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Departments of
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Medicine and
Microbiology, and Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455
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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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Inactivation of T cells by the tolerance mechanism called clonal anergy
was originally described using a tissue culture system of cloned T
cells (11, 12). Clonal anergy has since been defined as a reversible,
induced tolerance state in which the T lymphocyte cannot produce its
autocrine growth factor IL-2 or proliferate in response to the Ag it
recognizes (13). In vitro, this unresponsive state is induced by
stimulation of the T cell through its TCR in the absence of
costimulatory signals, such as those occurring as a result of the
interaction of B7 molecules on the APC with CD28 receptors on the T
cell (14, 15). In the absence of such costimulatory signals, T cells
fail to proliferate, and TCR occupancy unaccompanied by proliferation
down-regulates the T cell’s responsiveness (16). Biochemical analyses
have suggested that anergic T cells lose the capacity to synthesize
IL-2 because of a defect in the coupling of the Ag receptor to
downstream p21Ras-dependent signaling cascades (17, 18, 19)
necessary for the induction of activating protein-1-dependent
IL-2 gene transcription (20, 21, 22). Importantly, the clonal
anergy observed in vitro has not been associated with a global defect
in T cell activation events. Specifically, the secretion of IFN-
by
anergic CD4+ Th cells is not blocked after the induction of
clonal anergy (23). Furthermore, anergic CD8+CTL are
capable of lysing target cells (24). Finally, anergy induction cannot
effectively interfere with the delivery of helper signals for the
stimulation of B cell polyclonal IgG secretion in vitro (25).
Consistent with these functional results, anergic T cells can be shown
to develop increases in intracellular calcium free ion concentrations
that result in the dephosphorylation and nuclear translocation of the
nuclear factor of activated T cells (NFAT) upon stimulation (22). Thus,
the clonal anergy mechanism appears to selectively interrupt signal
transduction to the nucleus, resulting in a defective proliferative
capacity, yet it may not eliminate the potential of a T cell to
participate in an effector cell response.
These findings of selectivity in the inhibitory effects of clonal anergy raise uncertainty as to how this mechanism might contribute to the development of immune tolerance in vivo. Proliferative T cell clonal anergy has previously been reported in animals made tolerant of foreign Ag by the systemic administration of aqueous peptides in the absence of infection or adjuvant (26). Clonal anergy has also been observed after the systemic exposure of animals to superantigens such as staphylococcal enterotoxin B (SEB)3 (27). Interestingly, TCR-Vß8 transgenic mice immunized against tetanus toxoid showed no evidence of tolerance within the tetanus toxoid-specific Th cell population following exposure to SEB, despite the ability of the SEB to induce proliferative unresponsiveness in the naive T cell population (28). Likewise, systemic exposure of naive mice to the lymphocytic choriomeningitis virus (LCMV) glycoprotein-derived peptide GP33 induced tolerance within CD8+CTL precursors, whereas mice that had first been infected with LCMV instead developed destructive spleen immunopathology in response to systemic administration of the aqueous GP33 peptide (29). These findings suggest either that memory T cells are insensitive to clonal anergy induction, or that the anergy mechanism cannot regulate all effector functions by an expanded T cell clone in the peripheral immune system.
Previously, in vivo models of T cell activation have not had the power to quantitate the effector cell capabilities of lymphocytes following the induction of an Ag-specific tolerance. Specifically, differences in the frequencies of Ag-reactive T cells that develop as a consequence of clonal expansion and/or activation-induced cell death have complicated the analysis. Therefore, we have developed an experimental system that both allows for the induction of T cell tolerance in mice using an aqueous peptide Ag, and that permits the subsequent recovery and identification of Ag-specific cells from these tolerant animals. Using this system, the functional capacities of equal numbers of normal and tolerant T cells can be directly compared following their adoptive transfer into normal recipient mice. In this study, we describe the results of experiments designed to assess the functional effects of clonal anergy induction on both naive and Ag-experienced Th cells. Our findings indicate that exposure to an aqueous peptide Ag in vivo results in the induction of a clonal anergy that limits the expansion of T cells responding to that Ag, and yet fails to interrupt the generation and delivery of Th1-like effector activities by these lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and BALB/c nu/nu (nu/nu) mice, 6 to 8 wk old, were purchased from Charles River (Wilmington, MA) through a contract with National Cancer Institute at National Institutes of Health (Frederick, MD), and housed under specific pathogen-free conditions. The DO11.10 TCR-transgenic (TCR-Tg) mouse line has been described previously (30) and was bred to homozygosity in our animal facility in accordance with the guidelines of National Institutes of Health. CD4+ T cells in these mice have been engineered to specifically recognize the chicken OVA peptide 323–339. All mice used for any given experiment were sex and age matched.
Immunizations and Ags
Normal BALB/c mice were immunized with 100 µg OVA peptide 323–339 (OVAp) (synthesized and purified within Microchemical Facilities at University of Minnesota, Minneapolis, MN) emulsified in incomplete Freund’s adjuvant (IFA) (Difco, Detroit, MI) by s.c. injection in a volume of 100 µl at the base of the tail. DNP-specific Abs were subsequently elicited by secondary immunization at the same site either with 10 or 100 µg DNP-conjugated OVA (Pierce, Rockford, IL) (DNP-OVA) emulsified in CFA (Difco), or with CFA alone as a negative control. Alternatively, anti-DNP Ab production was determined in some experiments after only primary s.c. immunization with DNP-OVA in CFA. BALB/c mice that received an adoptive T cell transfer were immunized 1 or 2 days later s.c. at the base of the tail with either 10 or 100 µg DNP-OVA emulsified in CFA, or with CFA alone as a negative control, to induce a T cell clonal expansion and the production of anti-DNP Ab. For the measurement of ex vivo intracellular IL-2 levels in Ag-stimulated T cells, 250 µg OVAp was injected i.v. into adoptive transfer recipient BALB/c mice 1 day after cell transfer. Delayed-type hypersensitivity (DTH) was also assessed 1 day after adoptive T cell transfer into normal BALB/c mice by intradermal (i.d.) injection of 10 µg OVA in 10 µl PBS into the pinna of one ear (with PBS alone injected into the other ear as a negative control). Finally, a regimen of three i.p. injections of 100 µg OVAp in PBS at 5-day intervals was utilized in some experiments to induce immune tolerance within the BALB/c mice.
TCR-Tg model of in vivo T cell tolerance
DO11.10 lymph node and spleen cells were harvested and treated
with ACK lysis buffer (Biofluids, Rockville, MD), followed by
anti-CD8 mAb 3.155 plus rabbit complement (Cedarlane, Westbury, NY)
to deplete RBC and CD8+ T cells. Subsequently, 
20
x 106 OVA-specific
KJ1-26+CD4+TCR-Tg T cells were injected i.p.
into -irradiated (200 rad) nu/nu mice. The number of
KJ1-26+ cells injected was calculated based on multiplying
the percentage of KJ1-26+CD4+ cells measured by
flow cytometry times the number of live cells purified. At days 15, 20,
and 25 after T cell transfer, some nu/nu mice were injected
i.p. with 100 µg OVAp to induce tolerance in the TCR-Tg cells.
Negative control mice received PBS injections without Ag. At day 30
after transfer, the nu/nu mice were sacrificed, lymph node
and spleen cells were isolated, and the
KJ1-26+CD4+ cells were enumerated by flow
cytometry. In some experiments, day 30 nu/nu mice were then
primed s.c. with 100 µg OVAp in CFA at the base of the tail and
sacrificed at day 40. Alternatively, nu/nu mice were primed
first with OVAp in CFA at day 15, and then tolerized at days 25, 30,
and 35 with i.p. OVAp injections before sacrifice at day 40. T cells
isolated from these mice were subsequently transferred i.v. into
unirradiated syngeneic BALB/c recipients, such that each host animal
received 0.25 to 2 x 106
KJ1-26+CD4+ donor cells in 0.5 ml PBS. Some of
the donor cells were also used immediately for in vitro proliferation
and lymphokine production assays.
Flow cytometry
Peripheral blood leukocytes, lymph node cells, or splenocytes were harvested, and 106 cells were incubated on ice with biotinylated anti-clonotypic mAb KJ1-26 (31), followed by streptavidin Cy-Chrome (PharMingen, San Diego, CA) together with anti-CD4-phycoerythrin (Caltag, Burlingame, CA). After several washes, cells were either immediately analyzed or were fixed in 0.5% paraformaldehyde before the acquisition of 10,000 lymphocyte-gated events using forward and side scatter. Isotype-matched irrelevant mAb dye conjugates served as negative controls in all experiments. Flow cytometry was performed using a FACScan and CellQuest Software (Becton Dickinson, Mountain View, CA).
Ex vivo intracellular IL-2 measurement
For intracellular IL-2 detection, peripheral lymph node
(inguinal, axillary, brachial, mesenteric, and periaortic) and spleen
cells were harvested 3 h after OVAp injection i.v., and 5 x
106 cells were immediately incubated on ice with
anti-FcR mAb 2.4G2 (American Type Culture Collection, Manassas,
VA) plus 10% rat serum (Sigma, St. Louis, MO), followed by
anti-CD4-FITC (Caltag) and the biotinylated KJ1-26 mAb, and then
streptavidin Cy-Chrome. Cells were fixed in 2% formaldehyde (Fisher,
Pittsburgh, PA) and permeabilized in 0.5% saponin (Sigma) before
staining with phycoerythrin-conjugated rat anti-murine IL-2 mAb
(PharMingen), as described (32). Following three washes, 10,000
lymphocyte-gated events in addition to 1,000
KJ1-26+CD4+-gated events were individually
collected. Results indicate the percentage of T cells with increased
IL-2-phycoerythrin staining in 1,000 cells from the
KJ1-26+CD4+ subpopulation. Data are expressed
as the mean ± SEM for replicate animals.
Anti-DNP Ab production
BALB/c mice were bled by retroorbital puncture, and sera collected both before immunization (preimmune) and 10 to 12 days after primary or secondary Ag challenge. Sera were stored at -20°C, and Ab titers were determined by ELISA using DNP-BSA as the capture Ag. DNP-specific IgG1 and IgG2a were detected using isotype-specific, horseradish peroxidase-labeled, goat anti-mouse IgG1 and anti-mouse IgG2a Abs, respectively (Southern Biotechnology, Birmingham, AL). The Ab titer is calculated based on that dilution of a serum that has a concentration of anti-DNP capable of eliciting a one-fourth maximal OD within the peroxidase assay. Titers shown typically represent the geometric mean ± SEM of replicate animals.
DTH reaction
Pinna thickness was determined for both ears using an engineer’s micrometer both before and 24 h after i.d. injection of 10 µg OVA into one ear and PBS into the opposite ear. The DTH response was determined as the difference between the pre- and postinjection measurements. Data shown are the mean ± SEM of replicate animals.
In vivo T cell clonal expansion
Five days after immunization with OVAp in CFA, animals were sacrificed, and draining lymph nodes (inguinal and periaortic) were harvested. The total viable lymph node cell number was determined by trypan blue exclusion microscopy. Lymph node cells were also analyzed by flow cytometry to detect the presence of KJ1-26+CD4+TCR-Tg T cells, as described above. The total number of KJ1-26+CD4+ T cells recovered was calculated by multiplying the percentage of KJ1-26+CD4+ cells times the total viable lymph node cell count. Data shown represent the mean ± SEM of replicate animals.
T cell proliferation assay
Harvested lymph node cells from nu/nu mice were cultured with 5 x 105 irradiated (3000 rad) normal BALB/c splenocytes and 1 µM OVAp for 48 h in 200 µl complete medium (1:1 Eagle’s Hanks’ amino acids (EHAA) medium (Biofluids) and RPMI 1640 (Celox, Hopkins, MN) containing 10% FCS (HyClone, Logan, UT), 2 mM L-glutamine, penicillin, gentamicin, and 5 x 10-5 M 2-ME). T cells were then pulsed with 0.25 µCi of [3H]thymidine (Dupont-NEN, Boston, MA) for 12 to 16 h, followed by harvesting of the cells on a PHD cell harvester (Cambridge Technology, Cambridge, MA) and liquid scintillation counting of the samples to determine the level of thymidine incorporation.
In vitro IL-2 and IFN- production
IL-2 secretion into the supernatant was monitored by bioassay at
48 and 72 h of stimulation using CTLL-2 cells (33) that express a
rBcl-x molecule (kindly provided by Dr. C. Thompson, University of
Chicago, Chicago, IL), as previously described (34). IFN- secretion
during the experiment was determined by ELISA using PharMingen capture
and detection mAbs. Lymphokine production results are expressed as the
amount secreted per OVA-specific KJ1-26+CD4+ T
cell present in the culture.
Statistical analysis
Data are expressed as the mean ± SEM of replicate animals. In some cases, experimental treatment groups were compared using Student’s paired or unpaired t tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, it had been observed that an immune tolerance to the
chicken OVA peptide 323–339 (OVAp) develops in animals following i.v.
exposure to the Ag in PBS because of the induction of T cell clonal
anergy (26, 35). To prove this, some of these experiments have relied
on an adoptive transfer of OVA-specific CD4+TCR-Tg DO11.10
T cells into normal BALB/c mice before the Ag infusion, and the
subsequent detection of these OVA-reactive T cells with the KJ1-26
anti-clonotypic mAb (26, 30, 31). In the experiments described in
this work, adoptive transfer of DO11.10-pooled lymph node and spleen
cells into lightly irradiated T cell-deficient BALB/c nu/nu
mutant mice was performed to allow for a detailed analysis of the
effects of tolerance induction on the effector functions of a
relatively large population of Ag-specific T cells in vivo (see
illustrations, Fig. 1
). Within 15 days of
i.p. transfer, significant numbers of clonotype-positive
(KJ1-26+) CD4+ T cells spontaneously left the
peritoneum and appeared in the peripheral blood (Fig. 2, A and B).
Recipient nu/nu mice were then challenged i.p. with OVAp in
PBS three times at 5-day intervals beginning on day 15 after transfer
(3xOVAp pretreatment) to induce tolerance within this TCR-Tg
OVA-responder population. Exposure to aqueous 3xOVAp i.p. did not
significantly alter the percentage of
KJ1-26+CD4+ T cells observed in either lymph
nodes (Fig. 2C) or spleen (data not shown) on day 30 after
transfer, and the total number of KJ1-26+CD4+
cells recovered from the 3xOVAp-treated mice averaged 8.5 ±
1 x 106 vs 8.7 ± 1.1 x 106 in
3xPBS-treated control mice (n = 10). Thus, an overall
decrease in OVA-responder cell frequency as a result of peripheral
deletion (36, 37, 38) did not accompany the exposure of T cells to aqueous
peptide administration in the nu/nu mice.
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A), as well as a markedly
reduced capacity to secrete their autocrine growth factor IL-2 (Fig. 3, B and C). In nine separate experiments, IL-2
production (corrected for the number of
KJ1-26+CD4+ T cells present) in cultures from
3xOVAp-treated nu/nu recipient animals was inhibited an
average of 93 ± 2% compared with control 3xPBS-treated animals.
This failure to detect the production of IL-2 by 3xOVAp-treated T
cells was not simply the consequence of a change in the kinetics of
secretion, as determined by time-course studies (data not shown);
furthermore, T cells recovered from nu/nu animals immunized
s.c. with OVAp in CFA retained the capacity to secrete IL-2 (Fig. 3C). Thus, exposure of CD4+TCR-Tg T cells in
nu/nu mice to repeated aqueous peptide-Ag infusions resulted
in clonal anergy induction.
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-producing phenotype
It previously has been observed that the s.c. immunization of
DO11.10 TCR-Tg T cells in BALB/c adoptive transfer animals with OVAp in
CFA results in the differentiation of these cells to an
IFN--producing Th1 phenotype (39, 40). Consistent with this,
increased IFN- production was observed in cultures containing
KJ1-26+CD4+ T cells recovered from
nu/nu mice after OVAp/CFA immunization in response to OVAp
rechallenge in vitro (Fig. 3
C). Cells from the 3xOVAp
i.p.-pretreated group, on the other hand, failed to secrete a
significant quantity of IFN- into the culture medium during
reexposure to Ag. Therefore, clonal anergy was induced in naive T cells
in the absence of a differentiation to a Th1-like phenotype. In
addition, no evidence for clonal diversion to a Th2-like phenotype was
obtained in these experiments, since 3xOVAp-treated T cells also
failed to develop the capacity to secrete either IL-4 or IL-5 (data not
shown).
To test whether systemic exposure to aqueous peptide-Ag precludes later
differentiation of an anergic T cell to a helper phenotype,
3xOVAp-pretreated adoptive transfer nu/nu mice were
challenged s.c. on day 30 with OVAp in CFA (Fig. 1B). When
recovered from these animals 10 days later (on day 40),
KJ1-26+CD4+ cells demonstrated little
improvement in their capacity to secrete IL-2 (13% of control
3xPBS-pretreated T cells; p = 0.025). On the other
hand, IFN- production in these cultures in response to Ag
restimulation was now greatly augmented and equal to that of a normal
OVAp/CFA-primed helper cell population (Fig. 3C). This
result suggested that the development of clonal anergy in a T cell in
vivo cannot prevent its differentiation to a Th1-like phenotype, and
indicated that defective IL-2 production can coexist with intact
IFN- gene inducibility. In support of this second
hypothesis, the induction of clonal anergy with 3xOVAp infusions i.p.
in animals previously primed s.c. with OVAp in CFA (Fig. 1C)
also failed to interfere with the secretion of IFN- in vitro by the
helper cells, even though IL-2 gene inducibility in the
KJ1-26+CD4+ T cells was reduced 97% relative
to animals treated only with OVAp in CFA s.c.
(p = 0.014) (Fig. 3C).
Clonal anergy induction blocks T cell clonal expansion, bystander lymph node cell recruitment, and IL-2 production in vivo
Our experiments using TCR-Tg T cells and nu/nu adoptive
transfer mice confirmed that the repeated exposure of naive
CD4+ T cells to aqueous peptide Ag in vivo leads to a
down-regulation of IL-2 gene inducibility and reduced
proliferative responsiveness, as measured by in vitro thymidine
incorporation. This predicted that the induction of clonal anergy would
also result in T cells with reduced capacity for clonal expansion in
vivo. We investigated this possibility by pretreating TCR-Tg T cells in
the nu/nu mice either with 3xPBS i.p. or the aqueous
3xOVAp i.p. infusions, and then transferring these T cells into normal
naive BALB/c recipients. These adoptively transferred BALB/c mice were
subsequently immunized s.c. either with CFA alone (as a negative
control) or with DNP-OVA in CFA to test the capacity of the
OVA-reactive T cells to undergo a clonal expansion during the next 5
days. As illustrated in Figure 4A,
KJ1-26+CD4+ T cells exposed to the aqueous
peptide-Ag pretreatment (3xOVAp) did indeed demonstrate defective
clonal expansion and accumulation within the draining lymph nodes, with
only about one-quarter the increase in cell number as that seen with
normal T cells transferred from PBS-treated nu/nu mice.
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B). Thus, the induction of
clonal anergy in vivo in response to aqueous peptide-Ag exposure led to
a stable (for at least 5 days) impairment in clonal expansion by
Ag-specific responder T cells, as well as to a defective capacity to
recruit other (Ag-nonspecific) leukocytes into the draining lymph node.
Defective clonal expansion in this system may reflect either decreased
production of or responsiveness to a growth factor such as IL-2, or
increased death in the Ag responders. Tolerant T cells survived well in
BALB/c adoptive transfer recipients for 5 days in the absence of Ag
stimulation (Fig. 4A). Likewise, reexposure of
3xOVAp-treated nu/nu mice to s.c. OVAp/CFA stimulation
failed to result in a significant reduction in the number of anergic
KJ1-26+CD4+ T cells recovered (data not shown).
IL-2 secretion is likely to contribute to the development of the
autocrine growth response, and may also act as a cell-survival factor
by regulating the death-repressor proteins Bcl-2 and Bcl-xL
(34, 41). On the other hand, IL-2 production during an Ag response also
appears to prime T cells for activation-induced cell death (37);
therefore, reduced IL-2 production by anergic T cells may actually
protect them from apoptosis. To address these issues, we examined
intracytoplasmic IL-2 levels by flow cytometry in
KJ1-26+CD4+ T cells ex vivo following i.v.
infusion of OVAp. No differences in cell survival after Ag stimulation
were noted between the 3xPBS and 3xOVAp pretreatment groups (data not
shown). Instead, it was observed that pretreatment of the TCR-Tg T
cells in nu/nu mice with 3xOVAp i.p. virtually eliminated
their subsequent capacity to synthesize IL-2 upon i.v. OVAp rechallenge
in the BALB/c adoptive transfer recipient mice (Fig. 5, A and B).
Therefore, poor clonal expansion by anergic T cells in vivo correlated
best with defective IL-2 production.
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, C and D).
Likewise, T cells initially primed s.c. with OVAp/CFA and subsequently
treated with 3xOVAp infusions i.p. to induce tolerance within the
nu/nu mice, failed to accumulate intracytoplasmic IL-2 when
adoptively transferred to normal BALB/c mice and rechallenged with OVAp
(data not shown). Taken together with the observed deficiency in clonal
expansion, these IL-2 data suggested that peptide-induced clonal anergy
leaves both naive and effector T cells defective for Ag-induced
autocrine growth in vivo. Furthermore, they indicated that exposure of
tolerant T cells to peptide Ag under immunogenic conditions neither
reverses the anergy nor allows for a significant outgrowth of normal
cells that may have escaped tolerance. Clonal anergy induction in vivo fails to regulate the delivery of helper signals for IgG2a production
In the next series of experiments, this nu/nu mouse
adoptive transfer system was used to investigate the effects of clonal
anergy induction on the capacity of a population of TCR-Tg T cells to
perform Th1-like helper functions in vivo. Normal BALB/c mice were
again used as final adoptive transfer recipents of TCR-Tg cells
initially parked in nu/nu mice, and these animals were then
tested for Th cell activity in the production of DNP-specific
anti-hapten IgG2a Abs. An anti-DNP response was utilized
because DNP-specific B cells are present in naive BALB/c animals at
relatively high frequency; therefore, no priming of the B cell
repertoire was necessary to elicit anti-hapten IgG2a responses when
adequate T cell help was available (42). The s.c. challenge of normal
BALB/c animals with the hapten-carrier conjugate DNP-OVA in CFA was
observed to elicit significant serum titers of anti-DNP IgG2a only
when these mice had first been primed s.c. with OVAp in IFA to increase
the frequency of OVA-reactive T cells (Fig. 6A). Thus, anti-DNP IgG2a
production in these BALB/c mice demonstrated the expected requirement
for a primed and clonally expanded Th cell population, presumably of a
Th1-like phenotype (43, 44).
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A). The ability of
these naive T cells to help in the production of anti-DNP IgG2a in
vivo apparently reflected their capacity to differentiate into potent
Th1-like effector cells during the 10- to 12-day course of the
DNP-OVA/CFA priming, since this population of cells displayed an
undifferentiated lymphokine production profile in vitro and
demonstrated no capacity to act as helpers in a 48-h DTH reaction in
vivo (Fig. 3C, and data not shown). Remarkably,
KJ1-26+CD4+ T cells recovered from tolerant
nu/nu mice (3xOVAp i.p. pretreated) were at least as potent
as the control naive cells in providing help for IgG2a anti-DNP Ab
(Fig. 6, A and B). In fact, immunization of
BALB/c adoptive transfer recipients with a low (10 µg) dose of
DNP-OVA in CFA revealed that the tolerant 3xOVAp-pretreated T cells
could be a more potent helper population than either
3xPBS-pretreated naive T cells, or Ag-experienced TCR-Tg T
cells that had initially been primed in the nu/nu mice with
OVAp in CFA (Fig. 6C). Therefore, under controlled
conditions in which T cells were shown to be anergic at the level of
the IL-2 gene, this peptide-induced tolerance mechanism
failed to eliminate the delivery of Th signals for the production of
IgG2a in vivo. Induction of clonal anergy in naive T cells does not prevent later Ag-induced differentiation to a Th1 phenotype capable of directing a DTH reaction
To determine whether this failure of in vivo tolerance
induction to regulate T cell participation in the production of the
anti-DNP IgG2a represents a general resistance of Th1 effector
functions to the effects of clonal anergy, T cells were also examined
for their ability to direct a DTH response after anergy induction. As
expected, naive KJ1-26+CD4+ T cells recovered
from 3xPBS i.p.-pretreated control nu/nu mice and
transferred into normal BALB/c recipients were incapable of inducing
DTH in response to i.d. ear injection with OVA (Fig. 7). Exposure of the T cells to 3xOVAp
i.p. administration in the nu/nu mice to induce tolerance
also failed to result in the development of a capacity to mediate DTH.
This was consistent with the finding that these cells also demonstrated
little ability to secrete IFN- (Fig. 3C). In contrast,
s.c. immunization with OVAp in CFA induced the generation of a potent
Th1 effector cell population in normal untransferred BALB/c mice that
was capable of eliciting a DTH reaction to OVA (data not shown).
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B). As
expected, positive control 3xPBS-pretreated
KJ1-26+CD4+ T cells recovered on day 40 from
these OVAp/CFA s.c. immunized nu/nu mice were found to be
fully capable of performing a DTH response within 24 h after
adoptive transfer into naive BALB/c mice and subsequent ear challenge
with OVA (Fig. 7). T cells pretreated with 3xOVAp i.p. also
demonstrated equally strong dose-dependent DTH reactivity if primed
s.c. with OVAp/CFA before adoptive transfer into the BALB/c mice (Fig. 7), consistent with their acquired ability to secrete IFN- (Fig. 3C), and despite a persistent inability to produce IL-2
(Fig. 5, C and D). These same T cells were also
potent Th cells for the production of anti-DNP IgG2a (Fig. 6C). The results indicate that exposure of naive T cells to
aqueous peptide Ag in the absence of infection or adjuvant is not
sufficient to cause their differentiation into DTH effectors, and
instead induces clonal anergy at the level of the IL-2 gene.
Furthermore, these experiments suggest that the development of
peptide-induced tolerance in vivo cannot interfere with the capacity of
anergic T cells to subsequently develop a Th1-like effector phenotype
if exposed to Ag in adjuvant, even though they never regain the ability
to secrete IL-2. The induction of clonal anergy in Th1-like effector cells fails to regulate their capacity to mediate a DTH reaction
The finding that differentiation to an IgG2a- and DTH-helper
phenotype could occur even in tolerant T cells implies that clonal
anergy induction in vivo does not regulate Th1 effector function per
se. To formally test this, KJ1-26+CD4+ T cells
were first primed s.c. in nu/nu mice with OVAp in CFA to
induce the differentiation of Th1 effector cells. Animals were then
either treated beginning on day 25 with 3xPBS injections alone, or
given 3xOVAp injections i.p. to induce T cell tolerance
(Fig. 1C). Indeed, the capacity of OVAp/CFA-primed
KJ1-26+CD4+ T cells to promote the development
of OVA-dependent DTH in BALB/c recipients was not found to be adversely
affected by three infusions of OVAp i.p. before the final adoptive
transfer (Fig. 7). Likewise, these same primed/tolerant T cells
remained fully competent to mediate an anti-DNP IgG2a response
(Fig. 6C), despite the fact that they had lost the capacity
to accumulate intracytoplasmic IL-2 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous reports have indicated previously that a systemic exposure of the mature peripheral immune system to deaggregated forms of a protein Ag or simple peptides, in the absence of adjuvant or infection, fails to productively engage the immune system and instead reduces the individual’s responsiveness to that Ag (45, 46, 47, 48, 49). Recently, several studies have taken advantage of TCR-Tg technology to probe the nature of this Ag unresponsiveness under conditions in which Ag responders can be carefully monitored. In one of these experiments, i.v. exposure of CD8+LCMV glycoprotein-specific T cells to a peptide derived from that Ag led to a tolerance as a result of the elimination of the majority of Ag responders and anergy induction within the survivors (36). Similarly, CD4+OVA-reactive T cells underwent an abortive clonal expansion following i.p. infusion of peptide Ag, and surviving cells were shown to have an IL-2 production defect in vitro, consistent with the induction of clonal anergy (26). This combination of activation-induced cell death and clonal anergy induction would be expected to hinder T cell clonal expansion in response to a later exposure to the Ag, and both most likely contribute to peripheral immune tolerance.
We speculate that naive T cells responding to aqueous peptide Ag
undergo an abortive differentiation as a result of TCR stimulation in
the presence of a relatively low level of B7 molecule expression. While
insufficient to induce T cell proliferation or the development of
DTH-helper activity, perhaps because of the counter-regulatory
influences of simultaneous CTLA-4 ligation, this activation event does
appear to leave the T cell susceptible to clonal anergy (39, 50). Upon
further TCR occupancy in the absence of infection or adjuvant, the
capacity of the T cells to produce IL-2 is progressively lost (Fig. 8).
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Peptide-induced anergy, if similar to in vitro models of Ag unresponsiveness, might be expected to inhibit the clonal expansion of Ag-reactive cells in vivo because of a block in signal transduction between the TCR and the IL-2 gene. Peptide-pretreated KJ1-26+CD4+ T cells did, in fact, demonstrate only poor clonal expansion in vivo in response to Ag rechallenge, following adoptive transfer into normal BALB/c recipients. Additionally, these tolerant T cells were incapable of recruiting other Ag-nonspecific leukocytes into the draining lymph node during the course of the Ag response. Consistent with these data, the in vivo induction of IL-2 synthesis within the tolerant T cells was found to be markedly defective. These results argue conclusively against a role for either peripheral elimination or suppression in this TCR-Tg immune tolerance model. While it is currently uncertain which factors are most responsible for the development of a strong clonal expansion response or recruitment of bystander cells in vivo, the results of these experiments suggest that tolerance develops because the elaboration of at least one critical factor by the responding T cells is blocked as a consequence of the induction of clonal anergy.
Does the induction of clonal anergy in vivo result in a complete
functional inactivation of the T cell? The evidence suggests otherwise.
First, peptide-pretreated T cells appeared fully competent to
participate in the production of anti-hapten IgG2a after challenge
with the hapten-carrier protein in adjuvant. Cognate help for IgG2a
isotype switching is thought to rely both on the delivery of the CD40
ligand as well as on the production of IFN- by Th cells responding
to carrier-derived peptide Ag presented on the surface of the
hapten-specific B cells (52, 53). Therefore, the production of high
titer anti-DNP IgG2a in animals transferred with high numbers of
peptide Ag-pretreated TCR-Tg cells implies that these T cells could be
induced to differentiate to a Th1-like phenotype upon exposure to
DNP-OVA in adjuvant, and provide help to DNP-specific B cells during
the course of the response. This helper activity was not simply the
result of an outgrowth of T cells that had escaped tolerance induction,
nor did it result from a reversal of the anergy: after transfer into
the BALB/c recipients, 3xOVAp i.p.-pretreated T cells maintained an
anergic phenotype based on their weak ability to undergo a clonal
expansion. Furthermore, aqueous peptide Ag-pretreated T cells
intentionally primed s.c. with OVAp in CFA within the nu/nu
mice never regained a normal ability to produce IL-2 in vivo.
Our own in vitro studies have also recently addressed this issue
and determined that under conditions in which T cell numbers were held
constant, the delivery of Th signals for the induction of B cell
polyclonal Ab production was only partially sensitive to clonal anergy
induction (25). The secretion of IL-4 and IL-5 by anergic T cells
responding to Ag on B cells was found to be moderately depressed in
these long-term tissue culture experiments. Nevertheless, CD40 ligand
was found to be inducible and fully functional after activation of
anergic T cells, and IFN- could still be secreted in a normal
fashion, consistent with these in vivo results.
Additional evidence that anergic T cells remain functionally responsive
to Ag stimulation includes the finding that aqueous
peptide-Ag-pretreated TCR-Tg T cells also developed the capacity to
direct a DTH response following priming s.c. with Ag in adjuvant within
the nu/nu mice. Neither naive cells nor T cells exposed to
the aqueous peptide Ag pretreatment alone had such a helper capacity.
The failure of 3xOVAp i.p.-pretreated T cells to directly help in the
development of a DTH response is consistent with their inability to
secrete IFN-, and may reflect insufficient IL-12 production by APC
or the effects of CTLA-4 ligation during the course of the clonal
anergy induction (40). Regardless, our data provide no support for the
notion that clonal anergy induction diverts differentiation away from
the Th1 phenotype, as anergic T cells responded to Ag stimulation in
the presence of adjuvant-induced costimulatory signals with the
development of Th1 helper activity.
Finally, these experiments demonstrate that clonal anergy at the level of the IL-2 gene can coexist in vivo with intact Th1 effector function. Specifically, we assessed the capacity of primed Th1-like memory cells to be made tolerant to Ag upon repeated i.p. exposure to the OVAp. It was shown that these Th1 cells lost the capacity to synthesize IL-2 following the 3xOVAp i.p. injections, consistent with the development of clonal anergy. Nevertheless, such tolerant T cells retained their capacity to immediately induce a DTH reaction, as well as participate in the production of anti-hapten IgG2a. Thus, primed effector cells are indeed susceptible to anergy induction at the level of the IL-2 gene, but this clonal anergy tolerance mechanism is incapable of interfering with the activation of T cells for the delivery of Th1-dependent activities important to the development of DTH and IgG2a Ab responses.
These findings suggest that the most important role for clonal anergy in the development of peripheral T cell tolerance may be the regulation of autocrine growth-factor production and clonal expansion, with the resultant maintenance of potentially deleterious T cells at low frequency. In pathologic states in which autoreactive T cells presumably reach high frequency, this mechanism may no longer have the inherent capacity to control their actions. Similarly, the design of future therapies for autoimmune disease based on this tolerance mechanism should anticipate that clonal anergy may not inhibit the delivery of all potentially dangerous Th1 activities.
It should be noted that Gilbert et al. (54) previously determined that the delivery of help for IgG production in vitro is sensitive to the induction of T cell clonal anergy. Likewise Finck et al. (55) demonstrated a capacity of the B7/CD28 antagonist CTLA-4Ig to interrupt the production of pathogenic autoantibodies and improve survival in (NZB/NZW)F1 lupus-prone mice, a therapy perhaps predicted to promote clonal anergy induction in the self Ag-specific Th cells. Both experiments, however, lacked the power to establish whether the development of tolerance stemmed from a reduced expansion of critical Ag-specific helper cells or from an ability of the tolerance mechanism to interrupt the delivery of Th activities.
Finally, Marusic and Tonegawa (56) recently showed that a single i.p.
injection of peptide Ag in IFA induced clonal anergy within a large
population of TCR-Tg T cells specific for myelin basic protein. In
their experiments, anergy was associated with a defect in IFN-
production, and was in fact sufficient to protect the mice from
experimental autoimmune encephalomyelitis. This apparent inconsistency
with the findings of our system may stem from their use of IFA in the
single peptide Ag infusion, since systemic Ag persists considerably
longer after i.p. injection with IFA than with PBS (M.K.J., unpublished
observation). The continuous presence of nonimmunogenic peptide most
likely desensitizes T cells to subsequent Ag stimulation, and may
promote the appearance of a more profound functional anergy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daniel L. Mueller, University of Minnesota Medical School, Box 334 FUMC, Minneapolis, MN 55455.
3 Abbreviations used in this paper: SEB, staphylococcal enterotoxin B; DTH, delayed-type hypersensitivity; i.d., intradermal; LCMV, lymphocytic choriomeningitis virus; OVAp, ovalbumin peptide 323–339; Tg, transgenic; IFA, incomplete Freund’s adjuvant.
Received for publication February 5, 1998. Accepted for publication April 30, 1998.
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References |
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OVAp/CFA s.c.) (see Fig. 1
