T Cell Effector Function and Anergy

Avoidance Are Quantitatively Linked to Cell

Division
This information is current as Andrew D. Wells, Matthew C. Walsh, David Sankaran and
of March 4, 2015. Laurence A. Turka
J Immunol 2000; 165:2432-2443; ;
doi: 10.4049/jimmunol.165.5.2432
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T Cell Effector Function and Anergy Avoidance Are
Quantitatively Linked to Cell Division1

Andrew D. Wells, Matthew C. Walsh, David Sankaran, and Laurence A. Turka2

We have shown previously that T cells activated by optimal TCR and CD28 ligation exhibit marked proliferative heterogeneity,
and ⬃40% of these activated cells fail entirely to participate in clonal expansion. To address how prior cell division influences the
subsequent function of primary T cells at the single cell level, primary CD4ⴙ T cells were subjected to polyclonal stimulation,
sorted based on the number of cell divisions they had undergone, and restimulated by ligation of TCR/CD28. We find that
individual CD4ⴙ T cells exhibit distinct secondary response patterns that depend upon their prior division history, such that cells

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that undergo more rounds of division show incrementally greater IL-2 production and proliferation in response to restimulation.
CD4ⴙ T cells that fail to divide after activation exist in a profoundly hyporesponsive state that is refractory to both TCR/CD28-
mediated and IL-2R-mediated proliferative signals. We find that this anergic state is associated with defects in both TCR-coupled
activation of the p42/44 mitogen-activated protein kinase (extracellular signal-related kinase 1/2) and IL-2-mediated down-reg-
ulation of the cell cycle inhibitor p27kip1. However, these defects are selective, as TCR-mediated intracellular calcium flux and
IL-2R-coupled STAT5 activation remain intact in these cells. Therefore, the process of cell division or cell cycle progression plays
an integral role in anergy avoidance in primary T cells, and may represent a driving force in the formation of the effector/memory
T cell pool. The Journal of Immunology, 2000, 165: 2432–2443.

T he events that drive the development of naive T cells into imidyl ester (CFSE),3 stimulated these cells in vitro, and sorted
effector and/or memory cells are unclear. While individ- them by flow cytometry based upon their proliferative history. Our
ual primary T cells exhibit a relatively uniform pattern of data demonstrate that the responsiveness of individual T cells to
early signal transduction and activation in response to optimal secondary TCR engagement is quantitatively tuned to the number
TCR and costimulatory receptor ligation (1, 2), a large degree of of mitotic events achieved during primary stimulation. We also
heterogeneity can be observed with respect to functional behavior define a T cell fate that is refractory to both TCR- and IL-2R-
such as cytokine production (3–7). A similar degree of diversity mediated signals, and is associated with the failure to proliferate
characterizes the proliferative behavior of individual activated T following primary activation in the presence of CD28 costimula-
cells. Several days following in vitro stimulation, one can detect tion. These data support a model in which T cells must divide after
individual T cells that have divided between one and eight times, Ag encounter to avoid anergy.
while as many as 40% of these activated cells do not participate at
all in clonal expansion (8). This limitation in the frequency of Materials and Methods
activated T cells that respond by proliferating is observed both in Mice, Abs, and reagents
vitro and in vivo in response to either mitogens or peptide Ag, and
occurs even when signals from TCR, CD28, and IL-2R are optimal Pooled spleen and lymph node cells from female BALB/c mice, aged 8 –12
wk, were used for all experiments. mAbs against CD3 (145-2C11) and
(8, 9). Interestingly, this limitation in responder frequency is not CD28 (37.51) were purified from hybridomas obtained from J. Bluestone
observed in TCR-transgenic T cell populations that have matured (University of Chicago, Chicago, IL) and J. Allison (University of Cali-
on a recombination-deficient background, suggesting that maximal fornia, Berkeley, CA), respectively. Purified, fluorochrome-conjugated
T cell proliferative potential requires allelic exclusion at the mAb against CD16/CD32 (Fc-block), Thy-1.2, CD4, CD25, CD122, com-
mon ␥-chain (␥c), IL-2, and IFN-␥, and biotinylated anti-CD3 and anti-
TCR-␣ locus (9).
CD28 were purchased from PharMingen (San Diego, CA). Abs reactive
This diversity observed in primary T cell responses has sug- with both the phosphorylated and unphosphorylated forms of extracellular
gested that an individual T cell’s proliferative behavior during pri- signal-related kinase (ERK)1, ERK2, and STAT5 were purchased from
mary culture might influence its eventual effector function (10 – Zymed Laboratories (San Francisco, CA). mAb specific for p27kip1 was
12). To further address this question, we have labeled T cells with purchased from Transduction Laboratories (Lexington, KY). Abs specifi-
cally reactive with only the phosphorylated forms of ERK1 and ERK2
the fluorescent dye 5- and 6-carboxyfluorescein diacetate succin- were purchased from New England Biolabs (Beverly, MA), and Ab reac-
tive with the phosphorylated form of STAT5 was purchased from Zymed
Laboratories (San Francisco, CA). Rabbit antiserum against actin was pur-
Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104 chased from Sigma (St. Louis, MO). CFSE, Indo-1 AM, FuraRed AM, and
Received for publication September 7, 1999. Accepted for publication June 15, 2000. TOPRO-3 were purchased from Molecular Probes (Eugene, OR). PMA
and ionomycin were purchased from Sigma and were used at 5 ng/ml and
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.
3
1 Abbreviations used in this paper: CFSE, 5- and 6-carboxyfluorescein diacetate suc-
This work was supported by National Institutes of Health Grants AI-37691, AI-
cinimidyl ester; [Ca2⫹]i, intracellular Ca2⫹ concentration; ERK, extracellular signal-
41521, and P30-CA16520-24. L.A.T. is an Established Investigator of the American
related kinase; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; MFI,
Heart Association. A.D.W. was supported by National Institutes of Health Training
mean fluorescence intensity; PDK, phosphoinositide-dependent kinase; PI3K, phos-
Grants CA 09140 and K01DK02771-01.
phatidylinositol 3-kinase; PKB, protein kinase B; PKC, protein kinase C; ␥c, common
2
Address correspondence and reprint requests to Dr. Laurence A. Turka, University ␥-chain; LAT, linker of activated T cells; SLP-76, SH2 domain-containing leukocyte
of Pennsylvania, 700 Clinical Research Building, 415 Curie Boulevard, Philadelphia, protein of 76 kDa; mTOR, mammalian target of rapamycin; FRAP, FK506-binding
PA 19104-6144. E-mail address: [email protected] protein-repamycin-associated protein.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

The Journal of Immunology 2433

0.25 ␮M, respectively. Murine rIL-2 was obtained from Genzyme (Cam- times with PBS containing 3% milk, and Cy5-conjugated donkey anti-
bridge, MA), and was used at 15–30 U/ml. mouse IgG F(ab⬘)2 (0.3 ␮g) was added for 30 min at room temperature.
Cells were washed twice in PBS and subjected to flow cytometric analysis.
Cell labeling and culture conditions
Cell isolation and fluorescent labeling of cells with CFSE were performed
as previously described (8). Briefly, pooled spleen and lymph node cells Results
were incubated with CFSE in PBS at a final concentration of 2 ␮M for The number of cell divisions achieved by an individual CD4⫹
3 min. CFSE-labeled cells were stimulated with soluble anti-CD3 mAb
(1 ␮g/ml, unless otherwise stated) at 2–5 ⫻ 106/ml in either 24-well plates T cell during primary activation predicts its capacity to
or 75-cm2 flasks. Where indicated, cultures contained anti-CD28 mAb (1 proliferate upon restimulation
␮g/ml) and/or IL-2 (15 U/ml). For primary cultures, the cells were stim-
ulated for 4 days, washed, and replated in fresh medium for 48 h, then Previously, we have shown that while optimal stimulation of
sorted based on CFSE fluorescence. Sorted T cells were restimulated in freshly isolated T cells by anti-CD3 ⫾ anti-CD28 mAb induces the
96-well round-bottom plates with soluble Abs, as indicated, for 4 days in activation of 95–98% of the T cell population, as assessed by
the presence of a 4-fold excess of irradiated syngeneic splenocytes, and CD25 and CD69 expression, a large proportion of the activated T
secondary proliferation (CFSE fluorescence) was analyzed by flow
cytometry. cells (up to 50%) fails to undergo even a single round of cell
division (8). This raised the question as to whether there might be

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Flow cytometry and cell sorting an association between the heterogeneity in proliferative behavior
Cell surface marker staining was performed as described (8), and flow observed within the CD4⫹ T cell pool and heterogeneity in sub-
cytometric analysis was performed on a FACScalibur flow cytometer using sequent effector function (i.e., cytokine production and prolifera-
CellQuest software (Becton Dickinson, San Jose, CA). Methods using tion). To test this hypothesis, murine spleen and lymph node cells
CFSE labeling to calculate the responder frequency (defined as the pro-
were labeled with CFSE and cultured in vitro with an optimal
portion of input T cells that undergo one or more cell divisions during the
culture period) and the absolute number of mitotic events occurring in the concentration of T cell mitogenic anti-CD3 Ab for 4 days. The
culture have been described (8). The vital dye TOPRO-3 was used to dis- cultures were then washed and rested for 48 h, and T cells that had
criminate live and dead cells (8). Live, Thy-1.2⫹, or CD4⫹ T cells were divided once, twice, or had remained undivided following primary
sorted based on CFSE fluorescence using a FACSvantage flow cytometer/ stimulation were purified by FACS and restimulated (Fig. 1A).
sorter (Becton Dickinson).
The experiment shown in Fig. 1 demonstrates this proliferative
Intracellular cytokine staining heterogeneity. After the peak of the clonal expansion phase (day
Cytokine expression was assessed at the single cell level, as described 4), 25.7% of the input T cells responded to primary stimulation by
previously (5), with some modifications. Cells that had been activated in dividing once, 18.7% divided twice, 11% divided three times, and
primary culture for 4 days and rested for 48 h were cultured for 5 h with less than 4% divided four or more times, while 41% remained
plate-bound anti-CD3 plus anti-CD28 mAbs (5 ␮g/ml each) in the presence undivided. Following cell sorting, T cells were cultured with fresh
of 2 ␮M monensin (Sigma). As primed T cells do not divide during this 5-h
restimulation period (data not shown), cytokine production can be assessed syngeneic accessory cells in medium alone, or in the presence of
as a function of primary proliferative history without the use of cell sorting. anti-CD3 mAb. As the cells remained labeled with CFSE, we were
able to monitor their proliferative response to restimulation. Sorted
Measurement of intracellular Ca2⫹ concentration ([Ca2⫹]i) CD4⫹ T cells cultured with feeders in medium alone did not divide
Primed T cells were loaded with either Indo-1 AM (2 ␮g/ml) or FuraRed during the 4-day restimulation period (Fig. 1B), confirming that all
AM (10 ␮g/ml) (Molecular Probes) for 30 min at 30°C in RPMI 1640 with of the T cells had exited the cell cycle following the initial phase
1% serum (13). Agonistic, biotinylated anti-CD3 and anti-CD28 Abs were
of activation and rest. When subjected to TCR reengagement by
also added at this time. Cells were washed, resuspended in RPMI 1640
lacking serum and sodium bicarbonate, and warmed to 37°C for 5 min, and the addition of anti-CD3 to the restimulation cultures, those CD4⫹
Indo-1 or FuraRed fluorescence before and after receptor cross-linking was T cells that had undergone at least a single round of cell division
assessed on a FACStarPlus or FACScalibur flow cytometer, respectively during primary activation initiated a second burst of proliferation
(13). Receptor cross-linking was achieved by the addition of streptavidin (Fig. 1C, middle and lower panels). In contrast, those cells that
(0.2 ␮g/ml, final concentration). Maximal [Ca2⫹]i flux was achieved by the
addition of ionomycin (1 ␮M). Kinetic analysis of [Ca2⫹]i was achieved failed to divide following primary activation were unable to pro-
using FloJo flow cytometry software (Tree Star Software, San Carlos, CA). liferate in response to TCR reengagement (Fig. 1C, upper panel).
Quantitative, single cell analysis of the dynamics of secondary
Assessment of ERK activation, STAT5 activation, and p27 kip1
clonal expansion shows that the proportion of input CD4⫹ T cells
degradation
that responded to restimulation by proliferating (i.e., the responder
CD4⫹ T cells sorted based on division cycle were rested at 37°C for 2– 4 frequency) was less than 5% in the undivided pool, whereas 65%
h in medium, and a portion of each pool was set aside for analysis of total of the input CD4⫹ T cells that divided once during primary acti-
ERK content. The remaining cells were restimulated for 10 min with poly-
styrene beads coated with anti-CD3 mAb ⫾ anti-CD28 mAbs (14), or with vation, and 75% of the CD4⫹ T cells that divided twice were able
PMA. Whole cell lysates (6 – 8 ⫻ 105 cell equivalents per lane) were sub- to reenter the cell cycle when subjected to TCR ligation again (Fig.
jected to SDS-PAGE, transferred to nitrocellulose membranes, and probed 1D). Measuring the accumulation of cell divisions within the
with anti-ERK or anti-phospho-ERK antisera (1/1000 dilution). Bands CD4⫹ subset during the restimulation period shows that 10,000
were quantified by densitometric analysis using NIH Image software, and
the phospho-ERK signals were normalized to the corresponding total ERK undivided CD4⫹ T cells cultured in the presence of anti-CD3 gave
signals to determine relative ERK activation. For the assessment of p27kip1 rise to ⬍1,500 total mitotic events. In contrast, the same number of
degradation, cells primed and rested as above were cultured for 48 h in the cells from the pool of CD4⫹ T cells that divided once gave rise to
presence of 50 U/ml IL-2. The cells were then sorted into divided and 50,000 mitotic events, while the CD4⫹ T cells that divided twice
undivided fractions, and the whole cell lysates were subjected to immu-
noblot analysis as above using a mAb against p27kip1. For the assessment
during primary activation gave rise to ⬎130,000 mitotic events
of STAT5 activation, splenic T cells were primed for 4 days with anti-CD3 (Fig. 1E). These absolute mitotic event values correspond to the
(1 ␮g/ml), rested for 24 h, and restimulated by the addition of IL-2 (100 generation of an average of between three and four daughter cells
U/ml) for 10 min. The cells were fixed for 5 min in cold 1% formaldehyde, per responder from the pool of precursors with a prior division
permeabilized for 10 min in cold methanol, and washed with PBS con-
taining 3% nonfat dry milk. The fixed/permeabilized cells were then
history of one, while the average CD4⫹ T cell that divided twice
stained for either total STAT5 or phosphorylated STAT5 using 1 ␮g of during the primary response generated 10 daughters during sec-
specific primary Ab for 1 h at room temperature. Cells were washed four ondary clonal expansion.
2434 CELL DIVISION AND ANERGY AVOIDANCE BY T CELLS

FIGURE 1. Quantitative assessment of

secondary T cell proliferative responses as
a function of prior cell division. CFSE-
labeled BALB/c T cells were stimulated
with soluble anti-CD3 Ab (2 ␮g/ml) for 4
days, washed, and rested in fresh medium
for 48 h. A, On day 6, Thy-1.2⫹ cells were
sorted based on CFSE fluorescence into
discrete populations corresponding to 0, 1,
or 2 rounds of cell division. A total of 2 ⫻
104 sorted T cells was cultured with 4 ⫻
104 irradiated syngeneic splenocytes in

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medium alone (B) or in the presence of
anti-CD3 Ab (1 ␮g/ml) (C), and cell di-
vision by the CD4⫹ T cell subset was as-
sessed 4 days later by flow cytometry. The
division profiles depicted in B and C were
used to calculate the frequency of CD4⫹
T cells that proliferated (D), as well as the
absolute number of mitotic events accu-
mulated (E) in response to medium alone
(䡺) or anti-CD3 (F). Values are shown
graphed as a function of primary division
cycle. The results shown are representa-
tive of six independent experiments.

Therefore, we find that both the probability that an individual tivation are subsequently unable to produce IL-2. This defect is a
CD4⫹ T cell will participate in the secondary phase of clonal ex- hallmark of T cell clonal anergy (15) and could possibly explain
pansion, and the number of rounds of cell division it subsequently the inability of the undivided cells to proliferate in response to
achieves are linked to its proliferative behavior during the primary restimulation.
response.
The integrity of TCR-coupled Ras-Raf-mitogen-activated protein
Relationship between primary proliferation and cytokine kinase (MAPK) activation is associated with prior cell division
production The failure of the undivided CD4⫹ T cells to produce IL-2 and
To explore the potential basis for the observed relationship be- proliferate in response to mitogenic anti-CD3 Abs, despite normal
tween primary and secondary proliferative behavior, we analyzed expression of the ␣␤ TCR (data not shown), suggested a biochem-
the production of IL-2 by individual CD4⫹ T cells as a function of ical defect in TCR-coupled signal transduction. As a first step to
their primary division status. CFSE-labeled splenocytes were localize this defect, we examined TCR-proximal signals involved
primed as described above, and then briefly (5 h) restimulated by in the elevation of [Ca2⫹]i following engagement of TCR/CD3.
coligation of TCR and CD28, after which cytokine production was All T cells exhibited an increase in [Ca2⫹]i in response to CD3
assessed by flow cytometry. As was the case for proliferation, the ligation (Fig. 3A), and the relative increase in [Ca2⫹]i was com-
frequency of CD4⫹ T cells that produced IL-2 upon secondary parable in T cells with varied proliferative history (Fig. 3B). The
TCR ligation increased with each successive division cycle (Fig. 2, ability of the nonproliferative cells to flux calcium and secrete
A and C), such that while only 3% of undivided CD4⫹ T cells IFN-␥ (Fig. 2) in response to TCR ligation emphasizes that these
could secrete IL-2, 12% of those cells that had divided three times cells are not globally unresponsive.
were able to produce IL-2. We have observed a similar relationship Next, we examined the Ras-coupled, Raf-dependent mitogen-
between IL-2 production and cell division in an MHC class II- activated protein kinase (Ras-Raf-MAPK) pathway, a particularly
restricted, TCR-transgenic T cell model, in which the frequency of important TCR-coupled signaling cascade involved in several as-
IL-2 producers is ⬍5% within the undivided Ag-specific CD4⫹ T pects of T cell activation, including IL-2 production (16 –20).
cell pool, but increases with each cell division such that up to 80% CFSE-labeled splenocytes were primed as described above, and
of the cells that have divided five or six times are able to produce the CD4⫹ T cells were sorted into a fraction that had divided one
IL-2 (9). This trend in cell division-associated IL-2 production or more times, and into another fraction that had remained undi-
does not generalize to all cytokines in this in vitro model, as all vided throughout primary activation. The cells were then restim-
primed CD4⫹ T cells exhibited a similar capacity to secrete IFN-␥, ulated with polystyrene beads coated with anti-CD3 Ab, and phos-
regardless of their previous proliferative behavior (Fig. 2, B and phorylation of the p42/44 extracellular signal-regulated kinase
D). These results suggest that T cells that fail to divide after ac- (ERK1/2) was analyzed as a reliable and quantitative indicator of
The Journal of Immunology 2435

FIGURE 2. Assessment of IL-2 and IFN-␥

production by CD4⫹ T cells as a function of
prior division history. Primary, CFSE-labeled T
cells were stimulated and rested as described in
Fig. 1, and intracellular cytokine expression was
detected by flow cytometry with PE-conjugated
anti-IL-2 (A) or anti-IFN-␥ (B) Abs. Cytokine
expression is shown as a function of CFSE flu-
orescence. CFSE fluorescence is shown also as
a frequency histogram above each plot. The dot-
ted lines in each plot represent the maximal flu-
orescence of CD4⫹ T cells stimulated, fixed,
and permeabilized as above, but stained with
PE-conjugated isotype control Ab. The data in A

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and B are also shown plotted as the specific pro-
portion of CD4⫹ T cells in each division secret-
ing IL-2 (C) or IFN- ␥ (D) following restimu-
lation with medium alone (E) or plate-bound
Abs (F). Background positive events, as defined
by isotype control staining (⬍1.5%), were sub-
tracted from the anti-cytokine Ab-stained sam-
ple values to obtain the specific proportion of
cytokine-positive CD4⫹ T cells. The data
shown are representative of two independent
experiments.

the activation of the Ras-Raf-MAPK pathway (21). The total levels ciently coupled to the TCR. Because maximal signals through
of both ERK isoforms (ERK 1 and 2) were reduced ⬃4-fold TCR and CD28 cooperated to overcome the defect in ERK acti-
(ERK1) and 2-fold (ERK2) in undivided CD4⫹ T cells compared vation, and because CD28 costimulation is also important for the
with the levels in divided CD4⫹ T cells (Fig. 4, A and B, upper activation of other signal transduction pathways required for op-
panels). In addition, we observed a ligand density-dependent de- timal IL-2 production and proliferation (28, 29), we tested whether
fect in the capacity to activate these ERK species in the undivided coligation of TCR and CD28 by agonistic Abs could overcome the
compared with the divided CD4⫹ T cell pool. In response to re- proliferative defect exhibited by previously undivided CD4⫹ T
stimulation with a relatively high density of TCR ligand (1 ␮g/ml cells. While the combination of agonistic anti-CD3 and anti-CD28
anti-CD3), the undivided CD4⫹ T cells exhibited a 3.5-fold re- Abs induced a nearly 4-fold expansion in the pool of previously
duction in the relative activation of ERK1 compared with the ac- divided cells, these signals failed to restore efficient clonal expan-
tivation of this isoform in divided cells, but ERK2 was activated to sion by the undivided CD4⫹ T cells (in this experiment, 20,000 T
a comparable degree (Fig. 4A, middle panel). At half this ligand cells gave rise to 76,100 mitoses within the previously divided
density (0.5 ␮g/ml anti-CD3), the defect was more profound: ac- pool vs 10,660 within the undivided pool; a 7-fold difference) (Fig.
tivation of ERK1 was not detected in the undivided CD4⫹ T cells, 4C). This is consistent with the fact that coligation of CD28 and
and ERK2 activation was reduced ⬃4-fold compared with the ac- TCR did not result in IL-2 production by the undivided cells (Fig.
tivation of this isoform in the divided cells (Fig. 4A, lower panel). 2). These results suggest that an additional defect(s) exists either
Therefore, we find that CD4⫹ T cells that fail to proliferate in downstream of ERK, or more likely, within a signal transduction
response to primary stimulation suffer from a quantitative defect in pathway distinct from the Ras-Raf-MAPK pathway, and that this
TCR-coupled activation of the Ras-Raf-MAPK pathway. This sig- pathway is required for T cell proliferation.
naling defect has been described previously in anergic T cell
clones (22–25), and could explain the inability of the undivided Nonproliferative CD4⫹ T cells are unable to use IL-2 as a
cells to produce IL-2. growth factor
Signals emanating from the CD28 costimulatory receptor nor- The inability of the previously undivided CD4⫹ T cells to activate
mally act in part to amplify TCR-coupled signals, including the the Ras-Raf-MAPK cascade, produce IL-2, and proliferate in re-
Ras-Raf-MAPK pathway (26, 27). Therefore, we examined sponse to restimulation constitutes a phenotype that is remarkably
whether provision of maximal Ag and costimulatory receptor similar to that of anergic T cell clones. However, another hallmark
cross-linking (10 ␮g/ml anti-CD3 and anti-CD28) could induce of T cell clonal anergy is that it can be readily reversed by the
maximal ERK activation in nonproliferative CD4⫹ T cells. Inter- addition of IL-2 (30). Therefore, we examined whether the hy-
estingly, coligation of TCR and CD28 induced comparable ERK poproliferative phenotype of the undivided CD4⫹ T cells could be
phosphorylation in both divided and undivided T cells (Fig. 4B, reversed by the addition of exogenous IL-2. Previously divided or
lower panel). This result suggests that the diminished activation of undivided CD4⫹ T cells were restimulated by TCR ligation in the
ERK in the undivided cells is not due to an intrinsic defect in the presence or absence of exogenous IL-2 (Fig. 5A). In response to
Ras-Raf-MAPK cascade, but rather that this pathway is ineffi- TCR ligation alone, the previously divided and undivided CD4⫹ T
2436 CELL DIVISION AND ANERGY AVOIDANCE BY T CELLS

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FIGURE 4. Assessment of MAPK phosphorylation by divided vs un-
divided CD4⫹ T cells following secondary receptor cross-linking. Primary,
CFSE-labeled T cells were stimulated and rested, as described in Fig. 1,
FIGURE 3. Kinetic analysis of TCR-coupled [Ca2⫹]i flux as a function and the CD4⫹ T cell population was sorted into a divided pool and an
of cell division cycle. Primary, CFSE-labeled T cells were stimulated and undivided pool. The cells were restimulated for 10 min with polystyrene
rested, as described in Fig. 1. A, The primed cells were then stained with beads coated with anti-CD3 (A) or anti-CD3 and anti-CD28 (B), and lysates
biotinylated anti-CD3 (1 ␮g/ml) and loaded with Fura Red, and resting were analyzed by immunoblotting for total ERK and phospho-ERK, as
[Ca2⫹]i was assessed by flow cytometry. During each kinetic experiment, described in Materials and Methods. The densitometric ratios of the phos-
TCR cross-linking was achieved by the addition of streptavidin (0.2 ␮g/ml; phorylated ERK to total ERK species for A are as follows: ERK1 undi-
solid arrow), and maximal [Ca2⫹]i flux was then induced by the addition of vided, 1 ␮g; 0.24, ERK1 divided, 1 ␮g; 0.82, ERK2 undivided, 1 ␮g; 3.05,
ionomycin (1 ␮M; open arrow). The plot shown is gated on the entire, live ERK divided, 1 ␮g; 2.9, ERK1 undivided, 0.5 ␮g; undetectable, ERK1
CD4⫹ T cell population. B, [Ca2⫹]i flux by CD4⫹ T cells was analyzed as divided, 0.5 ␮g; 0.73, ERK2 undivided, 0.5 ␮g; 0.44, ERK2 divided, 0.5
a function of time and division cycle by gating on discrete CD4⫹ CFSE ␮g; 1.7. Extracts from unstimulated T cells contained no phosphorylated
populations, corresponding to cells that divided 0, 1, 2, or 3 times during ERK (data not shown). C, Assessment of secondary proliferation in re-
the primary stimulation, and comparing the median traces of each popu- sponse to TCR and CD28 coligation. Sorted T cells that had divided two
lation over time in response to TCR cross-linking (solid arrow), followed times (u) or had remained undivided throughout primary stimulation (䡺)
by ionomycin (open arrow). Similar results were obtained when CD3 and were cultured with irradiated syngeneic splenocytes in medium, with anti-
CD28 were cocross-linked (data not shown), and experiments in which CD3 (1 ␮g/ml), or anti-CD3 and anti-CD28 (1 ␮g/ml each). Four days
[Ca2⫹]i flux was measured ratiometrically using the calcium probe Indo-1 later, the absolute number of mitotic events accumulated by each sorted
AM also generated similar results (data not shown). The data shown are CD4⫹ T cell population was quantified by flow cytometry. The data shown
representative of six independent experiments. are representative of three separate experiments.
The Journal of Immunology 2437

FIGURE 5. Assessment of high-affinity IL-2R ex-

pression and IL-2 utilization by CD4⫹ T cells as a
function of prior division history. A, Primary CFSE-
labeled T cells, stimulated and rested as in Fig. 1,
were sorted into fractions that had divided twice (u)
or had remained undivided during primary activation
(䡺). The cells were then cultured with irradiated, syn-
geneic splenocytes in the presence of either anti-CD3
Ab (1 ␮g/ml), IL-2 (15 U/ml), or anti-CD3 and IL-2.
Four days later, the absolute number of mitotic events
accumulated by the CD4⫹ T cell subset was quanti-
fied by flow cytometry. The data shown are represen-
tative of three independent experiments. B, Primary
CFSE-labeled T cells were primed, rested, and then
restimulated with anti-CD3 and irradiated splenic ac-
cessory cells. Cultures were harvested after 24 h, and
CD25 expression on the CD4⫹ T cell subset was as-

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sessed by flow cytometry as a function of CFSE flu-
orescence. No further division of CD4⫹ T cells was
observed during this 24-h period. The data shown are
representative of two independent experiments. C, In
a separate experiment, primary CFSE-labeled T cells
were primed, rested, and sorted into fractions that had
divided twice (right panel) or had remained undivided
(left panel). The sorted cells were then cultured in
medium with (solid lines) or without (dashed lines)
exogenous IL-2 (20 U/ml). After 24 h, CD25 expres-
sion was assessed by flow cytometry. The data shown
are representative of two independent experiments.

cell pools exhibited differences in responder frequency and prolif- the undivided CD4⫹ T cell pool suffers from a biochemical de-
erative capacity similar to those shown in Fig. 1, which together fect(s) in signal transduction downstream of IL-2R.
translated to a greater than 20-fold difference in the number of total Two major IL-2R-coupled signaling pathways have been re-
mitotic events. The previously undivided CD4⫹ T cells were like- ported to be absolutely necessary for the transduction of IL-2-
wise refractory to IL-2 alone. Surprisingly, even the combination mediated proliferative signals in T cells. One pathway involves the
of TCR or TCR/CD28 ligation and exogenous IL-2, which induced Janus kinase (JAK)3-mediated activation of the transcription fac-
a 30-fold expansion of the previously divided T cell pool, failed to tor STAT5 (31), while the other pathway involves the phosphati-
induce comparable expansion of the previously undivided popu- dylinositol-3 kinase (PI3K)-mediated activation of protein kinase
lation (Fig. 5A). Therefore, the apparent link between primary T B (PKB), also known as Akt (32). Interestingly, up-regulation of
cell proliferative behavior at the single cell level and subsequent CD25 in response to IL-2, which occurs normally in both the di-
proliferative responsiveness upon reactivation could be explained vided and undivided T cell populations (Fig. 5, B and C), requires
by the fact that the capacity of an individual CD4⫹ T cell to not the functional activity of STAT5 (33). This suggests that the
only produce IL-2 (Fig. 2), but also to respond to IL-2 (Fig. 5), is JAK3/STAT5 pathway is not compromised in the undivided
quantitatively associated with cell division. The inability of the CD4⫹ T cell pool. To confirm this biochemically, we assessed the
undivided cells to respond to IL-2 represents a distinction between activation of STAT5 in IL-2-stimulated CD4⫹ T cells by measur-
this division-associated hyporesponsive state and anergy in T cell ing STAT5 phosphorylation. A relatively large amount of total,
clones. intracellular STAT5 could be detected by flow cytometry in all
CD4⫹ T cells (Fig. 6A). Specific phosphorylation of STAT5 could
The integrity of IL-2R-coupled signal transduction is influenced likewise be detected, in an IL-2-dependent manner, in all CD4⫹ T
by prior cell division cells (Fig. 6A). Quantitative analysis of phospho-STAT5 content
To understand the basis of the association between cell division as a function of cell division (Fig. 6, B and C) showed that all
and IL-2 responsiveness, we first assessed the expression of IL-2R CD4⫹ T cells could activate STAT5 to a similar degree in response
chains on CD4⫹ T cells of varying proliferative histories. The to IL-2, regardless of their proliferative behavior during the pri-
majority of primed CD4⫹ T cells, regardless of prior division his- mary stimulus. These data suggest that the inability of the previ-
tory, were induced to express IL-2R ␣-chain (CD25) upon TCR ously undivided CD4⫹ T cells to proliferate in response to IL-2
reengagement (Fig. 5B, F), although the relative level of CD25 per results from a biochemical defect in a pathway distinct from the
cell was successively higher on cells that had undergone more JAK3/STAT5 pathway. To test the integrity of the PI3K/Akt path-
rounds of cell division (Fig. 5B, E). Flow cytometric analysis of way as a function of cell division, we compared the ability of
IL-2R ␤-chain (CD122) and ␥c-chain expression on primed CD4⫹ divided vs nondivided CD4⫹ T cells to down-regulate the cyclin-
T cells showed no significant differences as a function of division dependent kinase inhibitor p27kip1 in response to IL-2. IL-2-me-
cycle (data not shown). Furthermore, both divided and undivided diated down-regulation of p27kip1 is crucial for the progression of
CD4⫹ T cells were able to up-regulate CD25 in response to IL-2 T cells from the G1 phase to the S phase of the cell cycle (34), and
(Fig. 5C). Therefore, the differential responsiveness of the divided activation of the PI3K/Akt pathway is both necessary and sufficient
vs the undivided CD4⫹ T cells to IL-2 cannot be explained simply to achieve p27kip1 down-regulation in T cell lines (35). Interest-
by the presence vs absence of high-affinity IL-2R, suggesting that ingly, we found that while those CD4⫹ T cells that participated in
2438 CELL DIVISION AND ANERGY AVOIDANCE BY T CELLS

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FIGURE 6. IL-2R-coupled STAT5 activation and p27kip1 down-regulation in CD4⫹ T cells as a function of division cycle. A, Primary CFSE-labeled
T cells were primed, rested, and then cultured for 10 min in the presence of 100 U/ml IL-2. The cells were fixed, permeabilized, and stained for either total
STAT5 (solid black line) or phosphorylated STAT5 (solid gray line). The dashed gray line depicts the phospho-STAT5 content of CD4⫹ T cells cultured
in medium alone for 10 min, and the dashed black line depicts the isotype control staining of the IL-2-stimulated CD4⫹ T cells. B, Density plot of
phospho-STAT5 vs CFSE content in the IL-2-stimulated CD4⫹ T cells. C, Quantitation of phospho-STAT5 relative to total STAT5 content in unstimulated
(E) and IL-2-stimulated (F) CD4⫹ T cells. The pSTAT5/STAT5 ratio was calculated by dividing the phospho-STAT5 MFI/isotype control MFI ratio by
the total STAT5 MFI/isotype control MFI ratio. The data shown are representative of two independent experiments. D, Primary, CFSE-labeled T cells were
primed with anti-CD3, rested, and restimulated with 50 U/ml IL-2 for 48 h. The live, CD4⫹ cells were then sorted into fractions that had divided two or
more times (right lane), or had remained undivided during the culture period (left lane). The cells were lysed, equal cell equivalents were subjected to
SDS-PAGE, and p27kip1 content was assessed by immunoblot analysis (top panel). The blot was then stripped and reprobed for actin as a loading control
(lower panel). Although the divided and undivided subsets contained similar levels of p27kip1 before restimulation (see Fig. 7), the relative level of p27kip1
(normalized to actin) in the undivided CD4⫹ T cell fraction following IL-2 stimulation is nearly 15-fold higher than the level of p27kip1 in the divided CD4⫹
T cell fraction. The data shown are representative of two independent experiments.

clonal expansion during the primary stimulus contained very little cade from the Ag receptor by directly activating PKC (36 –38).
p27kip1 after restimulation with IL-2 (Fig. 6D, right lane), CD4⫹ Receptor-independent activation of PKC using PMA resulted in
T cells that failed to divide were unable to down-regulate this cell comparable ERK1/2 phosphorylation in both divided and undi-
cycle inhibitor (Fig. 6D, left lane). These results suggest that the vided CD4⫹ T cells (Fig. 7A). PKC also lies downstream of PI3K
inability of the undivided CD4⫹ T cells in this model may be due in the IL-2R signal transduction pathway (39, 40), and is normally
to a defect in IL-2R-coupled signal transduction involving the activated in response to IL-2 (41). To test whether receptor-inde-
PI3K, PKB/Akt pathway. pendent activation of PKC could overcome the defect in IL-2-
mediated p27kip1 down-regulation exhibited by the undivided
Receptor-independent activation of protein kinase C (PKC)- and CD4⫹ T cell subset, we restimulated primed cells with PMA and
Ca2⫹-mediated signal transduction pathways restores MAPK ionomycin instead of IL-2. This combination of phorbol ester and
activation, p27 kip1 down-regulation, and proliferative capacity calcium ionophore was able to induce comparable p27kip1 down-
of the previously undivided CD4⫹ T cell subset regulation in both the divided and the undivided CD4⫹ T cells
The data above suggest that the integrity of both TCR-coupled and (Fig. 7B). We next compared the ability of the divided vs the
IL-2R-coupled signal transduction pathways is linked to cell divi- undivided CD4⫹ T cells to proliferate upon restimulation with
sion, and that activated T cells that fail to divide suffer from de- anti-CD3 vs PMA/ionomycin. As seen before, the previously un-
fects in both these pathways that render them unable to proliferate divided CD4⫹ T cells exhibited little proliferation in response to
upon further mitogenic stimulus. We next tested whether these TCR engagement compared with the divided population (Fig. 7C).
functional defects could be overcome by bypassing T cell surface However, the combination of PMA and ionomycin was able to
receptors. The combination of the phorbol ester PMA and the cal- stimulate a large and equal degree of proliferation by both the
cium ionophore ionomycin can uncouple the Ras-Raf-MAPK cas- divided and the undivided subsets (Fig. 7C). These results suggest
The Journal of Immunology 2439

more likely to produce IL-2, and respond with a greater degree of

proliferation, upon restimulation. Furthermore, we show that the
integrity of both TCR- and IL-2R-coupled signal transduction
pathways is quantitatively tuned to cell division, such that the ac-
tivated cells that fail to divide are unable to respond to receptor-
coupled mitogenic signals. The failure of this subset of CD4⫹ T
cells to participate in the primary clonal expansion phase is not due
to limitations in CD28 costimulation or IL-2, as a similar propor-
tion of the activated CD4⫹ T cells fails to divide whether or not
agonistic anti-CD28 or exogenous IL-2 is included in the primary
cultures (8). Furthermore, CD4⫹ T cells that remain undivided
following primary stimulation in the presence of agonistic anti-
CD28 Ab are also unable to proliferate in response to restimulation
(our unpublished results). Finally, primary stimulation of T cells
with PMA and ionomycin does not induce all of the activated T

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cells to proliferate, suggesting that at least a significant component
of this pool fails to divide despite sufficient receptor-proximal sig-
nal transduction (8).
An association between cell division and cytokine production
has been observed previously during the primary phase of clonal
expansion in CD4⫹ T cells in vitro (10, 11) and in vivo (9), leading
to the hypothesis that multiple rounds of cell division may be re-
quired to render chromosomal loci associated with T cell effector
function accessible to specific transcription factors induced during
earlier activation (10, 12, 42). Our data show that this division-
associated trend in T cell effector differentiation (i.e., cytokine pro-
duction and proliferation) is stably maintained within the pool of
relatively long-lived cells present after a period of primary stim-
ulation and rest in vitro, and is again operative when these cells
reencounter Ag.
We show that those CD4⫹ T cells that fail entirely to divide in
response to primary activation exist in a hyporesponsive state that
is refractory to TCR ligation, and is associated with a quantitative
FIGURE 7. PMA and ionomycin induce normal MAPK activation, defect in TCR-coupled Ras-Raf-MAPK signal transduction. A rest
p27kip1 down-regulation, and proliferation in the previously undivided period of at least 2 days following primary stimulation is required
CD4⫹ T cell subset. A, Primary CFSE-labeled T cells, stimulated and for the development of this MAPK defect (our unpublished obser-
rested as above, were sorted into fractions that had divided twice (right vations), suggesting either that this defect arises only after cessa-
lane), or had remained undivided during primary activation (left lane). The
tion of primary signal transduction, or possibly that cell death due
cells were then restimulated by the addition of PMA and ionomycin for 10
to growth factor withdrawal may select for cells that exhibit this
min, and the phospho-ERK content of the cell lysates was assessed as in
Fig. 4. B, In a separate experiment, primed and rested cells were sorted by phenotype. However, this defect is selective, as although TCR-
division cycle and either assessed immediately for p27kip1 content (upper coupled MAPK activation is attenuated in these cells, the TCR-
panel; resting), or were restimulated with PMA and ionomycin for 48 h coupled calcium response appears intact. In normal T cells, the
before assessment of p27kip1 content (middle panel; PMA/IONO). The blot most TCR-proximal events following Ag recognition are initiated
was stripped and reprobed for actin as a loading control (lower panel). by the nonreceptor tyrosine kinases Lck and ZAP-70 (43). The
C, In a separate experiment, cells were primed, rested, and sorted as above, Ras-Raf-MAPK cascade is coupled to the TCR via adaptor mol-
and the undivided (䡺) and divided (u) fractions were restimulated for 4 ecules such as linker of activated T cells (LAT), SH2 domain-
days with either anti-CD3 Ab (1 ␮g/ml) or PMA/ionomycin (3 ng/ml and containing leukocyte protein of 76 kDa (SLP-T6), and Grb2/Sos
250 ␮M, respectively). The data shown are representative of two indepen-
(44), and involves initial events mediated by the SH3 domain of
dent experiments.
Lck (45). TCR-induced increase in [Ca2⫹]i, which is mediated by
phosphatidylinositol, is dependent on the activation of PLC-␥, and
is coupled to the TCR through ZAP-70 and Rho/Vav (45, 46).
TCR-coupled PLC-␥ activation, generation of phosphatidylinosi-
that receptor-independent activation of PKC is able to bypass the tol, and [Ca2⫹]i elevation are not dependent on the SH3 domain of
defect in PI3K-mediated signal transduction normally exhibited by Lck (45). The selective defect in MAPK activation, but not PLC-␥
the undivided CD4⫹ subset. activation, in the undivided CD4⫹ T cells suggests a defect in the
coupling of the SH3 domain of Lck to the Ras-Raf-MAPK path-
Discussion way. Also, we observe in this study that signal transduction
Previously, we have found that even with optimal TCR ligation through the costimulatory receptor CD28, which has been shown
and CD28 costimulation, a large proportion of the activated (i.e., to augment TCR-coupled ERK activation in normal T cells (27),
CD25⫹CD69⫹) T cells fails to divide both in vitro (8) and in vivo and phorbol ester, which bypasses the most proximal TCR-coupled
(9). We demonstrate in this work that individual, primed CD4⫹ T events through the direct activation of PKC, both restored maximal
cells exhibit distinct secondary response patterns that depend upon activation of the MAPK cascade in the undivided T cells. This also
their prior division history. Specifically, CD4⫹ T cells that un- suggests that the defect is not in the MAPK pathway itself, but
dergo more rounds of cell division during primary stimulation are rather is in a pathway that couples Ras-Raf-MAPK to the TCR.
2440 CELL DIVISION AND ANERGY AVOIDANCE BY T CELLS

PI3K, Raf, and mitogen-activated protein/extracellular signal-re- liferate in response to restimulation with IL-2, suggesting that
lated kinase kinase are capable of binding to the SH3 domain of PI3K and/or PKB/Akt are not coupled properly to the IL-2R, or
Lck (47– 49), suggesting that uncoupling of these factors from Lck that another biochemical step(s) leading to p27kip1 is not coupled
in undivided cells could result in this selective defect in MAPK properly to PKB/Akt in these cells. In this sense, the defect in IL-2
activation. The ability of PMA/ionomycin to induce proliferation responsiveness exhibited by the nonproliferative CD4⫹ T cell sub-
in these cells (see below) may also suggest that PKC-mediated set resembles the phenotype of T cells treated with the pharmaco-
activation of the NF-␬B pathway, which is required for the pro- logic agent rapamycin (34), which blocks IL-2R signal transduc-
duction of IL-2, could be defective in these cells. Alternatively, tion by binding to mammalian target of rapamycin (mTOR)/
such negative regulatory factors as Ras-GAP and c-Cbl also bind FK506-binding protein-rapamycin-associated protein (FRAP)
to SH3 domain of Lck (50, 51), suggesting that selective coupling (58), a downstream target of PKB/Akt.
of these factors to the TCR in undivided cells, but not divided Interestingly, receptor-independent activation of PKC in the un-
cells, could likewise explain this selective MAPK defect. Differ- divided CD4⫹ T cells using phorbol ester and calcium ionophore
ential coupling of negative regulatory molecules to the TCR has resulted in efficient down-regulation of p27kip1 and normal prolif-
been demonstrated previously in anergic human T cell clones, in erative capacity. These results suggest that the IL-2 refractory phe-
which the TCR is coupled not to Ras, but to Rap1 (25), a Ras notype of the previously undivided CD4⫹ T cells is due to a bio-

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homologue that binds Raf, but is unable to activate the MAPK chemical defect that lies somewhere upstream of the action of PKC
cascade. Interestingly, the Ras-Raf-MAPK defect exhibited by the within the PI3K-PKB/Akt pathway. PKC is normally coupled to
undivided cells in this model appears to preferentially affect the the IL-2R through PI3K, as PKC is activated by PDK1-dependent
ERK1 isoform, as TCR-coupled activation of ERK2, but not phosphorylation (40), and a role for PKC in IL-2-mediated signal
ERK1, remains relatively intact. The presence of a significant transduction has been defined using the IL-2-dependent cell line
amount of activated ERK2 in the undivided pool following TCR CTLL (59, 60). Interestingly, full activation of the PKC-␦ isoform
ligation might suggest that the Ras-Raf-MAPK pathway is not sig- requires the activity of mTOR/FRAP (61), which is in turn depen-
nificantly compromised in these cells; however, recent studies in dent on PKB/Akt (62). These two distinct PI3K-dependent path-
ERK1⫺/⫺ mice have defined an obligatory role for ERK1 in T cell ways leading to PKC activation, which can be bypassed using
activation that cannot be fulfilled by ERK2 (52). Together, these phorbol esters, represent a set of biochemical events that could
results support a model in which a selective defect in ERK1 might potentially be defective in the undivided pool. Additionally, PKC
result in a highly attenuated response to TCR ligation. can phosphorylate other downstream targets of PKB/Akt, includ-
The inability of maximal TCR-coupled calcium and Ras-Raf- ing GSK-3 and cAMP response element binding protein (56).
MAPK signaling to overcome the proliferative defect in the undi- Therefore, the defect in IL-2-mediated signal transduction exhib-
vided CD4⫹ T cells suggests the presence of further defect(s) in ited by the undivided CD4⫹ T cells in this model might result
growth factor-mediated signal transduction. Unlike the defect in specifically from the failure to activate downstream targets of
TCR-coupled MAPK activation, a rest period following primary PKB/Akt or PKC.
stimulation is not required for the development of this IL-2 refrac- The phenotype of the CD4⫹ T cells that fail to divide following
tory phenotype (our unpublished observations). CD4⫹ T cells from primary activation, i.e., an inability to secrete IL-2 and proliferate
both the divided and undivided pools were able to express all com- upon TCR reengagement, closely resembles that of clonal anergy,
ponents of the high-affinity IL-2R in response to both TCR en- a phenomenon normally associated with TCR occupancy in the
gagement and IL-2 itself, demonstrating that the inability of the absence of CD28 costimulation (15, 63, 64). The cell division-
undivided subset to proliferate is not due to a lack of growth factor associated hyporesponsive state that we observe also shares sim-
receptor expression. This suggests that the integrity of signal trans- ilarities with clonal anergy at the molecular level. Similar to the
duction from the IL-2R must be coupled to cell division. Two undivided CD4⫹ T cells described in this work, both mouse and
major IL-2R-coupled signaling pathways have been reported to be human T cell clones rendered anergic by TCR engagement in the
absolutely necessary for the transduction of IL-2-mediated prolif- absence of CD28 costimulation exhibit a normal increase in intra-
erative signals in T cells. One pathway, which is specifically cou- cellular calcium following TCR engagement (15), but suffer from
pled to the ␥c-chain of the IL-2R, involves the JAK3-mediated quantitative defects in Ras-coupled MAPK activation (22–25).
activation of the transcription factor STAT5 (31), while the other Also, the cyclin-dependent kinase inhibitor p27kip1, which we
pathway is coupled to the IL-2R ␤-chain and involves the PI3K- show is highly elevated in the CD4⫹ T cells that fail to divide after
mediated activation of PKB/Akt (32). All T cells were able to activation, has recently been shown to function as a molecular
phosphorylate STAT5 in response to IL-2 stimulation, regardless anergy factor in human T cell clones stimulated in the absence of
of their prior division history, suggesting that the defect in IL-2R- CD28 costimulation (65). In addition, the authors discovered that
coupled signal transduction does not reside in this JAK/STAT p27kip1 could actively inhibit IL-2 gene transcription by seques-
pathway. PI3K is an integral component of many growth factor tering the AP-1 coactivator JAB1, an activity not previously at-
receptor signal transduction pathways (53), and is absolutely re- tributed to this cyclin-dependent kinase inhibitor. Therefore, the
quired for normal T cell responses (54). D3 phosphoinositides gen- inability of the undivided CD4⫹ T cells in our model to down-
erated by PI3K recruit PKB/Akt and phosphoinositide-dependent regulate p27kip1 in response to IL-2 could explain not only why
kinase-1 (PDK1) to the cell membrane, in which PDK1 phosphor- these cells are unable to progress from the G1 phase to the S phase
ylates and activates PKB/Akt (55). The PKB/Akt kinase is also a of the cell cycle, but when paired with the coincident defect in
crucial signal transduction component for many growth factor re- TCR-coupled ERK1 activation, could also explain why these cells
ceptors (56, 57), and has been shown to be both necessary and are unable to produce IL-2 as well. Finally, the combination of
sufficient for the down-regulation of p27kip1, hyperphosphorylation phorbol ester and calcium ionophore, which was able to overcome
of the retinoblastoma tumor suppressor protein, and release of ac- the molecular and functional defects exhibited by the undivided T
tive E2F in T cell lines (35). However, the mechanism by which cells in this study, has been shown to reverse anergy in T cell
PKB/Akt activity leads to these downstream effects is unclear. Our clones (15).
studies show that CD4⫹ T cells that fail to divide following acti- While these two refractory states share many functional and
vation are neither able to down-regulate p27kip1 nor able to pro- biochemical similarities, they differ in at least two respects. As
The Journal of Immunology 2441

mentioned above, classical T cell clonal anergy results from TCR the peripheral CD4⫹ T cell repertoire with previous antigenic ex-
occupancy in the absence of CD28 costimulation, whereas the di- perience, or that has otherwise been rendered anergic in vivo be-
vision-associated unresponsiveness described in this work occurs fore our analysis. However, several lines of evidence argue against
despite the presence of sufficient CD28 costimulatory signals. Sec- this deterministic model. First, the use of mitogenic Abs against
ond, the failure of anergic T cell clones to proliferate in the models the monotypic CD3 component of the TCR effectively bypasses
described above is due to a defect in the production, but not in the the Ag specificity, and therefore the affinity, of the individual T
utilization, of IL-2. The provision of IL-2 during restimulation in cells. We have also detected this nonproliferative subset in popu-
both models restored clonal responsiveness (30, 65). This is in lations of TCR-transgenic T cells stimulated in vitro and in vivo
contrast to the phenotype of the undivided CD4⫹ T cells described with specific peptide (9). These data suggest that the nonprolifera-
in this work, which not only fail to produce IL-2 during restimu- tive population is not merely a subset of the peripheral repertoire
lation, but are also refractory to IL-2 provided exogenously. This with reduced TCR affinity. We have addressed the possibility that
difference may point to an important distinction between the divi- the nonproliferative pool represents a subset of T cells with pre-
sion-associated, hypoproliferative state and clonal anergy induced vious antigenic exposure by fractionating the peripheral T cell rep-
by TCR occupancy in the absence of costimulation. Alternatively, ertoire of normal mice and D011.10 TCR transgenic mice into
these two phenotypes could arise from analogous biochemical cir- naive and memory subsets based on surface phenotype. In these

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cumstances, and the differential IL-2 responsiveness may reflect experiments, both pools exhibited a similar degree of proliferative
differences in the capacity of IL-2R-coupled signal transduction heterogeneity and gave rise to both divided and undivided cells
pathways (e.g., PI3K or PKB/Akt) to remain poised in primary T after stimulation through TCR/CD28 (our unpublished observa-
cells vs long-term clones. Notably, IL-2 unresponsiveness has also tions). This suggests to us that the nonproliferative pool is not
been observed in T cells from mice chronically treated with staph- comprised preferentially of cells with any given antigenic history.
ylococcal superantigen (66). However, in this case, IL-2 unrespon- Our previous studies using Ag-specific T cells from TCR-trans-
siveness apparently resulted from a defect in ␥c-coupled activation genic Rag2⫺/⫺ mice also argue against this possibility. These cells
of the JAK3-STAT5 pathway. represent a population of purely clonal, naive T cells, and despite
These data show that cell division or a process associated with this, these cells still exhibit marked heterogeneity in proliferative
cell cycle progression controls the ability of a T cell to respond to behavior at the single cell level, and although they exhibit higher
both Ag and growth factor by modulating the integrity of the sig- responder frequencies than T cells from recombination-competent
nals transduced through both the Ras-Raf-MAPK cascade and the mice, the few Rag2⫺/⫺ D011 T cells that fail to divide in response
PI3K, PKB/Akt pathway. This further suggests that activated T to antigenic peptide also fail to produce IL-2 upon restimulation
cells must proliferate to avoid the induction of anergy. The hy- (9). Finally, the studies described above using cell cycle inhibitors
pothesis that cell cycle progression may be required for anergy (butyrate and rapamycin) show that T cells that would normally
avoidance by T cells was originally proposed by Schwartz and proliferate in response to TCR/CD28 stimulation can be rendered
Jenkins (15, 67). These investigators have suggested that CD28 unresponsive by overtly blocking their cell cycle progression. For
costimulation promotes anergy avoidance indirectly by mediating these reasons, we favor a stochastic model in which any given
efficient IL-2 production. In this scenario, it is the subsequent IL- naive T cell has the potential to respond to an antigenic stimulus by
2-driven cell division that allows T cells to escape anergy. Several dividing, but that the probability that any given cell will achieve
lines of evidence, including our results in this study, support this this goal is ⬍1; from our data, we estimate this number to be
model. Partial agonist ligands, which signal through the TCR, but between 0.5 and 0.7. The failure to divide in this scenario fixes the
do not cause proliferation, induce anergy in T cells despite the cell in a state that is refractory to further stimulation. Support for
presence of CD28 costimulatory signals (68). Furthermore, this stochastic model can be observed in our data in this study,
whether a given peptide ligand induces productive activation or which show that even among previously divided cells, a sizeable
anergy correlates with its capacity to induce IL-2 (and prolifera- proportion (⬃25%) fails to divide upon restimulation; i.e., a pure
tion), not earlier signaling patterns such as TCR-␨ phosphorylation population of responders can give rise to both responders and non-
(69). Second, overt blockade of IL-2-mediated signal transduction, responders (see Fig. 1). This indicates that the responsive pheno-
using either anti-IL-2/IL-2R Abs (67) or the pharmacologic agents type can be transient, and leaves open the possibility that random
butyrate (70) or rapamycin (71), induces anergy. Interestingly, pri- events that control gene expression in complex systems may reg-
mary T cells arrested with butyrate during in vitro stimulation with ulate proliferation (72). However, whether the origin of this non-
agonistic anti-CD3 and anti-CD28 Abs are also rendered anergic, responsive population is stochastic or deterministic, its presence is
and like the T cells that naturally fail to divide during primary remarkable, as it represents approximately one-third of the periph-
stimulation, these butyrate-treated cells are also refractory to IL-2 eral CD4⫹ T cell pool in a normal mouse.
(our unpublished results). These results are consistent with previ- The results reported in this work, together with several recent
ous studies using T cells clones; however, not all pharmacological studies (9 –11), suggest that secondary T cell responses are influ-
inhibitors of cell cycle induce anergy in T cells (70, 71) (our un- enced by a mitotic clock (73, 74). Specifically, T cells that fail to
published observations). More specifically, anergy avoidance may divide following activation are refractory to secondary stimulation,
be associated with the G1 to S phase transition, as opposed to while T cells that do proliferate remain responsive. Additionally,
mitosis per se, as drugs that block mitosis, but allow G1 to S phase those cells that progress through many division cycles during pri-
transition (e.g., hydroxyurea), do not induce anergy (71). mary stimulation tend to exhibit better secondary responses than
The use of primary T cells isolated from normal mice has al- those T cells that divide fewer times. Thus, the role of T cell clonal
lowed us to assess the size of the nonresponsive pool within a expansion in generating effective Ag-specific immunity may be
normal T cell repertoire. Surprisingly, this pool represents between 2-fold: The progression of naive T cells through a phase of expo-
30 and 40% of the mature, peripheral T cells in the mouse. Al- nential growth not only increases the frequency of Ag-specific T
though we believe that this population is generated during in vitro cells, but may also ensure that the resultant T cell pool consists
stimulation (see below), because our analysis involves a heteroge- preferentially of those T cells that have undergone multiple rounds
neous starting T cell population, we cannot eliminate the possibil- of cell division and therefore carry the greatest potential to respond
ity that the nonproliferative cells in our study represent a subset of to future antigenic challenge.
2442 CELL DIVISION AND ANERGY AVOIDANCE BY T CELLS

Acknowledgments 26. Avraham, A., S. Jung, Y. Samuels, R. Seger, and Y. Ben-Neriah. 1998. Co-
stimulation-dependent activation of a JNK-kinase in T lymphocytes. Eur. J. Im-
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Cancer Center Flow Cytometry and Cell Sorting Resource for excellent 27. Calvo, C. R., D. Amsen, and A. M. Kruisbeek. 1997. Cytotoxic T lymphocyte
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and Jun NH2-terminal kinase (JNK) activation, but does not affect phosphoryla-
advice concerning calcium experiments, M. Carroll for technical advice
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ligation in T-cell activation: evidence for two signal transduction pathways.
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