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*
Center for Immunology and Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455;
Departments of Medicine and Pathology, University of Chicago, Chicago, IL 60637; and
Department of Medicine, University of Connecticut, Farmington, CT 06030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Following antigenic stimulation and acquisition of effector functions, most activated T cells die (4, 5), while a small population survives, some of which are thought to contribute to the memory pool (5, 6, 7, 8). Currently, there are no data concerning the reexpression of LKLF after Ag stimulation and whether or not this factor is expressed in the memory T cell pool. Given the importance of this factor in survival of naive T cells, these questions are relevant to understanding whether LKLF is relevant only to virgin T cell lifespan, or whether this factor might play a role in maintenance of Ag-experienced T cells.
In this paper, we report that LKLF expression can be reinduced in Ag-activated CD8+ T cells by exposure to certain cytokines. We present evidence that expression of the factor is not likely to be due to expansion of a small population of T cells that failed to down-regulate LKLF, but rather appears to be genuine reexpression of the transcription factor by the bulk population. Furthermore, we show that after prolonged culture of Ag activated CD8 T cells in IL-7, these cells express a phenotype identical to memory CD8 T cells and stably maintain LKLF expression. Finally, we show that LKLF down-regulation following Ag activation and LKLF expression in memory T cells can be demonstrated in freshly ex vivo populations, suggesting that the changes in expression observed in vitro are physiologically significant.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD8+ cells from OT-I mice (9) were enriched by passage of lymph node (LN) cells over CD8 Cellect columns (Cytovax, Alberta, Canada); this protocol typically yielded >90% purity of CD8+ cells.
LN and spleen cells isolated from RAG-1o/o OT-I
mice were used without purification. By flow cytometry, LN cells were
>90% CD8+, V
2+, and
spleen cells were >80% CD8+,
V2+ in these animals.
Memory T cells were generated in vivo as described by Kim et al. (10).4 Briefly, LN cells from OT-I, Ly5.2+ congenic mice were adoptively transferred into normal C57BL/6 hosts (Ly5.1+). Donor OT-I cells were stimulated by infection with recombinant vesicular stomatitis virus expressing chicken OVA protein (10). Four weeks after infection, the mice were sacrificed, LN and spleens removed, and a single cell suspension generated. After depletion of RBC, the cells were incubated with anti-Ly5.2-biotin, washed, and stained cells recovered using magnetic streptavidin-microbeads (Miltenyi Biotec, Auburn, CA). Ly5.2+ cells were recovered using a midiMACS magnet, according to manufacturer’s instructions (Miltenyi Biotec). The enriched population was stained with anti-Ly5.1-FITC and anti-CD8-PE, and the long-lived donor OT-I cells (Ly5.1-, CD8+) were sorted to a purity of 99% by FACS on a FACStarPlus (Becton Dickinson, San Jose, CA). These cells were of memory (CD44high) phenotype.
T cell stimulation
All cell culture was performed in RPMI media supplemented with 10% FCS, L-glutamine, 2-ME, HEPES, and antibiotics (RP10 media).
Most stimulations were conducted using 5AKb cells as APC and purified OT-I CD8 T cells as responders. 5AKb cells were recovered using trypsin/EDTA, washed, and irradiated (20,000 rad). Next, these cells were pulsed with or without OVA peptide (OVAp; 10 nM) (Research Genetics, Huntsville, AL) as indicated for 1 h at 37°C and washed extensively. Two to three hundred thousand 5AKb were mixed with 6 x 105 OT-I cells and cultured in RP10 media in 24-well plates for the indicated period of time.
Labeling with 5- and 6-carboxyfluorescein diacetate succinimidyl ester
(CFSE) was based on the protocol of Weston and Parish
(11). Unpurified OT-I spleen cells (5 x
107/ml in PBS) were incubated with 1 µM CFSE
(Sigma, St. Louis MO) for 10 min at 37°C with gentle rotation. The
reaction was terminated by addition of ice-cold RP10 media and the
cells washed. These cells were suspended to
106/ml in RP10 and stimulated by addition of free
OVAp (10 nM) directly to the culture. By 2–3 days later, the majority
of the remaining viable cells were CD8+
V2+ blasts (data not shown). Activated cells
were sorted on a FACSvantage (Becton Dickinson) on the basis of high
forward scatter and loss of CFSE (compared with unactivated CFSE loaded
cells, fixed after CFSE labeling). In control staining of the sorted
cells, they were >95% CD8+ (data not
shown).
For in vivo stimulation, RAG-1o/o OT-I mice were
injected i.v. with either OVAp (200 nmoles) or P815p (200 nmoles) in a
volume of 
200 µl. Mice were sacrificed at various time points
after immunization, as indicated, at which point, spleen and LN were
recovered and prepared for FACS analysis or lysed for western blot
analysis.
Western blot analysis
Cells were lysed in standard Laemmli buffer containing SDS and 2-ME. Lysates were resolved by PAGE on a 10% gel and electroblotted onto nitrocellulose membranes (Micron Separations, Westborough, MA). The rabbit anti-mouse LKLF antisera has been described (1) and was detected with donkey anti-rabbit Ig-HRP (Jackson ImmunoResearch, West Grove, PA) followed by SuperSignal Chemiluminescent substrate (Pierce, Rockford, IL).
Blotting with antisera to -tubulin or ß-tubulin (Sigma) was based
on the manufacturer’s recommendations. Detection of these Abs was via
goat anti-mouse Ig-HRP (Southern Biotechnology Associates,
Birmingham, AL) and chemiluminescent detection.
Abs and cytokines
Abs to CD4, CD8, CD44, CD25, Thy1.1, and Thy1.2 were purchased
as FITC-, PE-, APC- or biotin-conjugates from PharMingen (San Diego,
CA). B20 (anti-mouse V2) and T3.70 were prepared and
biotinylated using standard procedures. Cells were stained as
previously described (9). The apoptotic marker annexin V
was obtained from PharMingen and used as recommended. 7-amino
actinomycin D (7-AAD) was obtained from Sigma.
Recombinant human IL-2 was from Boehringer Mannheim (Indianapolis, IN) and was used at 2 U/ml with daily replenishment of the cultures. Recombinant mouse IL-7 was from R&D Systems (Minneapolis, MN) and was used at 10 ng/ml. Recombinant mouse IL-12 was obtained from Genetics Institute (Cambridge, MA) and was a kind gift of Dr. Matt Mescher (University of Minnesota, Minneapolis, MN).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous experiments showed that LKLF is rapidly down-regulated in bulk T cells following treatment with anti-CD3. Since this treatment can lead to induction of anergy rather than reactivity among T cells (12), we first determined whether stimulation of T cells with a physiological ligand under activating conditions induced similar changes in LKLF expression.
We used OT-I TCR transgenic mice that bear a receptor specific for
OVAp/Kb and show positive selection into the CD8 subset
(9). LKLF protein was abundantly expressed in fresh LN CD8
T cells purified from these mice (Fig. 1
,
upper panel). These cells could be activated using OVAp
presented on 5AKb, a
Kb-expressing B7-1+
fibroblast cell line, which does not express LKLF (Fig. 1). Twenty-four
hours after initiation of the stimulation, LKLF protein expression had
dramatically decreased. If unpurified OT-I LN cells were used in the
starting population, the decrease in LKLF protein was slightly less
marked, presumably because of a few nonresponding CD4 T cells and B
cells remaining in these cultures. In contrast to these results with Ag
activation, if the OT-I CD8 T cells were exposed to APC without Ag,
there was still abundant LKLF protein 24 h later. The Ag-activated
T cells showed high viability, and were low/undetectable for LKLF
protein by 24 h poststimulation. This radical change in expression
was not a reflection of general protein levels, since ß-tubulin
levels were roughly equivalent in all cell populations (Fig. 1, lower panel). Analysis of unstimulated OT-I T cells beyond
24 h is difficult, since cell viability drops precipitously in the
absence of exogenous cytokines, as has been reported previously by
others for T cells in culture (13).
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) but was
maintained in unstimulated OT-I cells for at least 24 h (Figs. 1
and 2). In other experiments, we saw that LKLF expression had not
decreased by 2.5 h postactivation (data not shown). As shown in
subsequent figures, these low levels of LKLF persisted for at least
48 h after Ag stimulation.
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Having shown that LKLF expression in OT-I T cells was decreased upon stimulation with cognate TCR ligand, we wished to explore whether and how the cells could be induced to reexpress LKLF.
Cytokines are required to permit expansion and survival of Ag-activated
T cells. As is typical for CD8 T cells, in vitro-activated OT-I cells
produce sufficient cytokines to proliferate for about 48 h without
exogenous cytokine, but beyond this time they stop proliferating and
rapidly decrease in viability (Fig. 3).
Similar to observations by others (14, 15), we found that
both IL-2 and IL-7 could induce OT-I proliferation and survival in
vitro (Fig. 3). In our system, IL-2 had to be added regularly to
maintain T cell viability and proliferation (Fig. 3b). In
contrast, addition of a single dose of IL-7 allowed for initial
proliferation and then stable in vitro survival of OT-I T cells (Fig. 3): Similar to the data of others, we observed that Ag-driven OT-I
cells cultured in IL-7 became small cells and could be maintained for
at least 1 mo in culture without readdition of cytokine (14, 15) (data not shown).
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A, LKLF protein was
undetectable 48 h after Ag stimulation and was not expressed if
the cells were cultured in the absence of cytokine for an additional
day. However, culture with IL-7 induced high levels of LKLF protein
expression. At this time point, cell numbers and viability in both cell
populations were similar (Fig. 3, and data not shown). Furthermore, few
of the cells have entered the early stages of the apoptotic cell death
pathway, as detected using annexin V and 7-AAD (Fig. 4B).
Thus, the lack of LKLF expression in the absence of exogenous cytokine
could not be ascribed to rapid cell death of the T cells at this time
point. Importantly, however, the cells cultured without cytokine
reproducibly started to die during the following 24–48 h, whereas
those cultured with IL-7 continued to grow and maintain viability for
several weeks (Fig. 3). Thus, reexpression of LKLF correlated with
survival of activated T cells.
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A). In marked contrast to
these data, IL-12 consistently failed to induce LKLF expression (Fig. 5B). IL-12 is known to synergize with IL-2 to promote CD8 T
cell effector function (16), but on its own is
insufficient to promote T cell differentiation and survival (16, 17). Indeed, in our experiments, IL-12 alone failed to maintain
OT-I CD8 T cell viability in vitro, similar to the "no cytokine"
control (data not shown).
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B and Fig. 7). This effect, which was extremely variable, may be due to production
of endogenous cytokines by the CTL themselves, which was not prevented
in our experimental system. The experiment shown in Fig. 5B
was selected because there was a higher than normal expression of LKLF
in the no cytokine control; addition of IL-12 reduced expression of
LKLF in a dose-dependent manner (note that there is less LKLF protein
in the sample cultured with 2 U/ml IL-12 vs 5 U/ml IL-12). The capacity
of IL-12 to counteract the activated T cell response to other
cytokines, such as IL-2 and IL-7, is currently under investigation.
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We assumed that the LKLF protein detected after cytokine culture was derived from T cells that had responded to Ag during the previous 48 h. However, it was possible that LKLF-expressing cells arose from a population of naive OT-I T cells that failed to respond to Ag and that grew out in response to cytokine. It is known that IL-7, for example, can enhance survival and even growth of naive, mature phenotype T cells in the absence of antigenic stimulation (13, 18), and since naive cells already express LKLF, enhanced in vitro survival of unstimulated cells could be the source of LKLF protein detected in our experiments.
To test this, we labeled unstimulated OT-I spleen cells with the dye
CFSE before Ag stimulation. This fluorescent dye is incorporated into
the cytoplasm of cells, and is progressively lost during cell division,
allowing for discrimination between cells that have and have not
undergone proliferation (19). Two days after Ag
stimulation, we sorted OT-I cells that were CFSE-negative and that also
showed high forward scatter profiles indicative of blasts. The majority
of live cells fell into these gates, indicating that most cells
remaining in these cultures had responded to Ag and, as expected,
analysis of these cells showed that they were >95%
CD8+ (data not shown). These sorted cells were
very low for LKLF, but LKLF expression was efficiently induced after
incubation in IL-7 for 24 h (Fig. 6). These data indicated that Ag
responsive cells had the capacity to up-regulate LKLF upon IL-7
exposure.
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, showed that following addition of
IL-7, an increase of LKLF protein could be seen as early as 2 h in
some experiments, with a marked increase in the protein expression by
6 h. This was not simply due to an increase in overall protein
synthesis, since probing for -tubulin protein showed roughly
equivalent amounts at all time points (Fig. 7). These data argue
strongly against outgrowth of a small LKLF+
subpopulation of Ag-activated T cells expanding in these cultures, but
rather indicated that the bulk population is induced to reexpress the
factor via cytokine stimulation. Down-regulation of LKLF in vivo
Thus far, changes in LKLF expression had only been assessed in
vitro. Next, we used the OT-I system to study changes in expression of
this factor after in vivo activation. To rapidly induce activation in a
large cohort of OT-I T cells, we injected
RAG-1o/o OT-I mice with either OVAp or a control
Kb binding peptide, P815p, which has no effect on
cells bearing the OT-I receptor (9, 20). The mice were
sacrificed 45 min later (OVAp-injected mice) or 4 h later (both
OVAp- and P815p-injected groups). Cells from LN and spleen were
prepared for LKLF and FACS analyses. Mice injected with P815p showed no
signs of activation, as indicted by low levels of CD69 (data not
shown). In contrast, 45 min after OVAp injection, cell surface
expression of CD69 was increased, and this was even more pronounced at
4 h, indicating that the OT-I T cells had been activated in vivo
(data not shown). LKLF expression in cells recovered from these mice is
shown in Fig. 8. LKLF expression was still
abundant in the spleen and LN of P815p-injected mice, but expression of
this protein was dramatically reduced after injection with OVAp, being
significantly diminished at 45 min and undetectable by 4 h. In a
separate experiment (see Fig. 10 below), LKLF expression was
undetectable in cells isolated 2 or 4 h after in vivo stimulation
with OVAp. In these experiment also, the OT-I cells expressed CD69
within 1 h of OVAp injection (data not shown).
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Expression of LKLF in memory T cells
Little is known about gene regulation in the transition from Ag-stimulated T cells to memory T cells. If LKLF were to play a role in survival of memory as well as naive T cells, we would expect to see LKLF reexpression in the memory population derived from the Ag-activated pool.
Activated OT-I CD8+ cultured with IL-7 for
several days stopped proliferating rapidly but maintained high
viability for several weeks (14, 15) (Fig. 3, and data not
shown). By day 12 after incubation in IL-7, the OT-I
CD8+ T cells became small T cells with the
phenotype CD44high, CD69-,
CD25- (data not shown), which is characteristic
of memory CTL phenotype (7, 21) and is consistent with the
observations of others studying activated CD8 T cells cultured in IL-7
(14). At these time points, LKLF protein was still
abundantly expressed (Fig. 9), suggesting
that the factor was stably expressed beyond the likely duration of the
cytokine signaling, and that it may be expressed in the memory
cell pool.
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, purified populations of
memory OT-I cells expressed similar amounts of LKLF protein to the
naive population. Similar results were observed with cells isolated in
another experiment. These data demonstrate that in vivo-generated
memory cells express LKLF, similar to naive T cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We describe in vitro conditions that lead to the down-regulation and subsequent reexpression of the LKLF transcription factor after TCR stimulation with Ag/MHC ligands. Incubation of cells with suitable cytokines (IL-2 or IL-7 but not IL-12) leads to a rapid and dramatic increase in LKLF protein.
IL-2 and IL-7 induce signaling through receptors that share the
common-
-chain (22) and signal through Jak-1 and -3,
together with Stat-3, -5a, and -5b (23). It is tempting to
predict that cytokine signaling through these pathways is responsible
for reexpression of LKLF. Indeed, while our data do not specifically
address whether IL-2 and IL-7 induce LKLF up-regulation through a
direct signaling cascade rather than simply enhancing T cell survival,
the rapidity of LKLF reexpression after IL-7 coculture strongly
supports the former hypothesis. Furthermore, we have performed
preliminary experiments in which we can preserve activated OT-I T cells
in the absence of cytokine via culturing these cells in a mixture of
caspase inhibitors designed to block the inductive and/or execution
phase of apoptosis (S.L.S and S.C.J., unpublished observations). Such
cells are viable by trypan blue exclusion and absence of staining with
annexin V or 7-AAD, yet LKLF was not reexpressed, supporting the model
that LKLF expression requires specific cytokine signaling. On the other
hand, IL-12 signals through an entirely distinct system involving
different Jak/Stat members (23, 24). We find that this
cytokine consistently fails to reinduce LKLF in activated CD8 T cells,
although it has no effect on constitutive LKLF expression by naive,
nonactivated OT-1 cells (S.L.S and S.C.J., unpublished observations).
The capacity of these and other cytokines to synergize or antagonize
for LKLF reexpression is currently being investigated.
Our data using cell sorting to isolate cells that responded to Ag indicate that the bulk population of these cells can reexpress LKLF if cultured with cytokine. It is still possible, however, that LKLF-expressing cells arise from a small subpopulation of Ag responders that, unlike the majority of T cells, do not down-regulate LKLF upon activation. Such cells would be biologically interesting, since they may represent precursors that were predestined to survive Ag stimulation and possibly contribute to the memory pool, as has been discussed (7). However, the kinetics of reexpression indicate that LKLF protein levels reach naive cell levels well within 24 h, during which time the population has only doubled in number with little apparent loss in viability, making it unlikely that expansion of an initial LKLF+ subpopulation could explain our data.
The experiments of Kuo et al. (1) showed that activation
of T cells caused loss of LKLF, and that loss of LKLF (by means of gene
targeting) produced mature T cells with an activated phenotype, and
this lead to the hypothesis that LKLF might be important in maintaining
the resting state of naive T cells. At first glance, our data
contradict this model, since activated OT-I cells proliferate and
maintain expression of activation markers during the period 48–72 h
after activation, regardless of whether exogenous cytokine was added,
yet LKLF expression is radically different depending on cytokine
inclusion. However, it is not clear from these observations whether
LKLF fails to affect the T cell activation state, or simply takes time
to mediate its effects. In support of the latter hypothesis, sustained
expression of LKLF by activated OT-I cells cultured in IL-7 correlates
with their becoming small, nondividing, memory phenotype cells (Fig. 3, and data not shown).
One concern with in vitro experiments is that LKLF down-regulation might be related to the lack of some suitable factor or cell contact in tissue culture. Hence, we investigated LKLF down- and up-regulation in vivo. We showed that OT-I T cells rapidly lose LKLF protein in as little as 45 min after injection of OVAp, indicating very rapid down-regulation kinetics in vivo. Hence, the kinetics for LKLF down-regulation are at least as quick in vivo as they are in vitro; indeed, other experiments suggest that in vitro down-regulation is not apparent before 2.5 h of stimulation (data not shown), indicating that LKLF down-regulation is even faster after in vivo challenge.
More importantly, we demonstrate that in vivo-derived memory OT-I T cells (and memory phenotype cells generated in vitro) express LKLF similarly to naive T cells. Together, these data show that the in vivo regulation of LKLF matches the in vitro results, and suggest that this factor may play a role in long-term survival of both naive and memory populations.
Based on the LKLF knockout, a role for LKLF in maintaining naive T cells (but not B cells) was proposed (1, 3). While its relevant DNA targets are being determined (C.T.K and J.M.L, unpublished observations), the exact function of LKLF will remain a mystery. However, the current data suggest that LKLF is tightly regulated in protein expression, and that expression of LKLF predicts survival of the T cell population. These data thus support a model in which LKLF is involved in the maintenance of naive, and probably memory, T cell viability, perhaps by preventing spontaneous entry into apoptotic cell death pathways, as previously discussed (1). In addition, cytokine-induced LKLF expression in activated T cells correlates with their survival, indicating that this factor may be involved in avoiding activation-induced cell death. Our results also suggest the intriguing possibility that one role of cytokines that promote naive T cell survival, for example IL-7 (18), may do so in part by maintenance of LKLF, although direct experiments will be needed to test this.
Recent data indicate that naive T cell survival requires interaction of the TCR with self ligands (25, 26, 27). An interesting question now is whether LKLF expression in resting T cells is influenced by such TCR interactions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen C. Jameson, University of Minnesota, Center for Immunology, Box 334 FUMC, 420 Delaware Street SE, Minneapolis, MN 55455. E-mail address:
3 Abbreviations used in this manuscript: LKLF, lung Krüppel-like factor; LN, lymph node; CFSE, 5- and 6-carboxyfluorescein diacetate succinimidyl ester; OVAp, OVA peptide; 7-AAD, 7-amino actinomycin D.
4 S.-K. Kim, K. S. Schluns, and L. Lefrancois. 1999. Induction and visualization of mucosal memory CD8 T cells following systemic virus infection. Submitted for publication.
Received for publication April 15, 1999. Accepted for publication July 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain in cytokine signaling and lymphoid development. Immunol. Rev. 148:97.[Medline]
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