Here we report the generation of what we believe to be a new mouse model in which both T and B cells are specific for the same myelin antigen, MOG. We demonstrated that there was active cooperation between T and B cells in these animals: the presence of MOG-specific T cells led to massive production of MOG-specific IgG1 antibody, and MOG-specific B cells also enhanced MOG-specific T cell proliferation and activation. Strikingly, this cooperation resulted in the development of a spontaneous and severe form of EAE characterized by the specific presence of inflammatory foci in the spinal cords and optic nerves of the animals. This lesion distribution pattern is very similar to that of human NMO.
B lymphocytes have been implicated in several autoimmune diseases, mostly because of their capacity to produce specific autoantibodies. B cells and plasma cells are often present in MS lesions (30
), and oligoclonal Ig bands have been observed in the cerebrospinal fluid of many but not all MS patients (32
). The presence of Ig and products of complement activation cascade deposited around the vessels suggests a pathogenic role for autoantibodies in NMO (8
). In addition, a serum IgG autoantibody (NMO-IgG) serves as a specific marker for NMO (34
). Recent work has shown that the NMO-IgG is not specific for myelin antigens but selectively binds to the aquaporin-4 water channel, a component of the dystroglycan protein complex located in astrocytic foot processes at the blood-brain barrier (35
The high expression of class II MHC and costimulatory molecules by activated B cells suggest that they could also play a role in driving the T cell responses as a result of antigen presentation to large numbers of antigen-specific T cells (36
). In vivo, depletion of B cells from normal mice can reduce the magnitude of the immune response, suggesting that B cells contribute to the T cell response (38
), although primary T cell responses can also develop in B cell–deficient mice (42
). Therefore, a number of reports suggested that antigen-specific B cells induce naive CD4+
T cell proliferation in vivo (43
). The importance of B cells as APCs in some autoimmune diseases such as proteoglycan-induced autoimmune arthritis has recently been demonstrated (46
). In contrast with this arthritis model, EAE is generally considered to be mainly T cell driven (28
). Nevertheless, it is important to note that B lymphocytes are required for the induction of EAE when MOG protein, but not MOG peptide, is used for the induction of EAE (19
). Using a double antigen-specific transgenic model, we demonstrate here that, in contrast to wild-type splenocytes containing the classical APCs, MOG-specific B cells were able to stimulate the proliferation of MOG-specific T cells in the presence of a limited amount of rMOG. Since IgHMOG
B cells only recognize conformationally dependent epitopes of MOG but not MOG aa 35–55 (47
), the enhanced T cell response in the presence of IgHMOG
B cells and MOG most likely resulted from the capture of MOG through B cell surface Ig that caused enhanced presentation of the antigen rather than direct presentation of MOG aa 35–55 epitope.
Ex vivo analysis of T cells from TCRMOG×IgHMOG mice revealed that unless mice exhibited clinical disease, there was no marked increase in the number of activated T cells in the periphery (Figure ). Development of Devic-like disease correlated with the infiltration of both T and B cells into the spinal cord (Figure B and Figure A). The disease was also associated with the presence of lymphoid follicle–like structures in the spinal cords of TCRMOG×IgHMOG mice with Devic-like disease, suggesting an active cooperation between T and B cells in situ (Figure C). Similar to actively induced EAE, we observed that both IL-17– and IFN-γ–producing T cells infiltrated the CNS of TCRMOG×IgHMOG mice with Devic-like disease. Because of the low frequency of spontaneous disease observed in the TCRMOG mice, it was not possible to perform parallel intracellular cytokine staining of CD4+ T cells infiltrating the brains of TCRMOG mice with spontaneous EAE. However, IL-17 mRNA expression was greater in the spinal cords of TCRMOG×IgHMOG mice with Devic-like disease than in TCRMOG mice with EAE. Therefore the increase in IL-17 expression in the CNS of TCRMOG×IgHMOG mice with Devic-like disease might reflect an increase in the number of pathogenic Th-17 cells. Alternatively, we cannot exclude the possibility that other cell types might be responsible for the enhanced IL-17 production. The role of the CNS cytokine milieu and MOG-specific B cells in the differentiation of Th-17 cells is currently under investigation.
Th cells are important for the differentiation of B cells into Ig-secreting plasma cells or long-lived memory B cells. In addition, interaction between antigen-specific T and B cells is required for B cell maturation and Ig isotype class switching. Consistent with the role played by Th cells in the maturation of B cells, we also observed that B cells from TCRMOG
mice underwent class switching from IgM to IgG1 in vivo. Ig switch is dependent on the subset of T cells providing help to B cells, and IgG1 is regarded as a Th2-dependent isotype (48
). In TCRMOG
mice, however, the T cell response seems to be biased toward Th1 cells, since we only detected IFN-γ and not Th2 cytokines in cultures of TCRMOG
mouse spleen cells. In addition, we observed an increase in the number of B7.1-expressing B cells in TCRMOG
mice with Devic-like disease. B7.1 is a costimulatory molecule associated with enhanced Th1 responses. Therefore, the requirement for Th2 responses in IgG1 class switching might not be absolute in this model. In support of this hypothesis are previously published results showing that mice deficient in the Th2-specific cytokine IL-4 have diminished but still robust production of IgG1 (48
). Alternatively, other factors present in the microenvironment might contribute to the IgG1 class switching observed in TCRMOG
mice. Taken together, the TCRMOG
model provides an interesting and, to the best of our knowledge, unique model to study the induction and cooperation of effector autopathogenic T and B cell responses in vivo and in autoimmunity.
mice it is unlikely that MOG-specific antibodies themselves are responsible for the initiation of spontaneous Devic-like disease, since we did not observe a substantial difference in the level of MOG-specific antibodies between healthy and diseased TCRMOG
mice (Figure C), and the transfer of serum from TCRMOG
mice to TCRMOG
mice did not induce EAE or Devic-like disease (data not shown). However, the combination of enhanced antibody production and a robust MOG-specific pathogenic T cell response might be responsible for the fulminant disease observed in TCRMOG
mice. In addition, the antibody and T lymphocyte specificity might play a role in determining the specific lesion distribution pattern as follows: First, the amount of antigen might be important. Previously we reported that there is relatively more MOG expressed in the optic nerves than in the spinal cords of C57BL/6 mice (23
). Here we additionally demonstrated that mRNA for MOG was more abundant in the spinal cords than the brains of these mice. It is therefore possible that a differential expression of MOG protein results in predominant attack of optic nerves and spinal cords in TCRMOG
mice because there are relatively higher levels of the target antigen. Second, the accessibility of the autoantigen might be a critical factor. While the white matter in the spinal cord is on the outside and bathed in the cerebrospinal fluid, the gray matter is on the outside in the cerebellum and cerebrum. In the TCRMOG
mice, the inflammatory infiltrates in the subarachnoid space surrounding the spinal cord and around the optic nerves were more numerous than generally observed in induced EAE models and TCRMOG
mice with spontaneous EAE. Therefore, because MOG antigen might be more accessible in the spinal cords and optic nerves, enhanced MOG-specific T and B cell responses generated in TCRMOG
mice might preferentially attack these CNS areas, resulting in selective lesion distribution similar to that of NMO.
Autoimmune diseases have a complex pathogenesis, and the TCR transgenic mice have proven to be valuable tools to investigate the behavior of self-reactive T cells, their interactions with their antigenic targets, and their induction of disease. Several TCR transgenic mice specific for autoantigens have been generated, and many of the autoantigen epitopes recognized by transgenic T cells have restricted expression in the CNS (49
). Despite the striking increase in the frequency of self-reactive T cells in most of the TCR transgenic mouse lines, however, it is noteworthy that spontaneous autoimmune diseases do not occur in 100% of the animals. For example, 60% of PLP TCR transgenic mice develop spontaneous EAE (50
), and the incidence of spontaneous EAE in MBP aa 1–11–specific TCR transgenic mice ranges from 0% to 14% depending on the line (51
). While 47% of TCRMOG
mice develop spontaneous clinical optic neuritis, only 4% of these animals develop spontaneous EAE (23
). The initiation of disease most likely requires adequate priming of the autoimmune response and/or control of regulatory T cell responses. In one of the MBP-specific TCR transgenic lines, spontaneous disease occurs in 15% of the animals housed in a pathogen-free facility and 43% of those housed in conventional facilities (51
), suggesting a role for infectious agents as triggers of the disease. At the present time, the environmental factor(s) in specific pathogen–free facilities that may initiate spontaneous EAE and are responsible for differences in incidence of spontaneous EAE remain unidentified. Moreover, the mechanisms underlying initiation of spontaneous EAE by infectious agents remain to be elucidated. The inflammatory response generated by infection could trigger the autoreactive T cell response and initiate autoimmune diseases. Alternatively, molecular mimicry between microbial and self antigens has been proposed as a mechanism for the development of EAE (53
). Of particular relevance in this regard to the present study is the fact that there are a large number of molecular homologs of the extracellular domain of MOG. Specifically, the butyrophilin gene products exhibit linear aa sequence homologies with MOG ranging from 35% to 50% and have in some cases been shown to exhibit molecular mimicry with MOG (54
). In TCRMOG
mice, autoreactive MOG-specific T cells may recognize an antigen mimicking MOG that is present in the peripheral immune compartment and then get further activated and amplified by MOG presented by large numbers of MOG-specific B cells. This might then lead to a high frequency of spontaneous disease. Since the restricted expression of MOG to the CNS has recently been challenged (55
), it is also possible that MOG itself expressed in the periphery or released into the peripheral circulation could be presented to autoreactive T cells by MOG-specific B cells very efficiently. Alternatively, the increased incidence of spontaneous EAE observed in MBP TCR transgenic mice crossed to RAG-deficient mice and therefore lacking regulatory T cells expressing endogenous TCR shows the importance of regulatory cells in keeping autoreactive T cells in check (49
). Since only 4% of TCRMOG
mice and 60% of TCRMOG
mice develop spontaneous EAE (23
), we compared the number of regulatory cells between these 2 strains. Although we found that the number of CD4+
T cells was low in TCRMOG
mice (around 2%), it was similar in TCRMOG
mice. Additional experiments are necessary to determine whether B cells can modulate the activity of regulatory cells.
In summary, TCRMOG×IgHMOG mice represent a useful model to determine the factors responsible for different lesion localization and to study the role of T cell/B cell cooperation in the induction of autoimmune pathologies. In addition, since TCRMOG×IgHMOG mice develop a high incidence of spontaneous disease involving both T and B cells, these mice may be useful for testing the biology of T cell/B cell cooperation in the genesis of autoimmune diseases and for testing novel therapies for MS and Devic disease.