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Center for Immunology and Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) or proliferation (human IL-2)
do not restore the ability to produce IL-2 in response to
costimulation. Although similar to the anergy described for
CD4+ T cell clones, AINR differs in that it results from an
initial stimulation with both signal 1 and signal 2. AINR appears to be
an aspect of the normal differentiation of fully stimulated
CD8+ T cells. It is probably important in regulating CTL
responses; it limits the initial T helper-independent response and
converts it to a response that requires T cell help to be sustained and
further expanded. When the initial helper-independent response is not
sufficient to clear Ag, and if help is not available, AINR likely
results in tolerance to the Ag. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is unclear why this autocrine IL-2 production is not sufficient to support a sustained CD8+ T cell response, and that, instead, the response often becomes dependent on help from CD4+ T cells (1, 2, 3, 4, 5). One possibility is that, as the CD8+ T cells develop lytic effector function, they eliminate the APC so that effective Ag presentation is no longer occurring. The decline that occurs following the initial response involves substantial deletion of the activated CD8+ T cells through death and could be consistent with growth factor deprivation in the absence of continued stimulation. It has also been shown, however, that some of the CD8+ T cells that are present during the decline appear to be anergic, unable to respond to the same stimulus that was effective in initially activating the response. In vivo induction of anergy in CD8+ T cells has been found in response to virus (11), HY Ag (12, 13), and superantigens (14).
The origin and status of these anergic CD8+ T cells is not well understood. Classical anergy, defined originally for CD4+ T cell clones, is the nonresponsive state that results when the cells receive a signal 1 stimulus via the TCR in the absence of signal 2 via a costimulatory receptor, and are thus rendered unable to subsequently produce IL-2 in response to full stimulation (15, 16, 17). Thus, the anergic CD8+ T cells present following an initial in vivo response could potentially be a subset of the Ag-specific cells that recognized Ag in a context where costimulation was lacking; it has been demonstrated that cloned CTL lines can be rendered anergic by exposure to just signal 1 (18). The role of anergy in limiting the helper-independent CTL response is also not understood. The anergic state might not be relevant if it simply represents a stage at which cells have lost responsiveness on their way to deletion. Alternatively, it might be essential for preventing further autocrine IL-2 production and converting the response to one that is now under the regulation of CD4+ T cells.
When CD8+ T cells are stimulated in vitro, proliferation and clonal expansion peak on day 3 or 4 and then decline, and this decline has been attributed to killing and elimination of the APC as the CD8+ T cells become lytically active by day 3 (19). However, when the stimulus was anti-TCR mAb and purified B7-1 coimmobilized on latex microspheres, a comparable time course was observed, even though the inert beads could not be "killed," and the stimulus thus could not be eliminated by the effector cells (8). This raised the possibility that CD8+ T cells may become anergic following a response to full stimulation with both signal 1 and signal 2, i.e., they may become unable to produce IL-2 and proliferate in response to subsequent stimulation. The results described here demonstrate that this is the case for cells responding both in vitro and in vivo, and this activation-induced nonresponsiveness (AINR)3 has important implications for the regulation of CTL responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells were obtained from 6- to 12-wk-old female mice of either the C57BL/6 strain (Charles River Breeding Laboratories, Wilmington, MA) or the OT-1 strain having a transgenic TCR specific for Kb/OVA ( (20); a kind gift from Dr. Frank Carbone, Monash Medical School, Victoria, Australia). Mice were maintained in the University of Minnesota (Minneapolis, MN) specific pathogen-free animal facilities according to National Institutes of Health guidelines. Lymph nodes were harvested, and CD8+ responders were purified as described (8). Responders were routinely assessed for purity by flow cytometry and were always >90% CD8+ with <1% CD4+ contamination.
A soluble form of ICAM-1 was purified, as described by Kuhlman et.al.
(21). Purification of B7-1 and preparation of microspheres
bearing optimal levels of the anti-TCR mAb F23.1, B7-1, and ICAM-1
have been described in detail (8, 22). Purified
CD8+ C57BL/6 responders were always found to be
20–23% positive for Vß8 TCR expression. For experiments using
responder cells from OT-1 mice, anti-V2 mAb B20 (PharMingen, San
Diego, CA) was used. Triplicate cultures were set up as described
(8) using Falcon (Franklin Lakes, NJ) flat-bottom 96-well
tissue culture plates . Primary cultures were initiated with 5 x
104 purified cells, and cultures of activated
cells were initiated with 2 x 104 viable
cells, or as indicated. F23.1/B7-1/ICAM-1 microspheres (F23/B/I) were
added to cultures at 1 x 105 per well, and
the final volume was 0.2 ml. For the experiments shown in
Figs. 5–8
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activated responders were pooled from multiple microwell cultures and,
viable cells were purified over Lympholyte-M (Cedarlane, Hornby,
Ontario, Canada), according to the instructions provided by the
supplier. For assessment of proliferation, cells were pulsed with 1
µCi of [3H]TdR for the last 6–8 h of culture, lysed
with distilled H2O, and the amount of
[3H]TdR incorporated into DNA was determined by liquid
scintillation counting. Results are expressed as the average ± SD
of triplicate samples. For quantitation of clonal expansion, cells were
counted using a hemacytometer, and viability was determined based on
trypan blue exclusion.
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A/D (Genzyme, Cambridge, MA). Anti-IL-2R mAb,
PC61.5.3, was purified from culture supernatant and added to cultures
at a final concentration of 10 µg/ml in blocking studies. CTLA-4
blocking was performed using purified anti-CTLA-4 mAb, clone 9H10
(PharMingen), at 50 µg/ml. In vivo CTL activation and ex vivo functional analysis
The procedure for adoptive transfer, stimulation, and analysis of 2C TCR transgenic CD8+ T cells has been described in detail (23, 24). Briefly, 5 x 106 adherence-depleted spleen and lymph node (LN) cells from 2C transgenic mice (25) (a kind gift of Dr. Dennis Loh Hoffman-La Roche, Nutley, NJ) were injected i.v. into C57BL/6 sex-matched recipients. After 2 days, recipient mice were challenged i.p. with 5 x 107 B7-1-transfected P815 tumor cells (26) (a kind gift from Dr. Thomas Malek, University of Miami School of Medicine, Miami, FL). After 6 days, spleen and peritoneal exudate cells were harvested from challenged mice, and the CD8+ T cells were purified. Resting CD8+ 2C T cells were similarly purified from the spleen of transgenic mice. Ex vivo restimulation for detection of proliferation by [3H]TdR uptake was performed essentially as described above for stimulation of C57BL/6 cells, with the exception that 2.5 x 104 responders were used and microspheres had the 1B2 anti-clonotypic mAb specific for the 2C receptor (1 µg/ml) immobilized instead of F23.1 mAb.
Clonal expansion of 2C transgenic T cells resulting from ex vivo stimulation was determined by flow cytometry. Purified responders were stained with anti-CD8 and 1B2 mAbs and quantitated as described (23) to determine input responder frequency. A total of 2.5 x 105 cells were then cultured in 2 ml of media in 24-well plates for 72 h. At the end of the culture period, cells were harvested, viability determined by trypan blue exclusion, and the frequency of 1B2+/CD8+ 2C cells determined by flow cytometry. For this analysis, viable lymphocytes were gated based on forward and side scatter characteristics and exclusion of 7-amino-actinomycin D (7-AAD).
Flow cytometry
Cells and microspheres were stained using identical protocols using the relevant Ab for 20 min at 4°C in 0.1 ml of HBSS with 2% FCS and 0.02% NaN3 (flow buffer) in the dark. Two washes (before analysis and between multistep stains) were performed using flow buffer. Flow cytometric analysis was performed on a Becton Dickinson (Mountain View, CA) FACScan using CellQuest software. Ligand densities on all microsphere preparations were quantitated as described elsewhere (22). To quantitate apoptosis, cells were stained with 1 µg of F23.1 mAb, washed, then stained with 1 µg FITC-conjugated goat anti-mouse Ig (Jackson ImmunoResearch, West Grove, PA) and washed. The cells were then fixed with 70% ethanol for 60 min on ice, washed with PBS, and resuspended in 1 ml PBS with RNase I-A at 0.5 µg/ml. An amount of 1 ml of propidium iodide at 100 µg/ml was then added and the cells incubated with periodic agitation for 1 h at room temperature in the dark. Vß8+ cells were gated, and the percent of F23+ cells having hypodiploid DNA content was determined based on the propidium iodide staining.
For the phenotypic analysis shown in Fig. 6
, cells were stained first
with 1 µg of F23.1 then with 1 µg PE-conjugated goat anti-mouse
Ig (Jackson ImmunoResearch) and either nothing or the indicated
FITC-conjugated mAbs. Anti-CD8-FITC (clone CT-CD8) and
anti-CD28-FITC (clone 37.51.1) were from Caltag (Burlingame, CA),
and anti-LFA-1-FITC (clone 2D7) was from PharMingen. A three-step
procedure was used for detection of CTLA-4 expression. Approximately
5 x 105 cells were stained first with
anti-CTLA-4 (clone 9H10; PharMingen), followed by biotin-conjugated
goat anti-hamster Ig (Jackson ImmunoResearch) and then
PE-conjugated streptavidin (PharMingen). Control staining was
performed with second- and third-step reagents only. Viable lymphocyte
responders were gated based on forward and side scatter characteristics
(22) and exclusion of 7-AAD.
Cytokine ELISAs
Sandwich ELISAs were used to detect murine IL-2 and IFN-
in
0.05 ml supernatants removed from cultures at the times indicated,
using mAbs obtained from PharMingen, according to the protocol provided
by the supplier. HRP-conjugated streptavidin was from Sigma (St. Louis,
MO). Standard curves using recombinant murine IL-2 or IFN-
(PharMingen) were used to quantitate production. For both cytokines,
the detection limit of the assay was 
0.04 ng/ml. Results are
expressed as the average ± SD of triplicate samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stimulation of resting C7BL/6 CD8+ T cells
with microspheres having just F23.1 anti-TCR mAb immobilized on the
surface results in only marginal proliferation, as measured by
[3H]TdR incorporation (Fig. 1A). In contrast, beads having
purified B7-1 coimmobilized with the F23.1 (F23/B) stimulate quite
effectively, and the most potent stimulation is obtained when both B7-1
and ICAM-1 are coimmobilized with the anti-TCR mAb (F23/B/I) (Fig. 1A). The cells initially produce IL-2 in response to
costimulation with B7-1 and ICAM-1, but levels decline dramatically by
day 4 (Fig. 1B). Proliferation is paralleled by clonal
expansion through days 3 and 4, but at longer times, IL-2 levels
decline, [3H]TdR incorporation ceases, total cell numbers
no longer increase, and cell viability rapidly declines (Fig. 1C) as cells die by apoptosis. Apoptotic death during this
period was demonstrated by analysis of subdiploid DNA content using
propidium iodide, and confirmed using 7-AAD staining to detect
apoptotic cells (data not shown). Candidates for mediating the death of
the activated T cells include TNF that is produced by activated CTL,
and Fas-dependent killing, since activated CD8+ T
cells express both Fas and Fas ligand. However, neither neutralizing
anti-TNF-ß mAb nor anti-Fas ligand mAb inhibited the
apoptotic death (data not shown). Death due to growth factor withdrawal
also occurs by apoptosis (27), and disappearance of IL-2
from the cultures appears likely to be responsible for the death that
occurs at later times, since addition of exogenous IL-2 can prolong
survival (see below).
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The decline in the CD8+ T cell response that begins after day 3 could not be attributed to elimination of the APC, since the microspheres cannot be killed. However, it was possible that activation ceased because the anti-TCR mAb and/or costimulatory ligands were no longer available on the microspheres due to loss or degradation. Analysis of the beads by flow cytometry after 4 days indicated that this was probably not the case, since only small decreases in the density of the surface ligands were found (data not shown). This raised the possibility that the cells were becoming nonresponsive as a result of activation, even though signals 1 and 2 were both being delivered, and experiments were done to directly examine this.
CD8+ T cell cultures were stimulated with
microspheres having F23.1, B7-1, and ICAM-1 on the surface, and the
cells responded with the expected time course of proliferation, clonal
expansion, and decline in viable cell numbers (Fig. 2A). Cells stimulated in
parallel were harvested from culture on day 4 (90 h), washed, and
viable cell number determined. The cells were then placed in culture in
fresh medium at a cell density comparable to that used at the
initiation of the cultures on day 0, stimulated in various ways, and
both proliferation and viable cell number determined over the next
several days (Fig. 2, B and C). In the absence of
stimulation, minimal [3H]TdR incorporation was found and
the number of viable cells declined. In contrast, exogenous IL-2
provided potent stimulation of proliferation, and the number of viable
cells expanded greatly over days 5–7. Although IL-2 could support
proliferation and clonal expansion, microspheres having F23.1, B7-1,
and ICAM-1 on the surface (F23/B/I beads) could not. Thus, F23/B/I
beads that can provide costimulatory activation to induce IL-2
production and proliferation by resting CD8+ T
cells could not support continued expansion of previously activated
cells. The experiment shown in Fig. 2 is representative of at least
five similar experiments. Additional experiments have been done with
cells stimulated 3 days (72 h) before washing and restimulation, and
similar results have been obtained (data not shown, and see below).
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A). As expected from the
experiments examining C57BL/6 CD8+ T cells that
are predominantly naive, the naive OT-1 cells proliferated strongly in
response to microspheres having anti-TCR mAb along with B7.1 and
ICAM-1 on the surface (Fig. 3B), but, by day 3, had beome
nonresponsive to costimulation, although they could still proliferate
in response to added IL-2 (Fig. 3C).
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Following adoptive transfer of 2C cells and challenge with P815-B7-1
tumor, cells were isolated from the spleen and peritoneal cavity on day
6 and stimulated in vitro. Microspheres used for in vitro stimulation
had the 1B2 anti-TCR mAb on the surface to insure that observed
responses were mediated by the transgenic T cells. Resting 2C cells
were examined in parallel and showed the expected pattern of response
(Fig. 4A). Beads with
anti-TCR, B7-1, and ICAM-1 stimulated proliferation, and the
response did not increase significantly when IL-2 was added, indicating
that the cells produce sufficient IL-2 upon costimulation to support a
maximal response. In contrast, IL-2 alone stimulated no response.
Responses by the 2C cells that had undergone an in vivo response to
P815-B7-1 were quite different. The 1B2, B7-1, ICAM-1 beads (1B2/B/I)
stimulated no response, while IL-2 alone stimulated a strong
proliferative response (Fig. 4A). Addition of both beads and
IL-2 resulted in a response essentially identical to that of IL-2
alone. When [3H]TdR incorporation is normalized to the
number of 2C cells in the different populations, it is readily apparent
that responses by resting cells to anti-TCR and costimulation are
much stronger than those of the cells that have responded in vivo
(Fig. 4B).
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C). In fact, it appears that stimulation of previously
activated cells with the beads in the absence of exogenous IL-2 may
increase the rate at which these cells die.
The results shown in Fig. 4 are representative of two experiments
examining responsiveness of cells from P815-B7-1-challenged mice.
Similar results were obtained when irradiated P815-B7-1 tumor cells
were used instead of microspheres as the in vitro stimulus, and in
experiments examining cells from mice challenged with normal P815 tumor
cells lacking B7-1 (data not shown). Thus, full activation (with
costimulation) results in subsequent nonresponsiveness of
CD8+ T cells, whether the cells are responding in
vitro or in vivo.
AINR is not a consequence of ongoing cell death
The above experiments demonstrated that previously activated CD8+ T cells were "anergic" in that they could no longer proliferate in response to costimulation. However, continued death of the previously activated cells was occurring during the period of restimulation with microspheres, raising the possibility that the inability to proliferate was simply a consequence of the induction of death. This appeared unlikely, since cells that could not respond to F23/B/I bead restimulation on day 3 still remained viable in sufficient numbers by day 4 to give a good response to exogenous IL-2 (data not shown). To more directly examine this issue, we took advantage of the fact that murine T cells can respond to human IL-2. Thus, human IL-2 could be added to cultures to support survival and proliferation, and, at the same time, allow the production of murine IL-2 to be examined by ELISA using a species-specific mAb for detection.
Activation of resting cells with F23/B/I beads results in clonal
expansion by day 3, and this is blocked by addition of anti-IL-2R
mAb, confirming that the response is IL-2-dependent (Fig. 5A). The
CD8+ T cells are stimulated to produce IL-2 (Fig. 5B) in sufficient amounts to fully support the response,
since addition of exogenous human IL-2 does not increase clonal
expansion (Fig. 5A) or alter the level of murine IL-2 that
is produced (Fig. 5B). In contrast, for cells that had been
activated 3 days earlier by F23/B/I beads, restimulation with F23/B/I
beads did not result in clonal expansion (Fig. 5D) or
significant IL-2 production (Fig. 5E). When both F23/B/I
beads and human IL-2 were added, the cells remained viable and expanded
in number (Fig. 5D), but even under these conditions,
production of murine IL-2 was minimal (Fig. 5E). Thus, even
when previously activated cells remain viable, they are unable to
produce IL-2 in response to costimulation by B7-1 and ICAM-1. When the
same experiment is done in the presence of anti-IL-2R mAb to block
consumption of murine IL-2, IL-2 levels in cultures of resting cells
are comparable at day 2 and remain about the same at day 3, indicating
that the majority of IL-2 production occurs in the first 2 days
following costimulation. Murine IL-2 production remains minimal in
cultures of previously activated cells, even when anti-IL-2R mAb is
added to prevent consumption (data not shown).
Stimulated CD8+ T cells acquire cytolytic
activity by day 3, and this activity usually reaches maximal levels by
day 4 (8). Thus, the cells can receive signals via the TCR
to mediate degranulation and cytolysis during the time that they are
anergic with respect to costimulation of IL-2 production. A similar
"split anergy" is seen when production of IFN- is examined.
Stimulation of resting cells results in production of IFN- by days 2
and 3 (Fig. 5C). When the cells are washed and placed back
into culture, no release of IFN- is detected in the absence of
further stimulation, indicating that production requires ongoing
signaling (Fig. 5F). In contrast to IL-2 production,
restimulation with F23/B/I beads results in IFN- production by the
previously activated cells that begins more rapidly and occurs to
higher levels than for resting cells (Fig. 5F). More IFN-
is detected when exogenous IL-2 is added to cultures of the previously
activated cells, since more cells remain viable for longer times to
produce the cytokine (Fig. 5F). In contrast, addition of
just IL-2 in the absence of a TCR stimulus does not cause IFN-
production (data not shown). Thus, although previously activated cells
are anergic with respect to IL-2 production, they can be stimulated via
the TCR to produce IFN-.
The effects of IL-7 and IFN- on previously activated cells were also
examined, since both can support prolonged survival of the cells but do
not stimulate proliferation to as great an extent as IL-2. When cells
were stimulated with F23/B/I beads, harvested on day 3, and
restimulated, F23/B/I beads alone caused no proliferation (Fig. 6A), and the number of viable
cells present was low by day 3 (Fig. 6B). As shown above,
addition of human IL-2 along with the beads stimulated proliferation
and clonal expansion (Fig. 6, A and B) but did
not allow production of significant amounts of murine IL-2 (Fig. 6C). IL-7 supported less proliferation or clonal expansion
than IL-2, and IFN- allowed the cells to survive without significant
expansion (Fig. 6, A and B). In neither case were
significant amounts of murine IL-2 produced in response to the F23/B/I
beads, while IFN- production was stimulated (Fig. 6, C
and D). Thus, previously activated
CD8+ T cells remain anergic with respect to IL-2
production when cell viability is maintained with or without further
proliferation.
AINR is not due to receptor down-regulation or negative signaling by CTLA-4
Ag or anti-TCR Abs can stimulate internalization of the TCR
(30, 31), and, in some in vivo systems,
CD8+ T cells that have been rendered anergic
following a strong in vivo response have dramatically decreased surface
expression of TCR and CD8 (12). This appeared unlikely to
be the case for the anergy examined here, since anti-TCR mAb could
still trigger IFN- production by the cells (Figs. 5 and 6). This was
confirmed using flow cytometry to examine surface receptor levels on
resting cells in comparison to cells stimulated 3.5 days previously
with F23/B/I beads. TCR expression was comparable on both resting and
activated cells, while CD8 expression was somewhat increased on the
activated cells (Fig. 7A).
Furthermore, both the CD28 and LFA-1 costimulatory receptors were
expressed at substantially higher levels on the activated cells (Fig. 7A).
Activated T cells express CTLA-4, a second receptor for B7-1 and B7-2
ligands, and recent evidence is suggesting that this receptor may
deliver a "negative" signal to down-regulate T cell responses
(32, 33, 34, 35). Stimulation of CD8+ T
cells with F23/B/I beads results in up-regulation of CTLA-4 expression
by 48 h, and expression persists at relatively high levels through
at least 4 days (Fig. 7B). Thus, negative signaling by the
up-regulated CTLA-4 receptor could potentially provide the mechanism by
which the cells are rendered anergic. This does not appear to be the
case, however, since addition on day 0 of anti-CTLA-4 mAb to block
binding to B7-1 did not prevent the decline in response to F23/B/I
beads that occurs after day 3 (Fig. 8A). Furthermore, addition of
anti-CTLA-4 mAb could not reverse the nonresponsiveness of cells
that had been activated 3 days previously with F23/B/I beads; no
response to restimulation with F23/B/I beads occurred in the absence or
presence of anti-CTLA-4 mAb, while, again, the cells responded well
to addition of IL-2 (Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although activated CD8+ T cells become
unresponsive to costimulation with respect to IL-2 production within
3–4 days, they remain responsive to stimulation through the TCR since
they can lyse target cells (8) and secrete IFN- (Figs. 4 and 5) in a TCR-dependent manner during this time. A phenomenon
similar to this has been observed in long-term cloned lines of murine
CTL and was termed "split-anergy" (18). The role of
costimulatory ligands was not directly assessed in that study, but it
was similar to anergy induction in CD4+ T cell
clone in that stimulation by fixed APC, but not irradiated APC, induced
the hyporesponsiveness (17, 18). Induction of a
nonresponsive state has also been described for human
CD8+ CTL clones (38). Ag-specific
stimulation with B7-expressing EBV-transformed B cells as APC resulted
in hyporesponsiveness with respect to IL-2 production and
proliferation, but not cytolytic activity. Thus, studies of long-term
cloned CTL lines have demonstrated nonresponsive states similar to
those shown here for normal resting CD8+ T cells;
whether the nonresponsive clones reflect the same physiological state
and mechanism(s) of nonresponsiveness as the normal cells is
uncertain.
Although CD8+ T cells begin to undergo apoptotic
cell death by days 3 and 4 after stimulation (Fig. 1), the inability to
produce IL-2 in response to costimulation cannot be attributed to the
fact that the cells are dying. If viability is maintained by the
addition of IFN-, IL-7 (Fig. 5), or human IL-2 (Fig. 4), the cells
remain unable to produce murine IL-2. Thus, AINR appears to be a
distinct phenomenon from that of activation-induced cell death
(39, 40, 41), and this conclusion is further supported by the
fact that nonresponsiveness is not prevented by addition of
anti-TNF-ß or anti-Fas ligand mAbs to either the primary or
restimulation cultures (data not shown). AINR is also distinct from the
effector T cell down-regulation observed by Liu et al.
(42), since addition of blocking anti-IFN- mAb also
failed to prevent its development (data not shown).
The mechanism(s) by which activated CD8+ T cells
become nonresponsive does not appear to involve down-regulation of the
receptors needed for delivering signals 1 or 2; TCR and CD8 levels are
comparable on resting and activated cells, while CD28 and LFA-1 are
expressed at higher levels on the activated cells (Fig. 6). CTLA-4 is a
second receptor that binds B7-1 and B7-2 ligands, and evidence is
accumulating that suggests that it can have a "negative" signaling
role, at least on CD4+ T cells
(32, 33, 34, 35). Resting T cells express little or no CTLA-4 on
the surface, but its expression is up-regulated upon activation
(43). This was confirmed to be the case for the
CD8+ T cell responses studied here, with CTLA-4
being readily detected within 48 h and persisting through at least
4 days (Fig. 6). Although expressed, "negative" signaling via
CTLA-4 did not account for the nonresponsiveness of activated
CD8+ T cell since addition of a blocking
anti-CTLA-4 mAb did not prevent or reverse the nonresponsiveness
(Fig. 7). This conclusion is further supported by the observation that,
while resting cells can respond to microspheres having just F23.1 mAb
and ICAM-1 on the surface, activated cells cannot (data not shown).
Thus, the cells remain nonresponsive even when no B7 ligand is present.
Anergic CD4+ T cell clones have been shown to
have a defect in the signaling pathways activated by the CD28 receptor
(44, 45), and it appears likely that this may also be the
case for activated CD8+ T cells. Costimulation
through CD28 and LFA-1 results in activation of some common signaling
events (29), and these are likely candidates for being
affected by AINR.
AINR may play an important physiological role in converting the CD8+ CTL response from a helper-independent mode to a helper-dependent mode. Thus, a CTL response could be initiated via an autocrine IL-2 pathway, with the cells undergoing clonal expansion and developing effector function. As part of the developmental program, a critical signaling pathway(s) linking costimulatory receptors to IL-2 production may be "disconnected." Thus, the CTL would be able to continue to carry out their Ag-specific, TCR-dependent effector functions, cytolysis, and cytokine production, but further expansion of the population would be prevented unless CD4+ T cell help became available. This could explain the importance of CD4+ T cell help for generating sustained CTL responses (1, 4, 5) and would be consistent with the observations that acute viral infections can be effectively controlled in the absence of CD4+ T cells (46, 47, 48), but chronic infections cannot (2, 3). AINR would insure that CD8+ cells do not continue to expand in an uncontrolled manner if the effector CTL generated initially are unable to rapidly eliminate Ag. In such cases, it would also lead to tolerance to the Ag if CD4+ T cell help is not available.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matthew F. Mescher, Center for Immunology, Box 334 Mayo, 420 Delaware Street S.E., Minneapolis, MN 55455. E-mail address:
3 Abbreviations used in this paper: AINR, activation-induced nonresponsiveness; LN, lymph node; 7-AAD, 7-amino-actinomycin D.
Received for publication October 7, 1998. Accepted for publication April 9, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain signaling cytokines regulate activated T cell apoptosis inresponse to growth factor withdrawl: selective induction of anti-apoptotic (bcl-2, bcl-xl) but not proapoptotic (bax, bcl-xs) gene expression. Eur. J. Immunol. 26:294.[Medline]
plays a critical role in induced cell death of effector T cell: A possible third mechanism of self-tolerance. J. Exp. Med. 172:1735.ß and 
T cell subsets in viral immunity. Annu. Rev. Immunol. 10:123.[Medline]
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 R. B. Jones, L. C. Ndhlovu, J. D. Barbour, P. M. Sheth, A. R. Jha, B. R. Long, J. C. Wong, M. Satkunarajah, M. Schweneker, J. M. Chapman, et al. Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection J. Exp. Med., November 24, 2008; 205(12): 2763 - 2779. [Abstract] [Full Text] [PDF]
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H. Zhang, K. M. Snyder, M. M. Suhoski, M. V. Maus, V. Kapoor, C. H. June, and C. L. Mackall 4-1BB Is Superior to CD28 Costimulation for Generating CD8+ Cytotoxic Lymphocytes for Adoptive Immunotherapy J. Immunol., October 1, 2007; 179(7): 4910 - 4918. [Abstract] [Full Text] [PDF]
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K. A. Casey and M. F. Mescher IL-21 Promotes Differentiation of Naive CD8 T Cells to a Unique Effector Phenotype J. Immunol., June 15, 2007; 178(12): 7640 - 7648. [Abstract] [Full Text] [PDF]
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L. Chiodetti, S. Choi, D. L. Barber, and R. H. Schwartz Adaptive Tolerance and Clonal Anergy Are Distinct Biochemical States J. Immunol., February 15, 2006; 176(4): 2279 - 2291. [Abstract] [Full Text] [PDF]
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Y. Huang, N. Obholzer, R. Fayad, and L. Qiao Turning On/Off Tumor-Specific CTL Response during Progressive Tumor Growth J. Immunol., September 1, 2005; 175(5): 3110 - 3116. [Abstract] [Full Text] [PDF]
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 M. Uhlin, M. G. Masucci, and V. Levitsky Regulation of lck degradation and refractory state in CD8+ cytotoxic T lymphocytes PNAS, June 28, 2005; 102(26): 9264 - 9269. [Abstract] [Full Text] [PDF]
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A. A. Filatenkov, E. L. Jacovetty, U. B. Fischer, J. M. Curtsinger, M. F. Mescher, and E. Ingulli CD4 T Cell-Dependent Conditioning of Dendritic Cells to Produce IL-12 Results in CD8-Mediated Graft Rejection and Avoidance of Tolerance J. Immunol., June 1, 2005; 174(11): 6909 - 6917. [Abstract] [Full Text] [PDF]
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A. Facchinetti, S. D. Santa, S. Mezzalira, A. Rosato, and G. Biasi A Large Number of T Lymphocytes Recognize Moloney-Murine Leukemia Virus-Induced Antigens, but a Few Mediate Long-Lasting Tumor Immunosurveillance J. Immunol., May 1, 2005; 174(9): 5398 - 5406. [Abstract] [Full Text] [PDF]
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 S. Glennie, I. Soeiro, P. J. Dyson, E. W.-F. Lam, and F. Dazzi Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells Blood, April 1, 2005; 105(7): 2821 - 2827. [Abstract] [Full Text] [PDF]
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W. L. Redmond, B. C. Marincek, and L. A. Sherman Distinct Requirements for Deletion versus Anergy during CD8 T Cell Peripheral Tolerance In Vivo J. Immunol., February 15, 2005; 174(4): 2046 - 2053. [Abstract] [Full Text] [PDF]
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 S.-H. Tseng, Y. Chen, C.-J. Chang, K.-F. Tai, S.-M. Lin, and L.-H. Hwang Induction of T-Cell Apoptosis in Rats by Genetically Engineered Glioma Cells Expressing Granulocyte-Macrophage Colony-Stimulating Factor and B7.1 Clin. Cancer Res., February 15, 2005; 11(4): 1639 - 1649. [Abstract] [Full Text] [PDF]
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V. Y. Taraban, T. F. Rowley, and A. Al-Shamkhani Cutting Edge: A Critical Role for CD70 in CD8 T Cell Priming by CD40-Licensed APCs J. Immunol., December 1, 2004; 173(11): 6542 - 6546. [Abstract] [Full Text] [PDF]
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A. Boissonnas, C. Combadiere, E. Lavergne, M. Maho, C. Blanc, P. Debre, and B. Combadiere Antigen Distribution Drives Programmed Antitumor CD8 Cell Migration and Determines Its Efficiency J. Immunol., July 1, 2004; 173(1): 222 - 229. [Abstract] [Full Text] [PDF]
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T. F. Rowley and A. Al-Shamkhani Stimulation by Soluble CD70 Promotes Strong Primary and Secondary CD8+ Cytotoxic T Cell Responses In Vivo J. Immunol., May 15, 2004; 172(10): 6039 - 6046. [Abstract] [Full Text] [PDF]
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Y. Zhang, G. Joe, J. Zhu, R. Carroll, B. Levine, E. Hexner, C. June, and S. G. Emerson Dendritic cell-activated CD44hiCD8+ T cells are defective in mediating acute graft-versus-host disease but retain graft-versus-leukemia activity Blood, May 15, 2004; 103(10): 3970 - 3978. [Abstract] [Full Text] [PDF]
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J. Kan-Mitchell, B. Bisikirska, F. Wong-Staal, K. L. Schaubert, M. Bajcz, and M. Bereta The HIV-1 HLA-A2-SLYNTVATL Is a Help-Independent CTL Epitope J. Immunol., May 1, 2004; 172(9): 5249 - 5261. [Abstract] [Full Text] [PDF]
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P. Bansal-Pakala, B. S. Halteman, M. H.-Y. Cheng, and M. Croft Costimulation of CD8 T Cell Responses by OX40 J. Immunol., April 15, 2004; 172(8): 4821 - 4825. [Abstract] [Full Text] [PDF]
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D. M. Richards, S. L. Dalheimer, B. D. Ehst, T. L. Vanasek, M. K. Jenkins, M. I. Hertz, and D. L. Mueller Indirect Minor Histocompatibility Antigen Presentation by Allograft Recipient Cells in the Draining Lymph Node Leads to the Activation and Clonal Expansion of CD4+ T Cells That Cause Obliterative Airways Disease J. Immunol., March 15, 2004; 172(6): 3469 - 3479. [Abstract] [Full Text] [PDF]
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R. A. Wilcox, K. Tamada, D. B. Flies, G. Zhu, A. I. Chapoval, B. R. Blazar, W. M. Kast, and L. Chen Ligation of CD137 receptor prevents and reverses established anergy of CD8+ cytolytic T lymphocytes in vivo Blood, January 1, 2004; 103(1): 177 - 184. [Abstract] [Full Text] [PDF]
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 K. A. Mitchell and B. P. Lawrence Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Renders Influenza Virus-Specific CD8+ T Cells Hyporesponsive to Antigen Toxicol. Sci., July 1, 2003; 74(1): 74 - 84. [Abstract] [Full Text] [PDF]
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Q.-W. Zhang, D. D. Kish, and R. L. Fairchild Absence of Allograft ICAM-1 Attenuates Alloantigen-Specific T Cell Priming, But Not Primed T Cell Trafficking into the Graft, to Mediate Acute Rejection J. Immunol., June 1, 2003; 170(11): 5530 - 5537. [Abstract] [Full Text] [PDF]
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C.-T. Huang, D. L. Huso, Z. Lu, T. Wang, G. Zhou, E. P. Kennedy, C. G. Drake, D. J. Morgan, L. A. Sherman, A. D. Higgins, et al. CD4+ T Cells Pass Through an Effector Phase During the Process of In Vivo Tolerance Induction J. Immunol., April 15, 2003; 170(8): 3945 - 3953. [Abstract] [Full Text] [PDF]
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R. L. Reinhardt, D. C. Bullard, C. T. Weaver, and M. K. Jenkins Preferential Accumulation of Antigen-specific Effector CD4 T Cells at an Antigen Injection Site Involves CD62E-dependent Migration but Not Local Proliferation J. Exp. Med., March 17, 2003; 197(6): 751 - 762. [Abstract] [Full Text] [PDF]
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C. Wiethe, K. Dittmar, T. Doan, W. Lindenmaier, and R. Tindle Provision of 4-1BB Ligand Enhances Effector and Memory CTL Responses Generated by Immunization with Dendritic Cells Expressing a Human Tumor-Associated Antigen J. Immunol., March 15, 2003; 170(6): 2912 - 2922. [Abstract] [Full Text] [PDF]
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Q. Yu, J. X. Gu, C. Kovacs, J. Freedman, E. K. Thomas, and M. A. Ostrowski Cooperation of TNF Family Members CD40 Ligand, Receptor Activator of NF-{kappa}B Ligand, and TNF-{alpha} in the Activation of Dendritic Cells and the Expansion of Viral Specific CD8+ T Cell Memory Responses in HIV-1-Infected and HIV-1-Uninfected Individuals J. Immunol., February 15, 2003; 170(4): 1797 - 1805. [Abstract] [Full Text] [PDF]
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P. Shrikant and M. F. Mescher Opposing Effects of IL-2 in Tumor Immunotherapy: Promoting CD8 T Cell Growth and Inducing Apoptosis J. Immunol., August 15, 2002; 169(4): 1753 - 1759. [Abstract] [Full Text] [PDF]
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R. E. Miller, J. Jones, T. Le, J. Whitmore, N. Boiani, B. Gliniak, and D. H. Lynch 4-1BB-Specific Monoclonal Antibody Promotes the Generation of Tumor-Specific Immune Responses by Direct Activation of CD8 T Cells in a CD40-Dependent Manner J. Immunol., August 15, 2002; 169(4): 1792 - 1800. [Abstract] [Full Text] [PDF]
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E. L. Tham and M. F. Mescher The Poststimulation Program of CD4 Versus CD8 T Cells (Death Versus Activation-Induced Nonresponsiveness) J. Immunol., August 15, 2002; 169(4): 1822 - 1828. [Abstract] [Full Text] [PDF]
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J. L. LaBelle, C. A. Hanke, B. R. Blazar, and R. L. Truitt Negative effect of CTLA-4 on induction of T-cell immunity in vivo to B7-1+, but not B7-2+, murine myelogenous leukemia Blood, March 15, 2002; 99(6): 2146 - 2153. [Abstract] [Full Text] [PDF]
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E. L. Tham, P. Shrikant, and M. F. Mescher Activation-Induced Nonresponsiveness: A Th-Dependent Regulatory Checkpoint in the CTL Response J. Immunol., February 1, 2002; 168(3): 1190 - 1197. [Abstract] [Full Text] [PDF]
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D. A. Carlow, S. Y. Corbel, M. J. Williams, and H. J. Ziltener IL-2, -4, and -15 Differentially Regulate O-Glycan Branching and P-Selectin Ligand Formation in Activated CD8 T Cells J. Immunol., December 15, 2001; 167(12): 6841 - 6848. [Abstract] [Full Text] [PDF]
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J. Hernandez, S. Aung, W. L. Redmond, and L. A. Sherman Phenotypic and Functional Analysis of Cd8+ T Cells Undergoing Peripheral Deletion in Response to Cross-Presentation of Self-Antigen J. Exp. Med., September 17, 2001; 194(6): 707 - 718. [Abstract] [Full Text] [PDF]
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S. Mirshahidi, C.-T. Huang, and S. Sadegh-Nasseri Anergy in Peripheral Memory Cd4+ T Cells Induced by Low Avidity Engagement of T Cell Receptor J. Exp. Med., September 17, 2001; 194(6): 719 - 732. [Abstract] [Full Text] [PDF]
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E. L. Tham and M. F. Mescher Signaling Alterations in Activation-Induced Nonresponsive CD8 T Cells J. Immunol., August 15, 2001; 167(4): 2040 - 2048. [Abstract] [Full Text] [PDF]
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F. M. Marelli-Berg, D. Scott, I. Bartok, E. Peek, J. Dyson, and R. I. Lechler Activated Murine Endothelial Cells Have Reduced Immunogenicity for CD8+ T Cells: A Mechanism of Immunoregulation? J. Immunol., October 15, 2000; 165(8): 4182 - 4189. [Abstract] [Full Text] [PDF]
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N. Bercovici, A. Heurtier, C. Vizler, N. Pardigon, C. Cambouris, P. Desreumaux, and R. Liblau Systemic Administration of Agonist Peptide Blocks the Progression of Spontaneous CD8-Mediated Autoimmune Diabetes in Transgenic Mice Without Bystander Damage J. Immunol., July 1, 2000; 165(1): 202 - 210. [Abstract] [Full Text] [PDF]
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). Unstimulated cells or cells
stimulated with beads having just F23.1 on the surface incorporated
<0.2 x 103 and <7 x 103 cpm,
respectively, at all time points, and IL-2 was undetectable in these
cultures. C, Numbers of live and dead cells recovered
from cultures at the indicated times. Fewer than 104 cells
were recovered by day 3 from cultures stimulated with nothing, BI
beads, or F23 beads.
). B and
C, Cells stimulated in parallel with those shown in
A were harvested and washed on day 4 of the primary
response, diluted 1:5 in fresh medium, and restimulated with media
alone (none), F23/B/I microspheres identical to those used in
A, or IL-2. Proliferation (B) and viable
cell numbers (C) of cells in these cultures were then
determined on the indicated days following restimulation.
