The role of CTLA-4 in tolerance induction and T cell differentiation in experimental autoimmune encephalomyelitis: i.p. antigen administration

Robert B. Ratts, Lachelle R. Arredondo, Patrice Bittner, Peter J. Perrin1, Amy E. Lovett-Racke and Michael K. Racke

Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, USA
1 Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

Correspondence to: M. K. Racke, Department of Neurology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9036, USA


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Recent evidence suggests that co-stimulation provided by B7 molecules through CTLA-4 is important in establishing peripheral tolerance. In the present study, we examined the kinetics of tolerance induction and T cell differentiation following i.p. administration of myelin basic protein (MBP) Ac1–11 in mice transgenic for a TCR Vß8.2 gene derived from an encephalitogenic T cell clone specific for MBP Ac1–11. Examination of the lymph node cell response after antigen administration demonstrated a dependence on CTLA-4 for i.p. tolerance induction. Examination of splenocyte responses suggested that i.p. antigen administration induced a Th2 response, which was potentiated by anti-CTLA-4 administration. Interestingly, i.p. tolerance was able to inhibit the induction of experimental autoimmune encephalomyelitis and anti-CTLA-4 administration did not alter this phenotype, suggesting that CTLA-4 blockade did not block tolerance induction. Thus, T cell differentiation and the dependence on CTLA-4 for tolerance induction following i.p. antigen administration differs between lymph node and spleen in a model of organ-specific autoimmunity.

Keywords: experimental autoimmune encephalomyelitis, co-stimulatory molecules, neuroimmunology, tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T cells require two signals to become activated and differentiate. Signal one occurs when the TCR complex recognizes antigen bound to MHC molecules on the surface of antigen-presenting cells (APC). This interaction determines the antigen specificity of the immune response. Signal two, termed co-stimulation, is provided by accessory molecules on the APC and T cell, and appears to be necessary for functional T cell activation. While there are several TCR–ligand pairs which can provide co-stimulation, the signal provided by the B7 family of surface molecules with its receptors on T cells, CD28 and CTLA-4, appear to predominate in T cell activation (13). Initial studies indicated that CTLA-4 provided a positive co-stimulatory signal in conjunction with CD28 (4). Recent evidence suggests that signaling through CTLA-4 mediates a negative regulatory function (5). Furthermore, mice deficient in CTLA-4 develop a lymphoproliferative disorder (6,7). Our own studies and those of others have demonstrated that anti-CTLA-4 administration can increase the production of proinflammatory cytokines such as IFN-{gamma}, IL-2 and tumor necrosis factor-{alpha}, and exacerbate clinical signs of experimental autoimmune encephalomyelitis (EAE) (810). These studies all suggest that B7-mediated co-stimulation, through its interaction with CTLA-4, provides important regulatory signals during an immune response. Early studies in vitro indicated that T cell recognition of antigen in the absence of co-stimulation led to anergy (11). Recent evidence suggests that co-stimulation provided by B7 molecules through CTLA-4 is also important in several different methods used to establish peripheral tolerance in vivo (1215).

EAE is a T cell-mediated autoimmune disorder characterized by central nervous system inflammation and demyelination, features reminiscent of the human disorder, multiple sclerosis (MS) (16,17). EAE can be induced in susceptible inbred strains of mice by immunization with whole myelin, proteins of the myelin sheath such as myelin basic protein (MBP), proteolipid protein or encephalitogenic peptides in complete Freund's adjuvant (CFA). Many organ-specific autoimmune diseases such as EAE are mediated by CD4+ T cells of the Th1 phenotype. These encephalitogenic T cells produce lymphotoxin and IFN-{gamma}, but little IL-4 (16,17). B7 co-stimulation can contribute to IFN-{gamma} secretion by Th1 clones (18).

Work in the EAE model has demonstrated several methods for inducing antigen-specific tolerance to encephalitogenic antigens, with the result that subsequent induction of disease is inhibited (1921). Several studies have examined i.p. administration of antigen in incomplete Freund's adjuvant (IFA) as a means of inducing tolerance in both neonatal and adult animals using the EAE model (22,23). Most of these studies used lymph node cell proliferation to antigen and disease induction as readouts for tolerance induction. More recently, studies examining splenocyte responses following i.p. antigen administration suggest the induction of a Th2 response rather than tolerization of antigen-specific splenocytes (22).

The co-stimulation requirements following i.p. antigen administration in the EAE model remain ill defined. In addition, examination of co-stimulation requirements in antigen-specific tolerance have largely been confined to lymph node responses and ignored antigen-specific responses in the spleen.

Recent studies examining the role of CTLA-4 in peripheral tolerance following i.p. antigen administration have used transgenic T cells that recognize ovalbumin (OVA) peptide 323–339 which have been transferred into naive BALB/c mice (12). We have previously used T cells from MBP-specific TCR transgenic mice to follow the fate of encephalitogenic T cells in vivo following i.v. tolerance (24,25). These mice express Vß8.2 and V{alpha}2.3 TCR that are specific for MBP Ac1–11, which is the immunodominant MBP peptide in B10.PL mice of the H-2u haplotype (25). Recent studies have shown that mice expressing the Vß8.2 TCR transgene and wild-type {alpha} chains have increased encephalitogenic precursors compared to wild-type B10.PL mice and are highly susceptible to EAE (26,27). In this study, we used the Vß8.2 TCR transgenic mice to examine the role of CTLA-4 in establishing tolerance following i.p. administration of antigen. In addition, we have used the EAE model as a means of determining the functional significance of CTLA-4 blockade in vivo following i.p. antigen administration in an autoimmune disorder.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Transgenic mice bearing the rearranged Vß8.2 gene specific for MBP Ac1–11 on the B10.PL background were provided by Dr Joan Goverman (University of Washington, Seattle, WA) (2527). These mice were bred and maintained in our animal colony at Washington University School of Medicine in compliance with the Animal Studies Committee. All mice were 6–8 weeks of age when experiments were initiated.

Reagents
Whole MBP was prepared from guinea pig spinal cords as previously described and purity assessed by SDS–PAGE (28). MBP peptide Ac1–11 was synthesized by the Protein Chemistry Laboratory at Washington University School of Medicine with purity assessed by mass spectrometry. Hamster anti-mouse CTLA-4 mAb UC10-4F10 was provided by Dr Jeff Bluestone (University of Chicago, Chicago, IL) (29). In the present study, intact mAb was used, since prior studies using this antibody or its Fab fragments have shown no fundamental differences (9,30). Hamster IgG was purchased from Jackson ImmunoResearch (West Grove, PA).

Antigenic challenges
At day 0 mice in the primed group were immunized s.c. with MBP Ac1–11 (30 µg) in IFA (Difco, Detroit, MI). Mice receiving i.p. tolerization protocols were injected with MBP Ac1–11 (200 µg)/IFA at day 0.

In vivo antibody treatments
Anti-CTLA-4 mAb (200 µg) or control hamster IgG (200 µg) were given on days –1, 0 and +1 relative to i.p. antigen administration.

Lymphocyte proliferation
Proliferative responses by LNC and splenocytes were measured 3 and 10 days after antigenic challenge. Results shown are representative of three experiments. Cells (2x105/well) were incubated with MBP or media alone as indicated. Cultures were maintained in 96-well, flat-bottom microtiter plates for 96 h at 37°C in humidified 5% CO2 air. The wells were pulsed with 0.5 µCi/well of [3H]methyl-thymidine for the final 16 h of culture. Cells were harvested on glass fibers and incorporated [3H]methyl-thymidine was measured with a Betaplate counter (Wallac, Gaithersburg, MD). Results were determined as means from quadruplicate cultures and are shown on log scale with SEM.

Measurement of cytokine production
An IL-2-dependent cell line, CTLL.EV (31), was generously provided by Dr Jonathan Katz (Washington University School of Medicine, St Louis, MO). Aliquots of 20 µl of supernatants from experimental cell cultures were assayed in quadruplicate. Results were compared with proliferation of the cell line to known amounts of IL-2 (R & D System, Minneapolis, MN) as standards. IFN-{gamma} and IL-4 ELISA (R & D Systems) were performed in duplicate according to the manufacturer's instructions. Cytokine results are representative of three to six experiments.

Induction of EAE
Ten days after receiving tolerizing antigen and antibody, mice were immunized with MBP Ac1–11/CFA (200 µg) s.c. over the flanks. Mice were examined daily for signs of disease and graded on the following scale: 0, no abnormality; 1, a limp tail; 2, moderate hind limb weakness; 3, severe hind limb weakness; 4, complete hind limb paralysis; 5, quadriplegia or premoribund state (24,32).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Vß8.2 TCR transgenic mice have increased frequency of MBP-specific T cells
To determine if naive B10.PL mice transgenic for the Vß8.2 TCR derived from an encephalitogenic T cell clone have a higher frequency of MBP-specific T cells than wild-type B10.PL mice, a proliferation assay was performed. As seen in Fig. 1Go, the LNC and splenocytes from Vß8.2 TCR transgenic mice demonstrated a robust proliferative response to MBP compared to cells from wild-type mice. Limiting dilution analysis estimated the frequency of MBP-specific LNC to be ~1/163,000 in Vß8.2 TCR transgenic mice, which is significantly higher than the frequency in naive, wild-type B10.PL LNC (<1/830,000), but not as high as that seen in Vß8.2, V{alpha}2.3 TCR transgenic mice (data not shown). Due to the high frequency of naive MBP-specific T cells in these mice, they provided an informative system for studying tolerance induction in the EAE model and were used for all subsequent experiments.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Naive LNC and splenocytes from B10.PL Vß8.2 transgenic mice (closed symbols) display increased proliferative response to MBP compared to wild-type B10.PL LNC and splenocytes (open symbols). Proliferation was measured by 3H-labeled thymidine incorporation assay as described in Methods. SEM are shown from quadruplicate cultures.

 
Tolerance induction after i.p. administration of antigen
Previously anti-CTLA-4 has been reported to block tolerance induction by i.p. administration of antigen (OVA peptide 323–339) (12). To determine if tolerance induction to a self-antigen was dependent on CTLA-4 engagement, we compared the proliferative responses of LNC from naive mice, mice primed with MBP Ac1–11 s.c., mice receiving MBP Ac1–11 i.p. and mice receiving MBP Ac1–11 i.p. with anti-CTLA-4. When B10.PL Vß8.2 TCR transgenic were mice primed with 30 µg MBP Ac1–11/IFA s.c., their LNC at day 3 and day 10 demonstrated an enhanced proliferative response compared to naive LNC (Figs 2A and 3GoGo). When 200 µg MBP Ac1–11/IFA was administered i.p., it resulted in dramatically reduced proliferative responses and IL-2 production by LNC, suggesting tolerance of MBP-specific T cells (Figs 2A and B, and 3GoGo). This reduction in proliferative response was not affected by the administration of anti-CTLA-4 antibody (4F10), when examined 3 days after i.p. antigen administration (Fig. 2AGo), but was partially reversed by anti-CTLA-4 10 days after the i.p. tolerizing protocol (Fig. 3Go). Tolerance induction of MBP-specific LNC following i.p. antigen administration was suggested by the dramatically reduced IL-2 and IFN-{gamma} production by LNC at day 3, with little IL-4 production detected by any of the LNC (Fig. 2BGo–D).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. MBP-specific LNC responses 3 days after i.p. antigenic challenge in vivo. LNC (pooled from two to three mice per condition) were removed from B10.PL Vß8.2 TCR transgenic mice 3 days after the mice were unmanipulated (closed squares), primed with 30 µg MBP Ac1–11/IFA s.c. (closed circles), tolerized with 200 µg MBP Ac1–11/IFA i.p. and hamster IgG (open circles) or tolerized with 200 µg MBP Ac1–11/IFA i.p. and anti-CTLA-4 antibody (4F10) (open squares). LNC were cultured with whole MBP as described in Methods. Supernatants were taken at 24, 48 and 72 h, and frozen at –20°C. IL-2 concentrations were determined by CTLL-2 assay. IL-4 and IFN-{gamma} production was determined by ELISA. Proliferation was measured by 3H-labeled thymidine incorporation assay as described in Methods. SEM are shown from quadruplicate cultures (A and B) and 1 SD is shown from duplicate wells (C and D).

 


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. MBP specific LNC proliferation 10 days after i.p. antigenic challenge in vivo. Lymph nodes were removed from B10.PL Vß8.2 TCR transgenic mice that were unmanipulated (closed squares), primed with 30 µg MBP Ac1–11/IFA s.c. (closed circles), tolerized with 200 µg MBP Ac1–11/IFA i.p. and hamster IgG (open circles) or tolerized with 200 µg MBP Ac1–11/IFA i.p. and anti-CTLA-4 antibody (4F10) (open squares). The resulting LNC were then cultured with MBP as described in Methods. Proliferation was measured by 3H-labeled thymidine incorporation assay as described in Methods. SEM are shown from quadruplicate cultures.

 
The kinetics of the immune response of MBP-specific splenocytes following i.p. antigen administration and their corresponding cytokine production was examined. At 3 days following a tolerogenic dose of antigen administered i.p., enhanced MBP-specific proliferation was observed by splenocytes, while a priming dose of antigen s.c. had no effect on antigen-specific proliferation by splenocytes (Fig. 4AGo). In addition, the administration of anti-CTLA-4 antibody dramatically reduced the enhanced MBP-specific splenocyte proliferative response observed at day 3. Splenocytes from mice receiving a tolerogenic dose of antigen produced similar amounts of IL-2 at 24 h compared to naive mice and those receiving a priming dose of antigen, which suggested that MBP-specific splenocytes were not tolerized following i.p. antigen administration (Fig. 4BGo). Administration of anti- CTLA-4 with the tolerogen reduced IL-2 production at 48 h compared to tolerogen alone (Fig. 4BGo). Splenocytes from tolerized mice produced more IFN-{gamma} than splenocytes from naive mice 3 days after antigen challenge, again suggesting that MBP-specific splenocytes were not tolerized (Fig. 4CGo). IL-4 production was highest by MBP-specific splenocyte mice receiving i.p. MBP Ac1–11 administration (Fig. 4DGo).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4. MBP-specific splenocyte responses 3 days after i.p. antigenic challenge in vivo. Spleens were removed from B10.PL Vß8.2 TCR transgenic mice 3 days after the mice were unmanipulated (closed squares), primed with 30 µg MBP Ac1–11/IFA s.c. (closed circles), tolerized with 200 µg MBP Ac1–11/IFA i.p. and hamster IgG (open circles) or tolerized with 200 µg MBP Ac1–11/IFA i.p. and anti-CTLA-4 antibody (4F10) (open squares). The resulting splenocytes were cultured with whole MBP as described in Methods. Supernatants were taken at 24, 48 and 72 h, and frozen at –20°C. IL-2 concentrations were determined by CTLL-2 assay. IL-4 and IFN-{gamma} production was determined by ELISA. Proliferation was measured by 3H-labeled thymidine incorporation assay as described in Methods. SEM are shown from quadruplicate cultures (A and B) and 1 SD is shown from duplicate wells (C and D).

 
In contrast to the proliferative response at 3 days post antigen administration, at 10 days the splenocytes show a dramatic reduction in proliferation following i.p. administration of MBP Ac1–11, which was partially reversed by administration of anti-CTLA-4 (Fig. 5AGo). At this time, MBP-specific splenocytes still produced significant amounts of IL-2 suggesting that they were not tolerized (Fig. 5BGo). Administration of anti-CTLA-4 with i.p. administration of antigen also dramatically reduced IFN-{gamma} production and increased IL-4 production, suggesting a shift to a more Th2-like response (Fig. 5C and DGo). Also at day 10 following antigenic challenge, splenocytes from mice primed with MBP Ac1–11/IFA s.c. produced more IFN-{gamma} and earlier production of IL-2, which also correlated with the proliferative response.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5. MBP-specific splenocyte responses 10 day after i.p. antigenic challenge in vivo. Spleens were removed from B10.PL Vß8.2 TCR transgenic mice 10 days after the mice were unmanipulated (closed squares), primed with 30 µg MBP Ac1–11 in IFA s.c. (closed circles), tolerized with 200 µg MBP Ac1–11/IFA i.p. and Hamster IgG (open circles) or tolerized with 200 µg MBP Ac1–11/IFA i.p. and anti-CTLA-4 antibody (4F10) (open squares). The resulting splenocytes were cultured with whole MBP as described in Methods. Supernatants were taken at 24, 48 and 72 h, and frozen at –20°C. IL-2 concentrations were determined by CTLL-2 assay. IL-4 and IFN-{gamma} production was determined by ELISA. Proliferation was measured by 3H-labeled thymidine incorporation assay as described in Methods. SEM are shown from quadruplicate cultures (A and B) and 1 SD deviation is shown from duplicate wells (C and D).

 
The effects of anti-CTLA-4 administration on tolerance induction and susceptibility to EAE
Because 100% of B10.PL Vß8.2 TCR transgenic mice develop EAE when challenged with MBP Ac1–11/CFA (Table 1Go), we decided to examine the effects of i.p. antigen administration with and without CTLA-4 blockade on EAE susceptibility. Mice underwent the i.p. tolerization protocol with MBP Ac1–11 as before and were then reimmunized 10 days later with 200 µg MBP Ac1–11/CFA s.c. Mice were then observed for clinical signs of EAE (Table 1Go). We hypothesized that if anti-CTLA-4 administration blocked tolerance induction, as has been previously suggested (12), then mice receiving i.p. antigen and anti-CTLA-4 would remain susceptible to EAE. However, mice that received antigen i.p. did not develop EAE, irrespective of anti-CTLA-4 administration. These results suggest that the Th2-promoting effect of anti-CTLA-4 administration associated with i.p. antigen administration results in mice remaining free from the development of EAE.


View this table:
[in this window]
[in a new window]
 
Table 1. Induction of EAE following i.p. administration of antigen with or without anti-CTLA-4
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CTLA-4 shares considerable homology with CD28 and initial studies indicated its engagement had stimulating effects on T cells (4). More recently, CTLA-4 engagement has been shown to negatively regulate T cell activation (5–10,29). In addition to down-regulating an ongoing T cell response, CTLA-4 is postulated to play a role in initial tolerance induction (1215). In this role, co-stimulation through CTLA-4, instead of CD28, leads to an aborted T cell response and ultimately T cell anergy or death. Although CTLA-4 is not detectable on resting T cells, its expression is up-regulated to detectable levels within hours of TCR engagement (29). Additionally, CTLA-4 has a higher avidity for B7-1 and B7-2 than CD28, and can compete for these two molecules with CD28 even though it is present at lower concentrations (33). Recently this model of CTLA-4 involvement in tolerance has been investigated using several methods of tolerance induction (1215).

Perez et al. (12) investigated tolerance induction in transgenic T cells, which were specific for OVA 323–339, and which had been adoptively transferred into naive recipients. Subsequently these mice were tolerized with i.p. injections of OVA and LNC responses were examined 3 days later (12). They reported that blocking B7 interactions, by administering CTLA-4–Ig on days –1, 0 and +1 with respect to tolerogen, resulted in cells that phenotypically appeared naive. Using anti-CTLA-4, which specifically blocks B7/CTLA-4 interactions, it was observed that a normally tolerizing dose of antigen (i.p. in IFA) resembled priming as measured by proliferation, IL-2 production and IFN-{gamma} production. The present study differs from the prior study in several aspects. The prior study only examined LNC responses 3 days after antigenic challenge. This study examined the response to an autoantigen, MBP, rather than a foreign antigen, OVA. In addition, our studies used an additional readout of tolerance induction, that being susceptibility to EAE. If anti-CTLA-4 administration blocks tolerance induction, then mice receiving i.p. antigen plus anti-CTLA-4 should have remained susceptible to EAE, but this was not the case. In our study, by examining both LNC and splenocyte responses 3 and 10 days after antigenic challenge, we were able to demonstrate that i.p. tolerance induces a Th2 phenotype in splenic T cells, which is enhanced by anti-CTLA-4 administration. This potentiation of a Th2 response by anti-CTLA-4 with i.p. antigen administration could explain why mice receiving this protocol remained resistant to EAE induction.

Another group has utilized oral tolerance to observe the effects of co-stimulation on tolerance induction (14). They fed antigen to mice, while at the same time injecting them with anti-CTLA-4 antibody. The mice were later immunized with the same antigen and 10 days later the splenocyte response was observed. Blocking B7/CTLA-4 interaction during oral tolerance induction prevented a decrease of antigen-specific IFN-{gamma} production, but did not prevent a decrease in antigen-specific proliferation or IL-4 production (14). When administering oral antigen and specifically blocking CTLA-4 with anti-CTLA-4 antibody, they observed splenocyte responses similar to the naive response. It should be noted that this study used OVA-specific TCR transgenic mice, which have a very high frequency of OVA-specific T cells.

In another system of tolerance induction, administering anti-CTLA-4 5 days after an intrathymic (i.t.) injection of antigen abrogated tolerance (15). Injections of antigen i.t. were associated with antigen-specific suppression of LNC proliferation. Interestingly, when anti-CTLA-4 was administered at the same time as tolerance induction, tolerance was still observed. In functional EAE studies, i.t. injections of MBP Ac1–11 were able to block development of EAE after immunization with MBP Ac1–11. Administering anti-CTLA-4 5 days after the tolerogen and 3 days post-immunization abrogated tolerance and mice were again susceptible to EAE (15).

In the present study, we examined the effect of i.p. antigen administration on the effect of cytokine production, lymphocyte proliferation and ability to induce the organ-specific autoimmune disorder, EAE. In particular, we addressed the role of CTLA-4 in these processes. We demonstrated that there was an increase in production of IL-4 and decrease in IFN-{gamma} production by MBP-specific splenocytes when anti-CTLA-4 was administered at the same time as i.p. antigen, consistent with a shift in the lymphocyte response from a Th1 to a Th2 phenotype. Interestingly, the fact that CTLA-4 blockade enhanced the differentiation toward a Th2 phenotype is consistent with a recent report showing secretion of Th2 cytokines by proliferating lymphocytes in CTLA-4 deficient mice (34). Administration of anti-CTLA-4 has demonstrated Th2-promoting effects in other systems of tolerance (13). Examination of this form of tolerance induction on EAE susceptibility confirmed that anti-CTLA-4 administration enhanced Th2 differentiation rather than returning lymphocytes to a naive or primed phenotype, since these mice remained resistant to the development of EAE (Table 1Go).

It should also be noted that studies with anti-CTLA-4 (4F10) and its Fab fragments have shown no fundamental differences (9, 30). It must be emphasized that while the Vß8.2 TCR transgenic mice used in these studies have elevated frequencies of MBP-specific T cells compared to wild-type mice, the majority of T cells are specific for other antigens, unlike the Vß8.2, V{alpha}2.3 TCR transgenic mouse. In addition, a recent study suggests that patients with multiple sclerosis may actually have similar frequencies of myelin-reactive T cells as high as 1/300 (35), thus the responses examined here may have relevance to human disease.

It is important to note that differences in LNC and splenocyte responses may reflect the different circulation patterns of naive and memory lymphocytes (36,37). Lymphocytes responding to i.p. antigen appear to home to the spleen and secrete IL-4, and it is likely that these cells do not circulate in lymph nodes unless an inflammatory response has occurred (37). Thus, following i.p. antigen administration, Th2-like cells would not home to lymph nodes and are not detected when LNC are stimulated with antigen in vitro (Fig. 3Go). Our laboratory has also examined the role of CTLA-4 in tolerance induction following i.v. antigen administration (38). In contrast to the findings in this study, i.v. antigen administration did not induce a MBP-specific Th2 response in splenocytes, and i.v. antigen administration (a single injection) was ineffective in protecting mice from EAE. Anti-CTLA-4 co-administration with i.v. antigen potentiated tolerance in terms of reduced IL-2 production and antigen-specific proliferation, and mice remained susceptible to induction of EAE. Thus, anti-CTLA-4 appears to have profoundly different effects in the EAE model when examining i.v. and i.p. antigen administration. Overall, our findings suggest that the route of antigen administration is important in determining the phenotype of the responding T cells and the contribution that CTLA-4 may play in that differentiation. By examining the antigen-specific responses of both LNC and splenocytes, we have demonstrated that while anti-CTLA-4 administration may `block' i.p. tolerance induction of LNC, it promotes Th2-like differentiation of splenocytes. That such an effect is important in vivo was supported by our EAE studies which showed that anti-CTLA-4 did not reverse the protective effect of i.p. antigen administration.


    Acknowledgments
 
This research was supported by grants from the National Multiple Sclerosis Society (M. K. R. and P. J. P.) and NIH (R29-AI-43296 to P. J. P. and RO1-NS-37513 to M. K. R.). M. K. R. is a Harry Weaver Neuroscience Scholar of the National Multiple Sclerosis Society, and the Young Investigator in Multiple Sclerosis of the American Academy of Neurology Education and Research Foundation.


    Abbreviations
 
APC antigen-presenting cell
CFA complete Freund's adjuvant
EAE experimental autoimmune encephalomyelitis
IFA incomplete Freund's adjuvant
i.t. intrathymic
LNC lymph node cells
MBP myelin basic protein
MS multiple sclerosis
OVA ovalbumin
Received 3 June 1999, accepted 12 August 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. June, C. H., Bluestone, J. A., Nadler L. M. and Thompson, C. B. 1994. The B7 and CD28 receptor families. Immunol. Today 15:321.[ISI][Medline]
  2. Jenkins, M. K. and Johnson J. G. 1993. Molecules involved in T-cell co-stimulation. Curr. Opin. Immunol. 5:351.
  3. Bretscher, P. 1992. The two-signal model of lymphocyte activation twenty-one years later. Immunol. Today 13:74.[ISI][Medline]
  4. Linsley, P. S., Greene, J. L., Tan, P., Bradshaw, J., Ledbetter, J. A., Anasetti, C. and Damle, N. K. 1992. Coexpression and functional cooperation of CTLA-4 and CD28 on activated T lymphocytes. J. Exp. Med. 176:1595.[Abstract]
  5. Green, J. M., Noel, P. J., Sperling, A. I., Walunas, T. L., Gray, G. S., Bluestone, J. A. and Thompson, C. B. 1994. Absence of B7-dependent responses in CD28-deficient mice. Immunity 1:501.[ISI][Medline]
  6. Waterhouse, P., Penninger, J. M., Timms, E., Wakeham, A., Shahinian, A., Lee, K. P., Thompson, C. B., Griesser, H. and Mak, T. W. 1995. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 170:985.
  7. Tivol, E. A., Borriello, F., Schweitzer, A. N., Lynch, W. P., Bluestone, J. A. and Sharpe, A. H. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3:541.[ISI][Medline]
  8. Perrin, P. J., Maldonado, J. H., Davis, T. A., June, C. H. and Racke, M. K. 1996. CTLA-4 blockade enhances clinical disease and cytokine production during experimental allergic encephalomyelitis. J. Immunol. 157:1333.[Abstract]
  9. Karandikar, N. J., Vanderlugt, C. L., Walunas, T. L., Miller, S. D. and Bluestone J. A. 1996. CTLA-4: a negative regulator of autoimmune disease. J. Exp. Med. 184:783.[Abstract]
  10. Hurwitz, A. A., Sullivan, T. J., Krummel, M. F., Sobel, R. A. and Allison, J. P. 1997. Specific blockade of CTLA-4/B7 interactions results in exacerbated clinical and histologic disease in an actively-induced model of experimental allergic encephalomyelitis. J. Neuroimmunol. 73:57.[ISI][Medline]
  11. Gimmi, C. D., Freeman, G. J., Gribben, J. G., Gray, G. and Nadler, L. M. 1993. Human T cell clonal anergy is induced by antigen presentation in the absence of B7 co-stimulation. Proc. Natl. Acad. Sci. USA 90:6586.[Abstract]
  12. Perez, V., Parijs, L., Biuckians, A., Zheng, X., Strom, T. and Abbas, A. 1997. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity. 6:411.[ISI][Medline]
  13. Walunas, T. L. and Bluestone, J. A. 1999. CTLA-4 regulates tolerance induction and T cell differentiation in vivo. J. Immunol. 160:3855.[Abstract/Free Full Text]
  14. Samoliova, E. B., Horton, J. L., Zhang, H., Khoury, S. J., Weiner, H. L. and Chen, Y. 1998. CTLA-4 is required for the induction of high dose oral tolerance. Int. Immunol. 10:491.[Abstract]
  15. Issazadeh, S., Zhang, M., Sayegh, M. H. and Khoury, S. J. 1999. Acquired thymic tolerance: role of CTLA-4 in the initiation and maintenance of tolerance in a clinically relevant autoimmune disease model. J. Immunol. 162:761.[Abstract/Free Full Text]
  16. Martin, R., McFarland, H. F. and McFarlin, D. E. 1992. Immunological aspects of demyelinating diseases. Annu. Rev. Immunol. 10:153.[ISI][Medline]
  17. Zamvil, S. S. and Steinman, L. 1990. The lymphocyte in experimental allergic encephalomyelitis. Ann. Rev. Immunol. 8:579.[ISI][Medline]
  18. Murphy, E. E., Terres, G., Macatonia, S. E., Hsieh, C. S., Mattson, J., Lanier, L., Wysocka, M., Trinchieri, G., Murphy, K. and O'Garra, A. 1994. B7 and interleukin 12 cooperate for proliferation and interferon gamma production by mouse T helper clones that are unresponsive to B7 co-stimulation. J. Exp. Med. 180:223.[Abstract]
  19. Su, X.-M. and Sriram, S. 1991. Treatment of chronic relapsing experimental allergic encephalomyelitis with the intravenous administration of splenocytes coupled to encephalitogenic peptide 91–103 of myelin basic protein. J. Neuroimmunol. 34:181.[ISI][Medline]
  20. Kennedy, M. K., Tan, L.-J., Dal Canto, M. C., Tuohy, V. K., Lu, Z., Trotter, J. L. and Miller, S. D. 1990. Inhibition of murine relapsing experimental autoimmune encephalomyelitis by immune tolerance to proteolipid protein and its encephalitogenic peptides. J. Immunol. 144:909.[Abstract/Free Full Text]
  21. Gaur, A., Wiers, B., Liu, A., Rothbard, J. and Fathman, G. C. 1993. Amelioration of autoimmune encephalomyelitis by myelin basic protein synthetic peptide-induced anergy. Science 258:1491.[ISI]
  22. Qin, Y., Sun, D., Goto, M., Meyermann, R. and Wekerle, H. 1989. Resistance to experimental autoimmune encephalomyelitis induced by neonatal tolerization to myelin basic protein: clonal elimination vs. regulation of autoaggressive lymphocytes. Eur. J. Immnunol. 19:373.
  23. Forsthuber, T., Yip, H. C. and Lehmann, P.V. 1996. Induction of Th1 and Th2 immunity in neonatal mice. Science 271:1728.[Abstract]
  24. Critchfield, J. M., Racke, M. K., Zuniga-Pflucker, J. C., Cannella, B., Raine, C. S., Goverman, J. and Lenardo, M. J. 1994. T cell deletion in high antigen dose therapy of autoimmune encephalomyelitis. Science 263:1139.[ISI][Medline]
  25. Goverman, J., Woods, A., Larson, L., Weiner, L. P., Hood, L. and Zaller, D. M. 1993. Transgenic mice that express a myelin basic protein-specific T cell receptor develop spontaneous autoimmunity. Cell 72:551.[ISI][Medline]
  26. Buenafe, A. C., Tsu, R. C., Bebo, B., Vandenbark, A. A. and Offner, H. 1997. Myelin basic protein-specific and TCR Vß8.2-specific T cell lines from TCR Vß8.2 transgenic mice utilize the same V{alpha} and Vß genes: specificity associated with the V{alpha}CDR3–J{alpha} region. J. Neurosci. Res. 47:489.[ISI][Medline]
  27. Siklodi, B., Jacobs, R., Vandenbark, A. A. and Offner, H. 1998. Neonatal exposure of TCR BV8S2 transgenic mice to recombinant TCR BV8S2 results in reduced T cell proliferation and elevated antibody response to BV8S2, and increased severity of EAE. J. Neurosci. Res. 52:750.[ISI][Medline]
  28. Deibler, G. E., Martenson, R. E. and Kies, M. W. 1972. Large scale preparation of myelin basic protein from central nervous system tissue of several mammalian species. Prep. Biochem. 2:139.[ISI][Medline]
  29. Walunas, T. L., Lenschow, D. J., Bakker, C. Y., Linsley, P. S., Freeman, G. J., Green, J. M., Thompson, C. B. and Bluestone, J. A. 1994. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1:405.[ISI][Medline]
  30. Kearney E. R., Walunas, T. L., Karr, R. W., Morton, P. A., Loh, D. Y., Bluestone, J. A. and Jenkins, M. K. 1995. Antigen-dependent clonal expansion of a trace population of antigen-specific CD4+ T cells in vivo is dependent on CD28 co-stimulation and inhibited by CTLA-4. J. Immunol. 155:1032.[Abstract]
  31. Yoshimoto, T. and Paul, W. E. 1994. CD4 pos, NK1.1 pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3. J. Exp. Med. 179:1285.[Abstract]
  32. Racke, M. K., Bonomo, A., Scott, D. E., Cannella, B., Levine, A., Raine, C. S., Shevach, E. M. and Rocken, M. 1994. Cytokine-induced immune deviation as a therapy for inflammatory autoimmune disease. J. Exp. Med. 180:1961.[Abstract]
  33. Linsley, P. S., Green, J. L., Brady, W., Bajorath, J., Ledbetter, J. A. and Peach. R. 1994. Human B7-1 (CD80) and B7-2 (CD86) bind with similar avidities but distinct kinetics to CD28 and CTLA-4 receptors. Immunity 1:793.[ISI][Medline]
  34. Khattri, R., Auger, J. A., Griffin, M. D., Sharpe, A. H. and Bluestone, J. A. 1999. Lymphoproliferative disorder in CTLA-4 knockout mice is characterized by CD28-regulated activation of Th2 responses. J. Immunol. 162:5784.[Abstract/Free Full Text]
  35. Bieganowska, K. D., Ausubel, J. L., Modabber, Y., Slovik, E., Messersmith, W. and Hafler, D. A. 1997. Direct ex vivo analysis of activated fas-sensitive autoreactive T cells in human autoimmune disease. J. Exp. Med. 185:1585.[Abstract/Free Full Text]
  36. Bradley, L. M., Atkins, G. G. and Swain, S. L. 1992. Long-term CD4+ memory T cells from the spleen lack MEL-14, the lymph node homing receptor. J. Immunol. 148:324.[Abstract/Free Full Text]
  37. Mackay, C. R., Marston, W. L. and Dudler, L. 1990. Naive and memory T cells show distinct pathways of lymphocyte recirculation. J. Exp. Med. 171:801.[Abstract]
  38. Ratts, R. B., Arredondo, L. R., Bittner, P., Perrin, P. J., Lovett-Racke, A. E. and Racke, M. K. (1999) The role of CTLA-4 in tolerance induction and T cell differentiation in experimental autoimmune encephalomyelitis: i.v. antigen administration. Int. Immunol. 11:1889.[Abstract/Free Full Text]