL-7R. In addition, leukocytes from the peripheral
lymph node used as donor-origin cells contain virtually no pro-
genitor/precursor cells. IL-7 also has been shown to inhibit apo-
ptosis (1). The data in this report rule out the possibility that the
increase in T cell numbers induced by IL-7 is due solely to the
accumulation of T cells by blocking cell turnover via an antiapop-
tosis mechanism. However, we speculate that the antiapoptotic ef-
fect of IL-7 may still be involved. Specifically, we hypothesize that
after the induction of proliferation by IL-7, the antiapoptotic ef-
fects of IL-7 may serve to maintain the survival of the proliferating
cells. This hypothesis fits with our previous results showing that T
cell numbers begin to return to normal levels by 1–2 wk following
cessation of IL-7 treatment in vivo (31). Further investigation is
necessary to address this issue.
These results demonstrate that in vivo IL-7 pretreatment in-
creases T cell numbers (particularly CD8
⫹
T cells) by increasing
basal proliferation via a nonactivating rather than an activation
type of mechanism. This increase in basal proliferation correlates
with the ability of T cells to respond in an enhanced fashion to a
subsequent proliferative stimulus as the cell cycle machinery is in
place from induction by IL-7 and may serve to potentiate Ag-
specific responses as indicated previously by in vitro studies (6, 7).
Moreover, the data clearly show that IL-7 has differential effects on
T cell subsets in that CD71 is up-regulated only on the CD8
⫹
T
cells and the increase in basal proliferation is more profound in this
subset as well. These results may be important for the clinical
development of IL-7 as a vaccine adjuvant or to ameliorate im-
munosuppression in that IL-7 expands T cell numbers and en-
hances their functional capacity via a nonactivating mechanism.
Thus, although IL-7 induces polyclonal priming of T cells, this
potential can be restricted to selected cells that can specifically
respond to a particular Ag of interest.
Acknowledgments
We thank Drs. Robert Wiltrout, John Ortaldo, and Scott Durum for critical
review of this manuscript; Tim Back, Kathy McCormick, Erin Parsoneault,
and John Wine for their excellent technical expertise; and Susan Charbon-
neau and Joyce Vincent for typing and editing this manuscript.
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