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Two Distinct Stages in the Transition from
Naive CD4 T Cells to Effectors, Early
Antigen-Dependent and Late
Cytokine-Driven Expansion and
Differentiation
Dawn M. Jelley-Gibbs, Nancy M. Lepak, Michael Yen and
Susan L. Swain
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2000; 165:5017-5026; ;
doi: 10.4049/jimmunol.165.9.5017
http://www.jimmunol.org/content/165/9/5017
Two Distinct Stages in the Transition from Naive CD4
T Cells to Effectors, Early Antigen-Dependent and Late
Cytokine-Driven Expansion and Differentiation1
Dawn M. Jelley-Gibbs, Nancy M. Lepak, Michael Yen, and Susan L. Swain2
Efficient peptide presentation by professional APC to naive and effector CD4 T cells in vitro is limited to the first 1–2 days of
culture, but is nonetheless optimum for effector expansion and cytokine production. In fact, prolonging Ag presentation leads to
high levels of T cell death, decreased effector expansion, and decreased cytokine production by recovered effectors. Despite the
absence of Ag presentation beyond day 2, T cell division continues at a constant rate throughout the 4-day culture. The Agindependent later stage depends on the presence of IL-2, and we conclude optimum effector generation depends on an initial 2 days
of TCR stimulation followed by an additional 2 days of Ag-independent, cytokine driven T cell expansion and differentiation. The
Journal of Immunology, 2000, 165: 5017–5026.
Trudeau Institute, Saranac Lake, NY 12983
Received for publication June 19, 2000. Accepted for publication August 10, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant AI26887.
2
Address correspondence and reprint requests to Dr. Susan L. Swain, Trudeau Institute, P.O. Box 59, Saranac Lake, NY 12983. E-mail address: [email protected]
3
Abbreviations used in this paper: AICD, activation-induced cell death; Tg, transgene; HA, hemagglutinin; HNT T cells, TCR Tg⫹ HNT (influenza HA-specific)
CD4⫹ T cells; CFSE, 5-carboxyfluorescein diacetate; PCCF, pigeon cytochrome c
fragment; AND T cells, TCR Tg⫹ AND (PCCF-specific) CD4⫹ T cells; PI, propidium iodide; DC, dendritic cell; MitC, mitomycin c; ICCS, intracellular cytokine
staining; FSC, forward scatter; SSC, side scatter; MFI, mean fluorescent intensity.
Copyright © 2000 by The American Association of Immunologists
drive polarization are most effective when present at the initiation
of T cell activation before the first round of cell division (15, 16).
Once cell proliferation commences, highly activated T cells divide
rapidly and can continue through multiple rounds of division, resulting in an impressive expansion of the initial population (17, 18). Little
is known about the critical sequence of external signals required for
driving cell division over the later phase of the 4-day period necessary
to generate large numbers of highly activated effectors.
Differentiation, leading to changes which effect the future functional and survival potential of the progeny cells, also occurs during
effector generation. The differentiation of naive CD4 T cells into polarized effectors in vitro is dependent on high doses of high avidity
Ag, the interaction of multiple costimulation receptor/coreceptor
pairs, growth-promoting cytokines, like IL-2, and polarizing cytokines (1–3, 16, 19, 20). IL-12 or IFN-␥, and the lack of IL-4, generate
Th1-polarized effectors (capable of IL-2, IFN-␥, TNF-␣, and TGF-␤
production), whereas the presence of IL-4 and the lack of IFN-␥ generate Th2-polarized effectors (capable of IL-4, -5, -10, and -13 production) (16). Although many of the signals required for effector differentiation are known, it is not clear whether these and other
differentiation signals are delivered early or late during a primary T
cell response to drive the development of functional and phenotypic
characteristics critical for optimal effector function. It is likely that
various differentiation signals and differentiation-associated changes
are available throughout in vivo effector generation at different times.
For example, for polarization, the presence of polarizing cytokines are
required only during the early phase of effector generation, supporting
that the signals required for polarized T cell differentiation begin immediately after stimulation of naive CD4 T cells (21–23). However,
the ability of effectors to become competent to produce large amounts
of polarized cytokines (i.e., IFN-␥ and IL-4) rapidly upon restimulation takes 2– 4 days (3, 15, 23, 24). Additionally, the growth-promoting cytokine IL-2 is only produced at maximal levels by the responding CD4 T cells during the later phase of effector generation (1, 2, 21,
24, 25). Another important change that appears to occur during the
late phase of effector differentiation includes the induction of susceptibility to activation-induced cell death (AICD),3 which requires early
exposure to IL-2 and at least 2 days of Ag/APC (26, 27).
Although effects of the duration of Ag stimulation on the ability
of naive T cells to proliferate has been studied previously, equally
important functional characteristics including cytokine production,
0022-1767/00/$02.00
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A
primary CD4 T cell response can be conceptually divided into three distinct phases involving activation,
proliferation, and differentiation of responding T cells
leading to the generation of large numbers of highly potent effectors with the capacity to rapidly produce high levels of multiple
cytokines after reencounter with Ag (1, 2). However, each of these
phases may in fact occur sequentially and/or simultaneously.
Which stimulatory signals are required to trigger each phase, and
the sequence in which they must be delivered to the T cell, are also
unclear. For the purpose of these studies, we have defined effectors
as activated T cells with the capacity to rapidly produce high levels
of multiple cytokines after restimulation (1, 2). We have found that
full development of effector function requires 4 days of in vitro
culture, (3), and we have also seen a similar requirement for 4 days
or more to generate fully activated effector T cells in vivo (4).
We consider the activation phase to be the first step that occurs
before division, which commits the cell to enter the cell cycle,
requiring a number of signals to promote progression through the
cell cycle. There is at least a 24-h lag while a resting (Go) T cell
becomes activated and thus competent to progress through the cell
cycle (5, 6). During this phase, many genes and their products are
expressed in preparation for DNA replication and cell division
(7–12). Epigenetic remodeling changes resulting in “on” configurations of polarized cytokine genes account for some of the very
earliest changes that occur during T cell activation as the cell undergoes its first round of DNA replication before the first cell division (10, 13, 14). Therefore, it is notable that cytokines which
5018
Ag-DEPENDENT AND IL-2-DRIVEN STAGES FROM NAIVE TO EFFECTOR
Materials and Methods
Animals
H-2k/k (B10.Br) and H-2b/b (C57BL/6) V␣11/V␤3 AND TCR transgene
(Tg; I-Ek restricted), B10.Br.HNT.B10.D2/F1 (V␤8.3/V␣2) TCR Tg (I-Ad
restricted), and H-2k/d B10.Br.B10.D2/F1 mice were used at 2– 6 mo of age
and were bred in the animal facilities at the Trudeau Institute.
Cell isolations
The isolation of pooled spleen and lymph node cells enriched for naive
CD4 T cells has been described previously (34). In brief, the pooled spleen
and lymph node cells were passed through a nylon wool column, and the
nonadherent cells treated with a panel of CD8, heat-stable Ag, and class II
MHC-depleting Ab and complement followed by Percoll gradient separation. The purified cell populations were routinely ⬎95% CD4⫹ cells, 90 –
95% of which had a naive phenotype (CD45RBhigh, CD62Lhigh, CD44low,
and CD25low) and expressed the TCR Tg.
Immunofluorescent staining
All staining was done at 4°C in PBS (Life Technologies, Grand Island,
NY) with 1% BSA (Sigma, St. Louis, MO) and 0.1% NaN3 (Sigma). FITClabeled Ab are produced in our laboratory, and PE-biotin- and APC-labeled
Ab were purchased where indicated. The following Ab and fluorescent
reagents were used: PE-labeled, CyChrome-labeled, and APC-labeled antiCD4 (clone RM4 –5; Caltag, Burlingame, CA), FITC- and PE-labeled antiCD44 (clone IM7; PharMingen, San Diego, CA), FITC- and PE-labeled
anti-CD62L (clone Mel 14; Caltag), FITC- and PE-labeled anti-CD45RB
(clone 23G2; PharMingen), FITC- and PE-labeled anti-CD25 (IL-2R␣
chain, clone PC61; PharMingen), FITC-labeled anti-V␣11 (clone RR8-1),
PE- and biotin-labeled anti-V␤3 (clone KJ25), PE-labeled anti-V␤8.3
(clone 1B3.3; PharMingen), streptavidin-APC (PharMingen), PE-labeled
antimouse IL-2 (clone S4B6; PharMingen), PE-labeled antimouse IFN-␥
(clone XMG.1; PharMingen), and PE-labeled antimouse IL-4 (clone BVD24G2; Caltag). All isotype control Abs were purchased from PharMingen:
FITC- and PE-labeled hamster Ig, FITC- and PE-labeled rat IgG1, FITCand PE-labeled rat IgG2a, and FITC- and PE-labeled rat IgG2b. Flow cytometry was conducted using FACScan or FACScalibur flow cytometers,
and the data were analyzed with CellQuest software (all from Becton Dickinson, San Jose, CA).
Labeling of cells with fluorescent dyes
T cell populations were stained with the vital fluorescent dye 5 (and 6-)carboxyfluorescein diacetate succinimidyl ester (CFSE) or BODIPY Red
(Molecular Probes, Eugene, OR) as previously described (25, 35). In brief,
cells were resuspended in serum-free RPMI 1640 (Life Technologies) at
10 ⫻ 106/ml. CFSE or BODIPY Red was added to the cell suspension at
1 ␮M final concentration and incubated in a 37°C shaking water bath for
13 min. Cells were then washed twice, recounted, and cultured as described
below.
Cell culture and effector generation
All in vitro cultured cells were cultured in RPMI 1640 supplemented with
penicillin (200 ␮g/ml; Sigma), streptomycin (200 ␮g/ml; Sigma), glutamine (4 mM; Sigma), 2-ME (50 ␮M; Sigma), HEPES (10 mM; Sigma),
and 8% FBS (Intergen, Purchase, NY).
DCEK-ICAM, fibroblast cell line that expresses B7.1 constitutively and
is engineered by stable transfection with ICAM-1 and class II MHC (I-Ek)
molecules, was used as APC at 2:1 T cell:APC. These cells do not express
other costimulatory molecules such as LFA-1, CD48, or heat-stable Ag and
were originally generated by R. Germain (National Institutes of Health,
Bethesda, MD). T-depleted spleen APC blasts were prepared as described
previously (22). Briefly, splenic cell suspensions were depleted of T cells
using anti-Thy1.2 (clones HO13.14 and F7D5), anti-CD4 (clone RL172.4),
anti-CD8 (clone 3.155), and complement. The resulting cells were small
and 90 –95% B220⫹. The T-depleted spleen APC were then stimulated at
5 ⫻ 105/ml for 3 days with LPS (25 ␮g/ml; Sigma) and dextran (25 ␮g/ml;
Sigma). All APC were treated with 100 ␮g/ml of mitomycin c (MitC;
Sigma) for 45 min at 37°C before use.
CD4 effectors were generated by culturing Tg⫹ CD4 cells as previously
described (22). Briefly, naive Tg⫹ CD4 AND T cells (3 ⫻ 106 cells/ml,
Fig. 1, and 3 ⫻ 105 cells/ml, Figs. 2– 6) were cultured with syngeneic
T-depleted splenic APC blasts (3 ⫻ 105 cells/ml, Fig. 2) or DCEK-ICAM
APC (1.5 ⫻ 106 cells/ml, Fig. 1, and 1.5 ⫻ 105 cells/ml, Figs. 3– 6) plus
5 ␮M KAERADLIAYLKQATAK pigeon cytochrome c fragment (PCCF
peptide) (Figs. 1– 6). We have compared stimulation of T cells by culture
with syngeneic T-depleted splenic APC blasts to that of DCEK-ICAM
APC and seen no differences (data not shown). In Fig. 2, 5 ␮M HNTNGVTAACSHE influenza hemagglutinin (HA peptide; New England Peptide, Fitchburg, MA) was added in addition to the PCCF peptide to stimulate the CD4 HNT T cells. Alternatively, Tg⫹ CD4 cells (3 ⫻ 105 cells/
ml, Fig. 5) were stimulated with plate-bound anti-V␤3 (10 ␮g/ml) and
soluble anti-CD28 (10 ␮g/ml). Recombinant murine cytokines IL-2 and
IL-4 were obtained from culture supernatant of X63.Ag8-653 cells transfected with cDNA for the respective cytokines (36). Recombinant murine
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activation status, and survival of the generated effectors are commonly overlooked. An additional weakness of these earlier studies
is that they involved the use of non-physiological plate-bound antiTCR/CD3 mAb plus anti-CD28 mAb as an Ag/APC surrogate,
which may not lead to an accurate picture of Ag-induced responses
in situ (16, 20, 28).
Here, we determine the impact of duration of TCR stimulation
on the quantity and quality of effectors generated. To directly assess the requirements of naive CD4 T cells for, and response to,
defined durations of Ag presentation, we used a TCR-transgenic
#mouse model system in which we isolate a homogeneous population of naive Ag-specific CD4 T cells. We evaluate several TCR
agonists, including plate-bound anti-TCR mAb and two different
physiologically relevant Ag/APC model systems. Because Rogers
and Croft (29) have previously reported on the effects of Ag concentration on T cell stimulation, we restricted these studies to optimal concentrations of Ag as previously determined for each Ag
to be used in these studies. We know that suboptimal or variable
levels of Ag, costimulation, and growth factors (i.e., IL-2) can
have major negative impacts on the generation of CD4 effectors;
therefore, we chose to limit the scope of these studies to determining the effects of Ag duration under conditions of controlled
and optimal Ag dose, costimulation, and IL-2 (1, 19, 29, 30, 31).
With the use of vital dyes and FACS analysis, we have examined
the impact of varied durations of TCR stimulation on both T cell
division and effector expansion, and confirm that a short duration,
1–2 days, of TCR stimulation promotes optimal proliferation and
effector expansion during culture. We also measured the functional
capacity of the resulting effectors as the ability to produce high
levels of polarized cytokines upon restimulation, and we find that
a short duration, 1–2 days, of TCR stimulation in the presence of
growth-promoting and polarizing cytokines is both sufficient and
optimal to promote stable polarized cytokine production. In fact,
increasing the duration of TCR stimulation beyond 2 days leads to
high levels of CD4 T cell death and a significant decrease in the
ability of the remaining viable effectors to produce polarized cytokines upon restimulation. We observed that a TCR stimulationindependent expansion phase exists during the late phase, days
2– 4, of effector generation and is dependent on IL-2. These studies
suggest that optimal CD4 effector generation occurs only when
there is an initial 2 days of Ag presentation followed by an Agindependent and IL-2-driven cell expansion phase lasting an additional 2 days. These observations predict that sequential compartmentalization and redistribution of T cells and Ag-bearing
APC during in vivo cognate interactions are necessary to achieve
this precise sequence of T cell stimulatory conditions, linking observations of compartmentalization of cognate T cell-APC interactions to the regulation of TCR stimulation and signals provided
by growth-promoting cytokines on the generation of optimally
functional effectors (18, 32, 33). These results have important implications for in vitro model systems in which TCR stimulation is
provided through plate-bound anti-TCR Ab. Our results would
predict that prolonged TCR stimulation may select for a subpopulation of less functional effectors and preclude recovery of the most
active populations of effectors.
The Journal of Immunology
5019
IL-12 was a gift from Stanley Wolf (Genetics Institute, Cambridge, MA).
IL-2-polarized effectors (Th0) were generated with IL-2 (80 U/ml). Th1
effectors were generated with IL-2, IL-12 (2 ng/ml), and anti-IL-4 (clone
11B11; 10 ␮g/ml). Th2 effectors were generated with IL-2, IL-4 (200 U/ml),
and anti-IFN-␥ (clone XMG1.2; 10 ␮g/ml). Culture durations varied as indicated in each individual experiment. At the end of culture, live T cells were
enumerated by direct cell counts of trypan blue-excluding cells.
Cytokine detection
Results and Discussion
Kinetics of CD4 effector expansion during the proliferative
phase
In order for naive CD4 T cells to progress from a resting (G0) state
to an activated effector state and initiate division, they must transit
from G0 into the cell cycle. Because the transition from a resting
G0 state into the cell cycle involves expression of multiple genes
and their products in a coordinated fashion, this often results in a
delay between entry into the cell cycle and the first round of cell
proliferation (5, 6). To analyze the rate of division of naive CD4 T
cells following Ag stimulation, we stained naive CD4 T cells with
the vital dye CFSE, which binds to intracellular proteins and is
partitioned to daughter cells with each division (35, 40). Analysis
of the CFSE profiles on each of the 4 days following culture initiation indicates that naive CD4 T cells have a delay of ⬃24 h
between initial TCR stimulation and the first round of cell division
(Fig. 1, a and b), but that after this initial lag period the cells
progress through multiple cell cycles at a constant rate of 7–10
h/cell division throughout the remaining 3 days of culture (Fig.
1b). After day 2, only cells that have divided are detected, indicating either that all of the cells have been stimulated to undergo
division or that cells which have not divided fail to survive (21).
Moreover, when we compare the predicted to the actual effector
recovery for each day during effector generation, with the calculations based on the starting cell number of 3 ⫻ 105 cells and the
average number of cell divisions (Fig. 1, a and b) for each day, we
find that the actual effector recovery is much lower than the predicted effector recovery (Fig. 1c). This suggests that there are significant levels of cell death which occur throughout effector generation, leading to lower effector recoveries than would be
predicted if all of the cells generated survived the 4-day culture
period. These results suggest that although the CD4 T cell prolif-
FIGURE 1. Kinetics of naive CD4 cell division. Naive CD4 AND T
cells were labeled with CFSE and cultured at 3 ⫻ 106 T cells/culture with
1.5 ⫻ 106 DCEK-ICAM, PCCF, and IL-2. a, Analysis of CFSE dye loss
was measured at days 1– 4 during effector generation by FACS analysis as
indicated. b, Number of cell divisions per 24-h time period was determined
by counting the number of cell divisions, as measured by CFSE dye loss,
that occurred between each successive 24-h interval. Duration per cell division was determined by dividing the number of cell divisions per 24-h
interval by 24 h. c, Actual effector recovery was measured by direct cell
counts. Predicted effector recovery was estimated on the basis of starting
cell number and average number of cell divisions. Data are representative
of three independent experiments.
erative phase has the potential to generate ⬃250-fold cell expansion, conditions in vitro are such that the viable effector expansion
is limited to ⬃10- to 20-fold (Fig. 1c). The nature of this cell death
is unclear, but we and others also observe that naive CD4 T cell
expansions in vivo in response to peptide Ag in adjuvant are also
less than would be predicted by the CFSE profiles (E. Roman, G.
E. Huston, and S. L. Swain, unpublished observations; Refs.
41– 43).
One possible explanation for limited CD4 expansion, despite the
high rate of cell division, is that Ag presentation is of limited
duration in vitro and cells need repeated or ongoing TCR triggering to persist after division. Several groups have reported that
DNA synthesis, measured via [3H]thymidine incorporation, or cell
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Culture supernatants collected after 24 h, from cultures of effectors (5 ⫻
105 cells/ml) restimulated with Ag/APC (2.5 ⫻ 105 cells/ml) or with platebound anti-V␤3 (10 ␮g/ml), were assayed for the presence of IL-2 in a
bioassay with NK-3 cells and for IL-4 by ELISA as previously described
(37). IL-2 concentrations are expressed in U/ml (1 U of IL-2 is equal to 1.2
ng). IL-4 concentrations are expressed in nanograms per milliliter.
Alternatively, intracellular cytokine staining (ICCS) was detected in
restimulated effectors as previously described (38, 39). In brief, effectors
(5 ⫻ 105 cells/ml) were restimulated for 24 h with DCEK-ICAM APC
(2.5 ⫻ 105 cells/ml) with PCCF (5 ␮M) or restimulated for 4 h with PMA
(10 ng/ml; Sigma) and ionomycin (50 ng/ml; Sigma). Negative controls
(indicated by dotted lines) indicate ICCS of T cells that are not restimulated
and thus do not express the indicated cytokine. Brefeldin A (10 ␮g/ml final
concentration; Sigma) was added 2 h after culture initiation and maintained
throughout the ICCS protocol. At the end of the specified restimulation
period, cells were collected and surface stained for CD4 and Tg expression
as described above. The cells were then divided into separate tubes,
washed, fixed in 75 ␮l of fresh 4% paraformaldehyde (Sigma) plus 25 ␮l
of PBS containing 10 ␮g/ml brefeldin A, and incubated for 20 min at room
temperature. The tubes were then washed once with PBS and resuspended
in 50 ␮l saponin buffer (PBS containing 1% FBS, 0.1% NaN3, and 0.1%
saponin (Sigma), pH 7.4 –7.6). PE-labeled antimouse IL-2, PE-labeled antimouse IFN-␥, or PE-labeled antimouse IL-4 was added and incubated
with the T cells for 30 min at room temperature. All samples were then
washed with PBS/BSA ⫹ 0.2% azide and analyzed on a FACScan or
FACScalibur cytometer.
5020
Ag-DEPENDENT AND IL-2-DRIVEN STAGES FROM NAIVE TO EFFECTOR
proliferation, measured via 5-bromo-2⬘-deoxyuridine incorporation, of naive T cells is largely determined by the strength and/or
duration of Ag (2, 16, 20, 44, 45). Although division of responding
lymphocytes is certainly an important step in the development of
an immune response, we would argue that the development of
increased numbers of highly potent effectors capable of rapidly
producing multiple cytokines upon restimulation with Ag/APC is
the most important criteria for measuring the generation of an effective immune response. We particularly wanted to address the
impact of prolonging TCR stimulation. To better understand what
happens normally in our in vitro cultures, we started by evaluating
how long Ag presentation was actually occurring.
Efficient Ag presentation is restricted to the first 1–2 days of
culture
FIGURE 2. Efficient Ag presentation only occurs for the first 1–2 days
of culture. Naive CD4 HNT T cells were BODIPY Red-labeled and cultured at 3 ⫻ 105 T cells/culture with 3 ⫻ 105 splenic APC blasts, 5 ␮M HA
peptide, 5 ␮M PCCF peptide, and under IL-2-polarizing (Th0), Th1-, or
Th2-polarizing cytokine environments. Fresh naive CD4 AND T cells were
CFSE labeled and added at 3 ⫻ 105 T cells/culture to the ongoing HNT T
cell:Ag/APC cultures at days 0 –3 as indicated. The AND T cells were
harvested 3 days after their addition to the cultures. a, Cell division was
measured by CFSE dye loss of live V␤3⫹ (AND)/CD4⫹ cells, and the
percent undivided cells are indicated by FACS gating. b, Live V␤3⫹
(AND)/CD4⫹ cell recoveries were determined by performing direct cell
counts of total cells and extrapolating them from the percentages of CFSElabeled live V␤3⫹ (AND)/CD4⫹ cells present in the whole culture. Starting AND T cell numbers were 3 ⫻ 105. Data are representative of three
independent experiments.
vision (Fig. 2a) and expansion (Fig. 2b) of naive AND T cells
declined during the first day of culture and was negligible by 2
days after the original Ag/APC cultures were initiated.
To determine whether cytokines present during effector polarization might prolong the ability of the splenic APC blasts to
present Ag to naive CD4 cells, we designed this experiment under
Th0 (IL-2 only)-, Th1-, or Th2-polarizing conditions. The ability
of the Ag/APC cultures to stimulate naive AND T cell proliferation was similar under all three polarization conditions (Fig. 2a).
Therefore, the cytokines present during in vitro effector polarization did not enhance the ability of the splenic APC blasts to present
Ag to naive CD4 T cells. We repeated this experiment using the
DCEK-ICAM fibroblast APC line and have seen no difference in
the ability of the two APC populations to stimulate naive CD4
AND T cell expansion and differentiation (data not shown; Refs. 1,
2, 30).
The recovery of the AND T cells after 3 days in culture mirrored
their division. Naive AND T cells added at the initiation of HNT
T cell:Ag/APC culture (day 0) expanded markedly by 3 days,
whereas AND T cells added after 1 or 2 days of Ag/APC culture
underwent little expansion or even declined over the 3 days (Fig.
2b). Th2-polarizing conditions supported slightly greater effector
recoveries as compared with IL-2-polarizing (Th0) and Th1-polarizing conditions, suggesting that the presence of IL-4 in the
cultures may enhance T cell survival. However, all AND T cell
effector recoveries had decreased to numbers below starting cell
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We know from previous studies that MitC-treated APC disappear
from culture by 48 h, as measured by surface staining for ICAM-1
and class II (17, 46). This phenomenon only occurs during culture
conditions where Ag presentation occurs. MitC-treated APC cultured with T cells and without Ag or MitC-treated APC cultured
with Ag and without T cells survive for 4 days of culture, whereas
MitC-treated APC cultured with T cells and Ag are rapidly deleted
from culture (data not shown). However, since it is possible that
membrane fragments of no longer intact APC might still present
Ag, we sought to determine the time at which cultures of MitCtreated APC are no longer capable of effectively stimulating naive
CD4 T cell proliferation or cytokine production by effectors in
standard in vitro cultures. We developed a cell mixing system
where we initiate cultures of BODIPY Red-labeled naive HNT T
cells, specific for influenza HA peptide, with an activated APC
population containing not only B cell blasts, but also a small number of other APC populations including macrophages and dendritic
cells. We then loaded the splenic APC blasts with HA and PCCF
peptides such that the APC are capable of efficient presentation of
both the influenza HA peptide to HNT T cells and the PCCF peptide to AND T cells within the same culture. At various time points
after initiation of the HNT T cell:Ag/APC culture, we isolated
fresh naive AND T cells from spleens and lymph nodes from AND
TCR Tg mice, labeled these cells with CFSE, and introduced them
to the ongoing HNT T cell:Ag/APC cultures. Three days after the
introduction of the AND T cells to the ongoing HNT T cell:Ag/
APC cultures, the T cells were harvested and analyzed for cell
division and effector expansion. With these experiments, we were
able to easily identify the two populations of T cells with anti-TCR
␤-chain-specific Ab, and based on their BODIPY Red vs CFSE
staining, measure the capacity of the newly added AND T cells to
respond to whatever APC were still actively presenting Ag at various time points after initiation of HNT T cell:Ag/APC cultures,
and verify that the proliferative capacity of the HNT T cells added
at the initiation of the T cell:Ag/APC cultures was not perturbed by
the addition of the fresh naive AND T cells.
When naive AND T cells were added to HNT T cell:Ag/APC
cultures at day 0, ⬍10% of the AND T cells could be detected as
nondivided cells under all three polarizing conditions (Fig. 2a),
indicating that these culture conditions are fully capable of stimulating optimal effector generation. However, when the naive
AND T cells were added after 1 day of Ag/APC culture, ⬃40% of
the AND T cells recovered after 3 days had not divided, and adding naive AND T cells to Ag/APC cultures at day 2 resulted in
little AND T cell division (Fig. 2a). Our results indicate that although the original HNT T cells proliferate normally, regardless of
the time of naive AND T cell introduction to the cultures (data not
shown), the ability of the splenic APC blasts to stimulate cell di-
The Journal of Immunology
FIGURE 3. Loss of Ag presentation to CD4 effectors by Ag/APC cultures. Naive CD4 AND T cells were stimulated for 4 days under IL-2polarizing (Th0) or Th2-polarizing conditions. The resulting Th0 and Th2
4-day effectors were CFSE labeled and then reintroduced to culture with
freshly prepared DCEK-ICAM APC without PCCF peptide (A, negative
control: dotted lines), 24-h cultured PCCF peptide-pulsed DCEK-ICAM
(B, solid lines), or freshly prepared PCCF peptide-pulsed DCEK-ICAM (C,
positive control: shaded histograms). ICCS for IL-2 and IFN-␥ was measured on CFSE⫹-, V␤3⫹-, CD4⫹-gated Th0 effectors after 24 h of restimulation. IL-4 was measured on CFSE⫹-, V␤3⫹-, CD4⫹-gated Th2 effectors
after 24 h of restimulation. Data are representative of between three and
four independent experiments.
they exhibit significantly reduced intracellular cytokine production
(Fig. 3, solid lines). These results indicate that the ability of Ag/
APC to stimulate cytokine production by even highly activated
effectors is lost as early as 1 day after in vitro culture of Ag/APC
with T cells. Together, these results reveal that efficient Ag presentation to both naive and effector T cells by professional APC is
limited to the first 1–2 days in standard in vitro culture systems
(Figs. 2 and 3), and any potential residual APC are incapable of
supporting naive T cell proliferation and cytokine production or
even the response of highly activated effectors.
Prolonged Ag stimulation leads to detrimental effects on effector
expansion and function
In all cases discussed thus far in these studies, the APC used were
MitC treated; therefore, their short life span was expected, but it is
important to note that under these standard in vitro culture conditions used by many groups of investigators, Ag presentation is of
short duration but nonetheless results in highly efficient T cell expansion and effector generation. However, in vivo there is the potential for APC to interact with T cells throughout the process of
effector generation and to potentially provide T cells with prolonged or repeated TCR stimulation. Since our in vitro Ag/APC
culture system results in the disappearance of APC between 1 and
2 days, to determine whether increased durations of exposure to
Ag/APC results in more efficient effector generation, we examined
the effects of increasing the duration of Ag presentation on effector
generation by supplementing cultures with additional Ag/APC
throughout the 4-day culture period. We incubated naive CD4 T
cells under IL-2-polarizing (Th0) and Th2-polarizing conditions
and supplemented the cultures with fresh peptide-pulsed APC once
at day 0 (normal culture conditions), twice at days 0 and 1, three
times at days 0 –2, and four times at days 0 –3. The effectors
present after 4 days were then analyzed for effector recovery and
their ability to secrete cytokines upon restimulation.
The results indicate that effector recovery in both the IL-2-polarized (Th0) (Fig. 4a) and Th2-polarized (Fig. 4b) subsets is significantly decreased when standard in vitro T cell cultures are supplemented with extended durations of Ag/APC exposure.
Although the Th2-polarized subset benefited slightly from one additional Ag/APC supplement, extended durations of Ag/APC exposure resulted in markedly decreased effector recoveries (Fig. 4b,
shaded bars). Interestingly, the viable cells recovered after 4 days
all maintained similar proliferation profiles, as measured by CFSE
dye loss (data not shown), suggesting that the decrease in effector
recovery which results from extended durations of Ag/APC exposure is not due to an impairment in proliferation potential of surviving cells. Therefore, we hypothesized that the decreased effector recovery (Fig. 4) indicates that effectors stimulated with ⬎2
days of Ag presentation undergo high-level death as they respond.
Using forward scatter and side scatter (FSC ⫻ SSC) FACS profiles
of the CD4 Tg cells, we were able to show that, in fact, the percentages of cell death increased from 11 to 55% for the IL-2 (Th0)polarized effectors and from 18 to 34% for the Th2 effectors as
Ag/APC exposure was extended. These numbers may seem too
low to account for the low cell recoveries, but the cell death measurements taken at day 4 do not reflect the integrated effect of cell
death during the culture. Dead cells typically disappear from culture within 24 –36 h of their death, and therefore a cell that dies at
days 2–3 of culture would not be detectable on day 4. Additionally,
if a cell dies on day 2 its potential capacity to expand had it lived
must be taken into account when adjusting for cell recovery at day
4. Thus, the cell recovery at day 4 has the potential to be much
lower than what would be expected based on calculations of percentages of cell death.
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numbers when AND T cells were added on either day 2 or 3 of
Ag/APC culture. Overall, these results suggest that MitC-treated
splenic APC blasts are no longer capable of supporting naive T cell
proliferation and effector generation when cultured for more than
2 days with Ag-specific T cells, regardless of the T cell-polarizing
cytokine milieu.
Since naive CD4 T cells require a significantly greater amount
of TCR stimulation and costimulation to enter into the cell cycle
and rapidly produce high levels of cytokines, as compared for
4-day effectors, we examined the ability of freshly prepared (day
0) and 1-day Ag/APC cultures to stimulate 4-day effectors to produce polarized cytokines (1, 30). We generated 4-day Th0-or Th2polarized effectors and restimulated them with freshly prepared
DCEK-ICAM APC loaded with PCCF peptide (Fig. 3, positive
control, filled histograms), freshly prepared DCEK-ICAM APC
without Ag (negative control, dotted lines), or 1-day cultures of
DCEK-ICAM APC loaded with PCCF peptide (solid lines). Twenty-four hours after the 4-day Th0 and Th2 effectors were added to
the various APC cultures, we harvested the T cells and measured
intracellular cytokine production by effectors. Because we needed
to culture APC with Ag and T cells in order for the APC to be
deleted from the cultures, and these T cells could potentially contribute secreted cytokines to culture supernatants used in ELISA
cytokine detection methods, we used ICCS to ascertain the cytokine production of the 4-day Th0 or Th2 effectors while gating out
any cytokine contribution of the naive T cells used to generate the
1-day Ag/APC cultures. When 4-day effectors were stimulated for
24 h with freshly prepared Ag/APC, they were induced to secrete
readily detectable levels of appropriate cytokines (positive control), whereas 4-day effectors stimulated for 24 h with freshly prepared APC without Ag do not produce detectable levels of cytokines (negative control). Th0 effectors typically produce high
levels of IL-2 and slightly lower production of IFN-␥, whereas Th2
effectors produce measurable levels of IL-4 as determined by ICCS
(Fig. 3, shaded histograms, positive controls). These results can be
mimicked by the recovery of cytokines in culture supernatants
from purified 4-day effector populations (data not shown). In contrast, when 4-day Th0 or Th2 effectors are added to Ag/APC cultures which have interacted for as little as 1 day with naive T cells,
5021
5022
Ag-DEPENDENT AND IL-2-DRIVEN STAGES FROM NAIVE TO EFFECTOR
In these studies, we provide exogenous IL-2 at the beginning of
T cell culture (day 0) to promote optimal T cell expansion. However, previous studies have shown that production and accumulation of autocrine IL-2 by naive T cells takes 20 –30 h following
stimulation with optimum levels of TCR stimulation and costimulation (Ref. 11; L. Haynes and S. L. Swain; unpublished data). IL-2
is known to regulate AICD such that T cells previously exposed to
IL-2 undergo apoptosis rapidly following Ag receptor stimulation
(26, 47). Moreover, the kinetics of IL-2 addition to culture has
been shown to have profound effects on the susceptibility and rescue of T cells from Fas-mediated AICD (17). Therefore, we examined the effects of increasing the duration of Ag presentation on
effector generation by supplementing cultures with additional Ag/
APC throughout the 4-day culture period, as described in Fig. 4,
and adding exogenous IL-2 on day 0 (normal culture conditions),
1, or 2. We found there to be no difference in the kinetics of
decreased effector recovery and increased cell death caused by
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 4. Negative effect of prolonged Ag stimulation with Ag/APC.
Naive CD4 AND T cells were CFSE labeled and cultured at 3 ⫻ 105 T
cells/culture with 1.5 ⫻ 105 DCEK-ICAM pulsed with PCCF peptide under IL-2-polarizing (Th0) or Th2-polarizing cytokine environments. A total
of 1.5 ⫻ 105 freshly prepared PCCF peptide-pulsed DCEK-ICAM was
added once at day 0 only, twice at days 0 and 1, three times at days 0 –2,
or four times at days 0 –3. a, Th0 effector recoveries of live V␤3⫹(AND)/
CD4⫹ T cells were determined at day 4 of culture by direct cell counts. Cell
death was measured by FSC ⫻ SSC FACS profiling and determined to be
11, 36, 52, and 55%, respectively. IL-2 production was measured by bioassay of supernatants recovered after 24 h of restimulation of 4-day Th0
effectors with PCCF peptide-pulsed DCEK-ICAM. b, Th2 effector recoveries of live V␤3⫹(AND)/CD4⫹ T cells were determined at day 4 of culture by direct cell counts. Cell death was measured by FSC ⫻ SSC FACS
profiling and determined to be 18, 13, 23, and 34%, respectively. IL-4
production was measured by ELISA of supernatants recovered after 24 h of
restimulation of 4-day Th2 effectors with PCCF peptide-pulsed DCEKICAM. Data are representative of four independent experiments.
prolonged Ag presentation when the time of exogenous IL-2 addition was delayed from day 0 to day 1 or 2 (data not shown).
The ability of effectors to rapidly produce high levels of multiple
cytokines after restimulation is an important functional attribute
which is critical to regulate the immune response to an invading
pathogen. Therefore, we also tested the ability of the surviving
viable 4-day effectors to produce polarized cytokines. Because T
cell recovery was much lower in the cultures with prolonged Ag/
APC exposure, we readjusted the 4-day effectors recovered to
equivalent cell concentrations before restimulation with Ag/APC
for 24 h. We measured IL-2 levels produced by IL-2-polarized
(Th0) effectors (Fig. 4a) and IL-4 levels produced by Th2-polarized effectors (Fig. 4b). Equivalent numbers of effectors obtained
from cultures with prolonged Ag/APC stimulation produced much
lower levels of cytokines compared with effectors resulting from
the standard (shorter) Ag/APC exposure (Fig. 4). Together, these
studies show that exposure to Ag/APC beyond 2 days during effector generation not only leads to decreased effector recovery,
which is most likely due to increased cell death, but also decreases
the ability of surviving T cells to function properly and produce
polarized cytokines upon restimulation. Thus, the susceptibility of
restimulated developing effectors to cell death may limit the cytokine accumulation upon restimulation or select for less differentiated T cells.
Supplementing T cell cultures with Ag/APC beyond 2 days was
detrimental to the recovery and function of resulting effectors;
however, it is possible that this result might be due to the effects of
culture crowding by the added APC or some other negative impact
of adding fresh APC to the culture. Therefore, we used platebound anti-TCR mAb stimulation plus soluble anti-CD28 mAb
costimulation conditions to independently define the optimal duration of Ag presentation. These studies were designed to provide
optimal stimulatory conditions throughout the culture period with
the only variable being the duration of TCR stimulation. Naive
AND T cells were incubated in anti-V␤3 mAb-coated wells with
soluble anti-CD28 mAb and IL-2 alone (Th0) or under Th2-polarizing conditions. The T cell cultures were then transferred to
noncoated tissue culture wells, without washing, over a broad
range of time points. Effector recovery, effector death, and polarized cytokine-producing capacity of the resulting effectors were
measured at the end of a total 4-day culture period.
Our results indicate that the highest level of effector recovery
results from 2 days of TCR stimulation in both the IL-2-polarized
(Th0) (Fig. 5a) and Th2-polarized (Fig. 5c) populations, with
shorter and longer durations of TCR stimulation resulting in low
numbers of effectors (Fig. 5, a and c). Stimulation with anti-TCR
mAb for ⬍2 days resulted in effectors that appeared poorly activated as indicated by changes in activation marker expression and
small cell size, whereas exposure to anti-TCR mAb beyond 2 days
resulted in effectors that did display a highly activated phenotype
(data not shown). We hypothesized that the decrease in effector
recovery resulting from increased durations of Ag/APC exposure
(Figs. 1c and 4) is due to AICD as effectors are reexposed to
Ag/APC or anti-TCR Ab, rather than due to a decrease in induction
of cell proliferation. Therefore, we examined effector death, as
measured by propidium iodide (PI) uptake or FSC ⫻ SSC FACS
profiles of the cultures. Indeed, longer durations of TCR stimulation resulted in high levels of effector death (Fig. 5, a and c). Taken
together, the decreased effector recovery and increased cell death
(Fig. 5, a and c) by cultures stimulated with ⬎2 days of TCR
stimulation support the hypothesis that prolonging Ag presentation
results in high levels of T cell death, which directly results in decreased effector recovery rather than increased effector responses.
The Journal of Immunology
5023
We also analyzed the ability of effectors generated in the antiTCR mAb cultures to be restimulated to produce polarized cytokines. We found that a slightly longer Ag presentation of 21⁄2 days
resulted in IL-2-polarized (Th0) effectors best capable of producing high levels of IL-2 upon restimulation, with lesser and longer
durations of Ag presentation resulting in lower levels of cytokine
production (Figs. 4a and 5b). Whereas 2 days of Ag presentation
resulted in Th2 effectors best capable of producing high levels of
IL-4 upon restimulation (Figs. 4b and 5d). The need for a shorter
TCR stimulation in the Th2-polarizing cultures could be due to the
presence of IL-4, which promotes a more rapid activation as compared with IL-2 alone that is present in Th0 cultures (D. M. JelleyGibbs and S. L. Swain, unpublished observations).
Cytokine dependence during Ag-independent late phase of
effector generation
Our next set of experiments was aimed at examining the mechanism responsible for driving the constant rate of cell division seen
in the late phase (days 2– 4) of our standard in vitro cultures (Fig.
1), despite the clear lack of Ag presentation beyond the early phase
of effector generation (days 0 –2). We hypothesized that the Agindependent constant rate of cell division observed in the late
phase of cell expansion is either driven by growth-promoting cytokines, like IL-2, or is a consequence of early and irreversible
programming events within the T cell, or a combination of both.
To test whether IL-2 is required to drive the late phase of effector
generation, naive CD4 cells were cultured with Ag/APC under
standard Th2-polarizing conditions for 2 days. The T cells were
then washed to remove any exogenous and secreted IL-2, CFSE
labeled, and recultured for an additional 2 days with either 1) culture supernatants added back, 2) fresh T cell media plus exogenous
IL-2, or 3) fresh T cell media plus anti-IL-2R␣-blocking Ab. To
obtain high levels of synchronized CFSE labeling, we CFSE la-
beled the T cells at day 2 of culture before dividing the cells into
the three respective groups. Because all of the T cells are already
cycling, we observed a relatively synchronized cell proliferation
profile unlike that seen when naive or resting T cells are CFSE
labeled and then cultured to proliferate. Naive or resting T cells
enter the cell cycle in an asynchronous manner which has a delay
of 24 –32 h, and then they divide at a relatively constant rate of
every 6 – 8 h (Fig. 1). Alternatively, actively cycling cells (those
seen on days 2– 4 of culture) appear to be relatively synchronized
in their cell cycles. This synchronized cell cycling results in CFSE
profiles which are less defined for specific division peaks. However, the numbers of divisions each cell has undergone can be
easily calculated by measuring the mean fluorescent intensity
(MFI) of the CFSE as dye loss in each daughter cell being equal to
exactly 50% of that of the parent cell. For example, if the MFI of
CFSE labeling on day 2 is 1000, the MFI of a cell that had divided
one time would be 500, twice would be 250, and three times would
be 125. Using these mathematical calculations, we were able to
determine the average number of divisions occurring between days
2 and 4 of effector generation. At the end of the 4-day culture
period, the effector recovery and CFSE profiles were examined,
and the ability of the resulting viable effectors to produce polarized
cytokines upon restimulation was measured by ICCS.
Under conditions where effectors received no ongoing IL-2
stimulation (blocking with anti-IL-2R␣ Ab for the last 2 days of
culture), there was a greatly reduced effector recovery (Fig. 6a),
which was paralleled by a decreased rate of cell division between
days 2 and 4 as determined by CFSE dye loss (Fig. 6b). The decreased effector recovery of ⬃50% of that found when IL-2 was
present was accounted for by a reduction of an average number of
cell divisions of 4.25 divisions for the cultures containing IL-2, to
2 divisions for the cultures without IL-2. We quantitated cell death
of these cultures by FSC ⫻ SSC FACS profiling and found no
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FIGURE 5. Negative effect of prolonged Ag
stimulation with anti-V␤3 (anti-TCR). Naive
CD4 AND T cells were CFSE labeled and cultured at 3 ⫻ 105 T cells/culture in antiV␤3-coated wells with soluble anti-CD28 under
IL-2-polarizing (Th0) or Th2-polarizing cytokine environments. Total T cell cultures and supernatants were transferred to noncoated tissue
culture wells without washing at the times indicated. a, Th0 effector recoveries of live
V␤3⫹(AND)/CD4⫹ T cells were determined at
day 4 of culture by direct cell counts. Percent
effector mortality was determined by PI uptake
(percent PI⫹) of V␤3⫹(AND)/CD4⫹ as measured by FACS analysis. b, IL-2 production was
measured by bioassay of supernatants recovered
after 24 h of restimulation of 4-day Th0 effectors
with PCCF peptide-pulsed DCEK-ICAM. ND,
Not determined because there were not enough
cells to restimulate. c, Th2 effector recoveries of
live V␤3⫹(AND)/CD4⫹ T cells were determined at day 4 of culture by direct cell counts.
Percent effector mortality was determined by
FSC ⫻ SSC FACS profiling of V␤3⫹(AND)/
CD4⫹ T cells. d, IL-4 production was measured
by ELISA of supernatants recovered after 24 h
of restimulation of 4-day Th2 effectors with
PCCF peptide-pulsed DCEK-ICAM. Data are
representative of five independent experiments.
5024
Ag-DEPENDENT AND IL-2-DRIVEN STAGES FROM NAIVE TO EFFECTOR
significant increases in cell death as a result of blocking IL-2.
Therefore, instead of proliferating every 8 h, effectors cultured
during the late phase of effector generation with antiIL-2R␣-blocking Ab only underwent one round of proliferation
approximately every 16 h (Fig. 6b). Moreover, effectors cultured
under conditions where IL-2 stimulation was blocked were capable
of producing little IL-2 and no IL-4, whereas cultures reconstituted
with their own supernatants or with exogenous IL-2 produced normal levels of IL-2 and IL-4 (Fig. 6c). Therefore, not only was the
rate of cell proliferation during the final 2 days of culture reduced
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FIGURE 6. Cytokine dependence during Ag-independent phase of effector generation. Naive CD4 AND T cells were cultured at 3 ⫻ 105 T
cells/culture with 1.5 ⫻ 105 PCCF peptide-pulsed DCEK-ICAM under
Th2-polarizing conditions for 2 days. To synchronize the CFSE labeling,
the cultured cells were then washed to remove exogenous and secreted
IL-2, CFSE labeled, and cultured an additional 2 days with either their
original culture supernatants added back, T cell media plus only exogenous
IL-2, or T cell media plus anti-IL-2R␣-blocking Ab. a, Live V␤3⫹(AND)/
CD4⫹ effector recoveries were determined at day 4 of culture by direct cell
counts. Cell death was determined by FSC ⫻ SSC FACS profiling and was
17, 14, and 25%, respectively. b, FACS analysis of CFSE dye loss of live
V␤3⫹(AND)/CD4⫹ effectors was used to measure cell division during the
last 2 days of culture. c, ICCS for IL-2 and IL-4 was measured on live
CFSE⫹V␤3⫹(AND)/CD4⫹ effectors restimulated for 4 h with PMA ⫹
ionomycin. Data are representative of three independent experiments.
by the lack of IL-2 stimulation, but the resulting effectors were
incapable of optimum effector function, as measured by cytokine
production. This supports the hypothesis that there are two distinct
phases of cell activation and expansion such that effectors first
undergo a 1–2-day Ag-dependent phase of cell activation and expansion, which is then followed by an Ag-independent and IL-2driven late phase of effector differentiation and expansion into
fully functional effectors (Figs. 1 and 6) (25). This pattern suggests
that mechanisms may well exist in situ to ensure that once fully
activated, responding CD4 T cells must be segregated from Agbearing APC. In these studies, which use highly purified naive
CD4 T cells and APC populations, autocrine IL-2 and exogenous
IL-4 are the only IL-2R␥ chain-binding, growth-promoting cytokines available. It is likely that other IL-2R␥ chain-binding cytokines, or other growth-promoting cytokines, may drive in vivo
responses (48, 49). Thus, it will be important to determine whether
other cytokines can promote this Ag-independent phase in vivo
and to determine what regulates their availability.
By supplementing T cell cultures with more prolonged Ag presentation, either in the form of Ag/APC or plate-bound anti-TCR
Ab, we found that Ag presentation beyond day 2 leads to the disappearance and death of responding CD4 T cells, thus resulting in
recovery of fewer effectors at day 4. Moreover cytokine production
by surviving effectors is decreased by the prolonged Ag exposure
(Figs. 4 and 5). Many responding CD4 T cells, which continue to
encounter Ag/APC, apparently undergo AICD, which results in the
selection of a population of cells which produce less cytokine,
perhaps reflecting a selection for the least differentiated cohorts of
cells. This may be a means to prevent uncontrolled effector expansion, which could lead to detrimental inflammatory responses.
These results augment many previous in vitro studies whereby
restimulated effector T cells undergo cell deletion via AICD (17,
50). We have recently confirmed that CD4 effectors transferred to
adoptive hosts also disappear when hosts are immunized with Ag
(E. Roman, G. E. Huston, and S. L. Swain, unpublished observations). As discussed below, we suggest that these findings may
indicate that productive in vivo responses must involve a process
which ensures responding CD4 T cells are not reexposed to Ag
after 2 days.
The results presented in these studies also have important implications for the popular in vitro model system in which TCR
stimulation is provided through plate-bound anti-TCR Ab. These
studies are commonly performed by incubating T cells in tissue
culture plates coated with anti-CD3 Ab, anti-TCR Ab, or tetramers
for the entire duration of the experiment, which typically extends
to 4 –7 days of culture. These results predict that prolonged TCR
stimulation, beyond the first 2 days of culture for naive T cells,
may select for a subpopulation of less functional effectors and
preclude recovery of the most active populations of effectors.
These results also predict that extended TCR stimulation would
promote the selection of a subpopulation of effectors resistant to
cell death. These results present a need to evaluate the functional
and phenotypic qualities of effectors generated using this particular
in vitro model system for T cell stimulation.
Since optimal CD4 effector generation and function is dependent on an initial 1–2 days of Ag presentation followed by the
absence of Ag presentation during an IL-2-driven cell expansion
phase lasting an additional 2 days (Fig. 6), it is important to consider the possibility that in vivo CD4 T cell responses may be
compartmentalized to achieve this set of conditions. Several published reports support the concept of chronologically and anatomically separate phases in CD4 T cell response. Ingulli et al. (18)
have shown that Ag (OVA)-specific CD4 T cells and Ag-pulsed
The Journal of Immunology
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dendritic cells (DC) accumulate to a maximal level in the T cellrich paracortical regions of spleen and lymph nodes within 24 h of
i.v. injection of the Ag-pulsed DC and that the accumulation of
DC:T cell clusters then declined by 48 h (18). This dissociation
could have been due to limitations of Ag or instead an active
process associated with the CD4 T cell response. By 48 h, Agpulsed DC rapidly disappeared from the lymph nodes, either because they migrate out of the lymph node or are killed by the
responding T cells. Two days after immunization, both Ag-specific
T cells and B cells moved toward each other from their separate
starting locations and cognate T cell-B cell interactions could be
visualized at the edge of the B cell-rich follicles at the border
between the follicles and the T cell areas in the spleen (33). By
days 3 and 4, the Ag-specific T cells are no longer found near the
Ag-presenting B cells (18, 33). Perhaps those T cells destined to
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to a separate location rich in growth and differentiation-promoting
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need for responding CD4 T cells to encounter IL-2 or an in vivo
surrogate for full expansion and effector development would suggest that they may do so in a defined niche.
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Ag-DEPENDENT AND IL-2-DRIVEN STAGES FROM NAIVE TO EFFECTOR
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