New genetic loci that control susceptibility and symptoms of experimental allergic encephalomyelitis in inbred mice

Experimental allergic encephalomyelitis (EAE), the principal animal model of multiple sclerosis, is a genetically determined phenotype. In this study, analyses of the cumulative disease frequencies in parental, F1 hybrid, and F2 mice, derived from the EAE-susceptible SJL/J strain and the EAE-resistant B10.S/DvTe strain, confirmed that susceptibility to EAE is not inherited as a simple Mendelian trait. Whole genome scanning, using 150 informative microsatellite markers and a panel of 291 affected and 390 unaffected F2 progeny, revealed significant linkage of EAE susceptibility to marker loci on chromosomes 7 (eae4) and 17, distal to H2 (eae5). Quantitative trait loci for EAE severity, duration, and onset were identified on chromosomes 11 (eae6, and eae7), 2 (eae8), 9 (eae9), and 3 (eae10). While each locus reported in this study is important in susceptibility or disease course, interactions between marker loci were not statistically significant in models of genetic control. One locus, eae7, colocalizes to the same region of chromosome II as Orch3 and Idd4, susceptibility loci in autoimmune orchitis and insulin-dependent diabetes mellitus, respectively. Importantly, eae5 and eae7 are syntenic with human chromosomes 6p21 and 17q22, respectively, two regions of potential significance recently identified in human multiple sclerosis genome scans.     

Inactivation of T cell receptor peptide-specific CD4 regulatory T cells induces chronic experimental autoimmune encephalomyelitis (EAE)

T cell receptor (TCR)-recognizing regulatory cells, induced after vaccination with self-reactive T cells or TCR peptides, have been shown to prevent autoimmunity. We have asked whether this regulation is involved in the maintenance of peripheral tolerance to myelin basic protein (MBP) in an autoimmune disease model, experimental autoimmune encephalomyelitis (EAE). Antigen-induced EAE in (SJL x B10.PL)F1 mice is transient in that most animals recover permanently from the disease. Most of the initial encephalitogenic T cells recognize MBP Ac1-9 and predominantly use the TCR V beta 8.2 gene segment. In mice recovering from MBP-induced EAE, regulatory CD4+ T cells (Treg) specific for a single immunodominant TCR peptide B5 (76-101) from framework region 3 of the V beta 8.2 chain, become primed. We have earlier shown that cloned B5-reactive Treg can specifically downregulate responses to Ac1-9 and also protect mice from EAE. These CD4 Treg clones predominantly use the TCR V beta 14 or V beta 3 gene segments. Here we have directly tested whether deletion/blocking of the Treg from the peripheral repertoire affects the spontaneous recovery from EAE. Treatment of F1 mice with appropriate V beta-specific monoclonal antibodies resulted in an increase in the severity and duration of the disease; even relapses were seen in one-third to one-half of the Treg-deleted mice. Interestingly, chronic disease in treated mice appears to be due to the presence of Ac1-9-specific T cells. Thus, once self-tolerance to MBP is broken by immunization with the antigen in strong adjuvant, TCR peptide-specific CD4 Treg cells participate in reestablishing peripheral tolerance. Thus, a failure to generate Treg may be implicated in chronic autoimmune conditions.     

Preferential but not exclusive T cell receptor V beta chain utilization of myelin basic protein and peptide-specific T cell clones in mice

The repertoire of T cell receptor (TCR) V beta chain utilization was investigated in PL/J, CXJ-1, SJL/J and B10.S-->SJL/J chimeric mice in response to either myelin basic protein (MBP) or the strain-specific encephalitogenic peptide. Our analysis showed that there was an overlapping predominance in the TCR V beta gene utilization in the MBP-specific responses, which were independent of the major histocompatibility complex (MHC) class II haplotype present, and the immunodominant peptide region recognized in these different strains. In those mice having the TCR V beta b haplotype (PL/J, CXJ-1, and the B10.S-->SJL chimera) either the TCR V beta 4, 8, and 13 or the TCR V beta 4, 6, and 13 predominated. In contrast, in mice with TCR V beta a haplotype (SJL/J) V beta 4, 6, and 17a were found. However, the quantitative distribution of these preferentially utilized TCR V beta chains in each strain was defined by the MHC class II haplotype and the immunodominant peptide recognized. The expression of the V beta 8 gene product in the peripheral TCR repertoire did not always correlate with predominant V beta 8 utilization in the MBP-specific response.     

Induction of a heterogeneous TCR repertoire in (PL/JXSJL/J)F1 mice by myelin basic protein peptide Ac1-11 and its analog Ac1-11[4A]

Experimental autoimmune encephalomyelitis (EAE) serves as a rodent model of the autoimmune disease multiple sclerosis. In mice, EAE is induced by immunizing with spinal cord homogenate, components of the myelin sheath, such as myelin basic protein (MBP) or proteolipid protein (PLP), or peptides derived from these components. EAE can be induced in H-2u or (H-2u x H-2s)F1 mice with the N-terminal peptide of MBP, Ac1-11. Coimmunization with Ac1-11 and Ac1-11[4A], an analog in which lysine at position four is substituted with alanine, prevents EAE. The mechanism of inhibition has not been elucidated, but probably does not work through MHC blockade, T cell anergy or clonal elimination of encephalitogenic T cells. We have isolated T cell clones and hybridomas from (PL/J x SJL/J)F1 mice immunized with either Ac1-11 alone or Ac1-11 and Ac1-11[4A] and analysed these cells for differences in their T cell receptor repertoire and in vitro response. Although T cells elicited by coinjection of Ac1-11 and Ac1-11[4A] expressed TCR that used V alpha and Vbeta gene elements similar to those elicited by Ac1-11 alone, they differed in the sequences of the junctional region of the alpha chain. Most of these T cells also responded less well to Ac1-11 in vitro, suggesting that coinjection of Ac1-11 and Ac1-11[4A] preferentially activates T cells bearing TCR of different affinity for Ac1-11 bound to I-A(u), and which may therefore be less encephalitogenic. Furthermore, our results show that a more diverse repertoire of V alpha and Vbeta genes are elicited by Ac1-11 in (PL/J x SJL/J)F1 mice compared to PL/J and B10.PL mice, providing further evidence that a restricted TCR repertoire is not required for the development of autoimmune disease.     

A TCR alpha chain transgene induces maturation of CD4- CD8- alpha beta+ T cells from gamma delta T cell precursors

The proportion of CD4- CD8- double-negative (DN) alpha beta T cells is increased both in the thymus and in peripheral lymphoid organs of TCR alpha chain-transgenic mice. In this report we have characterized this T cell population to elucidate its relationship to alpha beta and gamma delta T cells. We show that the transgenic DN cells are phenotypically similar to gamma delta T cells but distinct from DN NK T cells. The precursors of DN cells have neither rearranged endogenous TCR alpha genes nor been negatively selected by the MIsa antigen, suggesting that they originate from a differentiation stage before the onset of TCR alpha chain rearrangements and CD4/CD8 gene expression. Neither in-frame V delta D delta J delta nor V gamma J gamma rearrangements are over-represented in this population. However, since peripheral gamma delta T cells with functional TCR beta gene rearrangements have been depleted in the transgenics, we propose that the transgenic DN population, at least partially, originates from the precursors of those cells. The present data lend support to the view that maturation signals to gamma delta lineage-committed precursors can be delivered via TCR alpha beta heterodimers.     

Regulatory CD4(+) T cells expressing endogenous T cell receptor chains protect myelin basic protein-specific transgenic mice from spontaneous autoimmune encephalomyelitis

The development of T cell-mediated autoimmune diseases hinges on the balance between effector and regulatory mechanisms. Using two transgenic mouse lines expressing identical myelin basic protein (MBP)-specific T cell receptor (TCR) genes, we have previously shown that mice bearing exclusively MBP-specific T cells (designated T/R-) spontaneously develop experimental autoimmune encephalomyelitis (EAE), whereas mice bearing MBP-specific T cells as well as other lymphocytes (designated T/R+) did not. Here we demonstrate that T/R- mice can be protected from EAE by the early transfer of total splenocytes or purified CD4(+) T cells from normal donors. Moreover, whereas T/R+ mice crossed with B cell-deficient, gamma/delta T cell-deficient, or major histocompatibility complex class I-deficient mice did not develop EAE spontaneously, T/R+ mice crossed with TCR-alpha and -beta knockout mice developed EAE with the same incidence and severity as T/R- mice. In addition, MBP-specific transgenic mice that lack only endogenous TCR-alpha chains developed EAE with high incidence but reduced severity. Surprisingly, two-thirds of MBP-specific transgenic mice lacking only endogenous TCR-beta chains also developed EAE, suggesting that in T/R+ mice, cells with high protective activity escape TCR-beta chain allelic exclusion. Our study identifies CD4(+) T cells bearing endogenous alpha and beta TCR chains as the lymphocytes that prevent spontaneous EAE in T/R+ mice.     

Thymocyte differentiation in gamma-irradiated severe-combined immunodeficient mice: characterization of intermediates and products of V(D)J recombination at the T cell receptor alpha locus

Treatment with DNA-damaging agents promotes rescue of V(D)J recombination, limited thymocyte differentiation, and development of thymic lymphomas in severe-combined immunodeficient (SCID) mice. One intriguing aspect of this system is that irradiation rescues rearrangements at the T cell receptor (TCR) beta, gamma and delta loci, but not at the TCR alpha locus. Current models posit that only those loci that are recombinationally active at the time of irradiation can be rescued. Here, we employ sensitive, semiquantitative ligation-mediated polymerase chain reaction assays to detect a specific class of recombination intermediates, hairpin coding ends, at the TCR alpha locus. We found that J alpha-coding ends are undetectable in unirradiated SCID thymocytes, but accumulate after irradiation at times coincident with the emergence of a CD4+ CD8+ thymocyte population. Coding joints produced by joining of these ends, however, are extremely rare. To test whether the presence of hairpin coding ends at TCR alpha is sufficient for irradiation-mediated rescue of coding joint formation, we administered a second dose of gamma-irradiation after abundant CD4+ CD8+ thymocytes and hairpin TCR alpha coding ends had accumulated. This treatment failed to stimulate rescue of TCR alpha coding joints. Thus, the presence of hairpin coding ends at the time of irradiation, while perhaps necessary, is not sufficient for rescue of V(D)J rearrangements. These results support a refined model for irradiation-mediated rescue of TCR rearrangements in SCID mice.     

The glutamate dehydrogenase, E74 and putative actin gene loci in the Drosophila montium subgroup. Chromosomal homologies among the montium species and D. melanogaster

Myasthenia gravis (MG) is a T-cell-regulated autoimmune disease in which a pathological autoantibody response is mounted against the nicotinic acetylcholine receptor of the neuromuscular junction. Our laboratory previously identified a T cell epitope, p195-212, derived from the human acetylcholine receptor alpha subunit, which triggered PBL to proliferate from about 70% of MG patients tested. p195-212 was also found to be an immunodominant T cell epitope in SJL mice and a cryptic epitope in C3H.SW mice. Inoculation of naive SJL mice with cells from a p195-212-specific syngeneic T cell line caused MG-related autoimmune manifestations in those mice. In these studies we analyzed TCR alpha and beta chain sequences used by T cell lines and clones from both high- and low-responder mouse strains in response to p195-212. T cell lines generated from either strain expressed single TCR V beta gene segments (V beta 17 in SJL mice and V beta 8 in C3H.SW mice). By deleting V beta 17-expressing T cells in p195-212-immunized SJL mice we established a T cell line that expressed the V beta 6 gene product, suggesting that SJL mice are not limited to using a single V beta gene segment in response to p195-212. In addition, we found that N- and/or C-terminal-truncated peptides of p195-212, presented under the same culture conditions to different clones bearing the same TCR alpha beta chain, could elicit very different proliferative responses from the clones. Thus, even within a constrained system, factors other than TCR sequence contribute to the differential stimulation of T cell responses.     

V-D-J rearrangements at the T cell receptor delta locus in mouse thymocytes of the alpha beta lineage

The T cell receptor (TCR) delta locus lies within the TCR alpha locus and is excised from the chromosome by V alpha-J alpha rearrangement. We show here that delta sequences persist in a large fraction of the DNA from mature CD4+CD8- alpha beta+ mouse thymocytes. Virtually all delta loci in these cells are rearranged and present in extrachromosomal DNA. In immature alpha beta lineage thymocytes (CD3-/loCD4+CD8+) and in CD4+CD8- alpha beta+ thymocytes expressing a transgene-encoded alpha beta receptor, rearranged delta genes are present both in chromosomal and extrachromosomal DNA. Thus, contrary to earlier proposals, commitment to the alpha beta lineage does not require recombinational silencing of the delta locus or its deletion by a site-specific mechanism prior to V alpha-J alpha rearrangement.     

The inflammatory response to nonfatal Sindbis virus infection of the nervous system is more severe in SJL than in BALB/c mice and is associated with low levels of IL-4 mRNA and high levels of IL-10-producing CD4+ T cells

SJL mice are susceptible to inflammatory autoimmune diseases of the central nervous system (CNS), while BALB/c mice are relatively resistant. To understand differences in immune responses that may contribute to autoimmune neurologic disease, we compared the responses of SJL and BALB/c mice to infection with Sindbis virus, a virus that causes acute nonfatal encephalomyelitis in both strains of mice. Clearance of virus was similar, but SJL mice developed a more intense inflammatory response in the brain and spinal cord and inflammation persisted for several weeks. Analysis of lymphocytes isolated from brains early after infection showed an absence of NK cells in SJL mice, while both strains of mice showed CD4+ and CD8+ T cells. During the second week after infection, CD4+ T cells increased in SJL mice and the proportion of CD8+ T cells decreased, while the opposite pattern was seen in BALB/c mice. Expression of IL-10 mRNA was higher and IL-4 mRNA was lower in the brains of infected SJL than in BALB/c mice, while expression of the mRNAs of IL-6, IL-1beta, TNFalpha, and the Th1 cytokines IL-2, IL-12, and IFN-gamma was similar. Lymphocytes isolated from the CNS of SJL mice produced large amounts of IL-10. CNS lymphocytes from both strains of mice produced IFN-gamma in response to stimulation with Sindbis virus, but not in response to myelin basic protein. These data suggest that IL-10-producing CD4+ T cells are differentially recruited to or regulated within the CNS of SJL mice compared with BALB/c mice infected with Sindbis virus, a characteristic that may be related to low levels of IL-4, and is likely to be involved in susceptibility of SJL mice to CNS inflammatory diseases.     

The role of IL-7 in thymic and extrathymic development of TCR gamma delta cells

IL-7-deficient (IL-7(-/-)) mice have reduced numbers of B and TCR alpha beta cells, but lack mature TCR gamma delta cells. Although most T cell development occurs in the thymus, some intestinal intraepithelial lymphocytes (IEL), including TCR gamma delta cells, can develop extrathymically. Epithelial cells in both thymus and intestine synthesize IL-7, suggesting that TCR gamma delta cell development could occur in either site. To evaluate the role of thymic IL-7 in development of TCR gamma delta cells, newborn TCR beta-deficient (TCR beta(-/-)) thymi were grafted to IL-7(-/-) mice. Donor- and host-derived TCR gamma delta cells were recovered from thymus grafts, spleen, and IEL. However, when IL-7(-/-) thymi were grafted to TCR beta(-/-) mice, no development of graft-derived TCR gamma delta cells occurred, indicating that extrathymic IL-7 did not support TCR gamma delta IEL generation from newborn thymic precursors. In contrast, TCR gamma delta IEL development occurred efficiently in adult, thymectomized, irradiated C57BL/6J mice reconstituted with IL-7(-/-) bone marrow. This demonstrated that extrathymic development of TCR gamma delta IEL required extrathymic IL-7 production. Thus, intrathymic IL-7 was required for development of thymic TCR gamma delta cells, while peripheral IL-7 was sufficient for development of extrathymic TCR gamma delta IEL.     

Minimum structural requirements for peptide presentation by major histocompatibility complex class II molecules: implications in induction of autoimmunity

The precise mechanisms of failure of immunological tolerance to self proteins are not known. Major histocompatibility complex (MHC) susceptibility alleles, the target peptides, and T cells with anti-self reactivity must be present to cause autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a murine model of a human autoimmune disease, multiple sclerosis. In EAE, residues 1-11 of myelin basic protein (MBP) are the dominant disease-inducing determinants in PL/J and (PL/J x SJL/J)F1 mice. Here we report that a six-residue peptide (five of them native) of MBP can induce EAE. Using peptide analogues of the MBP-(1-11) peptide, we demonstrate that only four native MBP residues are required to stimulate MBP-specific T cells. Therefore, this study demonstrates lower minimum structural requirements for effective antigen presentation by MHC class II molecules. Many viral and bacterial proteins share short runs of amino acid similarity with host self proteins, a phenomenon known as molecular mimicry. Since a six-residue peptide can sensitize MBP-specific T cells to cause EAE, these results define a minimum sequence identity for molecular mimicry in autoimmunity.     

The effect of TCR V beta 8 peptide protection and therapy on T cell populations isolated from the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

Vaccination or treatment of Lewis rats with TCR V beta 8 peptides can prevent or reverse the clinical signs of experimental autoimmune encephalomyelitis (EAE) which is mediated predominantly by V beta 8.2+ CD4+/CD45R lo T cells. However, rats protected or treated with V beta 8 peptides still developed histological lesions in the spinal cord (SC), even though they remained clinically well. We sought to discern phenotypic changes characteristic of these SC infiltrating lymphocytes. In particular, we focused on whether the immunoregulatory mechanism induced by TCR peptides caused a reduction of V beta 8.2+ T cells, or induced changes in CD45R lo or hi/CD4+ subpopulations that have been associated respectively with EAE induction or recovery. In the V beta 8 peptide vaccinated rats there was a dramatic decrease in the number of V beta 8.2+ T cells isolated from the SC early in disease. During the recovery phase, however, the number of V beta 8.2+ SC T cells was similar in protected and control groups; in contrast, there was striking reduction in the number and size of CD45R hi/CD4+ T cells in the protected animals. In rats treated with V beta 8.2 peptide, no changes were observed in the number of SC V beta 8.2+ T cells or expression of V beta 8.2 message, but similar to vaccinated rats, there was a marked decrease in the number of CD45R hi/CD4+ T cells. These data suggest that vaccination with TCR peptides prevented the initial influx of encephalitogenic V beta 8.2+ T cells into the central nervous system (CNS), whereas treatment appeared to inactivate V beta 8.2+ T cells already present in the CNS. In both cases, TCR peptide-induced inhibition of the encephalitogenic T cells apparently preempted the need for CD45R hi/CD4+ T cells that may normally be necessary to resolve the disease.     

Requirement for CD8+ cells in T cell receptor peptide-induced clonal unresponsiveness

T cell receptor (TCR) vaccination in rats prevents the development of experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. The mechanism of this potential immunotherapy was examined by vaccinating mice with an immunogenic peptide fragment of the variable region of the TCR V beta 8.2 gene. Another immunogen that usually induces an immune response mediated by V beta 8.2+ T cells was subsequently inhibited because specific clonal unresponsiveness (anergy) had been induced. Depletion of CD8+ cells before TCR peptide vaccination blocked such inhibition. Thus, the clonal anergy was dependent on CD8+ T cells, and such immunoregulatory T cells may participate in the normal course of EAE.     

TCR-gamma delta cells in CD3 zeta-deficient mice contain Fc epsilon RI gamma in the receptor complex but are specifically unresponsive to antigen

Unlike TCR-alpha beta cells, TCR-gamma delta cells express a distinct member of the zeta family, the gamma-chain of Fc epsilon RI (Fc epsilon RI gamma) within the TCR complex. To study the role of the Fc epsilon RI gamma-chain in TCR-gamma delta cells, a TCR-gamma delta transgenic mouse (G8) has been crossed with CD3 zeta-chain-deficient mice (G8.zeta-/-). Thy-1+ spleen and lymph node cells of these animals expressed low levels of CD3/TCR. These results suggested that the zeta-chain is required for effective TCR transport to the cell surface. In contrast, intraepithelial TCR-gamma delta cells of G8.zeta-/- mice expressed high levels of TCR. Immunoprecipitation with anti-CD3 showed that Fc epsilon RI gamma-chains were associated with the TCR complex in T cells isolated from zeta-deficient mice. Although the Fc epsilon RI gamma-expressing T cells proliferated in response to stimulation by TCR-specific Abs including anti-CD3 epsilon, anti-pan gamma delta, and anti-V gamma 2 mAb, the G8.zeta-/- T cells did not respond to the G8-specific Ag (T10b), anti-Thy-1 mAb, or Con A. The unresponsiveness to the Ag was not due to the reduced TCR expression, because intraepithelial TCR-gamma delta cells from the zeta-deficient mice did not respond to Ag. The inability of the G8.zeta-/- T cells to respond to Ag could not be overcome by providing an anti-CD28 costimulatory signal or by adding exogenous rIL-2. Taken together, our data suggest that the Fc epsilon RI gamma-chain associates with the TCR-gamma delta complex in the absence of the zeta-chain, but it is not able to substitute for the zeta-chain for effective transport of TCR to the cell surface or functional responses to Ag.     

The size heterogeneity of human lysyl oxidase mRNA is due to alternate polyadenylation site and not alternate exon usage

Theiler's murine encephalomyelitis virus (TMEV) produces a chronic disease in its natural host, the mouse, characterised by primary inflammatory demyelination of the spinal cord. This viral infection is considered a very good model for human MS because the pathogenesis of myelin injury is mediated through the host immune response. Susceptibility and/or resistance to the demyelinating disease depend on multiple genes both in and outside the major histocompatibility complex (MHC). The pathological lesions in animals with different degrees of susceptibility vary in both their severity and in their ability to become remyelinated. In general, animals with intermediate levels of susceptibility show the best potential for remyelination. Most crosses of susceptible animals with resistant strains carrying the H-2b haplotype are resistant with only a couple of exceptions. One such exception is the (SJL/J x C57L/J)F1 hybrid, which is susceptible to the disease. To study whether the resistant genotype of C57L/J mice could modify the phenotypic expression of pathological lesions characteristic of the highly susceptible SJL/J mouse, we performed a light microscopical and ultrastructural study of the spinal cord of both parental strains and their F1 progeny. We focused particularly on the relationship between severity of inflammation, and especially macrophage infiltration, and the subsequent remyelinating potential of lesions. The results show a dramatic difference between the ability to remyelinate lesions by infected SJL/J mice vs similarly infected (SJL/L x C57L/J)F1 hybrids, and suggest an important influence by resistant genes in modulating the phenotypic expression of disease, including the ability to stimulate oligodendroglia-mediated remyelination.     

Activation and peripheral expansion of murine T-cell receptor gamma delta intraepithelial lymphocytes

BACKGROUND & AIMS: The intestinal epithelial compartment is populated by CD8(+) alpha beta and gamma delta intraepithelial lymphocytes (IELs), which monitor the integrity of the epithelial barrier. alpha beta IELs are activated by peptide antigens presented by class I major histocompatibility complex (MHC) molecules, but it is unclear how gamma delta IELs are activated. METHODS: G8 T-cell receptor (TCR) gamma delta transgenic (Tg) mice (specific for the class I MHC alloantigen, T22/10(b)) were crossed to class I MHC-deficient beta2-microglobulin-knockout (beta2m degrees) mice, and Tg+ IELs were examined for relative yields and surface and functional phenotype. RESULTS: Evidence for class I MHC-induced activation of Tg+ IELs was supported by the detection of 4-fold greater numbers of Tg+ IELs in G8 x beta2m+ mice that proliferated at 15-fold higher levels than IELs from G8 x beta2m degrees mice. However, expression of CD69, production of cytokine (interleukin 2 and interferon gamma), and detection of cytolytic function for IELs in G8 x beta2m degrees mice suggested that class I MHC was not required for gamma delta IEL development or maturation. CONCLUSIONS: These results suggest that CD8(+) TCR gamma delta IELs do not require class I MHC for development but support the notion that antigens presented by class I MHC molecules are involved in the peripheral expansion and differentiation of this subset.