The major histocompatibility complex influences myelin basic protein 63-88-induced T cell cytokine profile and experimental autoimmune encephalomyelitis

Polymorphism of the major histocompatibility complex (MHC) influences susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP) in rats. Current concepts relate such influences to the capacity of class II molecules to present relevant peptides to autoreactive T cells. We have here analyzed the MHC influence on the immune response and the development of EAE after immunization with the immunodominant peptide MBP-63–88. Analysis of MHC-congenic LEWIS strains showed that RT1^a, RT1^c and RT1^l haplotypes are permissive for disease induction, whereas RT1^d and RT1^u are resistant. All EAE responding strains showed peptide-specific proliferation and interferon (IFN)-γ secretion, but no early significant tendency to express interleukin (IL-4) or transforming growth factor (TGF)-β mRNA in lymphocytes in response to the MBP 63–88, 7 days post immunization (p.i.). Later, 14 days p.i., peptide-specific induction of IL-4 and TGF-β occurred in RT1^l rats. Among the EAE non-responders strains, only the RT1^u rats showed an immune response to MBP 63-88. This response, however, was qualitatively different from the immune response in the EAE-susceptible strains. Thus, there was no proliferation and only moderate IFN-γ production in response to peptide, but in contrast, a significant and early peptide-induced IL-4 and TGF-β response was observed. The data suggest that the MHC-associated susceptibility to EAE is partly related to the ability to mount a TH1-like immune response while the MHC-associated EAE resistance may either be related to MBP peptide non-responsiveness or to peptide recognition and induction of a qualitatively different and disease down-regulatory immune response.     

Regulatory T cells in experimental allergic encephalomyelitis. III. Comparison of disease resistance in Lewis and Fischer 344 rats

Rats of the Fischer 344 (F344) strain are resistant to experimental allergic encephalomyelitis (EAE) induced by active immunization with guinea pig myelin basic protein (MBP) in complete Freund's adjuvant whereas Lewis (LEW) rats are susceptible even though both strains share the same I-A-like class II alleles of the MHC RT1.B locus. To determine factors that might contribute to this difference in disease susceptibility, we have compared in these two strains (1) the frequency of MBP-reactive T cells in the lymph nodes and spleens of MBP-immunized animals, (2) the dominant MBP epitopes recognized by responding T cells, (3) the ability of MBP-reactive T cells to enter the central nervous system (CNS), and (4) the frequency of CD8+ regulatory T cells (RTC) whose activity is functionally antagonistic to MBP-reactive T cells. The results indicate that MBP-reactive T cell numbers are similar in MBP-immunized F344 and LEW rats, they both recognize p68-88 as the dominant encephalitogenic epitope of MBP, and MBP-reactive T cells isolated from immunized rats and adoptively transferred to naive animals are similarly effective in penetrating the blood-brain barrier and entering the CNS, leading to pathogenesis in EAE. However, the frequency of RTC that functionally inhibit MBP-reactive T cells is greater in F344 rats.     

Serum IgM repertoire reactions to MBP/CFA immunization reflect the individual status of EAE susceptibility

Lewis rats develop experimental allergic encephalomyelitis (EAE) in response to immunization with myelin basic protein (MBP) in CFA, while Fischer rats are usually resistant. These strains, while comparably producing anti-MBP antibodies, also differ in their repertoire reactions to immunization, as measured by patterns of serum IgM reactivity with various autologous proteins. We have now scored IgM repertoire reactions to MBP/CFA immunization after treatments that alter EAE susceptibility in either strain. The results show that abrogation of EAE susceptibility in Lewis rats by a previous experience of T cell-induced passive EAE provoked a novel set of IgM reactivities that otherwise characterized the Fischer's repertoire reaction. Conversely, these reactivities were delayed in the response of Fischer rats that had been rendered EAE-susceptible by cyclophosphamide. Another IgM reactivity with a significant association to individual EAE severity in Lewis rats behaved reciprocally. Together with previous results, these observations suggest that putative regulatory mechanisms concordantly affect EAE resistance and IgM repertoire reactions, operating naturally in Fischer rats and abrogatable by cyclophosphamide treatment, whereas naturally suppressed, but restorable in Lewis rats. Other treatments altering EAE susceptibility, however, did not share these characteristics and may thus be mediated by other mechanisms.     

The myelin basic protein-specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential Vβ8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment

In the Lewis rat, myelin basic protein (MBP)-specific, encephalitogenic T cells preferentially recognize sequence 68–88, and use the Vβ8.2 gene to encode their T cell receptors. To analyze the structural prerequisites for the development of the MBP-specific T cell repertoire, we reconstituted severe-combined immunodeficient (SCID) mice with fetal (embryonic day 15–16) Lewis rat lymphoid tissue, and then isolated MBP-specific T cell lines from the adult chimeras after immunization. Two types of chimera were constructed: SCID mice reconstituted with rat fetal liver cells only, allowing T cell maturation within a chimeric SCID thymus consisting of mouse thymic epithelium and rat interdigitating dendritic cells, and SCID mice reconstituted with rat fetal liver cells and rat fetal thymus grafts, allowing T cell maturation within the chimeric SCID and the intact Lewis rat thymic microenvironment. Without exception, the T cell lines isolated from MBP-immunized SCID chimeras were restricted by MHC class II of the Lewis rat (RT1.B¹), and none by I-A^d of the SCID mouse. Most of the T cell lines recognized the immunodominant MBP epitope 68–88. In striking contrast to intact Lewis rats, in SCID mice reconstituted by rat fetal liver only, MBP-specific T cell clones used a seemingly random repertoire of Vβ genes without a bias for Vβ8.2. In chimeras containing fetal Lewis liver plus fetal thymus grafted under the kidney capsule, however, dominant utilization of Vβ8.2 was restored. The migration of liver-derived stem cells through rat thymus grafts was documented by combining fetal tissues from wild-type and transgenic Lewis rats. The results confirm that the recognition of the immunodominant epitope 68–88 by MBP-specific encephalitogenic T cells is a genetically determined feature of the Lewis rat T cell repertoire. They further suggest that the formation of the repertoire requires T cell differentiation in a syngeneic thymic microenvironment.     

Essential role of TGF-beta in the natural resistance to experimental allergic encephalomyelitis in rats

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease induced in susceptible rat strains by a single immunization with myelin basic protein (MBP). The Lewis (LEW) strain is susceptible to disease induction while the Brown Norway (BN) strain is resistant. This resistance involves non-MHC genes since congenic BN-1L rats, with LEW MHC on a BN-derived background, are also resistant. In the present study we show that, upon immunization with MBP, the non-MHC-encoded resistance to develop clinical EAE in BN-1L rats is associated with a decreased production of IFN-gamma. This may be due to a difference between LEW and BN-1L rats in their ability to produce regulatory cytokines such as IL-4, IL-10 and TGF-beta. In comparison to LEW rats, immune lymph node cells from BN-1L rats express an increased amount of IL-4 mRNA but produce less IL-10. Furthermore, the sera from BN-1L rats contain higher amounts of active TGF-beta1. Therefore, we have investigated the involvement of IL-4 and TGF-beta in the resistance of BN-1L rats to develop EAE using neutralizing mAb. Neutralization of TGF-beta, but not IL-4, renders BN-1L rats susceptible to clinical EAE without affecting the proliferation or the cytokine repertoire of immune lymph node cells. With respect to the origin of the endogenous TGF-beta production, we excluded the involvement of CD8 T cells and discuss a possible role of platelets and of CD4 T cells exhibiting the CD45RC(low) phenotype.     

Minor lymphocyte stimulating (Mls) gene products in mice influence their genetic resistance or susceptibility to induction of autoimmune encephalomyelitis.

The role of the major histocompatibility complex (MHC) gene products in the genetics of experimental autoimmune encephalomyelitis (EAE) is well established. Here we demonstrate how non-MHC gene products, stimulatory to T cells specific to myelin basic protein (MBP), can affect the MHC control in determining genetic susceptibility or resistance to induction of EAE. I-As-restricted MBP-specific T cells derived from SJL/J mice are shown to cross-react with Mls-2a gene products. The Mls-2a gene product expressed by (SJL/J X BALB/c)F1 mice tolerize T cells recognizing I-As/MBP and favor the development of I-Es/d-restricted MBP-specific T cells mediating EAE in the (SJL/J x BALB/c)F1 mice. These I-Es/d/MBP-specific T cells, cross-reactive with Mls-1a, and the I-As/MBP-specific T cells, cross-reactive with Mls-2a gene products, are both eliminated by self tolerance mechanisms in the H-2-matched (SJL/J X DBA/2)F1 mice, expressing Mls-1a2a gene products, and thereby confer genetic resistance to EAE on the (SJL/J X DBA/2)F1 mice bearing EAE-permissive MHC alleles. These results reflect a developmental selection of a T cell repertoire to the self antigen MBP, imposed by self tolerance to self Mls gene products, which affect the genetic susceptibility to EAE. These studies also demonstrate that self tolerance to Mls gene products can strengthen the tolerance to organ-specific self antigens such as MBP, which may not be expressed or which are absent in the thymus at the time of thymic selection.     

The myelin basic protein-specific T cell repertoire in Lewis rats: T cell receptor diversity is influenced both by intrathymic milieu and by extrathymic peptide presentation

In the Lewis rat, the T lymphocyte response to guinea pig myelin basic protein (MBP) is focused almost exclusively on epitopes nested in the MBP peptide sequence p68 – 88, and is dominated by T cell receptors (TCR) using Vβ8.2 gene elements, together with short N(D)N regions. Here we analyzed MBP-specific TCR from Lewis T cells differentiating in chimeric thymuses of Lewis rat/SCID mouse chimeras, in the absence of an intact rat thymic microen vironment (SCID_FL mice). In these T cells, the TCR Vβ repertoire is broad, N(D)N regions are significantly longer, and contain regular rates of template-independent N nucleotides. In striking contrast, a Vβ8.2 biased TCR repertoire and few N-region inserts are seen in p68 – 88-specific, Lewis rat-derived T cells differentiating in the complete rat thymic microen vironment provided by chimeric SCID mice bearing embryonic Lewis thymus grafts (SCID_FL/FT mice). A T cell repertoire resembling the one in SCID_FL mice is used by T cells of intact Lewis rats following immunization with a truncated epitope of MBP, p69 – 86. Also this selection generates a broad TCR Vβ pattern with long N(D)N regions, and higher numbers of N nucleotides. These results show that both intrathymic repertoire selection, and extrathymic peptide priming exert profound effects on the TCR usage in the anti-MBP response of Lewis rats.     

Normal immunoglobulin G protects against experimental allergic encephalomyelitis by inducing transferable T cell unresponsiveness to myelin basic protein

Normal human IgG for intravenous use (IVIg), administered intraperitoneally, protected Lewis rats against experimental allergic encephalomyelitis (EAE) induced by immunization with myelin basic protein (MBP). We demonstrate that protection was associated with an acquired unresponsiveness of lymphocytes to MBP and a decreased ability of the cells to produce IL-2, IFN-γ and TNF-α and, to a lesser degree, IL- 4 and IL-10, in the presence of the antigen. Lymph node (LN) cells of protected rats failed to passively transfer EAE to naive syngeneic animals. Our observations indicate that, rather than inducing selective immune deviation, IVIg induces preferential MBP unresponsiveness of Th1 cells. Whereas LN and splenic cells of IVIg-treated rats did not proliferate nor secrete IL-2 in the presence of the antigen, proliferation was restored by adding exogeneous recombinant IL-2. In contrast, LN cells of IVIg-treated rats proliferated normally and produced IL-2 in the presence of concanavalin A, indicating the selectivity for MBP of the anergy induced by IVIg when given at the time of immunization with the antigen. Treatment with IVIg also allowed a resistance to the secondary induction of EAE, indicating that IVIg protects from EAE but does not interfere with the processes that eventually lead to resistance to re-challenge. These data document the immunomodulatory effects of IVIg in T cell-dependent experimental autoimmune disease and further suggest a role for normal Ig in the selection of functional T cell repertoires.     

Detection of autoreactive T cells in H-2u mice using peptide-MHC multimers

Myelin basic protein (MBP)-specific T cells play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a prototype for T cell-mediated autoimmunity. In PL/J and B10.PL mice (H-2(u) haplotype), the immunodominant epitope of MBP is represented by an N-terminal nonameric peptide, MBP1-9. To date, the MBP1-9-specific T cell repertoire has not been analyzed in quantitative terms. In the present study we demonstrate, using MHC class II tetramers, that 15,000-70,000 self-antigen-specific T(h) cells accumulate in the draining lymph nodes following immunization with spinal cord homogenate or MBP1-9. In contrast, MBP1-9-specific T cells are undetectable in unimmunized H-2(u) mice and represent >60% of the CD4 cells in naive mice transgenic for a TCR specific for this epitope. The results suggest that the extremely low affinity of the N-terminal peptide for I-A(u) does not limit the MBP1-9-specific T cells from expanding into a sizeable pool of autoreactive T cells. Therefore, the primary immune response to MBP1-9 does not differ quantitatively from previously reported CD4(+) T cell responses to foreign antigens.     

Major histocompatibility complex-controlled protective influences on experimental autoimmune encephalomyelitis are peptide specific

The myelin basic protein (MBP) peptide 63–88-induced experimental autoimmune encephalomyelitis (EAE) and its associated T cell cytokine profile are influenced by the rat major histocompatibility complex (MHC). There is an allele-specific protective influence of the MHC class I region, whereas the MHC class II region display either disease-protective or -promoting effects. To investigate if the MHC-associated protection is dependent on certain combinations of MBP peptide and MHC molecules, we have now used another peptide (MBP 89–101). A broader and different set of rat MHC alleles were associated with EAE induced with MBP 89–101 as compared to MBP 63–88. All EAE-susceptible strains mounted peptide-specific strong T helper (Th) 1-like immune responses in vitro. Immunization of rats with an extended peptide (MBP 87–110) induced EAE associated with the same MHC haplotypes as the 89–101 peptide, except in LEW.1N (RT1ⁿ) rats which were relatively resistant. Only this strain responded with additional Th2-like and transforming growth factor-β responses to the peptide in vitro. In vivo depletion of CD8⁺ cells aggravated the disease in this strain. We conclude that both MHC-controlled promoting and protective influences on EAE are dependent on certain MHC/MBP peptide combinations, and that the 87–110 region of MBP contains a major MHC-associated encephalitogenic epitope in the rat.     

Self and non-self peptides treat autoimmune encephalomyelitis: T cell anergy or competition for major histocompatibility complex class II binding?

In susceptible strains of mice, myelin basic protein (MBP) peptide Ac1-11 induces experimental autoimmune encephalomyelitis (EAE) providing a useful model for human multiple sclerosis. Ac1-11 binds major histocompatibility complex (MHC) class II molecules Aα^uAβ^u. Here, we show that the Ac1-11 peptide, when administered intraperitoneally in incomplete Freund's adjuvant (IFA) emulsion, can effectively treat Ac1-11-induced EAE in mice. Treatment with Ac1-11/IFA 9 days after initial immunization with Ac1-11 in complete Freund's adjuvant (CFA) results in a loss of T cell proliferation to MBP Ac1-11. This lack of T cell proliferation is not due to T cell anergy and is not specific. A similar lack of T cell proliferation and inhibition of EAE is observed when an ovalbumin peptide OVA323–339 or a sperm whale myoglobin peptide SWM110-121 are used to treat mice immunized with Ac1-11. Interestingly, we show that previously unresponsive lymph node cells from treated mice respond normally if Ac1-11 is presented by fresh antigen-presenting cells taken from normal mice. These results argue that the lack of T cell proliferation and inhibition of EAE is not due to specific T cell anergy as suggested by others. Instead this appears to be due to blocking of MHC class II molecules Aα^uAβ^u by the treating peptides.     

Partial NK cell tolerance induced by radioresistant host cells in rats transplanted with MHC-mismatched bone marrow

We have studied the effect of radioresistant host cells in inducing tolerance and adaptation of the MHC recognition repertoire of donor-derived NK cells in stem cell allotransplanted (allo-SCT) rats. Sub-lethally irradiated PVG.1AV1 rats (RT1(av1)) were transplanted with bone marrow from fully MHC-mismatched allotype-marked PVG.7B (RT1(c)) rats; MHC-identical PVG (RT1(c)) controls were transplanted in parallel. In the PVG.7B → PVG.1AV1 allogeneic chimeras, NK cells were donor derived and showed partial tolerance toward host cells. Allogeneic chimeras failed to efficiently reject PVG.1AV1 cells by an NK-mediated mechanism in vivo (allogeneic lymphocyte cytotoxicity), and IL-2-cultured NK cells derived from these chimeras showed diminished cytolytic activity against PVG.1AV1 cells in vitro. There were corresponding changes in the phenotype and function of the highly alloreactive Ly49i2(+) NK cells, which are specifically inhibited by a donor MHC class I ligand, RT1-A1(c). The ligand-negative host MHC haplotype apparently induced expression of a second uncharacterized inhibitory MHC receptor responsible for the partial tolerance toward host-derived cells, along with a modest increase in Ly49i2 receptor levels. The host MHC haplotype did not induce a general hyporesponsiveness in Ly49i2(+) NK cells, which showed normal activation responses in a panel of MHC congenic strains. The data suggest that the MHC constitution of radiation-resistant host cells can have permanent, albeit not fully tolerogenic, effects on the development of a functional NK repertoire following allo-SCT.     

Regulatory T cells in experimental allergic encephalomyelitis. I. Frequency and specificity analysis in normal and immune rats of a T cell subset that inhibits disease

We have shown previously that administration of myelin basic protein (MBP)-reactive T cells to naive Lewis rats induces not only autoimmune encephalomyelitis (EAE) but also a near total resistance to subsequent disease. By isolating the effector cells that are responsible for the resistance, we demonstrated that disease protection paralleled with increased numbers of a CD8+ regulatory T cell (RTC) subset and that co-injection of this RTC subset with encephalitogenic T cells aborted the pathogenic activity of the latter cells. Here, we show that a radio-sensitive splenic population of RTC also exists in naive rats that can be recruited and activated to inhibit the onset of secondary episodes of adoptive EAE. In co-transfer experiments, this protective RTC subpopulation can be isolated to neutralize the pathogenic activity of stimulatory MBP-reactive T cells in vivo. We show that the frequency of RTC with specificity for MBP-reactive T cells in naive rats is two orders of magnitude higher than the frequency of MBP-specific precursors, the activity of RTC increases substantially with age and RTC frequencies increase as a consequence of immunization with MBP-reactive cells lines. In specificity studies, we show that RTC isolated from naive rats and RTC from animals primed with one MBP-reactive cell line show cross-reactive responses to a variety of different MBP-reactive T cell lines. However, following repeated stimulation with a given MBP line, these RTC display a more limited, clonotypic response to the selecting line and assume a uniform CD8 phenotype. Finally, functional studies with RTC indicate that proliferative and lytic specificities do not necessarily correlate and that activated rat RTC are especially lytic for a Fas-sensitive murine cell line.     

Diversity of the B cell repertoire to myelin basic protein in rat strains susceptible and resistant to EAE

Myelin basic protein (MBP) is a major protein of central nervous system myelin which can induce experimental autoimmune encephalomyelitis (EAE) in susceptible laboratory animals. The role of T cells in the induction of EAE has been extensively studied, but the antibody response to MBP has not been well characterized. In the present work, we immunized rats with encephalitogenic guinea-pig MBP and mapped autoreactive antibodies binding to peptides in the rat MBP sequence. We studied the responses of the Lewis rat strain, susceptible to EAE, and the responses of the Fischer and Brown-Norway (BN) rats, resistant to EAE. We found that Lewis rats immunized to guinea-pig MBP develop antibodies to a diversity of MBP epitopes with a dominance of MBP peptide p11-30 and peptides in the 71-140 region. Fischer rats showed a similar pattern of antibody specificities, but with higher titers than the Lewis rats. BN rats, in contrast, developed a very low titer of antibodies and lacked a response to p11-30. Thus, there is no clear correlation between the nature of the anti-MBP antibody response and the state of susceptibility or resistance to EAE induction in the different rat strains.     

Limiting-dilution analysis of the frequency of myelin basic protein-reactive T cells in Lewis, PVG/c and BN rats. Implication for susceptibility to autoimmune encephalomyelitis.

Susceptibility to experimental autoimmune encephalomyelitis (EAE), which is an autoimmune disease inducible by immunization with a brain-specific antigen in complete Freund's adjuvant (CFA), is different among strains. In an attempt to resolve the immune mechanisms by which the difference in susceptibility to EAE is regulated, we re-estimated susceptibility of several strains of rats, and the frequency of antigen-reactive T cells in each strain was determined by limiting-dilution analysis. EAE was induced in Lewis (LEW), PVG/c and BN rats using four different methods: (i) active immunization with guinea-pig myelin basic protein (GPBP) in CFA; (ii) immunization with GPBP in CFA that had been further supplemented with Mycobacterium tuberculosis H37Ra (supplemented CFA); (iii) adoptive transfer of GPBP-activated spleen cells into syngeneic rats; and (iv) transfer of a GPBP-specific T-cell line. The LEW strain was susceptible to all four methods. The PVG/c strain was resistant to immunization with GPBP in conventional CFA (GPBP/conv. CFA), but was susceptible to immunization with GPBP in supplemented CFA (GPBP/suppl. CFA) and to transfer of activated spleen cells. The BN strain was resistant to all methods. Limiting-dilution analysis using T cells from LEW, PVG/c or BN rats has revealed that each strain of rat displays a different pattern of frequencies of GPBP-reactive or the 68-88 sequence (GP68-88)-reactive T cells. LEW rats showed relatively high frequencies of GPBP-reactive and GP68-88-reactive T cells after immunization with either GPBP/conv. CFA or GPBP/suppl. CFA, symptomatic rats showing higher values than asymptomatic rats. In asymptomatic PVG/c rats, the frequency of GP68-88-reactive T cells was lower than that of GPBP-reactive T cells. In PVG/c rats with clinical EAE, however, GP68-88-reactive T cells increased in frequency and were almost the same as GPBP-reactive T cells. BN rats, on the other hand, responded very poorly not only to the GP68-88 sequence but also to the whole GPBP molecule, even after immunization with GPBP/suppl. CFA. These findings, obtained by limiting-dilution analysis, strongly suggest that the development of EAE in LEW, PVG/c and BN rats is closely related to the frequency of GPBP-reactive T cells. Furthermore, it is shown that resistance to EAE found in PVG/c and BN rats may be generated by different immune mechanisms.     

Age-dependent T cell tolerance and autoimmunity to myelin basic protein

Experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, is induced by activating a subset of myelin basic protein (MBP)-specific T cells that have escaped tolerance induction. Here, we define the tolerance mechanisms that eliminate the majority of MBP-specific T cells from the periphery. We show that MBP-specific T cells undergo central tolerance mediated by bone marrow-derived antigen-presenting cells presenting exogenously derived MBP epitopes. The efficiency of tolerance is age dependent, reflecting the developmentally regulated expression of MBP. Dependence of tolerance on the amount of MBP expressed in vivo results in an age window of susceptibility to EAE in mice that peaks during puberty. These results suggest that factors regulating expression of self-antigens in vivo can influence susceptibility to autoimmunity.     

Acquired thymic tolerance to autoimmune encephalomyelitis is associated with activation of peripheral IL-10-producing macrophages/dendritic cells

Antigen injection into the thymus of adult animals induces systemic tolerance and protects animals from subsequent challenge for the autoimmune disease. However, its mechanisms are not well understood. In this study, we analyzed tolerance to experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by intrathymic (i.t.) injection of myelin basic protein (MBP). Intrathymic injection of MBP 7 days before immunization with MBP/complete Freund's adjuvant resulted in complete suppression of clinical signs of EAE in most animals and markedly reduced the histological severity in the central nervous system lesion. However, immunohistochemical examination and the TCR repertoire analysis revealed that there was no significant difference in the T cell composition in the lesion and the TCR spectratype pattern between MBP and saline i.t. rats, suggesting that encephalitogenic T cell activation occurs equally in both protected and symptomatic rats. In contrast, quantitative analysis of cytokine mRNA and flow cytometry revealed a marked increase of IL-10 production in the splenic macrophages/dendritic cell (M?/DC) population of MBP i.t. rats. Adoptive transfer of this population significantly suppressed the clinical course of EAE in recipients. Taken together, IL-10-secreting M?/DC in peripheral lymphoid organs activated by MBP i.t. injection may play a critical role in the induction and maintenance of tolerance.     

Genetic influence on disease course and cytokine response in relapsing experimental allergic encephalomyelitis

A protracted and relapsing form of experimental allergic encephalomyelitis (EAE) develops in the DA rat after immunization with rat spinal cord homogenate (SCH) emulsified in incomplete Freund's adjuvant (IFA). The genetic influence on this model has been analyzed by immunizing MHC congenic strains on both LEW and DA genetic backgrounds, and recombinant inbred strains between DA and E3 rats. An in situ hybridization assay was used to examine the expression of mRNA for IFN-gamma, IL-4, IL-10 and transforming growth factor (TGF)-beta both in sections of spinal cords and the antigen-induced expression for these cytokines by splenocytes after in vitro stimulation with encephalitogenic MBP peptides. The susceptibility of relapsing EAE after immunization with SCH in IFA in the DA strain, but not the E3 strain, was correlated with a lack of expression for TGF-beta in the spinal cord. The recombinant inbred DXEB rats developed a severe EAE while surprisingly no signs of disease were observed in the DXEA strain, which shares the MHC region with the DXEB strain, after immunization with the MBP 63-87 peptide. Resistance to relapsing EAE in the DXEA strain correlated with increased non-MHC controlled expression for TGF-beta and lack of IFN-gamma in the spinal cord. The same pattern of cytokine expression was seen in splenocytes after stimulation in vitro with the MBP 63-87 peptide. A spreading of the immune response to the MBP 87-110 peptide was seen. Non-MHC genes controlled the quality of this response: splenocytes from MBP 63-87 immunized DXEB rats responded in vitro towards the MBP 87-110 peptide by expressing mRNA for IFN-gamma, IL-10 and IL-4, whereas in the DXEA strain the corresponding response involved IL-4 and TGF-beta. Taken together these data show that non-MHC controlled expression of mRNA for TGF-beta is associated with resistance to EAE.      

Characterization of brain-isolated rat encephalitogenic T cell lines

In the present study, we have isolated and characterized five myelin basic protein (MBP)-reactive T cell lines directly from the brains of Lewis rats during the early paralytic phase of experimental autoimmune encephalomyelitis (EAE). Each T cell line responded to the dominant encephalitogenic epitope spanning residues 68–88, and did not react against the conserved encephalitogenic epitope [MBP(87–99)] or the nonencephalitogenic MBP epitope [MBP(50–69)]. We determined the T cell receptor (TcR) β chain usage by polymerase chain reaction, DNA sequencing analysis and by generation of MBP-reactive hybridomas from one of the T cell lines (BT74). The results revealed that brain-infiltrating, MBP-reactive T cells freshly isolated early in the course of the disease exhibit TcR diversity.     

Dynamics of serum IgM autoreactive repertoires following immunization: strain specificity,inheritance and association with autoimmune disease susceptibility

Immunization of Lewis rats with myelin basic protein (MBP) in complete Freud's adjuvant (CFA) provokes experimental allergic encephalomyelitis (EAE). Here we compare, irrespective of antigen specificity, the structure and dynamics of serum IgM autoreactive repertoires following immunization with MBP/CFA in EAE-susceptible Lewis and relatively resistant Fischer rats. Prior to the appearance of clinical symptoms, Lewis rats developed a specific modification of serum IgM autoreactivities that, scored on other determinants than MBP itself, showed a prognostic association with EAE symptoms. Although comparable in their production of MBP-specific serum IgM and IgG antibodies, Fischer rats did not share these MBP/CFA-induced IgM autoreactivities of Lewis rats when immunized in the same manner. Moreover, while the Lewis-type repertoire reaction was specific for MBP/CFA alone, the respective Fischer reaction was not qualitatively different from that observed in this strain upon non-pathogenic immunization with self-related or -unrelated antigens. In general, the repertoire reactions differed qualitatively between the strains, consisting of components with typical behavior and strain preferences. The EAE-associated, as well as the other components of both Lewis- and Fischer-type repertoire reactions were usually co-dominantly inherited in F1 animals. These results indicate that a global antibody repertoire analysis may serve as a tool to describe prototypical response structures, possibly involved in immune regulation and susceptibility to pathogenic autoimmunity.