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The Journal of Immunology, 2003, 170: 4389-4396.
Copyright © 2003 by The American Association of Immunologists

B7-1 Costimulatory Molecule Is Critical for the Development of Experimental Autoimmune Myasthenia Gravis1

Mathilde A. Poussin2, Erdem Tüzün, Elzbieta Goluszko, Benjamin G. Scott, Huan Yang, Juan U. Franco and Premkumar Christadoss3

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Following immunization with acetylcholine receptor (AChR), MHC class II-restricted, AChR-specific CD4 cell activation is critical for the development of experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 mice. To study the contributions of B7-1 and B7-2 costimulatory molecules in EAMG, B7-1, B7-2, and B7-1/B7-2 gene knockout (KO) mice were immunized with Torpedo AChR in CFA. Compared with wild-type C57BL6 mice, B7-1 and B7-1/2 KO mice were resistant to EAMG development. B7-1 KO mice had reduced anti-AChR Ab compared with C57BL/6 mice. However, neither B7-1 nor B7-2 gene disruption impaired AChR-induced or dominant {alpha}146–162 peptide-induced in vitro lymphoproliferative responses. Blocking of the B7-1 or B7-2 molecule by specific mAbs in vivo led to a reduction in the AChR-specific lymphocyte response, and the reduction was more pronounced in mice treated with anti-B7-2 Ab. The findings implicate B7-1 molecules as having a critical role in the induction of EAMG, and the resistance of B7-1 KO mice is associated with suppressed humoral, rather than suppressed AChR-specific, T cell responses. The data also point to B7-2 molecules as being the dominant costimulatory molecules required for AChR-induced lymphocyte proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Myasthenia gravis (MG)4 is a chronic autoimmune disease characterized by muscular fatigability resulting from a disturbance of the neuromuscular transmission. The main immunological feature appears to be, in most cases, the presence of serum autoantibodies directed against acetylcholine receptor (AChR). Abs to AChR and complement mediate the neuromuscular damage.

EAMG is induced in susceptible strains of mice by s.c. immunizations with an emulsion of AChR and CFA. Mice subsequently produce serum anti-AChR Ab and develop muscle weakness and fatigability mimicking the human disease. This model has been extensively studied to determine the factors that could be involved in the development of this autoimmune disease (1). The production of autoantibodies to AChR is dependent on the help of T cells and results from complex, prior cellular activation events. MHC class II-restricted CD4 cells are crucial in EAMG pathogenesis (2, 3), which has also been shown to require complex cytokine regulation, with TNF, IL-6, and IL-18 playing a dominant role (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Activation of T cells requires two signals. The first is provided by the cognate TCR/MHC peptide interaction. A second costimulatory signal is mediated through ligation of CD28 or CD152 (CTLA-4) by the costimulatory B7 molecules. Mouse B7-1 (CD80) and B7-2 (CD86) molecules have respective molecular masses of 55 and 80 kDa and are expressed mainly on APC. The mechanisms by which B7-1 and B7-2 molecules regulate the immune system at the cellular and molecular levels are becoming clearer. A study using CD28-knockout (CD28-KO) and CD40 ligand-KO mice has revealed the differential requirement of B7/CD28 and CD40/CD40 ligand interactions in EAMG development (16). Another study reported that the immune response to AChR was suppressed by blocking the B7-1 and B7-2 costimulatory molecules with CTLA4 Ig (17). However, the respective involvement of B7-1 and B7-2 molecules in the pathogenesis of EAMG is still not understood.

In the present study we combined two complementary approaches to assess the respective roles of B7-1 and B7-2 in the immune response to AChR and EAMG. First, three genetically altered mouse strains (B7-1 KO, B7-2 KO, and B7-1/2 double KO) were used to examine and compare the contributions of the costimulatory molecules in the development and progression of EAMG. Second, B7-1 and B7-2 molecules were blocked in vitro or in vivo with Ab to transiently suppress the function of these molecules and further study their influence. Our findings implicate the B7-1 molecule as having a critical role in the development of the humoral immune response to AChR and clinical EAMG. Although B7-2 molecules are less critical for the development of clinical EAMG, they are important for the activation of AChR-specific T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 and B6.C-H2bm12/KhEg (bm12) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B10.TgE{alpha}k (B10.A{alpha}b:{beta}b.E{alpha}k:{beta}b) mice, expressing both I-A and I-E molecules, were a gift from Dr. C. David (Mayo Clinic, Rochester, MN) (18). B7-1-deficient (B7-1 KO) and B7-2-deficient (B7-2 KO) mice as well as mice simultaneously deficient in B7-1 and B7-2 (B7-1/2 double KO) mice (19, 20) were provided by Dr. A. Sharpe (Harvard University, Boston, MA). Mice were bred and maintained under viral Ab-free conditions in the animal facilities of the University of Texas Medical Branch. Mice were 8–10 wk old when used in the experiments. Animal experimental procedures were in compliance with institutional animal care and use committee guidelines.

AChR

AChR was purified from Triton X-100 extracts of electric organs from Torpedo californica by affinity chromatography on a conjugate of neurotoxin coupled to agarose as previously described (21, 22).

Induction and evaluation of clinical EAMG

Groups of mice (n = 7–10) were immunized s.c. in both hind footpads and on two shoulder regions with 20 µg of AChR/CFA (Difco, Detroit, MI). Each site received ~5 µg of AChR/CFA in a volume of 50 µl of emulsion. Mice were boosted on day 30 with 20 µg of AChR/CFA in the shoulder and thigh regions (four sites). These mice were assessed for the characteristic symptoms of EAMG. Muscle weakness is graded as follows. Grade 1 is defined as normal at rest, but weak with the chin on the floor and inability to raise the head and hunched back after exercise consisting of 20–30 consecutive paw grips. Grade 2 is defined as grade 1 weakness at rest. Grade 3 is defined as a moribund, dehydrated, and paralyzed condition (22, 23). Sera were collected on days 14 and 44 after the first immunization for the measurement of anti-AChR Ab. At termination, lymph node cells (LNC) were collected, and their proliferative and cytokine responses to AChR and dominant peptide {alpha}146–162 were assessed in vitro.

RIA for anti-muscle AChR Ab

The serum levels of autoantibodies to mouse muscle AChR (M-AChR) were measured using an established RIA (21, 22). M-AChR (1 x 10-9 M) was incubated in Triton buffer with [125I]{alpha}-bungarotoxin (2 x 10-9; Amersham Pharmacia Biotech, Arlington Heights, IL) for 4 h at 4°C. To 1 ml of a solution of M-AChR, 1 µl of serum from experimental mouse and 10 µl of normal mouse serum (as a carrier) were added. Samples with normal mouse serum alone were used as controls. After overnight incubation at 4°C, rabbit serum (50 µl) containing anti-mouse Igs was added. After incubation for 4 h at room temperature the tubes were centrifuged, and the pellets were washed with 1 ml of Triton buffer. The tubes were centrifuged again, the supernatants were discarded, and the pellets were counted in a gamma counter (Packard, Downers Grove, IL). The anti-AChR Ab level (moles of {alpha}-bungarotoxin binding sites bound per liter of serum) was calculated from the difference in the counts of M-AChR precipitated in the experimental and control samples.

Ag-specific lymphoproliferative assay

Mice were immunized at the base of tail with 20 µg of AChR in 200 µl of CFA emulsion (AChR/CFA). Seven days after immunization the mice were euthanized, and draining para-aortic and inguinal LNC were collected. LNC were cultured in 0.2 ml of RPMI at 4 x 105 cells/well in 96-well, flat-bottom microtiter plates with and without AChR (2.5 µg/ml) or AChR {alpha}-chain peptide {alpha}146–162 (20 µg/ml). This peptide was synthesized in the Protein Chemistry Laboratory at University of Texas Medical Branch. Proliferation was evaluated by measuring [3H]thymidine incorporation as previously described (8, 23). Readings were performed using a Packard beta counter Matrix 9600.

Cytokine ELISA

LNC from AChR-immunized mice were collected and cultured in the presence of medium alone, AChR, or {alpha}146–162 peptide. After 3 days of culture, supernatants were collected and tested for cytokine production. ELISA plates (Dynatech, Chantilly, VA) were coated with 0.1 µg/well mAb to IFN-{gamma} and IL-10 (BD PharMingen, San Diego, CA) according to the manufacturer’s recommendations. Plates were blocked with 10% FBS in PBS. Supernatant samples were then added to the plates in duplicate and incubated for 2 h at room temperature. A secondary biotinylated Ab was then added (0.1 µg/well). Development of coloration was conducted using avidin-peroxidase (Sigma-Aldrich, St. Louis, MO), followed by the substrate consisting of 0.3 mg/ml ABTS (Roche, Indianapolis, IN). Color development occurred at room temperature in the dark. Serially diluted recombinant cytokines were used to establish a standard curve. Plates were read at OD405, and results were expressed as cytokine concentration (picograms per milliliter) (23).

Mitogenic stimulation of splenocytes

Splenocytes from five naive B7-1 KO, B7-2 KO, or C57BL/6 mice were seeded into 96-well cell culture plates at a concentration of 4 x 105 cells/well and stimulated for 72 h in triplicate with 10 µl of PBS (control), 0.25–1.0 µg/ml LPS, or 1.25–5 µg/ml Con A. At 18–20 h before harvesting, cells were pulse-labeled with 0.1 µCi [3H]thymidine/well. 3H incorporation was measured using a beta scintillation counter (Beckman, Fullerton, CA).

In vitro B7 blockade assay

C57BL/6 mice were immunized at the base of tail with 20 µg of AChR/CFA. Seven days after immunization the mice were euthanized, and LNC were collected. LNC were cultured with 2.5 µg/ml AChR, and supernatants were added from hybridomas producing Abs to either B7-1 (clone 1G10) or B7-2 (clone GL-1) or to mouse IgD (clone 11-26C) as a control. The crude supernatants were diluted serially from 1/4 to 1/20 in culture medium. Proliferation was measured as described above using [3H]thymidine incorporation, and the inhibitory effect of the Abs was expressed as the percentage of specific inhibition (% inhibition with Ab to B7 - % inhibition with 11-26C).

In vivo B7 blockade of the response to AChR

C57BL/6 mice were immunized at the base of tail with 20 µg of AChR/CFA. Mice were injected i.p. with 100 µg of purified Ab to B7-1 (clone 1G10), B7-2 (clone GL-1), or mouse IgD (clone 11-26C) on days 0 and 3 after immunization. Seven days after immunization the mice were euthanized, and LNC were collected. LNC were cultured with no Ag, 2.5 µg/ml AChR, or 20 µg/ml peptide {alpha}146–162. Proliferation was measured by [3H]thymidine incorporation.

Flow cytometric analysis

Mice were immunized s.c. at the base of the tail with 20 µg of AChR/CFA in a final volume of 200 µl of emulsion. One week after immunization the mice were killed, and their draining para-aortic and inguinal LNC were collected and stained for the expression of CD4, CD25, CD28, CD80 (B7-1), or CD86 (B7-2) molecules (all Ab from BD PharMingen) as described previously (8, 9).

Statistical analysis

To determine the significance of the observed results, two statistical tests were used. Incidences of EAMG were compared using Fisher’s exact test. Proliferative responses, cytokine production, and cell surface marker expressions were compared using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
B7-1 gene deficiency protects H-2b mice from developing AChR-induced clinical EAMG

To determine the effects of B7-1 and B7-2 costimulations on the development of AChR-induced EAMG, we immunized C57BL/6, B7-1 KO, B7-2 KO, and B7-1/B7-2 KO mice with 20 µg of AChR in CFA on day 0 and again on day 28. As shown in Table I, the incidence of clinical EAMG was suppressed in B7-1 KO mice in both experiments. In the first experiment B7-1 KO mice were significantly more resistant than B7-2 KO mice (p = 0.0204), and their incidence of disease was not different from that of B7-1/2 double KO mice (p = 0.5294). In the second experiment B7-1 KO mice were more resistant than B7-2 KO mice (p = 0.00497) or C57BL/6 mice (p = 0.0196), and the susceptibility of B7-2 KO mice was not different from that of C57BL/6 mice (p = 0.4235). However, B7-2 KO mice had less severe disease compared with B6 mice. The data indicate that B7-1 costimulation has a critical role in the pathogenesis of EAMG.


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  		Table I. B7-1 gene disruption reduces the incidence and severity of T-AChR-induced clinical EAMG

 
B7-1 and B7-2 gene disruption impairs the AChR-specific Ab response

Anti-AChR Abs are a central factor in EAMG pathology. To assess whether the reduction of clinical signs of EAMG was associated with lower levels of anti-AChR Ab, sera from AChR-immunized mice were collected 14 days after the first and 14 days after the second immunization. As shown in Fig. 1, we detected very little serum anti-AChR Ab, and there was no difference in the levels of anti-AChR Ab between strains 14 days after the first AChR immunization (p > 0.05 on day 14). However, 44 days after the first AChR immunization (14 days after the second), B7-1 KO mice had lower circulating anti-AChR Ab than B7-2 KO (p = 0.0178) or C57BL/6 mice (p = 0.0044). Also anti-AChR Ab were lower in B7-2 KO mice compared with C57BL/6 mice. However, this difference did not reach significance (p = 0.0756). The findings indicate that the increased resistance of B7-1 KO mice is associated with a decreased Ab response to AChR.



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  		FIGURE 1. Anti-AChR Ab response in AChR-immunized B7-1 KO, B7-2 KO, and C57BL/6 (B6) mice. Sera were collected 2 wk after the first immunization (day 14) and 2 wk after the second immunization (day 44). Results were compared using Student’s t test (*, p < 0.05). The data in the figure represent the cumulative results of two independent experiments.

 
Disruption of either the B7-1 or B7-2 gene does not impair the AChR- or peptide-induced in vitro lymphoproliferative response, but differentially affects the cytokine response

To determine whether the decreased susceptibility to EAMG in B7-1 KO mice results from the inability to evoke T cell responses to AChR and its dominant {alpha}146–162 peptide, which is involved in EAMG pathogenesis (23), draining LNC were collected from AChR-immunized mice at the termination of the experiment and restimulated in vitro with AChR or the dominant peptide {alpha}146–162. Fig. 2A shows that B7-1 KO and B7-2 KO mice exhibited a similar lymphoproliferative response to AChR (p = 0.935) and peptide {alpha}146–162 (p = 0.191). However, B7-1/2 double KO mice had a totally suppressed response compared with B7-1 KO and B7-2 KO mice (p < 0.001). Since B7-1 KO and B7-2 KO mice exhibited a similar proliferative response, the data suggest that the resistance of B7-1 KO mice compared with that of B7-2 KO mice does not result from the absence of a T cell response to AChR or to dominant peptide {alpha}146–162. To further assess the cellular response, LNC from AChR-immunized B7 KO mice were stimulated with AChR {alpha}146–162 peptide, and supernatants were collected after 72 h of incubation. Levels of IFN-{gamma} and IL-10 released in the supernatant were measured by ELISA. As shown in Fig. 2, B and C, B7-1/2 double KO mice had a suppressed AChR and {alpha}146–162 peptide-specific IFN-{gamma} and IL-10 responses (p < 0.05), compared with the strains with isolated B7-1 or B7-2 gene deficiency. Additionally, B7-1 KO mice exhibited reduced IFN-{gamma} production compared with B7-2 KO mice after in vitro stimulation with the dominant peptide {alpha}146–162 (p = 0.023) or AChR (p = 0.047).



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  		FIGURE 2. Effect of a deficiency in B7 costimulatory molecules on the response to AChR in vitro. Mice deficient in B7-1 (B7-1 KO), B7-2 (B7-2 KO), or both (Dble KO) were immunized s.c. with AChR/CFA to induce clinical EAMG. At termination, draining LNC were collected and restimulated in vitro with immunodominant peptide {alpha}146–162 or whole AChR molecule. The parameters studied were the lymphoproliferative response (A), IFN-{gamma} release (B), and IL-10 release (C). Results are expressed for individual mice as a stimulation index for the lymphoproliferative response and in picograms per milliliter for cytokine production (the numbers of mice for each group (B7-1 KO (n = 8), B7-2 KO (n = 9), and Dble KO (n = 9) mice), and each stimulation type (medium, AChR, or {alpha}146–162) in all panels (A–C) is the same. Results were compared using Student’s t test.

 
Lymphocytes from B7-1 KO mice exhibit normal mitogenic responses

A possibility to consider was that the B7-1 and B7-2 gene deletions could somehow confer a generalized reduction in lymphocyte responsiveness. To rule out this possibility, the splenocytes of B7-1 KO, B7-2 KO, and C57BL/6 mice were stimulated in vitro with the T and B cell mitogens Con A and LPS. B lymphocytes from both B7-1 and B7-2 KO mice displayed intact proliferative responses following stimulation with varying doses of LPS. This implies that the reduced production of AChR-specific Ab in B7-1 KO mice cannot be explained by a deficit in B cell responsiveness. T lymphocytes from B7-1 KO and C57BL/6 mice proliferated vigorously in response to Con A stimulation, although the T lymphocyte responses of B7-2 KO mice were partially suppressed (Fig. 3), revealing that B7-1 gene deletion does not generate lymphocyte dysfunction. Therefore, since both their B and T lymphocytes exhibit normal responsiveness in vitro, the resistance to EAMG exhibited by B7-1 KO mice is specifically due to the defect of costimulatory signaling between B and T lymphocytes.



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  		FIGURE 3. Splenocytes of B7-1 KO and B7-2 KO mice respond normally to mitogenic stimulation. Splenocytes from naive KO or wild-type mice were collected and stimulated in vitro for 72 h with PBS (control) or varying concentrations of LPS or Con A. Proliferation was determined by measuring thymidine incorporation. Results were evaluated by Student’s t test (*, p < 0.05). SE bars are shown. B7-1 and B7-2 KO mice display an intact response to B mitogen (LPS) stimulation, but B7-2 KO mice appear to possess a partially reduced response to T cell mitogen (Con A) stimulation.

 
B7-2 has a leading costimulatory role in regulation of the proliferative response to AChR in vitro

In KO mice a targeted deficiency can sometimes be compensated for by the use of a molecule with a similar activity. To evaluate the respective influences of the costimulatory molecules B7-1 and B7-2 in the lymphocyte response to AChR in mice with B7-1 and B7-2 molecules intact from birth, we first used Ab to B7-1 and B7-2 to block the in vitro lymphoproliferative response to AChR. Draining LNC were collected 1 wk after immunization of C57BL/6, bm12, or B10.TgE{alpha}k mice with AChR/CFA and stimulated in vitro with AChR in the presence of B7-1 or B7-2 blocking Ab or isotype-matched control Ab. As shown in Table II, although B7-1 blocking had an efficient inhibitory effect on the lymphoproliferative response to AChR, blocking of B7-2 had a more pronounced inhibitory effect in EAMG-susceptible C57BL/6 mice. Even mouse strains (bm12 and B10.TgE{alpha}k) that are relatively resistant to EAMG demonstrated a similar suppression of their AChR-specific lymphocyte response in the presence of B7-1 or B7-2 blocking Ab, with a major effect for B7-2 suppression. This also indicates that although B7-1 costimulatory molecules exhibit a central role in the pathogenesis of EAMG, B7-2 costimulation is mainly involved in the proliferative response of AChR-specific T cells.


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  		Table II. In vitro inhibition of the lymphoproliferative response to AChR from LNC from AChR-immune mice by antibodies to B7-1 and B7-2

 
Blocking of B7-1 or B7-2 in vivo causes a reduction in the lymphocyte proliferation response

The in vitro blocking of the B7 molecule only has an effect on the late response to AChR. It allows study of the effect of antigenic restimulation of AChR-immune cells. However, priming of lymphocytes to AChR occurs in vivo. To counteract the influence of B7-1 and B7-2 in vivo, immediately after immunization with AChR/CFA C57BL/6 mice were treated i.p. with B7-1 or B7-2 blocking Ab on days 0 and 3 after AChR immunization. After 1 wk draining LNC were collected, and the in vitro lymphocyte responses to AChR and peptides of the {alpha}-chain of AChR were evaluated. As shown in Fig. 4, blocking of B7-1 or B7-2 in vivo led to a significant reduction in the lymphoproliferative response to AChR or peptide {alpha}146–162 in vitro (p < 0.001). However, there was no difference in the inhibition induced by Ab to B7-1 or B7-2. These results clearly indicate that B7 receptors play a crucial role in the priming of lymphocytes.



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  		FIGURE 4. Effect of in vivo blocking of B7 costimulatory molecules on the short term immune response to AChR. C57BL/6 mice were treated i.p. with 100 µg of Ab to B7-1 or B7-2 or with isotype-matched control Ab on the day of immunization (D0) and on day 3. The lymphoproliferative response was assessed on day 7. One representation of three independent experiments is shown.

 
Administration of anti-B7-1 or anti-B7-2 Abs reduces CD4+ and CD4+CD25+ cells in lymph nodes

To further elucidate the cellular effects of Ab directed against B7 receptors, we immunized C57BL/6 mice at the base of the tail with 20 µg of AChR/CFA in a final volume of 200 µl of emulsion and treated them i.p. with blocking Ab to B7-1 or B7-2 on days 0 and 3 after immunization. After 1 wk draining LNC were collected and evaluated by FACS analysis for the expression of CD4, CD25, CD28, CD80 (B7-1), or CD86 (B7-2) molecules. As illustrated in Fig. 5, mice treated with B7 blocking Ab revealed significantly reduced percentages of CD4+ (p = 0.018 with anti-B7-1 and p = 0.019 with anti-B7-2) and CD4+CD25+ (p = 0.039 with anti B7-1 and p = 0.048 with anti-B7-2) cells compared with mice treated with control Ab (control). The treatment with anti-B7-1 or anti-B7-2 Ab induced a statistically identical effect. Interestingly, blocking Ab did not cause a significant change in B7-1, B7-2, or CD28 molecule expression, which might suggest that the Ab only transiently inhibit the functions of B7 molecules (probably inducing functional blocking or an internalization) without depletion of the B7-1- or B7-2-expressing cells. It is interesting to note that B7 blocking in vivo led to a reduced number of CD4+ CD25+ cells implicated in autoimmune disease regulation.



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  		FIGURE 5. Effect of in vivo treatment with anti-B7-1 or anti-B7-2 Ab on the expression of the cell surface markers CD28, CD4, CD25, B7-1, and B7-2. Treatment of C57BL/6 mice with anti-B7-1 or B7-2 Ab causes a reduction in CD4+ and CD4+CD25+ T cells without affecting B7-1, B7-2, or CD28 expression. Results were compared using Student’s t test. One representation of three independent experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The critical role of CD28/CTLA4 molecules has been demonstrated in experimental autoimmune encephalomyelitis (EAE) (24, 25, 26, 27) and in a variety of other autoimmune diseases, including collagen-induced arthritis (28), thyroiditis (29), and MG (17). There is an accumulated body of evidence that B7-1 and B7-2 molecules have differential roles in autoimmunity. For instance, anti-B7-1 Ab, but not anti-B7-2 Ab, decreases disease severity during the first phase of EAE (25, 26, 27, 28, 29, 30). Also, treatment of relapsing-remitting EAE with anti-B7-1 Ab reduces disease severity, whereas treatment with anti-B7-2 Ab aggravates the disease (27, 30). A similar discrepancy exists in experimental murine diabetes and lupus, whereas anti-B7-2 Ab prevents disease expression, and treatment with anti-B7-1 Ab worsens the clinical course (31, 32).

Our finding indicates that B7-1 KO and B7-1/2 double KO mice are resistant to AChR-induced EAMG, while B7-2 deficiency has little effect on the development of EAMG (Table I). It is also evident from our results that the deficiency of B7-1 alone is sufficient to prevent disease initiation. This suggests that B7-1 costimulation must intervene earlier than B7-2 costimulation in the immune response to AChR. Therefore, like other autoimmune diseases, B7-1 and B7-2 are differentially required for EAMG induction. Although B7-1, but not B7-2, molecule deficiency suppressed the anti-AChR Ab response significantly following immunization with AChR (Fig. 1), it did not affect the in vitro lymphocyte responses to AChR (Fig. 2A). Therefore, the reduction of the susceptibility to AChR-induced EAMG in B7-1 KO mice cannot be explained by an absence of primary response to AChR. Thus, B7-1 and B7-2 molecules must be involved in the cellular communication between T and B cells, leading to anti-AChR Ab production, with a more important role for B7-1. T and B cells of B7-1 KO mice gave responses as powerful as the responses of C57BL/6 mice to nonspecific mitogen Con A or LPS. This indicates that there is no functional deficiency in B and T cells from B7-1 KO mice, as evaluated by the proliferative response. This finding further supports the assumption that the B7-1 molecule is critical for the costimulation events leading to the production of pathogenic anti-AChR Ab by B cells.

In contrast with congenital deficiency of the B7-1 receptor, both in vivo (Fig. 4) and in vitro (Table II) mAb blocking of B7-1 in C57BL/6 mice efficiently prevented lymphocyte proliferation. This inconsistency between acquired and congenital forms of B7-1 dysfunction may be explained by some compensatory mechanisms taking place in B7-1 KO mice. It is probable that in B7-1 KO mice the B7-2 molecule acts to replace part of the function of the B7-1 molecule. This is corroborated by the fact that B7-1/2 double KO mice exhibit a totally suppressed proliferative as well as a AChR-specific cytokine in vitro response.

Additionally, in vitro evaluation of the proliferative responses of lymphocytes might not genuinely reflecting the long term, in vivo status of an ongoing autoimmune disease. B7-1 and B7-2 molecules have been shown to have different expression kinetics (31, 33, 34). B7-2 seemed to be the dominant B7 molecule in the early stages of immunological response, whereas B7-1 had a more significant role in the continuation and preservation of the T cell response to immunogens (35). Accordingly, administration of anti-B7-2 Ab prevented experimental diabetes only when they were given in the earlier stages of autoimmune disease (32). Moreover B7-1 production was not restricted to immunological cells, and various different tissues, including endothelial cells, expressed this molecule (35). Other than lymphocyte interactions, B7-1 expression might also play a role in tissue response (from Ag presentation with MHC molecules to inflammatory events) (35, 36). Therefore, a reduction in AChR-specific B cell stimulation might not be the only disturbance in B7-1 KO mice. As indicated by their reduced IFN-{gamma} response (Fig. 2B), a diminished inflammatory tissue response in B7-1 KO could also explain the reduction of EAMG susceptibility as well as why CD28 KO (16) and B7-2 KO mice (both strains lacking essential molecules in lymphocyte activation) are only partially resistant to EAMG, whereas B7-1 deficiency renders mice almost totally resistant. Although it is highly possible that the suppressed IFN-{gamma} production of B7-1 KO mice might have contributed to disease resistance, we do not believe that this has constituted a major effect on EAMG induction. In our experiments, B7-1 KO mice were almost completely resistant to disease. However, even complete deficiency of IFN-{gamma} does not render mice entirely resistant to EAMG (4, 6, 7). Therefore, we do not suggest that a relatively small decrease in IFN-{gamma} production could have made a major contribution to resistance against EAMG.

Another interesting finding was that in vitro blockade of B7-2 prevented the proliferation of AChR-immune LNC to AChR challenge more efficiently than B7-1 in a few mouse strains, with different MHC class II alleles. In contrast to B7-1, which in our system appears to play a more crucial role in the initiation of AChR-induced pathogenesis, B7-2 seems to be more important for in vitro lymphocyte proliferation of AChR-immune cells. This is possibly because this molecule is more important in memory T cell functions and the recall response to AChR.

One surprising observation was that CD4+CD25+ immunoregulatory T cells appear to be decreased substantially after anti-B7-1 Ab or B7-2 Ab treatment (Fig. 5). CD4+CD25+ T cells are a subset of CD4+ cells that are essential in the maintenance of tolerance to Ags. The absence of these cells results in several autoimmune diseases, such as thyroiditis, gastritis, and diabetes (37). CTLA-4/B7 interactions have also been demonstrated to be important in the regulation of peripheral tolerance (38), and CTLA-4 expression is dependent on the interaction of CD28 and B7 molecules (38). Since we did not test the in vivo effects of anti-B7-1 Ab treatment on the induction of EAMG, we are not certain whether this down-regulation of CD4+CD25+ T cells may adversely affect the clinical course of the autoimmune disease. However, to the best of our knowledge this is the first paper reporting a decrease in CD4+CD25+ cells following anti-B7 Ab treatment (although a similar decrease has been reported in B7 KO mice), which may indicate why anti-B7 treatment is detrimental in animal models of type 1 diabetes (31).

Although by the time the patients present, MG is at an advanced stage, and B7-1 is expected to play an important role at the initiation of the immune response, it is also known that MG patients frequently present with exacerbations, and these exacerbations may continue for months. Therefore, it might be expected that AChR is presented to the immune cells via B7-1 molecule not only at the initiation, but also several times throughout the course of the disease. However, we only demonstrated that blockade of the B7-1 molecule may affect the induction of EAMG and did not examine the effects of anti-B7 Ab treatment on the course of established disease. Therefore, we strongly recommend that the role of B7 molecules in the clinical course and the exacerbations of MG be investigated in future studies.

In this study we have shown that the B7-1 molecule is required for Ab production after AChR immunization and in an AChR-reactive immune system response, B7-2 molecule plays an important role in reactivation of T cells against AChR. Whatever the reasons for disease resistance of B7-1 KO mice are, our results present B7-1 as a critical molecule in the initiation and development of AChR-induced EAMG. The significance of B7 molecules in both T cell (EAE) (26, 27) and immune complex (lupus) (32)-mediated autoimmune diseases has been previously reported. To our knowledge this report is the first study of the importance of B7 molecules in the induction of a classical Ab-mediated disease, MG.


   Acknowledgments
 
We thank Dr. Arlene Sharpe (Harvard University, Boston, MA) for providing us B7-1, B7-2, and B7-1/B7-2 KO mice for this study.


   Footnotes
 
1 This work was supported by the Muscular Dystrophy Association. M.A.P. was supported by consecutive postdoctoral fellowships from the Association Française contreles Myopathies, Myasthenia Gravis Foundation of America (Kermit E. Osserman, Blanche McClure, and Hilbert Sosin), and The McLaughlin Foundation and a Muscular Dystrophy Association Research Career development award recipient. E.T. is a Myasthenia Gravis Foundation Osserman/Sosin/McClure postdoctoral fellow. B.G.S. is a Myasthenia Gravis Foundation Henry Viets fellow. H.Y. is a recipient of the Muscular Dystrophy Association Research Career Development Award. Back

2 Current address: University of Pennsylvania Medical Center, Department of Microbiology, 425 Johnson Pavillion, Philadelphia, PA 19104-6076. Back

3 Address correspondence and reprint requests to Dr. Premkumar Christadoss, Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1070. E-mail address: pchrista{at}utmb.edu Back

4 Abbreviations used in this paper: MG, myasthenia gravis; AChR, acetylcholine receptor; EAE, experimental autoimmune encephalomyelitis; EAMG, experimental autoimmune myasthenia gravis; KO, knockout; LNC, lymph node cells; M-AChR, muscle AChR. Back

Received for publication July 18, 2002. Accepted for publication February 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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