Studies in B7-deficient mice reveal a critical role for B7 costimulation in both induction and effector phases of experimental autoimmune encephalomyelitis

Abstract

The importance of B7 costimulation in regulating T cell expansion and peripheral tolerance suggests that it may also play a significant regulatory role in the development of autoimmune disease. It is unclear whether B7 costimulation is involved only in the expansion of autoreactive T cells in the periphery, or if it is also required for effector activation of autoreactive T cells in the target organ for mediating tissue injury and propagating autoimmune disease. In this study, the role of B7-CD28 costimulation and the relative importance of B7 costimulators for the induction and effector phases of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide were examined. Wild-type, B7-1/B7-2-deficient mice, or CD28-deficient C57BL/6 mice were immunized with MOG 35-55 peptide. Mice lacking both B7-1 and B7-2 or CD28 showed no or minimal clinical signs of EAE and markedly reduced inflammatory infiltrates in the brain and spinal cord. However, mice lacking either B7-1 or B7-2 alone developed clinical and pathologic EAE that was comparable to EAE in wild-type mice, indicating overlapping functions for B7-1 and B7-2. Resistance to EAE was not due to a lack of induction of T helper type 1 (Th1) cytokines, since T cells from B7-1/B7-2(-/-) mice show reduced proliferative responses, but greater interferon gamma production compared with T cells from wild-type mice. To study the role of B7 molecules in the effector phase of the disease, MOG 35-55-specific T lines were adoptively transferred into the B7-1/B7-2(-/-) and wild-type mice. Clinical and histologic EAE were markedly reduced in B7-1/B7-2(-/-) compared with wild-type recipient mice. These results demonstrate that B7 costimulation has critical roles not only in the initial activation and expansion of MOG-reactive T cells, but also in the effector phase of encephalitogenic T cell activation within the central nervous system.

Figure 1

EAE disease course in C57BL/6 B7-1^−/− mice (A; n = 34), B7-2^−/− mice (B; n = 22), B7-1/B7-2^−/− mice (C; n = 18), and CD28^−/− mice (D; n = 13). Filled diamonds in A–D, wild-type (Wt) mice. Mice were immunized with 100 μg of MOG 35-55 subcutaneously and injected twice with 200 ng of pertussis toxin intravenously. Mice were observed daily and scored as described in Materials and Methods. Data are pooled from three to five experiments and represent mean clinical score of each group plotted against time.

Figure 2

CNS histopathology. Representative fields from the anterior lumbar spinal cord of wild-type (A), B7-2^−/− (B), and B7-1/B7-2^−/− (C) mice from day 35 after immunization. In A and B, there are many mononuclear inflammatory cells in the leptomeninges and infiltrating the parenchyma. In C, there is a small meningeal inflammatory focus (arrow), but the parenchyma is not infiltrated. All are stained with Luxol fast blue–hematoxylin and eosin. Original magnifications: 280×.

Figure 3

Proliferation and cytokine production of lymph node cells from B7-1^−/− and B7-2^−/− mice challenged with MOG 35-55. Mice were immunized with 100 μg of MOG 35-55 in CFA. 10 d later, draining lymph node cells from wild-type (Wt, diamonds), B7-1^−/− (squares), or B7-2^−/− (triangles) mice were restimulated in vitro with a titration of MOG 35-55 (filled symbols) or MOG 92-106 (open symbols) as control. Culture supernatants were collected after 48 h from cultures stimulated with 50 μg/ml of MOG 35-55 or with media alone. Cytokines were measured by sandwich ELISA, and background levels from media controls were subtracted. Data are representative of eight independent experiments.

Figure 3

Proliferation and cytokine production of lymph node cells from B7-1^−/− and B7-2^−/− mice challenged with MOG 35-55. Mice were immunized with 100 μg of MOG 35-55 in CFA. 10 d later, draining lymph node cells from wild-type (Wt, diamonds), B7-1^−/− (squares), or B7-2^−/− (triangles) mice were restimulated in vitro with a titration of MOG 35-55 (filled symbols) or MOG 92-106 (open symbols) as control. Culture supernatants were collected after 48 h from cultures stimulated with 50 μg/ml of MOG 35-55 or with media alone. Cytokines were measured by sandwich ELISA, and background levels from media controls were subtracted. Data are representative of eight independent experiments.

Figure 4

Proliferation and cytokine production of lymph node cells from B7-1/B7-2^−/− mice challenged with MOG 35-55. Mice were immunized with 100 μg of MOG 35-55 in CFA. 10 d later, draining lymph node cells from wild-type (diamonds) or B7-1/B7-2^−/− (circles) mice were restimulated in vitro with a range of MOG 35-55 peptide concentrations. Supernatants were collected after 24, 48, and 72 h from cultures stimulated with 50 μg/ml of MOG 35-55 or with media alone. Cytokines from wild-type (Wt, solid bars) and B7-1/B7-2^−/− (hatched bars) cultures were measured by sandwich ELISA, and background levels from media controls were subtracted. Data are representative of eight independent experiments.

Figure 4

Proliferation and cytokine production of lymph node cells from B7-1/B7-2^−/− mice challenged with MOG 35-55. Mice were immunized with 100 μg of MOG 35-55 in CFA. 10 d later, draining lymph node cells from wild-type (diamonds) or B7-1/B7-2^−/− (circles) mice were restimulated in vitro with a range of MOG 35-55 peptide concentrations. Supernatants were collected after 24, 48, and 72 h from cultures stimulated with 50 μg/ml of MOG 35-55 or with media alone. Cytokines from wild-type (Wt, solid bars) and B7-1/B7-2^−/− (hatched bars) cultures were measured by sandwich ELISA, and background levels from media controls were subtracted. Data are representative of eight independent experiments.

Figure 5

EAE induction in wild-type and B7-1/B7-2^−/− mice by adoptive transfer of wild-type T cells. Wild-type mice were immunized with MOG 35-55, and draining lymph nodes were harvested 10 d later. Lymphocytes were cultured in MOG 35-55 and IL-12 for 4 d and then transferred into wild-type (Wt, n = 16) and B7.1/B7.2^−/− (n = 13) recipients. Data are pooled from two experiments and represent mean clinical score plotted against time. B7-1/B7-2^−/− mice developed significantly less severe disease compared with wild-type. *P < 0.05 by the Mann-Whitney test.