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*
Laboratory of Immunology and
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, and
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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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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The two-signal theory of T cell activation, originally postulated by Bretscher and Cohn (5) and expanded by other investigators (6, 7), states that interaction of antigenic peptide/MHC complex with the TCR delivers signal 1 to the T cell. A second, Ag-independent costimulatory signal delivered by ligands on the surface of APCs to the T cell is required for the up-regulation of IL-2R chains, cytokine transcription, Bcl-xL expression, differentiation, proliferation, and effector function. B7.1 (CD80) and B7.2 (CD86) on the APC are the major and most extensively studied costimulatory molecules. Their interaction with the counterreceptor CD28 on the T cell constitutes a costimulatory pathway that is essential for facilitating optimal T cell activation (reviewed in Ref. 8).
Studies with murine and human T cell clones have indicated that antigenic stimulation in the absence of costimulatory signaling through B7/CD28 causes the T cells to enter a state of long-term unresponsiveness, known as anergy (9, 10, 11, 12, 13). Anergy is connected to a failure to induce IL-2 gene transcription and may be reversed by exogenous IL-2 (14, 15, 16, 17 ; reviewed in Ref. 8). T cells also express a second receptor for B7 ligands, CTLA-4 (CD152). Studies with anti-CTLA4 Ab and CTLA-4-deficient mice have led to the conclusion that this molecule negatively regulates T cell activation and is central to immune homeostasis (18, 19, 20, 21, 22).
In view of these data and because it has been reported that blockade of costimulation can ameliorate autoimmunity and inhibit epitope spreading in some animal models (23, 24, 25, 26, 27, 28 ; reviewed in Ref. 8), we wanted to investigate whether blockade of the B7 pathway could protect mice against EAU. We show here that administration of anti-B7.1 and anti-B7.2 or of anti-CD28 mAb during a primary Ag challenge inhibited disease and suppressed associated immunologic responses. However, after the Abs had been cleared from the system, Ag rechallenge of the protected animals precipitated severe EAU accompanied by elevated cellular responses equivalent to those of rechallenged control mice and higher than those of control mice receiving only a single immunization. Thus, although the costimulatory blockade was effective in preventing the development of EAU, it did not result in long-term tolerance, and it appears to have permitted generation of immunological memory.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 10-wk-old B10.A mice were supplied by The Jackson Laboratory (Bar Harbor, ME) and the Frederick Cancer Research Facility (Frederick, MD). For experiments requiring adult-thymectomized mice, surgery was performed on 7-wk-old animals, which were then allowed to recover for 14 days before being used in an experiment. Animals were housed under specific pathogen-free conditions and were given water and chow ad libitum. The care and use of the animals were in compliance with institutional guidelines.
Reagents
Bovine interphotoreceptor retinoid-binding protein (IRBP) was purified from retinas by Con A-Sepharose affinity chromatography and HPLC (29). Pertussis toxin and CFA were purchased from Sigma (St. Louis, MO). Mycobacterium tuberculosis strain H37RA was purchased from Difco (Detroit, MI). Anti-B7.1 mAb (hybridoma 16-10A1) and anti-B7.2 mAb (hybridoma GL-1) were generated in hollow fiber Ab production systems and purified over a protein G-Sepharose column (Pharmacia, Piscataway, NJ). Anti-CD28 hybridoma PV.1 was a gift from Carl June (University of Pennsylvania, Philadelphia, PA). mAb from the cells were produced from ascites and were purified by ion exchange chromatography. Hamster Ig from Jackson ImmunoResearch Laboratories (West Grove, PA) was used as the control Ab for anti-B7.1. Control rat mAb (hybridoma 111/10), used as the control for anti-B7.2, was produced on a hollow fiber production system and purified by a protein G-Sepharose column (30).
Ab treatment
All Abs were administered i.p. on days -1, 0, 1, 3, 7, 10, and 13. For B7-blocking experiments, mice were treated with 150 µg of anti-B7.1 and/or 150 µg of anti-B7.2. Control groups received identical doses of hamster or rat Ig controls. For CD28 blocking, mice were treated with 0.5 mg of PV.1.
EAU induction and scoring
Mice were immunized s.c. with 50 µg of IRBP emulsified (1/1, v/v) with CFA that had been supplemented with M. tuberculosis strain H37RA (Difco) to a final concentration of 2.5 mg/ml. Concurrent with immunization, 0.5 µg of pertussis toxin was injected i.p. For single-challenge experiments, mice were immunized with 0.2 ml of emulsion split among the base of tail and both thighs. For Ag rechallenge experiments, mice were immunized with 50 µg of IRBP in 0.1 ml at the base of the tail for the primary challenge and were reimmunized with 50 µg of IRBP in 0.1 ml split between both thighs.
To assess the severity of eye disease during the course of an experiment, a fundoscopic examination was performed. For this procedure, mice were anesthetized briefly, and their pupils were dilated using AK-dilate (Akorn, Buffalo Grove, IL) and 1% Mydriacyl (Alcon, Humacao, Puerto Rico) ophthalmic solutions. The fundus was observed using a stereoscopic microscope. Disease severity was scored on a scale of 0 (no disease) to 4 (maximum disease) depending on the degree of inflammation and retinal damage using established criteria (31). At the end of an experiment, eyes were enucleated, fixed for 1 h in 4% phosphate-buffered glutaraldehyde, and transferred into 10% phosphate-buffered formaldehyde. Fixed and dehydrated tissue was embedded in methacrylate, and 4- to 6-µm sections, cut through the pupillary-optic nerve plane, were stained with standard hematoxylin and eosin. Quantitation of disease was performed in a masked fashion by one of us (C.-C.C., who is an ophthalmic pathologist). Eyes were assigned a score ranging from 0 to 4 depending on the extent of inflammation and tissue damage as described previously (31). Briefly, the minimal criterion to score an eye as positive by histopathology was inflammatory cell infiltration of the ciliary body, choroid, vitreous, or retina (EAU grade 0.5). Progressively higher grades were assigned for the presence of discrete lesions in the tissue, such as vasculitis, granuloma formation, retinal folding and/or detachment, photoreceptor damage, etc.
Lymphocyte proliferation
Draining lymph nodes (inguinals and iliacs) and spleens were
collected at the termination of an experiment (day 21, 49, or 54) and
were pooled within each group. Triplicate 0.2-ml cultures were
stimulated with 50 µg/ml IRBP in 96-well round-bottom plates at a
concentration of 5 x 105 cells/well in RPMI
1640 (BioWhittaker, Walkersville, MD) supplemented with 2-ME,
glutamine, nonessential amino acids, sodium pyruvate, and antibiotics
as previously described (1), 1% fresh-frozen normal mouse
serum, and 20 mg/ml 
-methylmannopyranoside (Sigma; to neutralize any
possible traces of Con A, which is used in the initial stages of IRBP
purification). In one experiment 25 U/ml of IL-2 (Proleukin; Midwest
Medical, Bridgeton, MD) was added to the cultures. The cultures were
incubated for 60 h and were pulsed with
[3H]thymidine (1.0 µCi/10 µl/well) for the
last 18 h. The data are presented as stimulation indexes (average
counts per minute in cultures with stimulus divided by those in control
cultures without stimulus) or 
cpm ( cpm = mean cpm in
cultures with stimulus - mean cpm in control cultures without
stimulus).
Cytokine assays
Lymph node cells and spleens prepared as described above were
cultured in 96-well flat-bottom plates at a concentration of 1 x
106 cells/well in 0.2 ml of medium. Cells were
stimulated with 50 µg/ml IRBP. ELISA for IL-2 was performed on 24-h
supernatants using Ab pairs from PharMingen (La Jolla, CA). ELISA
minikits from Endogen (Boston, MA) were used to measure IFN-
, IL-4,
and IL-10 levels in 48-h supernatants.
Assay for IRBP-specific Ab production
Serum levels of anti-IRBP IgG1 and IgG2a subclasses were determined by ELISA as described previously for another Ag (32). Briefly, 96-well microtiter plates (Costar, Corning, NY) were coated with IRBP (1 µg/ml). After blocking the plates with BSA (Sigma) and incubating overnight with the serum samples, plates were developed using HRP-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL) or rat anti-mouse IgG2a (PharMingen). The concentration of anti-IRBP Ab was estimated using standard curves generated by coating wells with anti-Ig Ab against the appropriate isotype (PharMingen) and adding polyclonal mouse IgG1 or IgG2a standards (PharMingen).
Statistical analysis
Each mouse (average of both eyes) was treated as one statistical event. Analysis of EAU scores was performed using Snedecor and Cochran’s test (33) for linear trend in proportions. This is a nonparametric test that generates its p values by frequency analysis of the number of individuals at each possible score, thus taking into account both severity and incidence of disease. Proliferation data were analyzed by independent Student’s t test. p < 0.05 was considered significant; significance is denoted by an asterisk in the figures.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the role of B7-mediated costimulation in the
development of a uveitogenic response, mice were treated with 150 µg
of anti-B7.1 and/or anti-B7.2 and were immunized with a
uveitogenic regimen of IRBP (Fig. 1
a). Histopathologic grading
of eyes collected 21 days after immunization, corresponding to 
9
days after EAU onset in controls, showed that the combination treatment
with both Abs completely abrogated disease in all 24 animals tested
(p < 5.31 x
10-9). All mice in the corresponding control
group (n = 22) developed EAU, averaging a disease score
of 2.0. Treatment with anti-B7.1 alone significantly down-regulated
disease (p < 0.001). A trend toward reduced
disease scores was observed in the group treated with anti-B7.2
alone, but it did not attain statistical significance (Fig. 1b).
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, top). Mice treated with
anti-B7.1 and anti-B7.2 Abs had completely normal retinal
architecture (Fig. 2, bottom).
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Proliferation to IRBP of draining lymph node cells from mice whose
EAU response is described above was tested 21 days after immunization.
The average stimulation indexes from three experiments revealed that
mice treated with both blocking Abs had a 10-fold reduction in response
compared with the control group (Fig. 1
c). Only a 1.5-fold
reduction in proliferation was observed in the anti-B7.2-treated
group. Although mice treated with anti-B7.1 had significant disease
suppression, there was no corresponding decrease in cellular
proliferation compared with that in the control group. Because the
proliferation assays were conducted using the primary cells harvested
from the Ab-treated and immunized animals, it was necessary to consider
the possibility that APCs in these cultures were functionally
compromised by the anti-B7 treatment, even though the Ab treatment
had been discontinued >1 wk before harvesting the cells. Therefore, we
conducted a duplicate experiment in which syngeneic T cell-depleted
splenocytes from naive donors were added to the cultures to serve as
exogenous APCs. T cell proliferation was only marginally better in the
cultures that received exogenous APC, confirming that the proliferative
defect in cultures from anti-B7-treated mice was not at the level
of APC (data not shown).
To test whether the depressed proliferation was due to lack of IL-2
production and could be reversed by exogenous IL-2, a situation
considered to be indicative of anergy, 25 U/ml of IL-2 was added to
duplicate cultures set up as described above. Under these conditions,
suppression of proliferation exhibited by lymphocytes from the combined
anti-B7 treatment group could not be reversed by exogenous IL-2
(Fig. 3). The same result was also
observed if exogenous APCs were included in the cultures (data not
shown). The inability of exogenous IL-2 to restore Ag-specific
proliferation is compatible with the interpretation that its absence
was not due to anergy.
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responses
without evidence for a Th2 shift
Draining lymph node cells of mice immunized for EAU and treated
with anti-B7 Abs were harvested 21 days after immunization, and
supernatants from IRBP-stimulated cells were assayed for cytokines.
Fig. 4a shows the results of
IL-2 production from two experiments. Individual treatment with either
anti-B7.1 or anti-B7.2 reduced IL-2 production in both
experiments 2-fold or more below the control groups. The combination
anti-B7.1 and anti-B7.2 treatment reduced the IL-2 response
below the detection limit of the assay (<1.5 U/ml). Reduction of
IFN- production by anti-B7.1 or anti-B7.2 treatment was
variable. However, similar to the IL-2 results, only trace amounts of
IFN- were measured after combination anti-B7.1 and anti-B7.2
treatment (Fig. 4b).
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IFN- and IL-4 promote Ig isotype switching to IgG2a vs IgG1,
respectively. The relative amounts of these Abs in the serum are
therefore reflective of the balance of Th1 vs Th2 responses. Assay of
anti-IRBP titers in sera collected from four experiments showed
that treatment with anti-B7.1 or anti-B7.2 alone did not affect
IgG2a titers and variably reduced IgG1 titers, whereas blocking both
B7.1 and B7.2 decreased IgG1 Abs by 3–4 orders of magnitude, and IgG2a
Abs by 4 orders of magnitude below control levels (data not shown).
Taken together with the results of cytokine production, the data
support immunological hyporesponsiveness rather than immune deviation
as the reason for inhibition of EAU.
B7 blockade does not result in long-term tolerance
The data reported to date showed that anti-B7.1 and
anti-B7.2 treatment protected mice from a primary episode of EAU.
To investigate whether long-term tolerance was achieved as a result of
this treatment, we reimmunized the mice for EAU induction on day 28
after the first immunization, corresponding to 2 wk after cessation of
Ab treatment. In these experiments we used adult-thymectomized mice to
eliminate the possibility of interference by new thymic emigrants. The
experimental setup is rather complex and is shown in Fig. 5. Briefly, mice treated with
anti-B7.1 and anti-B7.2 or control Abs for 14 days were
immunized on day 0, rechallenged on day 28, and harvested on day 49 (21
days after rechallenge). Appropriate controls included mice that were
treated but immunized only on day 28 (to confirm that Abs had been
cleared from the system by the time of rechallenge) and mice that were
not given the secondary immunization (to assess the effect of primary
immunization and treatment on expression of disease at 49 days). The
time line (Fig. 5a) illustrates the progression of the
different groups through the experiment. Assessment of disease in live
mice during the course of the experiment was performed by fundoscopic
examinations on days 21 and 28 (before rechallenge).
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b). It is notable that
the disease developed by the initially protected and rechallenged group
1 was more severe than that in control group 5, which received its
primary immunization on day 28 and exhibited typical day 21 scores.
Draining lymph nodes harvested on day 49 from all mice were tested for
proliferation and IFN- production in response to IRBP. As with EAU
scores, the initially protected and rechallenged group 1 and its
corresponding control (group 4) had the highest proliferative responses
and produced the most IFN-. Notably, the responses of these groups
were again higher than those demonstrated by control group 5, which
received its only immunization on day 28 (Fig. 5, c and
d).
These results consistently show that regardless of whether costimulatory blockade was administered, Ag rechallenge not only failed to confirm induction of unresponsiveness, but actually generated more severe EAU and greater immunological responses than a primary Ag challenge. These data suggest that despite prevention of the primary EAU episode, a memory response may have been generated.
CD28 blockade does not induce long-term tolerance
B7 blockade can prevent interaction not only with CD28, but also
with CTLA-4. This could, in theory, yield a net zero balance of
positive and negative costimulatory signals, resulting in no apparent
response, but also no induction of anergy. We therefore treated
adult-thymectomized mice with anti-CD28 Ab rather than with
anti-B7, so as to block the positive, but permit the negative,
costimulation signal. The experimental setup in terms of treatment and
rechallenge schedules was similar to that described above and is
illustrated in Fig. 6a.
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EAU pathology on day 54 (3 wk after rechallenge) showed that the
initially protected and rechallenged group 1 had very high disease
scores, equivalent in severity to their corresponding control (group
2). In this experiment, control group 4 that had received its primary
immunization only on day 33 also developed high EAU scores (Fig. 5b).
Draining lymph nodes harvested on day 54 were tested for proliferation
and IFN- production in response to IRBP. The initially protected
group 1 again had higher proliferative responses and produced more
IFN- than groups 3 and 4, which received their only immunization on
day 33 (Fig. 5, c and d). Thus, the pattern of
disease scores and cellular responses indicated that, similar to what
was shown above for B7 blockade, anti-CD28 blockade protected from
a primary episode of EAU, but did not result in long-lasting tolerance
and was followed by enhanced responses upon Ag rechallenge.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Investigations in other autoimmune disease models have yielded differential results, depending on the model and the schedule of intervention (reviewed in Ref. 8). Our finding that B7/CD28 blockade affords protection from a primary EAU episode is in partial agreement with studies showing that disruption of this pathway can prevent or ameliorate EAE (24, 28, 34). However, unlike in EAE, where protection was reported by some investigators to result from a Th2 response shift (24, 26), the changes in cytokine responses and IgG isotypes in the EAU model did not indicate immune deviation.
We next considered the possibility that protection by B7 blockade resulted from induction of anergy. However, IRBP-specific proliferation in the protected mice was not restored by exogenous IL-2, an accepted criterion for the presence of anergy. Furthermore, rechallenge after the Abs had cleared induced severe disease and strong Ag-specific cellular responses. In fact, responses of treated and rechallenged mice were higher than those of untreated control mice given only a primary challenge. Notably, this held true when B7/CD28 interaction was blocked at the level of CD28, ensuring that there would be no interference with CTLA-4 engagement, which may be important for induction of anergy. Our data are thus compatible with the interpretation that 1) disruption of CD28-B7 interaction at the time of Ag exposure did not induce long-term unresponsiveness; 2) prevention of the primary episode of EAU was due to inhibition of effector generation rather than to induction of anergy or immune deviation; and 3) enhanced responses to rechallenge suggest that immunological memory had been induced during primary Ag exposure in the face of costimulatory blockade.
These interpretations raise the questions of whether IRBP-specific memory cells could be generated in the absence of B7 costimulation, perhaps by using other costimulatory molecules, and whether memory cells can be generated despite a virtual lack of response to the primary immunization, i.e., without evidence of first inducing effector function. Some recently published reports suggest that the answers to both questions may be affirmative. A study by Cross et al. (27) suggested that memory T cells could be generated in vivo despite CD28 blockade in the form of CTLA-4 Fc administration. These investigators reported that although CTLA-4 Fc treatment was protective against an initial episode of EAE, cells from protected animals were encephalitogenic after in vitro restimulation and adoptive transfer. As a more direct approach, using mice deficient in costimulatory molecules, Lui et al. showed that induction of CD8+ effector cells required costimulation by B7, but generation of memory could be costimulated by either B7 or heat-stable Ag (35, 36). Importantly, in the hands of these investigators, costimulation through heat-stable Ag rather than CD28 appeared to favor partial activation and conversion of naive T cells to a memory phenotype, without transition through an effector stage (36). Contribution by other costimulatory molecules also cannot be excluded. It should be pointed out, however, that our in vivo system cannot distinguish between the possibility that Ag-specific T cells acquired a memory phenotype without first passing through an effector stage from the possibility that most Ag-specific T cells remained naïve, but a small number of effectors were induced that, while insufficient in number to bring about disease, gave rise to a memory population.
The lack of anergy induction in our model by CD28 blockade, which disrupts the CD28/B7 interaction ("on" signal) but permits the CTLA-4/B7 interaction ("off" signal) needs to be reconciled with the data reported by Perez et al. (37), who under similar conditions of costimulatory blockade demonstrated induction of anergy to a CD4-restricted OVA peptide in an OVA-TCR transgenic model. Our experimental system differs from the OVA system in two regards. 1) IRBP is a self Ag, and therefore the IRBP-specific T cell repertoire is likely to bear low-affinity TCRs that escaped the checkpoint of negative selection in the thymus. This is supported by recent data from our laboratory showing that IRBP knockout mice have much higher proliferative responses to IRBP than do wild-type mice (D. Avichezer et al., unpublished observations). It is conceivable, therefore, that the strength of TCR signaling that was insufficient to delete these low-affinity T cells during ontogeny might also be insufficient to anergize them in the absence of costimulation. A recent report that low-affinity TCR signals are particularly likely to lead to immunological memory while bypassing full T cell activation (that in the absence of costimulation might result in anergy), lends support to this reasoning (38). 2) Induction of EAU requires the use of bacterial adjuvants that are strong inducers of innate immunity. Inflammatory cytokines can under some conditions rescue T cells from apoptosis and/or anergy (39, 40, 41, 42, 43, 44, 45). Because ongoing autoimmunity creates an inflammatory milieu, it can be argued that newly recruited autoreactive cells are primed in an environment that is conducive to generation of memory rather than anergy. Our results also support and help explain the recent observations of Girvin et al. (46), who reported the appearance of delayed disease after the Abs had cleared in anti-B7-treated nonobese diabetic (NOD) mice immunized for EAE and concluded that long-term tolerance had not been induced. In the aggregate, these results underscore that model Ag systems do not necessarily predict the outcome of costimulatory blockade in an autoimmunity setting.
Several recent studies have investigated the role of costimulation in autoimmunity using gene knockout mice. Although valuable insights were gleaned from those studies, their interpretation is limited by the experimental system itself. As an example, a study by Oliveira-dos-Santos et al. (34) that examined EAE induction in CD28-/-xrecombinase-activating gene 1-/- mice showed that lymphocytes from these mice become primed, but the animals do not develop EAE. This led to the conclusion that although CD28 is necessary for disease pathogenesis, it controls the threshold of stimulation rather than anergy. However, a congenital lack of CD28 might lead to increased use of alternative costimulatory pathways, causing CD28-/- mice to prime their cells more readily than would a wild-type mouse under CD28 blockade. In addition, recombinase-activating gene deficiency causes lack of regulatory cells expressing endogenous TCRs, which might well affect the responses of such mice. Thus, conclusions drawn from knockout systems may not always be representative of the wild type. This point is further underscored by the work of Salomon et al. (47), who examined the effects of B7 or CD28 deficiency in the NOD vs EAE models. Opposite effects were seen on the two diseases, with EAE being inhibited and diabetes being exacerbated. Interestingly, both these knockout strains turned out to be profoundly deficient in CD25+ regulatory cells, and replacement of these cells from the wild-type animals caused the CD28-deficient NODs to become diabetes resistant. These data thus emphasize the importance of examining the issues in wild-type animals that possess all the requisite components of the immune system and in diverse autoimmune disease models to determine the possible consequences of costimulatory blockade in a therapeutic setting.
In conclusion, the idea that chronic autoimmunity must involve continuous recruitment and priming of Ag-specific T cells would support the idea that costimulatory blockade might be useful as a therapeutic approach to autoimmune disease. Our data in the mouse EAU model indicate that although this treatment can prevent generation of effector T cells, it does not result in anergy, nor does it prevent induction of immunological memory to the autoantigen. These results point out the potential limitations of costimulatory blockade as a therapy for autoimmune uveitis and related diseases, because the Ag-specific T cells are likely to represent a low-affinity TCR population that may not be easily tolerized, and because the preexisting inflammatory milieu might counteract the induction of tolerance. Finally, even though activation of naive T cells into effector cells appears to be prevented by B7/CD28 blockade, the question remains of whether activation of memory cells into effectors would also be blocked under these conditions. Several studies have indicated that memory cells are less dependent for activation on CD28 signals than are naive T cells (48, 49). Because in ongoing autoimmunity, memory cells are already present, this will need to be taken into account in a therapeutic setting.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: EAU, experimental autoimmune uveitis; EAE, experimental autoimmune encephalomyelitis; IRBP, interphotoreceptor retinoid-binding protein; NOD, nonobese diabetic.
Received for publication May 31, 2000. Accepted for publication August 10, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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