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Laboratory of Experimental Immunology, Division of Basic Sciences, and
Intramural Research Support Program, Science Applications International Corp. Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo administration of IL-7 results in the increase of B lineage cell and T cell numbers with a preferential increase in CD8+ T cells (13, 14, 15). Although this increase in T cell numbers appears to be predominantly a thymic independent event (15), the mechanism 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 increased 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 simultaneously occurs.
The results presented in this report demonstrate that T cells from IL-7-treated mice acquire enhanced functional capacity, as measured 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 enhanced 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 stimulation. This IL-7-induced proliferation appears to be nonactivating in that these T cells are not producing cytokine. Moreover, administration of IL-7 in vivo does not induce alterations in most activation 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 cycle. These results are important for the potential use of IL-7 clinically in that IL-7 increases T cell numbers and functional capacity 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 vaccines encoding these Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 Institute-Frederick Cancer Research and Development Center (Frederick, MD). Breeding pairs of C57BL/6 (CD45.2)-IL-7R knockout mice were purchased 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 Recherche (Gentilly, France). The IL-7 had a biologic activity of 5.4 x 107 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 Ca2+, Mg2+, or phenol red; BioWhittaker, Walkersville, MD) plus 0.1% normal mouse serum (NMS)3 as a vehicle control or with IL-7 at 10 µg/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 resuspended in PBS at 20 x 106 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 inactivate 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 x 105 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 µg/ml of anti-CD28 (BD PharMingen, San Diego, CA). Cell cultures were pulsed with 0.5 µCi of [3H]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). Proliferation was assessed in triplicate by measuring the amount of cellular incorporation of [3H]thymidine in cpm with a 1450 MicroBeta TRILUX liquid scintillation and luminescence counter (Wallac, Turku, Finland). Cells (2 x 106/ml) also were stimulated in vitro with PMA (20 ng/ml) and ionomycin (1 µg/ml) or Con A (5 µg/ml).
Surface phenotyping of cells
Single-cell suspensions of peripheral lymph nodes or spleens
were prepared in HBSS plus 0.5% BSA. RBC were lysed using ACK lysing
buffer (BioWhittaker). Cells were labeled with optimally titered Abs,
and 104 cells were analyzed for the percentage of
cells bearing a particular marker(s) by using a FACScan flow cytometer
affixed with a doublet discrimination module (Becton Dickinson,
Mountain View, CA) as described previously (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 Technologies,
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-conjugated
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
PharMingen) 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 Associates, 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.
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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 x 106 cells/ml followed by centrifugation at 1500 rpm for 5 min at 4°C. The supernatant was decanted and the pellet was resuspended in 0.5 ml/106 cells of saponin buffer containing 200 µg/ml of propidium iodide (Sigma, St. Louis, MO) and 50 µg/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 106 cells/ml in medium alone or anti-CD3 and anti-CD28 as indicated above. For each culture condition, cells were cultured either with or without 10 µg/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 distinguish T cell subsets at 106 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 x 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 incubation 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 centrifugation 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 cytometer. 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-nitrophenylphosphate, 1 mM PMSF) at 4 x
107cells/ml. Cell lysates were clarified by
centrifugation (5000 x g for 20 min at 4°C), and the
protein concentration was determined with the bicinchoninic acid
protein detection kit (Pierce, Rockford, IL). For Cdk2 kinase assay,
500 µg of protein from clarified cell lysates were incubated with 1.5
µg of anti-Cdk2 (Santa Cruz Biotechnology, Santa Cruz, CA); after
3 h, 50 µl 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 resuspended in 50 µl of
kinase buffer (10 mM HEPES, pH 7.2, 10 mM MgCl2,
10 mM MnCl2, 50 µCi/ml
[
-32P]ATP, and 3 µg of histone H1, a
substrate for cyclin-dependent kinases). The reaction was terminated
after 20 min at room temperature by the addition of 3x SDS sample
buffer, and the kinase mixture was separated through a 10%
polyacrylamide SDS gel and transferred to an Immobilon-P membrane
(Millipore, Bedford, MA). Autoradiography 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
MgCl2; 0.1% Triton X-100; 10 mM
-glycerophosphate, 1 mM
Na3VO4; 1 mM PMSF; and 1
µg/ml aprotinin/leupeptin). Insoluble material was removed by
centrifugation (5000 x g for 20 min at 4°C), and 20
µg 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
secondary Ab (anti-rabbit or mouse, as appropriate), then with
peroxidase-conjugated streptavidin, and developed by ECL.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 14, 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 µg/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. 1
A 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 enhancement 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 treatment 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 responding 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 G2/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 determine 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.
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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
increase, respectively, in [3H]thymidine
incorporation when cultured 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 increase
activity/levels of these components critical for the movement of cells
from G0 through G1 and
toward the S phase of cell cycle.
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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
G2/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
G2/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/G2/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/G2/M at a given time point compared with
CD4+ T cells. In contrast, cells from
control-treated mice have an approximately equal proportion of
CD4+ and CD8+ T cells in
S/G2/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.
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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 markers 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.
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were
produced. No detectable levels of any of the cytokines examined were
found in the supernatants from either group. In contrast, supernatants
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
proliferation 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 congenic 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 fluorescence halves accordingly. CFSE
labeled cells were injected i.v. into C57BL/6-CD45.2-IL-7 receptor
knockout mice. After allowing 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
demonstrates that 71.5% of the donor-origin CD4+
T cells from a representative mouse treated with HBSS were in the
nonproliferating 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). 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 categories 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 administration 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A) and before the alteration in the CD4:CD8 ratio (Fig. 1B) and the increase in pre-B cell numbers (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 (Fig. 2). Furthermore, the ability of in vivo
administration of IL-7 to enhance T cell function is not merely due to
the reported costimulatory effects of IL-7 (2, 3, 4, 5) as a
result of carry-over of IL-7 from the in vivo treatment into the ex
vivo culture. This is evidenced 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 enhance the proliferative response of T cells to a subsequent ex vivo stimulus, results presented here demonstrate that IL-7 administration 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 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 levels; components involved in the G1/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 induces 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/memory markers CD44 or CD62L (27, 28, 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 disproportionate increase in the number of CD8+ T cells after 7 days of IL-7 administration reported previously (15) support this hypothesis. Although the disproportionate increase in the basal proliferation level of CD8+ T cells could be attributable to the observation that a higher percentage of CD8+ T cells express the IL-7R compared 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 administration 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 unless 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, 14, 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 extrathymic
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 examined
(13, 14, 15). In addition, the increase in T cell numbers is
not attributable to increased exportation from the thymus, as
demonstrated 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
numbers via induction of proliferation. The data presented here
demonstrate that IL-7 increases basal proliferation in both T cell
subsets 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
progenitor/precursor cells. IL-7 also has been shown to inhibit
apoptosis (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
antiapoptosis mechanism. However, we speculate that the antiapoptotic
effect of IL-7 may still be involved. Specifically, we hypothesize that
after the induction of proliferation by IL-7, the antiapoptotic effects
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 increases 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 immunosuppression in that IL-7 expands T cell numbers and enhances 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kristin L. Komschlies, Intramural Research Support Program, Science Applications International Corp. Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Building 560, Room 31-93, Frederick, MD 21702-1201.
3 Abbreviations used in this paper: NMS, normal mouse serum; BrdU, 5-bromo-2'-deoxyurindine; Rb, retinoblastoma.
Received for publication August 7, 2000. Accepted for publication December 18, 2000.
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