T Cells in G1 Provide a Memory-Like
Response to Secondary Stimulation
Ivana Munitic, Philip E. Ryan and Jonathan D. Ashwell
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The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2005 by The American Association of
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References
J Immunol 2005; 174:4010-4018; ;
doi: 10.4049/jimmunol.174.7.4010
http://www.jimmunol.org/content/174/7/4010
The Journal of Immunology
T Cells in G1 Provide a Memory-Like Response to Secondary
Stimulation
Ivana Munitic, Philip E. Ryan, and Jonathan D. Ashwell1
D
uring the adaptive immune response, T cell priming
takes place in secondary lymphoid organs, where T cells
encounter Ag-bearing dendritic cells (DC)2 that have
migrated from peripheral tissues. T cells whose TCR recognizes its
cognate MHC-peptide ligand become activated and proceed to
proliferate and differentiate into effector and memory populations
(1–3). The specificity of the immune response is ensured by TCR
recognition of only cognate MHC peptide, but because the affinity
of this interaction is very low (typically having a micromolar Kd)
(4, 5), a variety of other molecules are necessary to stabilize and
prolong the contact between the T cell and an APC (6). The area
of contact has been extensively examined by visualizing T cells
bound to APC or MHC-peptide complexes inserted in artificial
lipid bilayers (7, 8). These studies have shown that stochastic T
cell movement in tissue culture comes to a complete stop upon
encountering a surface containing the appropriate MHC peptide (7,
9), and certain T cell molecules redistribute to the site of contact
and form an organized structure called a supramolecular activation
cluster (7) or immune synapse (10, 11).
The requirement for a highly organized molecular interface at
the site of Ag encounter may in part explain the observation that
activation of T cells requires a substantial period of contact with
the APC. In studies using naive CD4⫹ TCR transgenic cells stimulated with plate-bound peptide-MHC II and anti-CD28 (12) or
polyclonal activation with a mixture of anti-CD3 and anti-CD28
(13), at least 20 h was needed to cause T cells to commit to pro-
Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of
Health, Bethesda, MD 20892
Received for publication November 5, 2004. Accepted for publication January
11, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Jonathan Ashwell, Building 37,
Room 3002, 9000 Rockville Pike, National Institutes of Health, Bethesda, MD,
20892. E-mail address: [email protected]
2
Abbreviations used in this paper: DC, dendritic cell; MCC, moth cytochrome c; PP2,
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine.
Copyright © 2005 by The American Association of Immunologists, Inc.
liferate by 72 h. The required period of stimulation can be shortened using APC, in particular very efficient ones like mature DC or
fibroblasts engineered to express high levels of I-Ek, ICAM-1, and
LFA-1 (14). When pulsed with high concentrations of peptide, DC
can induce naive TCR transgenic CD4⫹ T cells to commit to proliferate in 6 h (15). A recent report has shown that even 2 h is
enough for DO11.10 TCR transgenic cells to commit after encountering APC pulsed with very high concentrations of antigenic peptide and then being adoptively transferred into congenic hosts or
maintained on non-Ag-bearing APC in vitro (16). Naive CD8⫹
cells were found to commit to proliferate within 2– 4 h of being
stimulated with artificial APC expressing peptide-MHC I, resulting
in six to seven cycles of division by 72 h (17). Once committed, T
cells will eventually proliferate without further stimulation and
multiply at a constant pace, dividing every 4 – 6 h (6).
Studies using two-photon and confocal technology for visualizing individual T cell:DC interactions in explanted lymph nodes
(18 –20) found that once the majority of T cells recognize peptideloaded DC they stop their “random walks” and linger on the DC
surface for prolonged periods, up to 15 h (19). However, there are
other data indicating that activation and commitment to proliferate
can occur even if a T cell encounters APC transiently but repeatedly (21, 22). For example, using a three-dimensional fibrillar collagen matrix system to mimic normal tissue topology, it was observed that TCR␣␤ transgenic CD4⫹ T cells did not stop moving
once a peptide-loaded DC was encountered but continuously rolled
on the DC surface, at times completely separating from it, later to
interact with the same or another DC (22). In this case, T cell:DC
contact was transient, being on average 6 –12 min. Despite the fact
that the cumulative time of interaction during the first 24 h was on
average less then 2 h, and that long-term stable immune synapses
were never attained, T cell proliferative capacity was preserved.
Another study using two-photon microscopy in lymph nodes of
anesthetized animals suggested that T cell priming occurs in three
distinct phases (23). An initial phase of 8 h includes multiple repetitive short encounters between T cells and DC. During the subsequent 12 h, T cells slow their movements and form longer lasting
contacts with DC. Finally, by the second day, most of the T cells
0022-1767/05/$02.00
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The commitment of naive T cells to proliferate is a function of the strength and duration of stimuli mediated by the TCR and
coreceptors. Ranges of 2–20 h of stimulation have been reported as necessary in vitro. Whether T cells actually experience
uninterrupted stimulation for such long periods under physiological conditions is controversial. Here we ask whether commitment
to proliferate requires continuous stimulation, or can T cells integrate intermittent periods of stimulation. T cells were stimulated
for two short-term (subthreshold) periods (5–7 h) either sequentially or separated by an interval of rest. Naive lymph node T cells
were able to integrate interrupted stimulation, even when the duration of rest was as long as 2 days. Furthermore, when shortterm-stimulated T cells were separated by density, three populations were observed: low density blasts, intermediate density G1
cells, and high density G0 cells. Low density cells progressed to division without further stimulation, whereas G0 and G1 cells
remained undivided. However, after a period of rest, a second subthreshold stimulation caused the G1 but not the G0 fraction to
quickly proceed through the cell cycle. We conclude that noncycling T cells in the G1 phase of the cell cycle remain in a state of
readiness for prolonged periods of time, and may represent a population of memory-like effectors capable of responding rapidly
to antigenic challenge. The Journal of Immunology, 2005, 174: 4010 – 4018.
The Journal of Immunology
Materials and Methods
Mice
C57BL/6 (B6) mice were obtained from Frederick Cancer Research Facility. TCR-Cyt-5CC7-I/Rag2 (5C.C7) mice, specific for pigeon/moth cytochrome c (MCC) presented by I-Ek (26), were purchased from Taconic
Farms. B10.A mice were obtained from The Jackson Laboratory.
Cell lines
P13.9 fibroblasts stably expressing I-Ek, ICAM-1, and B7.1, and the parental cell line DAP.3 that lacks I-Ek, were used as APC (14).
Reagents and Abs
CFSE and Hoechst 33342 were obtained from Molecular Probes. MCC
peptide 88 –103 was purchased from Macromolecular Resources. Purified
anti-mouse CD3⑀ NA/LE (145-2C11), anti-mouse CD28 NA/LE (37.51),
and directly conjugated mAbs for CD4 (RM4.5), CD8a (53-6.7), TCR␤
(H57-597), CD62L (MEL-14), CD44 (IM7), and IL-2 (JES6-5H4) were
obtained from BD Pharmingen. Mouse mAbs to p27Kip1 (F8) and cyclin D3
(D-7) were obtained from Santa Cruz Biotechnology. Propidium iodide,
Tween 20, aprotinin, leupeptin, 4-(2-aminoethyl)benzenesulfonyl fluoride
hydrochloride, sodium azide (NaN3), monensin, and mouse monoclonal
anti-␤-actin (AC-15) were purchased from Sigma-Aldrich. Mouse T cell
and CD4⫹ cell purification columns (Cellect) were obtained from Cedarlane Laboratories. Goat anti-mouse IgG magnetic beads (Dynabeads
M-450) and a magnet for 96-well plates were obtained from Dynal. Percoll,
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2),
pyronin Y, and HRP-linked anti-mouse IgG were purchased from Amersham Biosciences, Calbiochem, and Polysciences, respectively. SuperSignal WestDura Enhancer system was obtained from Pierce.
T cell stimulation and proliferation assays
Lymph nodes and spleens were obtained from 5- to 16-wk-old mice. T cells
were purified by negative selection of B cells on mouse T Cellect columns.
CD4⫹ cells were obtained by mouse CD4 Cellect columns to negatively
deplete of CD8⫹ and B cells. The purity was ⬎98% in both cases. Naive
CD4⫹ (CD4⫹CD44low) and CD8⫹ cells (CD8⫹CD44low) were obtained by
sorting with a FACSVantage cell sorter. In some experiments, naive CD4⫹
cells were enriched from purified CD4⫹ cells by depletion of CD44high
cells by magnetic separation. CFSE labeling was done by incubating cells
for 10 min at 37°C with 1 ␮M CFSE in HBSS with 2% FCS. Residual dye
was washed out with complete medium. For stimulation with Ab, 96- or
24-well plates were coated with anti-mouse CD3 and anti-mouse CD28,
each at concentration of 10 ␮g/ml in PBS. To remove unbound Abs, plates
were washed three times before use. A total of 0.25– 0.3 ⫻ 106 purified T
cells from B6 mice were added in 200 ␮l of RPMI 1640 (Biofluids) sup-
plemented with 10% heat-inactivated FCS, 250 ␮g/ml gentamicin, 100
U/ml penicillin, 4 mM glutamine, and 5 ␮M 2-ME (complete medium). T
cells were stimulated for the indicated periods of time and stimulation was
stopped by transferring them to uncoated wells (27). In restimulation experiments, after the indicated periods of rests in uncoated wells, T cells
were transferred to Ab-coated wells. Unless otherwise indicated, continuously stimulated cells were transferred immediately from one Ab-coated
well to another to control for cell loss during transfer and make them as
similar as possible to discontinuously stimulated cells. For Ag stimulation
experiments, P13.9 and DAP.3 fibroblasts were fed with magnetic beads
for 2–3 days in complete DMEM (Biofluids). Cells that had ingested the
beads were selected by magnet and irradiated to prevent division (40 Gy).
A total of 4 ⫻ 104 fibroblasts were plated per well in flat-bottom 96-well
plates and pulsed with 0.1– 0.5 ␮M MCC88 –103 peptide overnight. Agpulsed fibroblasts were washed three times before before use. A total of
0.25– 0.3 ⫻ 106 5C.C7 T cells were added per well in complete DMEM. To
end the stimulation, fibroblasts were removed by magnetic selection and
the remaining T cells were transferred to empty wells. For restimulation, T
cells were transferred to a new well containing Ag-pulsed fibroblasts. Proliferation was assessed by CFSE dilution. In some experiments, to interrupt
TCR proximal signaling, PP2 was added at a concentration of 10 ␮M.
Cell staining and flow cytometry
Lymph node T cells were stained for surface markers for 45 min on ice in
the presence of Fc␥III/IIR blocking Abs and analyzed either with a FACScan or FACSCalibur (BD Biosciences) in a buffer consisting of HBSS
containing 0.1% sodium azide and 0.5% BSA (FACS buffer). In some of
the experiments with unfixed cells, dead cells were excluded by staining
with propidium iodide. DNA and RNA staining was performed with
Hoechst 33342 and pyronin Y, respectively (28, 29). To do this, cells were
surface stained and then fixed with 5% formaldehyde. These cells were
incubated with 10 ␮g/ml Hoechst 33342 in FACS buffer for 10 min, at
which time pyronin Y was added at a concentration of 4 ␮g/ml for another
30 min. The cells were washed and analyzed with an LSR II (BD Biosciences) using DiVa software. Bandpass filters were 440 ⫾ 40 for Hoechst
33342 and 585 ⫾ 26 for pyronin Y. All data were analyzed with FlowJo
software (Tree Star). Doublet discrimination was applied for cell cycle
analysis. For intracellular cytokine staining, cells were cultured alone or
stimulated with plate-bound anti-CD3/anti-CD28 for 5 h in the presence of
2 ␮M monensin. Following surface staining, intracellular staining was performed using the BD Cytofix/Cytoperm kit according to the manufacturer’s
instructions (BD Biosciences).
Percoll gradient fractionation
Percoll gradient separation was performed as described (30, 31). Briefly, a
discontinuous 50:60:70% Percoll gradient was assembled by underlaying.
Activated or resting CFSE-labeled cells were resuspended in HBSS and
layered onto the gradient. Centrifugation was performed at 3000 rpm for 12
min at 6°C. After centrifugation, cells were retrieved from three interfaces:
low density (above 50% step), intermediate density (50 – 60% interface),
and high density (60 –70% interface).
SDS-PAGE and Western blotting
Cell lysates were prepared in Triton X-100 lysis buffer (1% Triton X-100,
150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM Na2EDTA, 2 ␮g/ml
aprotinin, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride,
and 10 ␮M leupeptin) and loaded (15 ␮g protein/lane) on 15% SDS PAGE
gels. Immunoblotting was performed in TBS (pH 8) with 1% milk and
washed in TBS with 0.1% Tween 20. Blots were incubated with HRPlabeled secondary anti-mouse IgG and developed with SuperSignal WestDura Enhancer (Pierce).
Results
Discontinuous stimulation is similar to continuous stimulation in
driving naive T cell proliferation
We assessed the minimum time needed in our hands to cause resting murine T cells to proliferate when stimulated with plate-bound
anti-CD3 and anti-CD28 Abs. Stimulation was stopped by transferring cells to uncoated plates (12, 27, 32), and proliferation was
assessed 2– 4 days later by CFSE dilution (33). In general, 10 –14
h of continuous stimulation resulted in most of the cells being
equally distributed into four to five CFSE peaks by 3 days (below
and data not shown). Having established minimum continuous
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resume their rapid motility and begin to proliferate. A recent study
has addressed the effect of interrupted stimulation on immune synapse formation in human T cell clones (24). Stimulation was
briefly interrupted by the addition of a Src family kinase inhibitor
for 10 min intervals, and stimulation was restored by washing it
away. Under these circumstances T cell clones could sum intermittent activation signals and respond with the same amount of
IFN-␥ production as cells that were continuously stimulated for the
same period of time. The “serial encounter” model supported by
these results holds that T cells can receive an activating stimulus
even when the interaction with an APC is intermittent (25), and
therefore, T cells must be able to “remember” previous transient
APC contacts and sum up multiple encounters to proceed to
proliferation.
To determine whether the duration of T cell “short-term memory” can be observed in primary resting T cells, we assessed T cell
proliferation under conditions of controlled activation. We find
that T cells that were stimulated continuously for defined periods
of time are comparable in their division potential to cells that were
exposed twice to a subthreshold stimulus with an interval of rest,
which can be remarkably long (days). The results demonstrate that
T cells that progress to, but not beyond, the G1 phase of the cell
cycle are rapidly induced to proliferate upon restimulation, and
thus have acquired a memory-like functional phenotype.
4011
4012
Inhibition of Src family kinases during rest does not prevent
recollection of previous subthreshold stimulation
To ensure that a low level of T cell signaling did not persist during
the interval of rest, proximal TCR-mediated signaling was inter-
rupted during this period with the Src family kinase inhibitor PP2
(34, 35). PP2 was added to the cells at the time of their transfer
from Ab-coated to uncoated wells, and was removed by washing
when the cells were returned to Ab-coated plates. As expected, T
cells stimulated for as long as 26 h in the presence of PP2 did not
proliferate when analyzed 41 h after removing PP2 (Fig. 2A).
CD4⫹ T cells stimulated for 5 h only (S5) did not proliferate (Fig.
2B). Cells that were stimulated for 5 h, rested for 20 h, and then
restimulated for 5 h, in contrast, proliferated extensively by 67 h.
The inclusion of PP2 during the 20-h rest period had no effect on
the extent to which the restimulated cells proliferated. This result
confirms the finding that subthreshold stimulation is “remembered” by T cells after prolonged periods in the absence of TCR
signaling.
T cells integrate discontinuous stimulation with physiologic
ligand
To determine whether integrated responses to discontinuous stimulation occur with a physiologic ligand, APC were used to present
cognate MHC-peptide to T cells of known Ag-specificity. T cells
were obtained from 5C.C7 mice, which are transgenic for an ␣␤
TCR recognizing MCC88 –103 in the context of I-Ek (26). The mice
were also deficient in RAG-2, ensuring that all of the T cells, the
large majority of which are CD4⫹, bore the transgenic TCR. The
fibroblast cell line P13.9, which stably expresses high levels of
I-Ek and the costimulatory molecules ICAM-1 and B7.1 (14, 36),
or its I-Ek-negative parental cell line DAP.3, was used as APC.
The APC were allowed to phagocytose magnetic beads before
their use so that they could be efficiently removed from the T cells
by magnetic separation. MCC88 –103 peptide-pulsed control DAP.3
fibroblasts were cultured with 5C.C7 T cells for up to 18 h, resulting in only a small number of cells that had divided by 67–73
h, thereby providing background values (Fig. 3). Stimulation with
Ag-pulsed P13.9 cells for 6 h resulted in negligible T cell proliferation, but continuous stimulation for 12 h was very effective in
inducing T cell proliferation. When the two periods of stimulation
FIGURE 1. Sorted naive CD4⫹ cells are able to integrate discontinuous stimulation. A, Schematic time-line of stimulation/restimulation experiments.
B, Purified naive (CD44low) CD4⫹ lymph node T cells were obtained by sorting. The CD44 expression profile of total (unfilled histogram) and sorted (filled
histogram) CD4⫹ cells is shown. C, Cells were stimulated on anti-CD3/anti-CD28-coated plates for the indicated periods of time (A) and analyzed by flow
cytometry for CFSE dilution at 62 and 69 h after the beginning of stimulation. Cells were continuously stimulated (S7 and S7/S7) or restimulated after 7and 14-h periods of rest (S7/07/S7 and S7/014/S7). The diagonal lines indicate cells that were analyzed at the same time after the last stimulation ended.
D, The percent of cells from the original population that had divided was calculated with FlowJo software. The data show the fraction of cells in the original
population that had divided by 48 (䡺) or 55 h (f) after stimulation had ceased, and were derived from the data obtained 62, 69, and 76 h (the first two
shown in C) after the experiment had begun. Representative data from one of three experiments is shown.
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stimulation periods required for proliferation, discontinuous periods of stimulation were used to test whether T cells can be integrate interrupted signals over time. Cells were stimulated for 7 h in
Ab-coated wells, and then either transferred to another set of Abcoated wells (S7/S7) or to uncoated wells for 7 h (depicted in Fig.
1A). One set of cells in uncoated wells was incubated without
further stimulation until analysis (S7), while another was subjected
to one more 7-h period of stimulation before finally being returned
to uncoated wells (S7/07/S7). To allow the comparison of groups
whose final stimulation ended at different times, proliferation was
assessed at several analyses that were each offset by 7 h. Sorted
naive CD4⫹ (CD44low) cells (Fig. 1B) were analyzed after 62 h
(Fig. 1C, upper row). Naive CD4⫹ cells stimulated for just 7 h
showed only a small amount of proliferation, whereas cells stimulated for two immediately consecutive 7-h periods (S7/S7) proliferated well. Strikingly, cells that were rested for 7 h between the
two 7-h periods of stimulation proliferated to a similar extent as
those whose stimulation was continuous. CD4⫹ T cells that were
restimulated after 14 h of rest had divided to a lesser extent at 62 h,
but if analyzed 7 h later (69 h) proliferated as well as the cells that
received their final stimulation earlier (compare S7/07/S7 at 62 h
to S7/014/S7 at 69 h). When the time after the final stimulation is
taken into account, the percentage of cells from the initial population that underwent one or more divisions was comparable between cells receiving continuous and those receiving discontinuous stimulation (Fig. 1D). Sorted naive CD8⫹ cells proliferated
more poorly than CD4⫹ cells due to the lower levels of IL-2 they
produce. Nevertheless discontinuously stimulated cells proliferated as well as continuously stimulated cells (data not shown).
Therefore, naive T cells can recall a subthreshold period of stimulation hours after its termination.
MEMORY-LIKE RESPONSE OF T CELLS IN G1
The Journal of Immunology
4013
were separated by one of rest (S6/06/S6), ⬃20% of cells had undergone division by 67 h. However, when assessed 6 h later so that
the time after the second stimulation was 55 h, the same as the S12
group when analyzed at 67 h, the discontinuously stimulated population had caught up and reached the same division profile as the
cells that received 12 h of uninterrupted stimulation (Fig. 3, compare S12 at 67 h to S6/06/S6 at 73 h, indicated by the diagonal
line). Therefore, T cells remember discrete periods of antigenic
stimulation separated by periods of rest.
Intermediate but not high density cells are responsive to
restimulation after rest
The progression of T cells from a resting to a proliferative state
requires the movement from the G0 phase of the cell cycle to G1,
and then through S and G2/M. Cell cycle progression involves
blast transformation, characterized by an increase in new RNA and
protein synthesis and cell size, and a corresponding decrease in cell
density. To determine the state of the T cells after subthreshold
periods of stimulation, CFSE-labeled resting T cells were either
incubated in uncoated wells or stimulated for 5 h in anti-CD3/antiCD28-coated wells and then transferred to uncoated wells. Both
groups were cultured for a total of 23 h, after which time the cells
were separated on a discontinuous density gradient consisting of
50, 60, and 70% Percoll; steps that have previously been extensively used to separate resting from activated B and T cells (30,
31). When unstimulated cells were separated after 23 h of culture
virtually no viable cells were recovered from the low density layer,
the large majority being of high density (Fig. 4A, upper row).
There were some apoptotic cells (low forward scatter, high side
scatter) present in all fractions after the overnight culture. The
densities of the different fractions of cells after 5 h of stimulation
and 18 h of rest were reflected in their forward vs side scatter
profiles. Some low density cells (blasts) were recovered, as well as
a large number of cells with high and intermediate density (Fig.
4A, lower row).
To determine the proliferative capacity of the different density
populations, one-half of each fraction was left untreated and the
other was stimulated for 5 h. All populations were analyzed 40 h
after Percoll fractionation (or 63 h from the beginning of the experiment). T cells that had never been stimulated (0/0) or that had
FIGURE 3. Integration of independent stimuli occurs for antigenic
stimulation. Peripheral lymph node T cells from 5C.C7 TCR transgenic
mice were stimulated with MCC88 –103-pulsed DAP.3 cells (which do not
express I-Ek) or P13.9 cells (stably expressing I-Ek) for the indicated times.
Stimulation was terminated by magnetic separation of T cells from APC.
The first row shows CFSE dilution profiles for cells incubated with DAP.3
cells for 10 or 18 h. The remaining rows show CFSE dilution profiles of T
cells stimulated with Ag-pulsed P13.9 cells for 6 or 12 h continuously (S6
and S12) or stimulated twice with an intervening 6-h period of rest (S6/
06/S6). The diagonal line indicates cells that were stimulated for 55 h after
the final stimulation ended. Duplicate samples are shown from one of three
experiments.
received only the second 5 h stimulation (0/5) never divided (insufficient numbers of low density cells were recovered under these
conditions for analysis) (Fig. 4B). A substantial number of the low
density blasts from the S5/0 group proliferated even when no second stimulus was applied, and virtually all underwent division after receiving a second 5-h period of activation. The high density
cells did not commit to proliferate whether or not they received a
second period of activation, and thus reflect the population that did
not remember the initial subthreshold stimulation. The most interesting group was that of the intermediate density cells. If they
received only one 5-h period of stimulation the cells did not
progress into division. However, a second 5-h stimulation caused
the majority of them to proceed through the cell cycle by 40 h.
Therefore, the intermediate density population comprises cells that
have not been adequately stimulated to divide after one stimulus,
but can sum two discontinuous periods of stimulation and respond
with vigorous proliferation.
To determine to what extent the rapid and robust response of
intermediate density cells to restimulation might be due to the
presence of preexisting memory T cells, similar experiments were
performed with CD4⫹ cells that had been depleted of CD44high
(i.e., memory) T cells. As above, cells that had been rested for 23 h
before Percoll density separation did not proceed to proliferate,
whether or not they received a 6-h period of stimulation before
analysis (Fig. 4C). As opposed to unfractionated CD4⫹ T cells,
naive (CD44low) CD4⫹ cells stimulated for 6 h and then recovered
in the low density fraction underwent only a small amount of division, although they proliferated robustly when given a second
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FIGURE 2. Inhibition of Src-kinase signaling during the period of rest
does not prevent recollection of previous stimulation. A, Purified CFSElabeled T cells were stimulated with anti-CD3/anti-CD28 for 26 h in the
presence of PP2, washed, and incubated for an additional 41 h. B, Purified
CFSE-labeled T cells were stimulated once with anti-CD3/anti-CD28 for
5 h (S5) or twice for 5 h with a 20-h interval of rest (S5/020/S5). In the
lower row, 10 ␮M PP2 was added during the rest period and washed away
20 h later, just before restimulation. One representative experiment of two
is shown.
4014
MEMORY-LIKE RESPONSE OF T CELLS IN G1
taminating intermediate density cells. In any case, the results
clearly show that naive T cells of intermediate density can integrate two subthreshold periods of stimulation separated by at least
18 h.
Intermediate density cells are in the G1 phase and rapidly
progress through the cell cycle upon restimulation
period of subthreshold stimulation. Importantly, the intermediate
density cells did not proliferate after only one round of stimulation,
but after 18 h of rest they responded very well to a second 6-h
stimulation. Only a few high density T cells proliferated after a
second stimulation, which may represent a small number of con-
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FIGURE 4. Intermediate but not high density CD4⫹ cells are responsive to restimulation after rest. A, Purified lymph node T cells were left
untreated or stimulated for 5 h on anti-CD3/anti-CD28-coated wells, followed by 18 h of rest. Gated CD4⫹ cells are shown. At 23 h, cells were
separated Percoll density gradient. Forward and side-scatter of unseparated
and Percoll density separated cells are shown. The Table indicates the
percentage of cells recovered in the different Percoll fractions. B, Following fractionation, cells were left untreated or subjected to a second 5-h
period of stimulation, and proliferation was assessed 40 h after fractionation. C, As in B, except that naive (CD44low) CD4⫹ cells were used and
the period of stimulation/restimulation was 6 h. CFSE dilution is shown
45 h after fractionation. One representative experiment of three is shown.
The density of T cells after activation decreases as they enter the
cell cycle, with cells in G0 being the densest. One can assign a cell
to a particular phase of the cell cycle based upon its RNA and
DNA content. RNA content is in direct proportion to cell’s metabolic potential, as the largest contributor is ribosomal RNA (37).
RNA levels increase in a continuum from the G0 phase to dividing
cells. G0 cells have 2N DNA (where N ⫽ haploid) and the lowest
RNA levels, G1 cells have 2N DNA, S cells have between 2N and
4N DNA, and G2/M cells have 4N DNA and high RNA levels
(28). DNA and RNA levels can be determined simultaneously by
using Hoechst 33342 and pyronin Y, respectively (28).
Following primary stimulation of purified CD4⫹ T cells by
plate-bound anti-CD3/anti-CD28 and 18 h rest (S5/018), cell cycle
status of the different populations recovered from the Percoll density gradient was determined by measuring RNA/DNA content.
Unfractionated unstimulated cells were almost entirely in G0, as
evidenced by 2N DNA and few cells with more than baseline RNA
levels (Fig. 5A). To determine the kinetics of cell cycle recruitment, S5/018 cells were either analyzed immediately after fractionation or subjected to continuous stimulation for 15 or 25 h
(S5/018-Percoll/S15 and S5/018-Percoll/S25). Immediately after
fractionation, the low density population was composed almost
entirely of cells that had progressed past G0 (Fig. 5B), with cells of
intermediate to high RNA content and ⬎2N DNA (i.e., in S or
G2/M). In contrast, there were almost no cells in the intermediate
or high density populations that had ⬎2N DNA. What distinguished the intermediate from the high density cells was their
RNA content, which was elevated in the former. Therefore, we
conclude that the intermediate density cells are largely in G1,
whereas the high density cells are in G0. After 15 or 25 h of
continuous secondary stimulation, the large majority of cells in the
low density fraction were actively cycling. In contrast, after 15 h
of continuous restimulation high density cells had just begun to
enter G1. Only after 25 h of restimulation did a substantial number
of high density cells progress into G1 (Fig. 5B) and go on to divide
by 48 h (data not shown), demonstrating that these cells are not a
“dead-end” population. Importantly, intermediate density cells accumulated large amounts of RNA much earlier, and by 25 h many
cells were in S ⫹ G2/M.
Cell cycle analysis was performed on the different density fractions that had received one or two subthreshold (5 h) periods of
stimulation (Fig. 5C). Cells in all three density populations retained their cell cycle phenotypes when simply cultured for 25 h
after Percoll separation, although the low density cells showed
some evidence of becoming less active (compare with Fig. 5B).
Notably, the majority of intermediate density G1 cells that received
a second subthreshold period of stimulation moved further into the
cell cycle, with many cells in late G1, S, and G2/M. The same
treatment caused a small percentage of high density G0 cells to
progress into early G1, but the majority remained in G0. Therefore,
both intermediate and high density fractions were able to respond
to a secondary stimulus, but only the intermediate density fraction
was able to respond to 5 h of restimulation by passing through the
whole cell cycle.
The Journal of Immunology
4015
T cells can remain in G1 and respond to subthreshold
stimulation for at least 2 days
Having observed that T cells were able to recall previous stimulation at least for 20 h, experiments were performed to test the
maximum duration of reset permissible (Fig. 6). After a 5-h subthreshold stimulation, T cells were rested for up to 46 h and restimulated another 5 h. Cells that received only one period of stimulation divided very little by 74 –92 h. Samples that were rested for
20 –25 h before restimulation, had divided extensively by 74 h,
with a similar division profile observed at 92 h, indicating that by
72 h they had divided to their full potential. Notably, cells that
were rested by periods as long as 46 h underwent some division by
74 h, but had “caught up” with the S5/020/S5 cells by 92 h. It was
not possible to analyze cells rested for ⬎2 days because of the
increasing amounts of cell death observed in prolonged in vitro
culture. Therefore, cells can remain in a responsive state for at
least 2 days after the initial subthreshold stimulation is withdrawn.
G1 but not G0 T cells produce IL-2 rapidly upon restimulation
FIGURE 5. Intermediate density cells are in the G1, and progress into
the cell cycle upon restimulation faster than high density (G0) cells. Purified CD44lowCD4⫹ T cells were either unstimulated or stimulated for 5 h
with anti-CD3/anti-CD28 and then rested for 18 h. At 23 h, DNA (Hoechst
33342) and RNA (pyronin Y) profiles were determined. A, Analysis of
unstimulated cells. B, Analysis of S5/018 and then Percoll-fractionated
cells. One set of cells was analyzed immediately after fractionation (first
row). A second and third set was restimulated for 15 or 25 h and then
immediately analyzed (second and third rows). The percentage of cells in
each gate is shown. Cells with hypodiploid DNA (not shown) accounted
for ⬍5% of the total. C, The same cells as in B were analyzed 25 h after
fractionation without (0) or with (S5) a 5-h period of restimulation.
FIGURE 6. Cells recollect stimulation for long periods of time. Purified T cells were stimulated with platebound anti-CD3/anti-CD28. CFSE profiles are shown for
gated CD4⫹ cells at 74 and 92 h. One set of cells was left
untreated. The remaining cells were stimulated either
once (S5) or twice, with periods of rest between stimulations lasting 20, 25, 41, or 46 h (S5/020/S5 to
S5/046/S5).
Rapidly responsive G1 T cells are CD44intCD62Lhigh and have
high cyclin D3 and low p27Kip1 expression
The expression of activation/memory markers on CD4⫹ T cell
populations of different density following a subthreshold period of
stimulation was assessed. Purified CD44low CD4⫹ T cells cultured
without any stimulation for 46 h had high L-selectin (CD62L) and
low CD44 expression (Fig. 8A, upper row). Cells that received
46 h of continuous stimulation with immobilized anti-CD3/antiCD28 down-regulated CD62L and up-regulated CD44. The same
initial CD4⫹ T cells were subjected to 5 h of subthreshold stimulation, rested for 18 h, density fractionated, and incubated for
additional 23 h in the absence of further stimulation (a total culture
time of 46 h). Unlike continuously activated cells, CD62L levels
remained high in all fractions, although it was slightly lower in the
low density cells (blasts). CD44 levels were more heterogeneous,
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
To determine whether other activation-induced T cell responses
are enhanced in G1 T cells, IL-2 production was measured. As a
control, CD4⫹ T cells were cultured for 23 h without stimulation
and then stimulated for 5 h in the presence of monensin (to prevent
cytokine secretion) and stained for intracellular IL-2 (38); almost
no IL-2⫹ cells were detectable (Fig. 7). In the experimental group,
cells were stimulated for 4 h (S4) and then rested for 19 h, at which
time they were Percoll-fractionated and rested or restimulated for
5 h in the presence of monensin. Cells that did not receive the
second round of stimulation were IL-2⫺. In contrast, after 5 h of
restimulation all fractions contained some IL-2⫹ cells. Notably,
the number of IL-2⫹ cells in the intermediate density G1 population was similar to that in low density cells, and 3- to 4-fold greater
than in the high density G0 fraction. Thus, G1 T cells can rapidly
respond to secondary stimulation not just by proliferating, but also
by producing IL-2.
4016
FIGURE 7. G1 but not G0 T cells produce IL-2 rapidly upon restimulation. Purified CD44low T cells were left untreated or stimulated with
plate-bound anti-CD3/anti-CD28 for 4 h (S4) and rested until 23 h, at
which time they were Percoll-fractionated. All populations were then stimulated (f) or not (䡺) for 5 h with plate-bound anti-CD3/anti-CD28 in the
presence of monensin. The cells were stained for intracellular IL-2, and the
percentage of IL-2⫹ CD4 cells are shown. The error bars represent the SD.
FIGURE 8. G1 T cells have some of the molecular features of memory
T cells. A, Upper row, CD44lowCD4⫹ T cells were untreated (dashed line)
or stimulated (solid line) for 46 h with plate-bound anti-CD3/anti-CD28
and stained for CD44 and CD62L. Lower row, CD44lowCD4⫹ T cells were
stimulated with anti-CD3/anti-CD28 for 5 h. After an 18-h period of rest,
they were Percoll-fractionated and incubated for another 23 h (total of
46 h). Analyses of high (dotted line), intermediate (filled line), and low
density (dashed line) cells are shown. B, CD44low T cells were cultured in
medium (unstimulated), constantly stimulated with anti-CD3/anti-CD28
for 23 h (const. stim.), or stimulated for 4 h and Percoll-fractionated and
lysed 19 h later (intermediate and high density fractions were analyzed).
Postnuclear extracts of the various populations were immunoblotted for
cyclin D3, p27Kip1, and ␤-actin. The data are representative of three independent experiments.
The rapid recruitment of memory T cells to divide has been
ascribed to faster advancement through the G1/S-phase transition
due to high cyclin D3 (G1/S progression factor) and low p27Kip1
(G1 cell cycle inhibitor) expression (39, 40). We compared cyclin
D3 levels in unstimulated and continuously stimulated purified naive T cells at 23 h to subthreshold-stimulated fractions (S4) obtained by Percoll gradient at 23 h (Fig. 8B). Cyclin D3 expression
was undetectable in unstimulated cells, and rose dramatically after
23 h of continuous stimulation. Notably, the level of cyclin D3 in
intermediate density G1 cells was also quite elevated, and it was
undetectable in high density G0 cells. Analysis of the cell cycle
inhibitor p27Kip1 levels gave a reciprocal result. As expected,
p27Kip1 levels were high in unstimulated cells and low in continuously stimulated cells (Fig. 8B). Among the experimental groups,
intermediate density cells had unmeasurable levels of p27Kip1
whereas high density cells had levels comparable to those of unstimulated cells. Thus, intermediate density cells were “relieved”
of cell cycle inhibition and had accumulated the cell cycle progression factor cyclin D3, while high density cells had not reached
that stage.
Discussion
Upon finding the appropriate peptide-MHC on DC, T cells do not
necessarily form a long-term sedentary interaction but can continue to “scan” other APC (21–23). Such observations have led to
the notion that T cells can integrate intermittent and short term
periods of stimulation. Indeed, our results show that the recollection of previous stimulation can last remarkably long (up to 2 days
in vitro). Moreover, upon subthreshold stimulation we can isolate
a population of T cells in G1 that are endowed with the capacity to
respond rapidly upon restimulation, although they cannot be classified as traditional memory cells.
Memory T cells emerge during the adaptive immune response to
provide a more rapid response to secondary Ag encounter (41).
They outlast the majority of cells that perish upon Ag clearance,
and are thought to arise after multiple cycles of division, long after
the effector population has reached its peak (42– 45). Different
populations of memory T cells have several traits in common:
relatively high TCR affinities for peptide-MHC (46), low activation threshold, and less stringent dependence on costimulation
(12), improved survival (47– 49), and a high capacity to expand
(50, 51). Unambiguous identification of memory cells in mice is
difficult because the widely used marker, high levels of CD44, is
also found on both activated and homeostatically proliferating
cells (52, 53).
Memory T cells can be divided into two broad groups, effector
and central memory cells, which are CD62L low and high, respectively (54). Although all memory populations continuously migrate (55), effector memory cells preferentially accumulate in peripheral nonlymphoid tissues, where if rechallenged they rapidly
release cytokines and/or exert direct cytotoxicity (51, 54). Central
memory T cells, in contrast, express the lymph node homing receptor CCR7 and largely reside in secondary lymphoid organs
(51). Provided with greater survival and proliferative capacities
than both naive and effector memory cells, central memory cells
can rapidly expand and seed the periphery with new generations of
effector cells. One of the requisites for a quicker response is rapid
entry into the cell cycle, which has been described for memory T
cells (39, 40). For example, memory cells are different from the
majority of resting lymphocytes in that they have preactivated cyclinD/CDK6 complexes in the cytoplasm and decreased levels of
the cell cycle progression inhibitor p27kip (39). Although there are
many nuances between different subsets of CD8⫹ and CD4⫹
memory cells, their single indisputable hallmark compared with
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being high on low density cells (although less than that for continuously activated cells; compare with Fig. 8, upper row), intermediate on the intermediate density (G1) fraction, and only slightly
increased above unstimulated levels on high density (G0) cells
(Fig. 8A, lower row). Therefore, the rapidly responsive G1 T cell
population is CD44intCD62Lhigh.
MEMORY-LIKE RESPONSE OF T CELLS IN G1
The Journal of Immunology
Acknowledgments
We are grateful to Barbara Taylor for cell sorting and expert help with flow
cytometric analysis, Ronald Germain for cell lines, Peter Mercado for expert technical help, and Paul Mittelstadt for critical review of the
manuscript.
Disclosures
The authors have no financial conflict of interest.
References
1. Ahmed, R., and D. Gray. 1996. Immunological memory and protective immunity:
understanding their relation. Science 272:54.
2. Lanzavecchia, A., and F. Sallusto. 2002. Progressive differentiation and selection
of the fittest in the immune response. Nat. Rev. Immunol. 2:982.
3. Jenkins, M. K., A. Khoruts, E. Ingulli, D. L. Mueller, S. J. McSorley,
R. L. Reinhardt, A. Itano, and K. A. Pape. 2001. In vivo activation of antigenspecific CD4 T cells. Annu. Rev. Immunol. 19:23.
4. Matsui, K., J. J. Boniface, P. A. Reay, H. Schild, B. Fazekas de St Groth, and
M. M. Davis. 1991. Low affinity interaction of peptide-MHC complexes with T
cell receptors. Science 254:1788.
5. Jorgensen, J. L., P. A. Reay, E. W. Ehrich, and M. M. Davis. 1992. Molecular
components of T-cell recognition. Annu. Rev. Immunol. 10:835.
6. Delon, J., and R. N. Germain. 2000. Information transfer at the immunological
synapse. Curr. Biol. 10:R923.
7. Monks, C. R., B. A. Freiberg, H. Kupfer, N. Sciaky, and A. Kupfer. 1998. Threedimensional segregation of supramolecular activation clusters in T cells. Nature
395:82.
8. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen,
and M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221.
9. Dustin, M. L., S. K. Bromley, Z. Kan, D. A. Peterson, and E. R. Unanue. 1997.
Antigen receptor engagement delivers a stop signal to migrating T lymphocytes.
Proc. Natl. Acad. Sci. USA 94:3909.
10. Norcross, M. A. 1984. A synaptic basis for T-lymphocyte activation. Ann. Immunol. 135D:113.
11. Paul, W. E., and R. A. Seder. 1994. Lymphocyte responses and cytokines. Cell
76:241.
12. Iezzi, G., K. Karjalainen, and A. Lanzavecchia. 1998. The duration of antigenic
stimulation determines the fate of naive and effector T cells. Immunity 8:89.
13. Viola, A., and A. Lanzavecchia. 1996. T cell activation determined by T cell
receptor number and tunable thresholds. Science 273:104.
14. Ding, L., P. S. Linsley, L. Y. Huang, R. N. Germain, and E. M. Shevach. 1993.
IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the
up-regulation of B7 expression. J. Immunol. 151:1224.
15. Lanzavecchia, A., G. Lezzi, and A. Viola. 1999. From TCR engagement to T cell
activation: a kinetic view of T cell behavior. Cell 96:1.
16. Lee, W. T., G. Pasos, L. Cecchini, and J. N. Mittler. 2002. Continued antigen
stimulation is not required during CD4⫹ T cell clonal expansion. J. Immunol.
168:1682.
17. van Stipdonk, M. J., E. E. Lemmens, and S. P. Schoenberger. 2001. Naive CTLs
require a single brief period of antigenic stimulation for clonal expansion and
differentiation. Nat. Immunol. 2:423.
18. Miller, M. J., S. H. Wei, I. Parker, and M. D. Cahalan. 2002. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science
296:1869.
19. Stoll, S., J. Delon, T. M. Brotz, and R. N. Germain. 2002. Dynamic imaging of
T cell-dendritic cell interactions in lymph nodes. Science 296:1873.
20. Bousso, P., N. R. Bhakta, R. S. Lewis, and E. Robey. 2002. Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy. Science
296:1876.
21. Underhill, D. M., M. Bassetti, A. Rudensky, and A. Aderem. 1999. Dynamic
interactions of macrophages with T cells during antigen presentation. J. Exp.
Med. 190:1909.
22. Gunzer, M., A. Schafer, S. Borgmann, S. Grabbe, K. S. Zanker, E. B. Brocker,
E. Kampgen, and P. Friedl. 2000. Antigen presentation in extracellular matrix:
interactions of T cells with dendritic cells are dynamic, short lived, and sequential. Immunity. 13:323.
23. Mempel, T. R., S. E. Henrickson, and A. U. H. Von. 2004. T-cell priming by
dendritic cells in lymph nodes occurs in three distinct phases. Nature 427:154.
24. Faroudi, M., R. Zaru, P. Paulet, S. Muller, and S. Valitutti. 2003. Cutting edge:
T lymphocyte activation by repeated immunological synapse formation and intermittent signaling. J. Immunol. 171:1128.
25. Friedl, P., and M. Gunzer. 2001. Interaction of T cells with APCs: the serial
encounter model. Trends Immunol. 22:187.
26. Seder, R. A., W. E. Paul, M. M. Davis, and B. Fazekas de St Groth. 1992. The
presence of interleukin 4 during in vitro priming determines the lymphokineproducing potential of CD4⫹ T cells from T cell receptor transgenic mice. J. Exp.
Med. 176:1091.
27. Beeson, C., J. Rabinowitz, K. Tate, I. Gutgemann, Y. H. Chien, P. P. Jones,
M. M. Davis, and H. M. McConnell. 1996. Early biochemical signals arise from
low affinity TCR-ligand reactions at the cell-cell interface. J. Exp. Med. 184:777.
28. Darzynkiewicz, Z., F. Traganos, and M. R. Melamed. 1980. New cell cycle compartments identified by multiparameter flow cytometry. Cytometry 1:98.
29. Glimm, H., I. H. Oh, and C. J. Eaves. 2000. Human hematopoietic stem cells
stimulated to proliferate in vitro lose engraftment potential during their S/G2/M
transit and do not reenter G0. Blood 96:4185.
30. Defranco, A. L., E. S. Raveche, R. Asofsky, and W. E. Paul. 1982. Frequency of
B lymphocytes responsive to anti-immunoglobulin. J. Exp. Med. 155:1523.
31. Louis, J. A., E. Moedder, H. R. MacDonald, and H. D. Engers. 1981. Recognition
of protozoan parasites by murine T lymphocytes. II. Role of the H-2 gene complex in interactions between antigen-presenting macrophages and Leishmaniaimmune T lymphocytes. J. Immunol. 126:1661.
32. Valitutti, S., S. Muller, M. Cella, E. Padovan, and A. Lanzavecchia. 1995. Serial
triggering of many T-cell receptors by a few peptide-MHC complexes. Nature
375:148.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
their naive counterparts is a rapid response to secondary Ag
encounter.
By providing a limited (subthreshold) period of stimulation, we
have identified a population of cells that are ready to rapidly commit to proliferate following a second subthreshold stimulus, meeting the fundamental requirement of classical memory cells. These
cells, although they have not committed to DNA synthesis or division, are characterized by a change in density coupled with increased RNA content (indicating higher metabolic potential), high
levels of cyclin D3, and low p27Kip1 expression. Moreover, they
can rapidly secrete cytokine upon restimulation. These results are
consistent with the observation that T cells undergo a G03 G1
commitment step after 3–5 h of stimulation with anti-CD3/antiCD28 (56). However, these G1 cells cannot be classified as either
central or effector memory cells because they remain CD62Lhigh
and only CD44int. Moreover, memory T cells are thought to develop only after multiple cycles of division, and the G1 cells arising during the course of subthreshold stimulation have not even
commenced S phase. It should be emphasized that although the
brief (4 –7 h) periods of stimulation used in this study can be considered “suboptimal” because they resulted in little proliferation,
this is not the same suboptimal stimulation that has been described
for cells activated by altered peptide ligands or low levels of costimulation (57, 58), because in those cases the initial stimulation
leads to tolerance/anergy rather than readiness to proliferate upon
restimulation. Hence, the processes initiated in cells that stopped at
G1 must have been qualitatively similar to those of continuously
stimulated cells.
How Ag-responsive G1 T cells might arise in vivo and contribute to an adaptive immune response remains to be determined.
Repeated intermittent contacts could be a way to avoid activationinduced cell death by preventing a sustained strong and thus lethal
stimulation. Cells have developed adapting mechanisms to control
excessive stimulation at many levels, from TCR down-regulation
and degradation to down-modulation of various signaling molecules (59 – 63), and expression of inhibitory receptors like CTLA-4
and lymphocyte activation gene-3 (64 – 66). Therefore, by transient
dissociation from APC, cells might escape both activation-induced
cell death and cell senescence as the consequences of vigorous
division (67, 68).
There is some evidence that T cell activation occurs after serial
encounters with different APC. For example, after s.c. Ag injection
the first APC to present the foreign Ag are DC residing in the
lymph nodes, and a second wave of Ag-delivering cells comes
from the inflamed tissues hours later (69). If the second set of DC
was denied access to lymph nodes, delayed hypersensitivity could
no longer be observed. Therefore, recollection of previous Ag encounter by G1 T cells could be a way to integrate two different
and/or similar stimuli given by different DC subpopulations.
The data in the present report demonstrate that Ag-responsive
G1 T cells represent a nonclassical source of memory-like responsiveness to secondary stimulation. Studies in which these cells are
adoptively transferred and then restimulated in host animals may
provide insights into how they function in vivo.
4017
4018
52. Goldrath, A. W., L. Y. Bogatzki, and M. J. Bevan. 2000. Naive T cells transiently
acquire a memory-like phenotype during homeostasis-driven proliferation.
J. Exp. Med. 192:557.
53. Murali-Krishna, K., and R. Ahmed. 2000. Cutting edge: naive T cells masquerading as memory cells. J. Immunol. 165:1733.
54. Lanzavecchia, A., and F. Sallusto. 2000. Dynamics of T lymphocyte responses:
intermediates, effectors, and memory cells. Science 290:92.
55. Klonowski, K. D., K. J. Williams, A. L. Marzo, D. A. Blair, E. G. Lingenheld,
and L. Lefrancois. 2004. Dynamics of blood-borne CD8 memory T cell migration
in vivo. Immunity 20:551.
56. Lea, N. C., S. J. Orr, K. Stoeber, G. H. Williams, E. W. Lam, M. A. Ibrahim,
G. J. Mufti, and N. S. Thomas. 2003. Commitment point during G03 G1 that
controls entry into the cell cycle. Mol. Cell Biol. 23:2351.
57. Sloan-Lancaster, J., A. S. Shaw, J. B. Rothbard, and P. M. Allen. 1994. Partial T
cell signaling: altered phospho-␨ and lack of zap70 recruitment in APL-induced
T cell anergy. Cell 79:913.
58. Constant, S. L., and K. Bottomly. 1997. Induction of Th1 and Th2 CD4⫹ T cell
responses: the alternative approaches. Annu. Rev. Immunol. 15:297.
59. Valitutti, S., S. Muller, M. Salio, and A. Lanzavecchia. 1997. Degradation of T
cell receptor (TCR)-CD3-␨ complexes after antigenic stimulation. J. Exp. Med.
185:1859.
60. D’Oro, U., M. S. Vacchio, A. M. Weissman, and J. D. Ashwell. 1997. Activation
of the Lck tyrosine kinase targets cell surface T cell antigen receptors for lysosomal degradation. Immunity 7:619.
61. Lee, K. H., A. R. Dinner, C. Tu, G. Campi, S. Raychaudhuri, R. Varma,
T. N. Sims, W. R. Burack, H. Wu, J. Wang, et al. 2003. The immunological
synapse balances T cell receptor signaling and degradation. Science 302:1218.
62. Naramura, M., I. K. Jang, H. Kole, F. Huang, D. Haines, and H. Gu. 2002. c-Cbl
and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR
down-modulation. Nat. Immunol. 3:1192.
63. Rao, N., I. Dodge, and H. Band. 2002. The Cbl family of ubiquitin ligases: critical
negative regulators of tyrosine kinase signaling in the immune system. J. Leukocyte Biol. 71:753.
64. Chambers, C. A., T. J. Sullivan, and J. P. Allison. 1997. Lymphoproliferation in
CTLA-4-deficient mice is mediated by costimulation-dependent activation of
CD4⫹ T cells. Immunity 7:885.
65. Rudd, C. E., M. Martin, and H. Schneider. 2002. CTLA-4 negative signaling via
lipid rafts: a new perspective. Sci. STKE 2002:PE18.
66. Workman, C. J., L. S. Cauley, I. J. Kim, M. A. Blackman, D. L. Woodland, and
D. A. Vignali. 2004. Lymphocyte activation gene-3 (CD223) regulates the size of
the expanding T cell population following antigen activation in vivo. J. Immunol.
172:5450.
67. Reed, J. R., M. Vukmanovic-Stejic, J. M. Fletcher, M. V. Soares, J. E. Cook,
C. H. Orteu, S. E. Jackson, K. E. Birch, G. R. Foster, M. Salmon, et al. 2004.
Telomere erosion in memory T cells induced by telomerase inhibition at the site
of antigenic challenge in vivo. J. Exp. Med. 199:1433.
68. Valenzuela, H. F., and R. B. Effros. 2002. Divergent telomerase and CD28 expression patterns in human CD4 and CD8 T cells following repeated encounters
with the same antigenic stimulus. Clin. Immunol. 105:117.
69. Itano, A. A., S. J. McSorley, R. L. Reinhardt, B. D. Ehst, E. Ingulli,
A. Y. Rudensky, and M. K. Jenkins. 2003. Distinct dendritic cell populations
sequentially present antigen to CD4 T cells and stimulate different aspects of
cell-mediated immunity. Immunity 19:47.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
33. Lyons, A. B., and C. R. Parish. 1994. Determination of lymphocyte division by
flow cytometry. J. Immunol. Methods. 171:131.
34. Molina, T. J., K. Kishihara, D. P. Siderovski, W. van Ewijk, A. Narendran,
E. Timms, A. Wakeham, C. J. Paige, K. U. Hartmann, A. Veillette, et al. 1992.
Profound block in thymocyte development in mice lacking p56lck. Nature
357:161.
35. Hanke, J. H., J. P. Gardner, R. L. Dow, P. S. Changelian, W. H. Brissette,
E. J. Weringer, B. A. Pollok, and P. A. Connelly. 1996. Discovery of a novel,
potent, and Src family-selective tyrosine kinase inhibitor: study of Lck- and
FynT-dependent T cell activation. J. Biol. Chem. 271:695.
36. Itoh, Y., B. Hemmer, R. Martin, and R. N. Germain. 1999. Serial TCR engagement and down-modulation by peptide:MHC molecule ligands: relationship to
the quality of individual TCR signaling events. J. Immunol. 162:2073.
37. Johnson, L. F., H. T. Abelson, S. Penman, and H. Green. 1977. The relative
amounts of the cytoplasmic RNA species in normal, transformed and senescent
cultured cell lines. J. Cell Physiol. 90:465.
38. Jung, T., U. Schauer, C. Heusser, C. Neumann, and C. Rieger. 1993. Detection
of intracellular cytokines by flow cytometry. J. Immunol. Methods 159:197.
39. Veiga-Fernandes, H., and B. Rocha. 2004. High expression of active CDK6 in the
cytoplasm of CD8 memory cells favors rapid division. Nat. Immunol. 5:31.
40. Latner, D. R., S. M. Kaech, and R. Ahmed. 2004. Enhanced expression of cell
cycle regulatory genes in virus-specific memory CD8⫹ T cells. J. Virol.
78:10953.
41. Dutton, R. W., L. M. Bradley, and S. L. Swain. 1998. T cell memory. Annu. Rev.
Immunol. 16:201.
42. Critchfield, J. M., M. K. Racke, J. C. Zuniga-Pflucker, B. Cannella, C. S. Raine,
J. Goverman, and M. J. Lenardo. 1994. T cell deletion in high antigen dose
therapy of autoimmune encephalomyelitis. Science 263:1139.
43. Zhang, X., T. Brunner, L. Carter, R. W. Dutton, P. Rogers, L. Bradley, T. Sato,
J. C. Reed, D. Green, and S. L. Swain. 1997. Unequal death in T helper cell (Th)1
and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated
apoptosis. J. Exp. Med. 185:1837.
44. Kaech, S. M., E. J. Wherry, and R. Ahmed. 2002. Effector and memory T-cell
differentiation: implications for vaccine development. Nat. Rev. Immunol. 2:251.
45. Rufer, N., A. Zippelius, P. Batard, M. J. Pittet, I. Kurth, P. Corthesy,
J. C. Cerottini, S. Leyvraz, E. Roosnek, M. Nabholz, and P. Romero. 2003. Ex
vivo characterization of human CD8⫹ T subsets with distinct replicative history
and partial effector functions. Blood 102:1779.
46. McHeyzer-Williams, M. G., and M. M. Davis. 1995. Antigen-specific development of primary and memory T cells in vivo. Science 268:106.
47. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, and
R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies
effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol.
4:1191.
48. Grayson, J. M., L. E. Harrington, J. G. Lanier, E. J. Wherry, and R. Ahmed. 2002.
Differential sensitivity of naive and memory CD8⫹ T cells to apoptosis in vivo.
J. Immunol. 169:3760.
49. Li, J., G. Huston, and S. L. Swain. 2003. IL-7 promotes the transition of CD4
effectors to persistent memory cells. J. Exp. Med. 198:1807.
50. Kaech, S. M., S. Hemby, E. Kersh, and R. Ahmed. 2002. Molecular and functional profiling of memory CD8 T cell differentiation. Cell. 111:837.
51. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two
subsets of memory T lymphocytes with distinct homing potentials and effector
functions. Nature 401:708.
MEMORY-LIKE RESPONSE OF T CELLS IN G1