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*
Center for Immunology, Departments of
Biochemistry, Molecular Biology, and Biophysics and
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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but they cannot
produce IL-2 in response to TCR ligation and costimulation, due at
least in part to an inability to up-regulate mitogen-activated protein
kinases and IL-2 mRNA. Exogenous IL-2 can drive continued proliferation
of AINR cells and nonresponsiveness is reversed within 1–2 days so
that Ag-driven proliferation can resume. Mitogen-activated protein
kinases and IL-2 mRNA can again be up-regulated, but "rewiring" has
occurred so that these events no longer depend upon costimulation; TCR
engagement is sufficient. Development of AINR appears to be a normal
part of the differentiation program of CD8 T cells, providing a
regulatory checkpoint to convert the initial helper-independent
response to one that depends upon CD4 T cell help for continued
expansion of the effector CTL. Once permission is given, in the form of
IL-2, to pass this checkpoint, the CTL can make a prolonged response to
persisting Ag in the absence of further CD4 T cell
help. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD4 help is necessary to initiate CTL responses in some cases. However, it is also clear that CD8 T cells can produce their own IL-2 in response to costimulation to drive activation in the absence of CD4 T cells (6, 7, 8). In a number of models of virus infection (9, 10, 11, 12, 13), tumor (14, 15, 16), and autoimmunity, (17) a CTL response occurs in the absence of help but the response only persists if CD4-dependent help is available. These observations would be consistent with there being more than one mechanism by which CD4 cells can contribute to the responses of CD8 T cells and suggest that different mechanisms may operate at differing stages of CD8 T cell activation, differentiation, clonal expansion, and survival. Conditions that determine whether and when CD4 help, in the form of IL-2, is required have not been well defined.
Costimulation of naive CD8 T cells through either CD28 or LFA-1
up-regulates IL-2 production, allowing the cells to undergo autocrine
IL-2-dependent clonal expansion (6, 7, 8, 18, 19). However,
we recently demonstrated that about 3 days after the response is
initiated, the cells lose the ability to make further IL-2 in response
to Ag and costimulatory ligands, and clonal expansion ceases
(20). This is not due to down-regulation of TCR or CD28
and is not due to CTLA-4 signaling. The cells can still be signaled
through the TCR as evidenced by their ability to kill and make IFN-
in response to Ag-bearing targets and can still proliferate if
exogenous IL-2 is present (20). This nonresponsiveness
resembles the "anergic" state originally described for CD4 T cell
clones following TCR engagement in the absence of costimulation
(21, 22, 23). However, it is distinct in that it occurs
following full stimulation through both the TCR and costimulatory
receptors. Therefore, we have termed this state of CD8 T cells
activation-induced nonresponsiveness
(AINR)4 to distinguish
it from the classical anergy that occurs when T cells receive just
signal one.
AINR is observed following an initial response of CD8 T cells both in vitro and in vivo (20, 24). Development of AINR should result in the conversion of an initially helper-independent CTL response into a helper-dependent response, because the AINR cells cannot further expand on their own but can continue to expand if they are provided with IL-2 (20, 24). This could account for the fact that a limited helper-independent CD8 T cell response frequently occurs upon challenge with virus or tumor, but expansion ceases after a few days unless help is available from CD4 T cells (9, 10, 11, 12, 13, 14, 15, 16).
We have further examined the effects of IL-2 on CD8 T cells that have become AINR. The results reported in this study demonstrate that the AINR state is reversed if continued proliferation is supported for a relatively short time (1–2 days) by provision of help in the form of IL-2. After this, the CD8 T cells regain the ability to produce IL-2 and continue to respond to Ag in an autocrine-driven manner for prolonged times. Furthermore, following reversal of AINR, the CD8 T cells become much less dependent upon costimulation for a continuing response. Thus, it appears that development of AINR provides a regulatory checkpoint and that the CD8 response again becomes helper-independent once the checkpoint has been passed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OT-I TCR-transgenic mice (25), originally a gift from Dr. F. Carbone (Monash Medical School, Victoria, Australia), were bred to Thy 1.1+ C57BL/6/PL mice. Lymph node cells from these mice were used for in vitro experiments and as the source of transgenic T cells for adoptive transfer. C57BL/6 were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were maintained under specific pathogen-free conditions. All experiments were performed in compliance with the relevant laws and institutional guidelines under a protocol approved by the Institutional Animal Care and Use Committee of the University of Minnesota (Minneapolis, MN). E.G7 tumor, EL-4 thymoma derived from the C57BL/6 mouse (H-2b) and transfected with the gene for OVA (26), was maintained in vitro in complete RPMI medium containing 400 µg/ml G418. Cell lines were periodically passaged in vivo.
Proliferation assays
Cell-size latex microspheres bearing H-2Kb-OVA257–264, H-2Kb- OVA257–264/B7-1, and H-2Kb- OVA257–264/B7-2 were prepared as described (27). Alternatively, microspheres were prepared by incubating 107 latex microspheres with 2.0 µg of DimerX I H-2Kb fusion protein (BD PharMingen, San Diego, CA) in 1 ml of PBS for 20 m at 4°C. Microspheres were then blocked with BSA, washed, and pulsed with OVA257–264 peptide at 2.0 µM in 1 ml of PBS for 2 h at 37°C. Lymph node OT-I cells were adherence depleted and the CD8 T cells were enriched by negative selection using CD8 Cellect columns (Cedarlane Laboratories, Hornby, Ontario, Canada). Purity of CD8 T cells was assessed by flow cytometry and was typically 90–95% with <1% CD4 T cells. A total of 5 x 104 CD8 T cells were cultured with 1 x 105 microspheres in 0.2 ml of complete RPMI medium. Cells were pulsed with 1 µCi of [3H]thymidine/well for the last 8 h of the indicated time and incorporation of radioactivity was determined. Results are expressed as the mean ± SD of triplicate samples. Anti-B7-1 (16-10A1) and anti-B7-2 (GL1) blocking Abs were used at 5 µg/ml. Anti-IL-2 receptor (PC61) blocking Ab was used at 10 µg/ml.
In vitro induction and reversal of AINR
Three to 4 days after initial stimulation of CD8 T cells, the AINR cells were harvested and dead cells were removed by Lympholyte M (Cedarlane Laboratories) gradient centrifugation. For all experiments, some of the cells were immediately restimulated to confirm that they were AINR, i.e., no longer responsive to stimulation with Kb-OVA/B7-1 microspheres, but still responsive to IL-2. Reversal of AINR was conducted by stimulating continued proliferation of the AINR CD8 T cells using 2.5 U/ml human rIL-2 for 1–2 days. In each experiment, a proliferation assay was again done at the end of the IL-2 culture period to confirm that AINR had been reversed. In all cases, proliferation assays were done using 5 x 104 CD8 T cells (naive, AINR, or reversed) with 1 x 105 microspheres in 0.2 ml of complete RPMI medium.
IL-2 mRNA measurements
CD8 T cells (naive, AINR, or reversed) and Kb-OVA/B7-1 microspheres were pelleted in 96-well V-bottom plates (1.5 x 105 cells and 3 x 105 microspheres/well) by centrifugation at 1250 x g for 10 min. The pellets were subsequently incubated at 37°C for 6–7 h. Trypan blue exclusion showed no significant change in cell viability at the end of the incubation period. Total RNA was isolated from the cells using the RNAqueous kit (Ambion, Austin, TX). IL-2 and actin mRNAs were determined by RT-PCR analysis using the same IL-2 and actin promoters and conditions as previously described, using a number of cycles confirmed to be within the linear amplification range for the assay (27). Experiments examining the effects of mitogen-activated protein kinase inhibitors on IL-2 mRNA expression were done by pretreating CD8 T cells with either DMSO (Fluka, Buchs, Switzerland), PD98059, or SB202190 (Calbiochem, San Diego, CA) at 37°C for 45 min before stimulation of the cells with microspheres.
In vitro kinase assays and immunoblotting
CD8 T cells (naive, AINR, or reversed) were pelleted with ligand-bearing microspheres by centrifugation at 1250 x g in a swinging bucket rotor for 10 min at 4°C. The pellets were then incubated in a 37°C water bath for 2.5 min (extracellular signal-regulated kinase (ERK) assay), 10 min (c-Jun N-terminal kinase (JNK) assay), or 7.5 min (phospho-p38 Western blot) and the cells were then lysed by the addition of ice-cold Triton-X buffer. ERK and JNK activities were assayed as previously described (27) following precipitation with GST-Elk-1 (aa 307–428) and GST-c-jun (aa 1–69), respectively. For immunoblotting, whole cell lysates were resolved on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). Blots were blocked with 5% skim milk powder and incubated with an anti-phospho-p38 Ab (Cell Signaling Technology, Beverly, MA) overnight at 4°C. Detection of a HRP-conjugated secondary Ab was done using the SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL).
Adoptive transfer, tumor challenge, and IL-2 administration
Adoptive transfer of Kb/OVA-specific
TCR-transgenic CD8 T cells from OT-I mice and analysis of responding
cells was done as previously described in detail (15, 24).
Briefly, lymph node cells from OT-I mice on the Thy
1.1+ C57BL/6/PL background were adoptively
transferred into C57BL/6 mice by i.v. injection. Mice were challenged 1
day later by i.p. injection of 3 x 106 E.G7
cells in 0.5 ml of PBS (day 0). At varying times, mice were sacrificed
and cells were isolated from the spleen, draining lymph nodes
(periaortic, mesenteric, axillary, and brachial) and the peritoneal
cavity. Adoptively transferred OT-I cells in the resulting cell
populations were identified and quantitated by flow cytometry by
staining with anti-Thy 1.1-PE and anti-CD8
-CyChrome mAbs and
CD25 levels were determined using a third anti-CD25-FITC mAb. Flow
cytometry was done using a FACSCaliber and CellQuest software (BD
Biosciences, San Jose, CA). When used, murine rIL-2 (R&D Systems,
Minneapolis, MN) was administered by i.v. (tail vein) injection of 2000
IU/day on days 8 and 9 after inoculation with tumor.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro induction of AINR was examined using CD8 T cells from
OT-I mice that have a transgenic TCR specific for
OVA257–264 bound to H-2Kb.
Stimulation of these cells with
Kb/OVA257–264 complexes
immobilized on microspheres (Kb-OVA) was not
sufficient to cause proliferation unless IL-2 was also added to the
cultures. However, the need for exogenous IL-2 was overcome if B7-1
ligand was immobilized along with Ag on the microspheres
(Kb-OVA/B7-1, Fig. 1
A); the B7-1 provides
costimulation to allow autocrine production and use of IL-2 by the
cells. Proliferation measured by [3H]thymidine
incorporation peaks on day 3 and then declines and maximal numbers of
OT-I cells are reached on day 4.
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in response to Ag
(20).
As discussed in more detail below, observations of in vivo responses of
OT-I T cells raised the possibility that the AINR state might be
reversible upon proliferation of the cells in response to exogenous
IL-2. This was examined using OT-I cells harvested after 3.5 days of
stimulation with Kb-OVA/B7-1 microspheres. Less
than 1% of the OT-I cells remain undivided at this time, as
demonstrated in experiments examining dye dilution in cells labeled
with CFSE before stimulation (data not shown). Following the initial
stimulation, cells were washed and placed back into culture and IL-2
was added. After varying times from 0 to 4 days in culture with IL-2,
cells were again harvested, washed, and placed into culture with
various stimuli. As expected, the cells were AINR at 3.5 days
and responded only if IL-2 was added; they did not respond to Ag or Ag
and costimulation alone (Fig. 2A). However, even after 1 day
in culture with IL-2, the AINR cells had regained significant
responsiveness that was further increased after 2 days and persisted
through at least 4 days. The responsiveness of the "reversed" cells
was distinctly different from that of naive cells in that Ag alone was
sufficient to stimulate a strong proliferative response and
coimmobilizing B7-1 to provide costimulation did not cause any further
increase in the response. Essentially identical results have been
obtained in experiments examining OT-I cells or CD8 T cells from
C57BL/6 mice stimulated with microspheres having anti-TCR mAb and
B7-1 immobilized on the surface (data not shown).
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Stimulation of the reversed cells with Ag alone suggested that
these cells had lost a requirement for costimulation to support
proliferation. However, activated T cells express B7 ligands and it was
important to determine whether transcostimulation was being provided by
cell-cell interactions in the culture. Anti-B7-1 and anti-B7-2 mAb
effectively block costimulation of naive cells when the respective
ligands are coimmobilized on microspheres along with Ag (Fig. 2
B). These same mAbs fail to block the response of reversed
cells to Ag alone on the microspheres (Fig. 2B), thus
demonstrating that costimulation provided by B7 ligands on the T cells
is not contributing to this response.
Because production of IL-2 by naive cells requires costimulation, the
independence from costimulation following reversal of AINR raised the
possibility that this response did not depend on IL-2. However,
proliferation of the reversed cells in response to Ag alone was
completely dependent on IL-2, as demonstrated by the effective blocking
of the response that occurred when anti-IL-2R mAb was added to the
cultures (Fig. 3). Consistent with this,
Ag alone is sufficient to up-regulate IL-2 mRNA expression in reversed
cells (see below).
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MAPKs are involved in regulating the transcription factors required for IL-2 gene expression in T cells and up-regulation of ERK, JNK, and p38 is defective in cells that have become AINR (27). Therefore, we examined cells that had recovered responsiveness following AINR to determine whether activation of the MAPKs was also restored. ERK and JNK activities were measured in lysates of stimulated cells using GST-Elk-1 and GST-c-jun fusion proteins as the substrates. Activation of p38 was assessed by Western blotting with a phospho-specific anti-p38 Ab to determine the amount of the active phosphorylated form of the enzyme.
ERK is activated in naive cells in response to Ag and its activity does
not increase further upon costimulation with B7-1 and/or ICAM-I (Fig. 4A). Activation was greatly
reduced in AINR cells, but restored in reversed cells to the same
levels as in naive cells. In contrast to ERK, JNK activation in naive
cells requires costimulation (Fig. 4B). Activation of JNK
was also greatly reduced in AINR cells and restored in reversed cells.
However, in the reversed cells, Ag alone was sufficient to fully
activate JNK, and the presence of costimulatory ligands caused no
further increase in activity. Active phospho-p38 increases in naive
cells in response to just Ag (Fig. 4C). AINR cells
were profoundly defective in phospho-p38 formation, but activation was
restored in reversed cells. Thus, activation of all three MAPKs is
defective in AINR cells and restored in reversed cells, and some
rewiring occurs during the reversal such that activation of JNK no
longer depends upon costimulation as it does in naive cells.
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, A and C) and
SB202190 (Fig. 5, B and D) blocked IL-2 mRNA
up-regulation and proliferation by reversed cells upon stimulation with
either Ag alone or Ag and B7-1. These results support the conclusion
that recovery from AINR involves, at least in part, recovery of the
ability to activate the MAPKs to signal for IL-2 production.
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Adoptive transfer of T cells from TCR-transgenic mice into normal recipients provides a means of directly visualizing and quantitating in vivo responses; the transgenic cells can be identified for flow cytometric analysis but are present in small numbers so that the immune response is not completely skewed as it is in the intact transgenic animal (28). We have used this approach to examine the response of transferred OT-I cells to i.p. E.G7 tumor that expresses OVA (15, 24). Within 3–4 days of i.p. injection of the tumor, OT-I cells migrate to the peritoneal cavity, undergo activation and clonal expansion, and control tumor growth. Clonal expansion peaks at about day 5 and the OT-I cells then migrate out of the peritoneal cavity and are found in the spleen and lymph nodes, even though the Ag-expressing tumor is still growing in the peritoneal cavity. The OT-I cells that have responded and migrated to the spleen have developed AINR; they exhibit direct ex vivo killing activity and can proliferate in response to exogenous IL-2 but not in response to Ag and costimulation.
The ability to reverse the AINR state in vitro by driving proliferation for 1–2 days with IL-2 predicted that it might also be possible to reverse the nonresponsive state in vivo with limited administration of IL-2 and achieve long-term responsiveness of the CD8 T cells once AINR was reversed. This was tested by challenging OT-I adoptive transfer recipients by i.p. injection of E.G7 tumor on day 0, allowing the initial response to occur unperturbed, and then administering low dose (2000 U/mouse) IL-2 systemically on days 8 and 9, the time at which the OT-I cells have become AINR (24). Mice were then sacrificed 10 days later (day 18 after tumor challenge) and the numbers of OT-I cells in the peritoneal cavity, spleen, and lymph nodes were determined, as well as the number of E.G7 tumor cells in the peritoneal cavity.
In adoptive transfer recipients that had not been challenged with
tumor, OT-I cells were present in the spleen and lymph nodes in low
numbers (Fig. 6A, OT-I) and
had a naive phenotype (data not shown). In mice challenged with tumor
and left untreated (Fig. 6A, OT-I/E.G7), some OT-I cells
were present in the peritoneal cavity but greater numbers were found in
the spleen and lymph nodes (Fig. 6A) and tumor load was high
(Fig. 6B). In contrast, mice treated with IL-2 on days 8 and
9 had a high number of OT-I remaining in the peritoneal cavity by day
18 (Fig. 6A, OT-I/E.G7 + IL-2, days 8–9), as well as
elevated numbers in the spleen and lymph nodes (Fig. 6A),
and tumor load was substantially reduced in comparison to that in
untreated mice (Fig. 6B). Essentially the same results have
been obtained in similar experiments done using recipient mice depleted
of CD4 T cells by in vivo mAb administration or CD4 knockout
recipients, thus ruling out a role for host CD4 T cells in reversal of
the AINR state by IL-2 administration (data not shown).
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) expression (Fig. 7). In
contrast, few of the day 18 OT-I cells from mice that had not received
IL-2 exhibited high forward scatter or high CD25 expression (Fig. 7).
Thus, brief exposure to IL-2 is sufficient to reverse AINR and allow
prolonged in vivo response to the tumor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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); they can still be signaled through the TCR to carry out
effector functions but can no longer produce IL-2 to sustain expansion
(20, 27). However, the CTL can continue to proliferate if
provided with IL-2. This appears very likely to be the basis for the
requirement for CD4 T helper cells to sustain effective CTL responses
in many instances.
We would suggest that AINR is an integral part of the CD8 T cell
developmental program, because it occurs both in vitro (Fig. 1; Ref.
20) and in vivo (20, 24), and occurs
irrespective of the nature of the primary stimulus. In vitro
stimulation results in the development of AINR within 3 days whether
peptide/class I Ag or high-affinity anti-TCR mAb are used to
provide signal one, and whether costimulation is provided by B7-1,
ICAM-1, or by both ligands which synergize to provide potent
costimulation (Fig. 1; Refs. 20 and 27). AINR
also develops when stimulation is with Ag or anti-TCR mAb in the
absence of costimulatory ligands, with exogenous IL-2 added to drive
proliferation (27). Thus, development of AINR does not
depend upon signals delivered through the costimulatory receptors. Once
AINR develops, costimulation by either B7-1, ICAM-1, or the combination
is ineffective (Fig. 1; Refs. 20 and 27).
AINR also develops in CD8 cells that have responded in vivo to
allogeneic tumor, even when the tumor cells express B7-1
(20), or to syngeneic tumor (24). Thus,
nonresponsiveness develops whether Ag is eliminated, as in the case of
the allogeneic tumor, or persists, as in the case of the syngeneic
tumor. Programmed development of AINR following the initial response of
CD8 T cells would provide a consistent explanation for the lack of
persistence of CTL responses in many situations, and is likely be the
basis for the phenomenon of clonal "exhaustion" that has been shown
to occur following a vigorous response to virus infection, thus
allowing persistent infection (29).
CTL that have developed AINR become helper-dependent; they can no
longer make IL-2, but can still respond if IL-2 is provided. However,
this is not a permanent conversion to helper dependence. Rather, if
proliferation is maintained by provision of exogenous IL-2 for a brief
period (1–2 days), the cells regain their ability to up-regulate IL-2
mRNA (Fig. 5) and continue to proliferate in response to the IL-2 they
produce (Figs. 2 and 3) without further exogenous growth factors being
provided. Following reversal of AINR, the CTL continue to respond for a
prolonged period of time, up to at least 14 days in vivo, the longest
times examined (data not shown). This is in marked contrast to the
initial response that ends in 3–4 days as the cells develop AINR (Fig. 1). Thus, it appears that development of AINR is not a mechanism for
tolerizing CD8 T cells. Rather, it is a normal part of the CD8
developmental program that serves as a regulatory checkpoint for the
potentially destructive CTL response, insuring that the cells can only
continue to expand if regulatory helper CD4 T cells give permission in
the form of IL-2. What determines whether a particular biological
system appears to be "helper-independent" or "helper-dependent"
may often depend upon whether or not the initial response is sufficient
to clear Ag before development of AINR, e.g., due to sufficiently high
precursor frequency, high avidity, or low Ag load.
Nonresponsiveness is not due to receptor down-regulation; AINR cells
express essentially the same levels of TCR, CD28, and LFA-1 as naive
cells (20). AINR cells also express CTLA-4, a receptor for
B7 ligands that can deliver an inhibitory signal to cells
(30, 31, 32, 33). However, CTLA-4 does not mediate the
nonresponsiveness that develops following initial stimulation. AINR
cells cannot be costimulated via LFA-1/ICAM-I interactions when B7
ligands are not present (20) and where CTLA-4 would,
therefore, not be engaged, and blocking anti-CTLA-4 mAb neither
prevents nor reverses AINR in vitro (20) or in vivo
(15). Rather, AINR appears to result from an intrinsic
inability of the cells to signal for up-regulation of expression of
IL-2 mRNA. Activation of MAPKs is required for IL-2 gene expression in
T cells and anergic CD4 T cell clones have a markedly reduced ability
to up-regulate ERK and JNK activities upon stimulation and cannot
produce IL-2 (34, 35, 36). CD8 T cells also exhibit reduced
up-regulation of ERK and JNK, as well as p38, upon development of AINR
(Fig. 4). The enzymes remain present and can be activated by treatment
of the AINR cells with phorbol ester and ionomycin (27),
but they are not up-regulated in response to Ag and costimulation. The
inability to up-regulate the MAPKs accounts, at least in part, for the
inability of AINR cells to make IL-2 because use of specific inhibitors
has demonstrated that activation of this pathway is essential for
up-regulation of IL-2 mRNA by naive cells (27). Upon
reversal of the AINR state, the ability to up-regulate the MAPKs is
restored (Fig. 4) and IL-2 mRNA up-regulation and proliferation by the
reversed cells depends upon these activities (Fig. 5). However, the
signaling requirements are different in naive and reversed cells.
Up-regulation of JNK in naive cells requires costimulation through
either CD28/B7 or LFA-1/ICAM-I interactions, while JNK up-regulation in
the reversed cells occurs when just the TCR is engaged (Fig. 4).
Consistent with this, the reversed cells exhibit a decreased dependence
on costimulation for increasing expression of IL-2 mRNA and
proliferating (Figs. 3 and 5). Thus, once AINR is reversed, the CTL can
more effectively respond to Ag that is not being expressed on
professional APC. Memory cells also exhibit a decreased dependence on
costimulation in comparison to naive cells; this may result from the
rewiring that occurs upon reversal of the AINR state. The molecular
basis for this change in signaling requirements remains to be
determined.
There is an increasing appreciation of the importance of CD4 T cell
help for generating effective anti-tumor responses by CD8 T cells
and development of AINR in the responding CTL in the absence of help is
likely to be an important factor in this. Evidence for this is
accumulating in studies using models that make it possible to directly
visualize the tumor-specific CTL during the course of a response. Using
an adoptive transfer system employing OT-I T cells having a transgenic
TCR specific for an OVA peptide bound to H-2Kb,
we showed that the OT-I cells could make an initial CD4-independent
response upon i.p. challenge with an OVA-expressing E.G7 tumor. The
OT-I cells clonally expanded in the peritoneal cavity developed
effector function and controlled tumor growth for a few days. However,
they then became AINR, migrated out of the peritoneal cavity, and
stopped responding and tumor expansion resumed (24). In
contrast, if OVA-specific CD4 helper cells were activated, the OT-I
cells continued to make a prolonged response at the site of the tumor
and control tumor growth and this was dependent on IL-2
(15). As shown in this study, brief administration of low
dose IL-2 results in reversal of the nonresponsiveness (Figs. 6 and 7)
and prolonged response of the OT-I cells at the site of the tumor (Fig. 6). A similar example is provided by studies of Marzo et al.
(16) using the influenza hemagglutinin gene as
a model tumor Ag. The tumor-specific CD8 T cells responded to tumor
challenge and on day 14 their numbers and functions were comparable
with or without Ag-specific CD4 T cells. However, more prolonged
maintenance of the CD8 response required the presence of Ag-specific
CD4 T cells and without them the number and function of the
tumor-specific CD8 T cells was substantially reduced by day
28.
Thus, it appears that there are two distinct mechanisms by which CD4 T cells can provide help for a CD8 T cell response. CD4 cells can interact with APCs via CD40 to condition them to provide more effective activation of CD8 T cells and, thus, help in the initiation of a response (3, 4, 5). The mechanism of this conditioning has not been defined, but may involve stimulation of the production of inflammatory cytokines by the APC as a result of CD40 ligation. This type of help is clearly not always required, because there are numerous examples of generation of CTL responses in the absence of CD4 T cells. The requirement for CD4-dependent conditioning of APC may be bypassed if microbial or viral components are present to activate the APC and stimulate inflammatory cytokine production. There is also evidence that priming of CD8 cells is less dependent on help when the peptide epitope binds class I with high affinity (37). The studies described here have defined the second mechanism by which CD4 T cells can deliver help by providing IL-2 to reverse the AINR state that develops following the initial CTL response. Whether there are conditions that bypass AINR and allow a prolonged CTL response in the absence of help from CD4 T cells remains to be determined.
The ability to reverse CTL nonresponsiveness by brief administration of
IL-2 (Figs. 6 and 7) has obvious therapeutic implications, but would
appear to be at odds with the limited therapeutic efficacy that has
been seen for IL-2 in animal models and in the clinic. However, in
addition to its ability to promote T cell growth, IL-2 can also induce
apoptotic death in activated T cells (38, 39). In another
study (P. Shrikant and M. Mescher, manuscript in preparation), we have
found that the reversal of AINR by low dose administration of IL-2 for
a brief period (Fig. 6) can result in significant therapeutic benefit
in the OT-I/E.G7 tumor model described above. However, when IL-2
administration is more prolonged or at high doses, as is often the case
in clinical studies, then the number of CTL declines dramatically, with
many of the cells dying by apoptosis, and the therapeutic benefit is
lost. It will be important to determine how long CTL can persist in the
AINR state and still be capable of having their nonresponsiveness
reversed. We have found that IL-2 administration as long as 18 days
after the peak of the primary response (the longest time examined in
the other study) can still result in reversal (P. Shrikant and
M. Mescher, manuscript in preparation). Some studies have shown that
CTL that have made a primary response and then stopped expanding even
though Ag remains present exhibit reduced effector function at longer
times (13, 16, 40, 41), suggesting that CTL that have
become AINR may undergo a gradual decline in function. Whether there is
a concomitant loss of reversibility will be of critical importance in
developing immunotherapeutic strategies for eliminating persistent
viral infections and residual tumors.
Although CTL that have become AINR in the face of persisting Ag require
help in the form of IL-2 to regain responsiveness (Figs. 6 and 7), the
situation appears to be different if Ag is eliminated as a result of
the primary response. When mice adoptively transferred with 2C
CD8+ T cells specific for
Ld alloantigen are challenged with allogeneic
tumor, the 2C cells respond and the tumor is rapidly rejected
(42). The 2C CTL that have responded have become AINR at
this time, as demonstrated by the fact that they are unable to respond
in vitro when restimulated with Ag and costimulation, but can respond
if exogenous IL-2 is provided (20). In vivo, the number of
these AINR 2C cells declines following elimination of Ag, but a
long-term memory population develops that is capable of mounting a
rapid secondary response upon rechallenge (43). These
observations suggest that the fate of AINR CTL differs depending upon
whether or not they continue to be exposed to Ag. Ehl et al.
(44) have similarly observed that CTL unresponsiveness can
be a consequence of persistent Ag. Clearly, further study will be
needed to understand the mechanisms that determine the ultimate fate of
CTL following a primary response to Ag and the programmed development
of AINR.
The CD40-dependent help provided by CD4 T cells to initiate a CTL response (3, 4, 5) has been termed the "license" to kill (45). However, the primary stimulation of a CTL response, whether helper-dependent or -independent, appears to result in only a "temporary" license. The "permanent" license is only issued when the CTL successfully eliminate Ag or when CD4 T cells provide IL-2 to reverse AINR.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263.
3 Address correspondence and reprint requests to Dr. Matthew F. Mescher, Center for Immunology, University of Minnesota, Mayo Mail Code 334, 420 Delaware Street SE, Minneapolis, MN 55455. E-mail address: mesch001{at}tc.umn.edu
4 Abbreviations used in this paper: AINR, activation-induced nonresponsiveness; ERK, extracellular-signal regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.
Received for publication October 15, 2001. Accepted for publication November 29, 2001.
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-herpesvirus in the absence of CD4+ T cells. J. Exp. Med. 184:863.
T lymphocytes for apoptosis. Nature 353:858.[Medline]
-chain in regulating IL-2-dependent, activation-induced CD8+ T cell death. J. Immunol. 163:3131.This article has been cited by other articles:
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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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|
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|
|
|
|
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|
|
|
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|
|
|
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|
|
|
|
|
|
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|
|
|
|
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|
M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, and R. W. Dutton Tc1 and Tc2 Effector Cell Therapy Elicit Long-Term Tumor Immunity by Contrasting Mechanisms That Result in Complementary Endogenous Type 1 Antitumor Responses J. Immunol., February 1, 2004; 172(3): 1380 - 1390. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
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D. D. Sloan, G. Zahariadis, C. M. Posavad, N. T. Pate, S. J. Kussick, and K. R. Jerome CTL Are Inactivated by Herpes Simplex Virus-Infected Cells Expressing a Viral Protein Kinase J. Immunol., December 15, 2003; 171(12): 6733 - 6741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Jackaman, C. S. Bundell, B. F. Kinnear, A. M. Smith, P. Filion, D. van Hagen, B. W. S. Robinson, and D. J. Nelson IL-2 Intratumoral Immunotherapy Enhances CD8+ T Cells That Mediate Destruction of Tumor Cells and Tumor-Associated Vasculature: A Novel Mechanism for IL-2 J. Immunol., November 15, 2003; 171(10): 5051 - 5063. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
 W. W. Overwijk, M. R. Theoret, S. E. Finkelstein, D. R. Surman, L. A. de Jong, F. A. Vyth-Dreese, T. A. Dellemijn, P. A. Antony, P. J. Spiess, D. C. Palmer, et al. Tumor Regression and Autoimmunity after Reversal of a Functionally Tolerant State of Self-reactive CD8+ T Cells J. Exp. Med., August 18, 2003; 198(4): 569 - 580. [Abstract] [Full Text] [PDF]
|
|
|
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 |
|
 E. J. Wherry, J. N. Blattman, K. Murali-Krishna, R. van der Most, and R. Ahmed Viral Persistence Alters CD8 T-Cell Immunodominance and Tissue Distribution and Results in Distinct Stages of Functional Impairment J. Virol., April 15, 2003; 77(8): 4911 - 4927. [Abstract] [Full Text] [PDF]
|
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|
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|
S.-i. Fujii, K. Shimizu, F. Koji, and F. Kawano Malignant counterpart of myeloid dendritic cell (DC) belonging to acute myelogenous leukemia (AML) exhibits a dichotomous immunoregulatory potential J. Leukoc. Biol., January 1, 2003; 73(1): 82 - 90. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. E. Cheng and P. D. Greenberg Selective Delivery of Augmented IL-2 Receptor Signals to Responding CD8+ T Cells Increases the Size of the Acute Antiviral Response and of the Resulting Memory T Cell Pool J. Immunol., November 1, 2002; 169(9): 4990 - 4997. [Abstract] [Full Text] [PDF]
|
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|
|
|
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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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