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Trudeau Institute, Saranac Lake, NY 12983
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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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, 8, 9, 10, 11, 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 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, 2, 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-ß 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, 22, 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, 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 anti-TCR/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 stimulation-independent 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 Ag-independent 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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H-2k/k (B10.Br) and
H-2b/b (C57BL/6) V11/Vß3 AND TCR transgene
(Tg; I-Ek restricted),
B10.Br.HNT.B10.D2/F1 (Vß8.3/V2) 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). FITC-labeled 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 anti-CD4 (clone
RM4–5; Caltag, Burlingame, CA), FITC- and PE-labeled anti-CD44
(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-V11 (clone RR8-1), PE- and biotin-labeled anti-Vß3
(clone KJ25), PE-labeled anti-Vß8.3 (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 BVD-24G2; Caltag). All isotype
control Abs were purchased from PharMingen: FITC- and PE-labeled
hamster Ig, FITC- and PE-labeled rat IgG1, FITC- and 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 x 106/ml. CFSE or BODIPY Red was added to the cell suspension at 1 µM 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 µg/ml; Sigma), streptomycin (200 µg/ml; Sigma), glutamine (4 mM; Sigma), 2-ME (50 µM; 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 x 105/ml for 3 days with LPS (25 µg/ml; Sigma) and dextran (25 µg/ml; Sigma). All APC were treated with 100 µg/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 x
106 cells/ml, Fig. 1
, and 3 x
105 cells/ml,
Figs. 2–6) were cultured with
syngeneic T-depleted splenic APC blasts (3 x
105 cells/ml, Fig. 2) or DCEK-ICAM APC (1.5
x 106 cells/ml, Fig. 1, and 1.5 x
105 cells/ml,
Figs. 3–6) plus 5 µM
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
µM 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 x 105 cells/ml, Fig. 5) were
stimulated with plate-bound anti-Vß3 (10 µg/ml) and soluble
anti-CD28 (10 µg/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 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 µg/ml). Th2 effectors were
generated with IL-2, IL-4 (200 U/ml), and anti-IFN- (clone
XMG1.2; 10 µg/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.
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Culture supernatants collected after 24 h, from cultures of effectors (5 x 105 cells/ml) restimulated with Ag/APC (2.5 x 105 cells/ml) or with plate-bound anti-Vß3 (10 µg/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 x 105 cells/ml) were
restimulated for 24 h with DCEK-ICAM APC (2.5 x
105 cells/ml) with PCCF (5 µM) 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 µg/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 µl of fresh 4% paraformaldehyde (Sigma) plus 25 µl of PBS
containing 10 µg/ml brefeldin A, and incubated for 20 min at room
temperature. The tubes were then washed once with PBS and resuspended
in 50 µl 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.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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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 x
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
proliferative 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, 42, 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 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
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 MitC-treated 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 ß-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 division (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 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 Th2-polarized 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, 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 x 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.
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 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 plate-bound 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-Vß3 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 x 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.
We also analyzed the ability of effectors generated in the anti-TCR
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. Jelley-Gibbs 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
Ag-independent 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 labeled 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 x SSC
FACS profiling and found no 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 anti-IL-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 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-2-driven 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
Ag-bearing 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 dendritic cells (DC)
accumulate to a maximal level in the T cell-rich 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, Ag-pulsed 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 expand and produce large
populations of effectors relocate as well to a separate location rich
in growth and differentiation-promoting factors. Indeed, a recent study
by Vasseur et al. (32) suggests that Ag-specific T cell
accumulation in the lymph nodes is very transient (1–2 days), and
proliferating cells recirculate in the blood rapidly after Ag
presentation and T cell activation (32). Thus, the concept
that the period where Ag is presented to CD4 T cells is of limited
duration seems to be mirrored by the in vivo migratory patterns of
responding CD4 T cells and APC. Together, these studies support a model
in which CD4 T cells are activated in the first 1–2 days of an immune
response by Ag-presenting DC and begin division. Then the T cells
migrate to either an area in the lymphoid compartment where they can
interact with Ag-presenting B cells or they may migrate away from the
Ag/APC environments altogether. Whether there is a defined site of
additional CD4 T cell expansion and differentiation is unclear
(18, 32, 33, 51, 52). However, the 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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Footnotes |
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2 Address correspondence and reprint requests to Dr. Susan L. Swain, Trudeau Institute, P.O. Box 59, Saranac Lake, NY 12983.
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.
Received for publication June 19, 2000. Accepted for publication August 10, 2000.
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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