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*
Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA 19104; and
Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, IL, 60637
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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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T cells that receive TCR-derived signals in the absence of
costimulatory signals enter a hyporesponsive state, termed anergy
(1, 2, 3). Anergic T cells fail to proliferate or secrete
IL-2 upon subsequent Ag receptor engagement, even in the presence of
costimulation, and have been reported to have defects in the Ras and
mitogen-activated protein kinase pathways (4, 5, 6), reduced
Ca2+ flux (7, 8, 9, 10), and altered
tyrosine kinase activity (7, 11). These signaling
deficiencies appear to lead to reduced NF-
B and AP-1 transcriptional
activities (12, 13, 14), and overexpression of c-Fos has been
found to prevent anergy induction by staphylococcal enterotoxin B or
Con A (15).
The TCR CTLA-4 has been proposed as a critical molecule for the initiation of anergy. CTLA-4, like CD28, binds to members of the B7 family but has an inhibitory effect on the T cell (16, 17), making CTLA-4 a logical candidate for initiating anergy. Furthermore, administration of anti-CTLA-4 Abs during anergy induction protocols appears to block the induction of Ag-specific tolerance in several in vivo systems (18, 19, 20, 21, 22), and CTLA-4-deficient CD4+ T cells were recently reported to be resistant to in vivo anergy induction (23).
Although a model with CTLA-4 as the anergy initiator molecule is an attractive one, there is a significant body of conflicting data. Anergy is not blocked by anti-CTLA-4 Abs in some in vivo systems (24, 25), despite close parallels to those in which CTLA-4 appears critical. Furthermore, B7.1/2-blocking agents, which prevent interaction with both CD28 and CTLA-4, can be used to induce anergy in vitro (26, 27, 28, 29) and long-term allograft tolerance in vivo (30, 31). Because interaction with B7 is required for CTLA-4 to signal, it is difficult to reconcile the model with these data. Furthermore, CTLA-4-deficient CD8+ T cells can be anergized in vitro (32). These apparent inconsistencies have led to the suggestion that the mechanisms of anergy induction may differ among systems, with details such as route of stimulus administration (in vitro/in vivo, i.p./i.v./oral/intranasal), differentiation state of the cells (naive/memory, primary cells/clones), and T cell subset (CD4+/CD8+, Th1/Th2) influencing the requirements. We therefore undertook a direct examination of the role of CTLA-4 in the initiation of T cell anergy in vivo in TCR-transgenic (Tg)3 mice, providing a very homogeneous population of naive T cells and control over many of the variables that may influence tolerance induction. Using CD8+ TCR-Tg, recombination-activating gene (RAG)2-deficient, CTLA-4-deficient T cells, we found that CTLA-4 signaling is unnecessary for in vivo anergy induction of naive CD8+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice (4–8 wk old) were purchased from The Jackson Laboratory (Bar Harbor, ME). 2C TCR-Tg (33)/RAG2-/-/CTLA-4+/- mice were kindly provided by Dr. T. Gajewski (University of Chicago, Chicago, IL). All mice were maintained in the University of Pennsylvania Animal Barrier Facilities (Philadelphia, PA). Animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (Bethesda, MD).
Abs and reagents
FITC-labeled hamster IgG, anti-Thy1.2, anti-CD3, anti-TCR, and anti-CD45RB, and PE-labeled hamster IgG, anti-Thy1.2, anti-CD44, anti-CD25, and anti-Fas were purchased from BD PharMingen (San Diego, CA). The H-2Kb-restricted 2C TCR-reactive peptide SIYRYYGL was purchased from Multiple Peptide Systems (San Diego, CA). Recombinant human IL-2 was purchased from Boehringer Mannheim (Mannheim, Germany).
Cell culture
All cells were maintained in DMEM (Life Technologies, Grand Island, NY) supplemented with glutamine, penicillin/streptavidin, HEPES buffer, MEM nonessential amino acids, 2-ME, and 10% FCS (Life Technologies) at 37°C in a 7% CO2 atmosphere.
T cell purification
Murine T cells were isolated from spleen using the StemSep negative-selection system (StemCell Technologies, Vancouver, British Columbia, Canada) following the manufacturer’s instructions, as previously described (32). The nonretained fraction typically contained >95% Thy1+ cells, as determined by flow cytometry. For separation of CD4high and CD4low cells, purified T cells were passed through the StemSep system a second time, using biotinylated anti-CD44 mAb (StemCell Technologies) as the negatively selecting agent, and both flow-through (CD44low) and retained (CD44high) fractions were collected.
In vivo anergy induction
2C TCR-Tg T cells were anergized in vivo by multiple peptide injections, modifying the protocol of Dubois et al. (8). Mice were injected i.p. one to three times, at 4-day intervals, with either 20–25 nM of peptide in PBS or PBS alone. Spleens were harvested 7 days after the final injection. T cells were purified as described above and tested for proliferation and cytokine secretion upon stimulation in vitro with irradiated C57BL/6 splenocytes plus titrated doses of peptide.
Cytokine and proliferation assays
IL-2, IFN-
, TGF
1, and IL-10 levels in 1-day restimulation
culture supernatants were measured by sandwich ELISA. Primary and
biotinylated secondary anti-cytokine Abs and recombinant cytokine
standards were purchased from BD PharMingen and used at the
concentrations recommended by the manufacturer. Alkaline
phosphatase-conjugated avidin was purchased from Jackson ImmunoResearch
Laboratories (West Grove, PA) and used at a 1/3000 dilution.
Colorimetric alkaline phosphatase substrate was purchased from
Sigma-Aldrich (St. Louis, MO) and used at 1 mg/ml in 10%
diethanolamine buffer, and quantitation was performed on a SpectraMax
190 spectrophotometer (Molecular Devices, Sunnyvale, CA). Data were
analyzed using SoftMax Pro software (Molecular Devices) by comparison
with a standard curve generated using recombinant cytokines at known
concentrations. Proliferation after 3 days of restimulation was
determined by [3H]thymidine incorporation.
Cells were pulsed for 6–8 h with 1 µCi/well
[3H]thymidine (ICN Pharmaceuticals, Costa Mesa,
CA), transferred to glass-fiber filters with a 96-well cell harvester
(Tomtec, Hamden, CT), and analyzed by liquid scintillation using a 1450
Microbeta scintillation counter (Wallac, Turku, Finland). Data points
for all analyses are presented as the mean of triplicate wells.
Flow cytometry
Levels of CD3, TCR, CD25, CD44, CD45RB, and Fas were determined
on T cells by two-color flow cytometry. Following harvest, cells were
washed once in flow cytometry buffer (1% BSA and 0.01% sodium azide
in PBS, pH 7.4) and incubated for 30 min on ice in 100 µl of flow
cytometry buffer containing FITC-conjugated (anti-CD3,
anti-TCR, anti-CD45RB) or PE-conjugated (anti-CD25,
anti-CD44, anti-Fas) mAb; FITC- and PE-conjugated hamster IgG
were used as negative controls. Cells were simultaneously stained with
PE- or FITC-conjugated anti-Thy1.2 to assess T cell purity. Cells
were resuspended in flow cytometry buffer and analyzed on either a
FACSCaliber or an LSR flow cytometer (BD Biosciences, Mountain View,
CA). Forward and side scatter gates were used to exclude dead cells.
Data from 
104 live cells were analyzed using
CellQuest software (BD Biosciences). For cell cycle analysis,
5 x 105 cells were fixed in 25% PBS/75%
ethanol overnight at 4°C. The fixed cells were resuspended in
propidium iodide staining solution (3.8 mM sodium citrate, 0.125 mg/ml
RNase A, and 0.01 mg/ml propidium iodide), incubated on ice for 30 min,
and then analyzed by flow cytometry as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine whether anergy could be induced in vivo in the absence
of CTLA-4, we developed an anergy induction system using mice Tg for
the 2C TCR on a RAG2-deficient background. Anergy was induced by
multiple i.p. injections of the 2C agonist peptide SIYRYYGL. Injecting
CTLA-4-positive, 2C TCR-Tg, RAG-deficient mice two or three times with
2C agonist peptide led to a significant depletion of splenic T cells,
ranging from 40 to 80% reduction in T cell recovery relative to
saline-injected controls (data not shown). Although the peptide-treated
T cells had a small (resting) size (Fig. 1
A) and showed no increase in
CD25 levels (Fig. 1B), they expressed increased CD44 (Fig. 1C) and decreased CD45RB (data not shown), a pattern
generally associated with Ag-experienced T cells. As previously
reported (8), TCR levels on T cells from peptide-injected
mice were consistently at least 80% of those on control T cells (Fig. 1D).
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A) and
IL-2 secretion (Fig. 2B). We consistently saw at least 90%
inhibition of both proliferation and IL-2 production with this
protocol. We were unable to detect IL-10 in any culture supernatants,
and TGF- production was comparable for T cells from control and
peptide-injected mice (data not shown). Viable T cells were recovered
after 2 days of in vitro stimulation and analyzed for cell cycle
distribution. Peptide-treated cells were arrested in the
G1 phase of the cell cycle, whereas the majority
of control T cells were in S or G2/M phase at
this point (Fig. 2C). Thus, injection of soluble peptide
induces a state of proliferative unresponsiveness in 2C TCR-Tg T
cells.
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C and data not shown). The presence of a sizable fraction
of nonresponsive, but apparently naive, cells suggested that anergic
cells might be suppressing the responses of
CD44low T cells (34, 35, 36, 37). To test
this possibility, T cells from peptide-injected mice were separated
into CD44high and CD44low
fractions (Fig. 3A) before
restimulation. Although proliferation of the
CD44low T cells was somewhat higher than that of
the CD44high T cells, responses in both fractions
were strongly inhibited (Fig. 3B), indicating that the
CD44low cells were independently anergic and were
not being suppressed by CD44high cells.
CD44low cells separated from control
(saline-injected) T cells responded identically to unseparated cells,
showing that the separation procedure itself did not inhibit subsequent
proliferation. This suggests that a fraction of T cells may have
encountered injected Ag and been rendered nonresponsive, but failed to
up-regulate CD44. This is supported by an analysis of CD25 and CD44
levels at various times after Ag injection. T cells from saline-treated
mice were CD25- (<1%
CD25+) and CD44low (<10%
CD44high). Within 24 h of peptide injection,
92% of splenic T cells were found to be
CD25+, indicating exposure to Ag, whereas the
CD44high fraction was 33% at this time and
reached a maximum of 62% at 48 h postinjection (data not
shown).
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We next compared anergy induction in 2C
TCR-Tg/RAG2-/-/CTLA-4+/+
and CTLA-4-/- mice. Mice were injected with
peptide as described above, and then T cells were isolated and
stimulated in vitro. Peptide-induced depletion of T cells from spleen
was comparable in CTLA-4+/+ and
CTLA-4-/- mice (41% depletion in
CTLA4+/+ and 56% depletion in
CTLA4-/- in one representative experiment). As
shown in Fig. 4, proliferation and IL-2 secretion were inhibited to
comparable levels in CTLA-4+ and
CTLA-4- T cells. As with CTLA-4-expressing
cells, TCR levels on peptide-treated CTLA-4-deficient T cells were at
least 80% of control TCR levels (Fig. 4C). CD25, CD44, and
CD45RB expression levels on CTLA-4+ and
CTLA-4- cells were also comparable (data not
shown).
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D), indicating that the cells were not being killed upon in
vitro stimulation. We also tested for anergy induction after only one
injection of peptide, as single-injection protocols were used in
several of the models implicating CTLA-4 (18, 19, 23) and
might be less likely to sensitize cells to apoptosis. A single
injection of peptide resulted in CD44 expression comparable to multiple
injections (data not shown). Although the level of inhibition was
somewhat more variable than with multiple injections, a one-time
peptide injection also significantly reduced T cell responsiveness and
affected CTLA-4+ and
CTLA-4- cells comparably (Fig. 5). Thus, naive
CD8+ T cells can be rendered anergic in vivo even
in the absence of CTLA-4 expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2C TCR-Tg/RAG2-deficient T cells isolated from mice multiply injected with soluble agonist peptide showed dramatically reduced proliferation and IL-2 secretion in response to in vitro stimulation and arrested in the G0/G1 phase of the cell cycle, indicating that they were rendered anergic, as has been reported for mice Tg for the F5 TCR (8). Surprisingly, we also found that a single peptide injection was sufficient to induce a high degree of unresponsiveness to restimulation. This is in contrast to several other systems, in which a single dose of soluble Ag is not able to induce anergy in intact TCR-Tg mice (8, 38, 39). It is unclear whether this is unique to the 2C TCR, or whether the peptide we used is simply particularly tolerogenic.
Using the 2C Tg/RAG2-deficient mice, we compared in vivo anergy induction in CTLA-4-expressing and CTLA-4-deficient T cells. With both single-injection and multiple-injection protocols, CTLA-4-deficient T cells were anergized as efficiently as cells expressing CTLA-4. Thus, there appears to be no difference between in vitro and in vivo anergy induction for CD8+ TCR-Tg T cells with regard to CTLA-4 requirement. However, these in vitro and in vivo results conflict with the recent report of Greenwald et al. (23). Using a CD4+ TCR-Tg system, DO11.10, Greenwald et al. found that i.p. injection of soluble Ag tolerized CTLA-4-positive, but not CTLA-4-negative, naive T cells. Similar results were obtained by Perez et al. (18), who showed that a blocking anti-CTLA-4 Ab could prevent anergy induction in the same DO11.10 Tg system. Understanding the difference between the 2C and DO11.10 models will be important for determining the role that CTLA-4 plays in regulating peripheral tolerance.
One distinction between the two models involves the details of the anergy induction protocols. The DO11.10 system uses adoptive transfer of a relatively small number of Ag-specific T cells into non-Tg mice, whereas the 2C system involves Ag injection into intact Tg mice. We would predict that this would make it more difficult to induce anergy in the 2C system due to the higher number of Ag-specific T cells. However, it is possible that the monoclonality of the 2C system leads to a massive immune response to peptide injection, causing systemic effects that result in anergy as a secondary consequence independent of CTLA-4 expression. We do not favor this explanation, as the strong systemic response induced by bacterial superantigen does not bypass the CTLA-4 requirement for anergy induction by staphylococcal enterotoxin B (19); however, the spectrum of cytokines induced by soluble peptide may differ from that induced by superantigen.
A second difference between the systems is that the DO11.10 TCR is MHC class II-restricted, giving rise to CD4+ T cells, while the 2C TCR is MHC class I-restricted, giving rise to CD8+ T cells. It has been found that there is a skewing toward preferential activation of CD4+ T cells in (non-TCR-Tg) CTLA-4-deficient mice, and that both the activated T cell phenotype and lymphoproliferative disease in these mice are dependent on CD4+, but not CD8+ T cells (40). This suggests that the inhibitory function of CTLA-4 plays a greater role in the regulation of CD4+ cells than that of CD8+ cells. Thus, induction of anergy in CD8+ T cells may depend on CTLA-4-independent signals, while CD4+ T cells require CTLA-4. Viewed in a larger context, this may reflect the fact that a wide variety of B7-negative cells in the body are potential class I MHC APC, allowing tolerization of CD8+ T cells without the need to cross-present tissue-specific Ags on "professional," B7-expressing APC.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Craig B. Thompson, Abramson Family Cancer Research Institute, University of Pennsylvania, 421 Curie Boulevard, Room 450, Philadelphia, PA 19104. E-mail address: drt{at}mail.med.upenn.edu
3 Abbreviations used in this paper: Tg, transgenic; RAG, recombination-activating gene.
Received for publication May 10, 2001. Accepted for publication August 24, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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