Soluble antigen therapy induces apoptosis of autoreactive T cells preferentially in the target organ rather than in the peripheral lymphoid organs

The administration of soluble myelin proteins is an effective way of down-regulating the inflammation in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. To shed more light on the mechanism of this antigen-specific therapy, we determined the effect of the intraperitoneal (i.p.) injection of soluble myelin basic protein (MBP) on T cell apoptosis in the CNS and peripheral lymphoid organs of Lewis rats with EAE induced by inoculation with MBP and complete Freund's adjuvant. In particular we assessed the level of apoptosis of Vβ8.2⁺ T cells, which constitute the predominant encephalitogenic MBP-reactive T cell population in the Lewis rat. The daily i.p. injection of MBP for 3 days from the onset of neurological signs inhibited the further development of neurological signs of EAE. Using two-color flow cytometry we found that a single i.p. injection of MBP increased the level of apoptosis of the Vβ8.2⁺ T cell population in the CNS to 26.2 % compared to 7.4 % in saline-treated rats and 7.6 % in ovalbumin-treated rats. In contrast, treatment with MBP did not increase the level of apoptosis of the Vβ8.2⁺ population in the popliteal lymph node draining the inoculation site (1.4 %) or in the spleen (1.6 %) above that occurring in saline-treated rats (1.6 % and 1.1 %, respectively). Limiting dilution analysis revealed that the frequency of T cells reactive to the major encephalitogenic epitope, MBP_{72 – 89}, was decreased in the CNS but not in the popliteal lymphnode by this treatment. Three-color flow cytometry in MBP-treated rats demonstrated that CNS Vβ8.2⁺ T cells expressing Fas (CD95) and Fas ligand were highly vulnerable to apoptosis compared to Vβ8.2⁺ T cells not expressing these proteins. We conclude that the i.p. injection of MBP increases the spontaneously occurring Fas-mediated activation-induced apoptosis of auto reactive T cells in the CNS in EAE and that this contributes to the therapeutic effect of the injection.     

Apoptotic elimination of Vβ8.2+ cells from the central nervous system during recovery from experimental autoimmune encephalomyelitis induced by the passive transfer of Vβ8.2+ encephalitogenic T cells

A CD4⁺Vβ8.2⁺ T cell clone specific for the peptide 72–89 of guinea pig myelin basic protein (GMBP) was used to induce acute experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To assess apoptosis in inflammatory cells infiltrating the central nervous system (CNS), we extracted cells from the spinal cord, enriched them for T cells and performed flow-cytometric analysis of their DNA stained with propidium iodide. The presence of apoptosis was confirmed by the demonstration of DNA fragmentation on gel electrophoresis. A gradual increase in the proportion of apoptotic cells was observed between 4 and 7 days after the transfer of the encephalitogenic T cells. The highest frequency of apoptotic cells (9.2 ± 1.2%) was observed 7 days after cell transfer, when clinical recovery commenced. Passive transfer of ovalbumin-specific cells resulted in only a background level (0.8%) of apoptosis in the CNS. We conclude that the apoptotic process selectively eliminates autoreactive T cells from the CNS as: (a) there was a selective disappearance of disease-relevant CD5⁺Vβ8.2⁺ cells from the CNS during the course of EAE; (b) there was a decrease in the frequency of CNS-infiltrating T cells reactive to the GMBP 72–89 peptide during the course of EAE, and in a standard proliferation assay there was a loss of in vitro reactivity of CNS-infiltrating cells to this peptide, but not to a non-CNS antigen (ovalbumin); (c) simultaneous surface labeling and DNA analysis of CNS-infiltrating cells revealed that the frequency of Vβ8.2⁺ cells was about sevenfold higher in the apoptotic T cell population than in the normal (non-apoptotic) T cell population; and (d) we were unable to detect recirculation of the Vβ8.2⁺ cells to lymphoid organs after their frequency decreased in the CNS. The selective apoptotic elimination of autoreactive T cells from the target organ of this spontaneously resolving autoimmune disease may have implications for the understanding of the mechanism by which an autoimmune attack is terminated and for the design of therapeutic strategies to facilitate this process.     

Anergy induction in encephalitogenic T cells by brain microvessel endothelial cells is inhibited by interleukin-1

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) which can be induced, in susceptible strains like Lewis rats, by transfer of activated myelin basic protein (MBP)-specific CD4⁺ T lymphocytes. The role of cerebral endothelium in the onset of EAE, with regard to adhesion, activation and infiltration in the CNS of encephalitogenic T lymphocytes, is not fully understood. When pretreated by interferon-γ, the immortalized Lewis rat brain microvessel endothelial (RBE4) cells expressed major histocompatibility complex class II molecules and stimulated MBP-specific proliferation and cytolytic activity of the syngeneic encephalitogenic T cell line, designated PAS. However, RBE4-stimulated PAS lymphocytes subsequently entered an unresponsive state, known as anergy. When inoculated in syngeneic animals, anergic PAS cells, although still cytotoxic, failed to induce EAE, and no cell infiltration was detectable within CNS. The addition of interleukin-1β (IL-1β) during MBP presentation by RBE4 cells prevented T cell anergy induction, and maintained T cell encephalitogenicity, although PAS cells stimulated in these conditions caused delayed and attenuated clinical signs of EAE, with only discrete inflammatory lesions in the CNS, compared with EAE induced by PAS cells fully activated by thymic cells. Altogether, our results indicate that MBP presentation by brain microvessel endothelial cells to encephalitogenic T cells induces T cell anergy and loss of pathogenicity. In addition, IL-1β co-stimulation of T cells prevents anergy induction in vitro and at least partially maintains encephalitogenicity in vivo.     

Characterization of CD4−CD8− T cell receptor αβ+ T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis

Inflammation of the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) starts in the subarachnoid space (SAS) and spreads later to the adjacent CNS parenchyma. To characterize the nature of lesion-forming T cells in situ in more detail, T cells were isolated from the SAS and their surface phenotype and the nucleotide sequence of the junctional region of the T cell receptor (TCR) was determined and compared with those of the lymph node (LN) and spinal cord (SC) T cells. Characteristically, more than 70% of SAS TCR αβ⁺ T cells isolated at the early stage of EAE lacked both CD4 and CD8 molecules, whereas those from LN and SC were either CD4⁺ or CD8⁺. Analysis of nucleotide sequences of the junctional region of TCR revealed that T cells bearing a sequence identical to that for encephalitogenic T cell clones were found in both SAS and SC. Furthermore, purified CD4^?CD8^? T cells expressed CD4 molecules after culture. At the same time, these T cells acquired reactivity to myelin basic protein and induced passive EAE in naive animals after adoptive transfer. Our results suggest that CD4^?CD8^? T cells in the SAS are precursors of lesion-forming T cells in the SC and that phenotype switching takes place during the process of T cell infiltration into the CNS parenchyma. The double-negative nature of these T cells may explain an escape of encephalitogenic T cells from negative selection in T cell differentiation.     

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.     

Delineation of the minimal encephalitogenic epitope within the immunodominant region of myelin oligodendrocyte glycoprotein: diverse Vβ gene usage by T cells recognizing the core epitope encephalitogenic for T cell receptor Vβb and T cell receptor Vβa H-2b mice

The nature of the autoimmune T cell response to myelin oligodendrocyte glycoprotein (MOG), recently recognized as a potential target antigen in multiple sclerosis (MS), has not yet been characterized, in contrast to the T cell reactivity to other potential target antigens in MS such as myelin basic protein and proteolipid protein. Here, we show that the encephalitogenicity of the recombinant Ig-like domain of human MOG is associated, in H-2^b mice, with an immunodominant T cell reactivity against a single region of MOG spanning amino acids 35–55, accounting for the previously reported strong encephalitogenic activity of pMOG 35–55. A single injection of pMOG 35–55 with or without administration of pertussis toxin was sufficient to induce severe clinical experimental autoimmune encephalomyelitis (EAE) in H-2^b mice. Encephalitogenic pMOG 35–55-specific T cell lines derived from C3H.SW (Vβ^b) mice were diverse in their TCR Vβ gene usage (Vβ1, Vβ6, Vβ8 and Vβ15), although Vβ8.2 was most predominantly expressed (48%). However, Vβ8⁺ T cells may only be part of the encephalitogenic MOG-specific T cell repertoire in H-2^b mice, as demonstrated by the susceptibility of C57L (Vβ^a) mice to disease induced by pMOG 35–55. Encephalitogenic T cell lines from Vβ^a mice were also diverse in their TCR Vβ gene usage (Vβ1, Vβ2, Vβ6, Vβ14 and Vβ16). Such a heterogeneous TCR Vβ gene expression by pMOG 35–55/I-A^b-reactive T cells from both Vβ^a and Vβ^b H-2^b mice suggested multiple epitopes within pMOG 35–55. Analysis of the pattern of reactivity by pMOG 35–55-reactive T cells to a set of truncated peptides was not commensurate with independent nested epitopes, but revealed a requirement for recognition of a core sequence, YRSPFSRVV (pMOG 40–48). However, optimal stimulation was obtained with longer peptides, with each additional amino acid flanking either the N or the C terminus differentially increasing the stimulatory capacity of pMOG 40–48. Nonetheless, pMOG 40–48 was the minimal encephalitogenic epitope for both Vβ^a and Vβ^b mice. Thus, the T cell reactivity against the immunodominant encephalitogenic region of MOG is characterized by a diverse Vβ gene usage and a requirement for the same core epitope. This pattern of reactivity may favor epitope-directed, rather than TCR-targeted, approaches to immunospecific therapy for MOG-related autoimmune disease.     

Prevention and treatment of Lewis rat experimental allergic encephalomyelitis with a monoclonal antibody to the T cell receptor Vβ8.2 segment

The predominance of T cell receptor (TCR) Vβ8.2 utilization by encephalitogenic T cells induced in Lewis rats by immunization with myelin basic protein (MBP) is controversial. Thus, both an almost exclusive usage of Vβ8.2 [Burns, F. R., Li, X., Shen, N., Offner, H., Chou, Y. K., Vandenbark, A. A. and Heber-Katz, E., J. Exp. Med. 1989. 169: 27; Chluba, J., Steeg, C., Becker, A., Wekerle, H. and Epplen, J. T., Eur. J. Immunol. 1989. 19: 279] and a quite diverse Vβ composition of CD4 T cells causing experimental autoimmune encephalomyelitis (EAE) [Sun, D., Gold, P. D., Smith, L., Brostoff, S. and Coleclough, C., Eur. J. Immunol. 1992. 22: 591; Sun, D., Le, J. and Coleclough, C., Eur. J. Immunol. 1993. 23: 494] have been reported. Using a recently developed monoclonal antibody (mAb) specific for TCR Vβ8.2, we show that postnatal treatment effectively eliminates Vβ8.2-bearing cells and prevents MBP-induced EAE in the majority of Lewis rats. Moreover, treatment of adult Lewis rats with Vβ8.2-specific mAb as late as on day 12 after MBP immunization suppressed the development of neurological symptoms. Thus, Vβ8.2-bearing cells do play a decisive role in Lewis rat EAE, and suppression of the small (5%) Vβ8.2-expressing T cell subset provides an effective therapeutic strategy.     

A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b mice: Fine specificity and T cell receptor Vβ expression of encephalitogenic T cells

A predominant response to myelin oligodendrocyte glycoprotein (MOG) was recently observed in patients with multiple sclerosis (MS). To study the possible pathogenic role of T cell response to MOG in MS, we have investigated the encephalitogenic potential of MOG. Synthetic MOG peptides, pMOG 1-21, 35–55, 67–87, 104–117 and 202–218, representing predicted T cell epitopes, were injected into C57BL/6J and C3H.SW (H-2^b) mice. The mice developed significant specific T cell responses to pMOG 1–21, pMOG 35–55 and pMOG 104–117. However, pMOG 35–55 was the only MOG peptide which could induce neurological impairment. The highly reproducible disease was chronic, with ascending paralysis and neuropathology comparable with those observed in experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein or proteolipid protein, except that in H-2^b mice the disease was consistently non-remitting. These features differ markedly from those which we recently observed in PL (H-2^u) mice with pMOG 35–55-induced disease. In PL mice, pMOG 35–55-induces atypical chronic relapsing EAE, the expression and progression of which are unpredictable. Hence, in different mouse strains, the same MOG peptide can induce typical EAE characterized by ascending paralysis, or atypical EAE with unpredictable clinical signs. pMOG 35–55-specific T cells from H-2^b mice recognized an epitope within amino acids 40–55 of the MOG molecule, and pMOG 40–55-reactive T cell lines were encephalitogenic upon transfer into syngeneic recipients. The encephalitogenic pMOG 35–55-reactive C57BL/6J T cell lines expressed Vβ1, Vβ6, Vβ8, Vβ14 and Vβ15 gene segments, and the pMOG 35–55-reactive C3H.SW T cell lines expressed Vβ1, Vβ2, Vβ6, Vβ8, Vβ10, Vβ14, and Vβ15 gene segments. However, in both mouse strains, the utilization of the Vβ8 gene product was predominant (40–43 %). The highly reproducible encephalitogenic activity of pMOG 35–55 strongly suggests a pathogenic role for T cell reactivity to MOG in MS and supports the possibility that MOG may also be a primary target antigen in the disease.     

Analysis of TCR β chains in Lewis rats with experimental allergic encephalomyelitis. II. Vβ8.2+ T cells with limited CDR3 N region additions derive from the adult thymus

Immunization of Lewis (LEW) rats with guinea pig myelin basic protein (MBP) induces a population of encephalitogenic CD4 T cells having specificity for the dominant immunogenic peptide of MBP, 68 – 86. The TCR β chains of these disease-causing T cells show three distinct features: they are almost exclusively Vβ8.2, they use AspSer as the first two amino acid residues of the third complementarity-determining region (CDR3) and these junctional region sequences show few if any non-germline N-region nucleotide additions. This last feature raises the possibility that these autoimmune T cell precursors derive from TCR gene rearrangements occurring during early, perinatal ontogeny, a period when the enzyme terminal deoxynucleotidyl transferase (TdT), responsible for N region additions, is not expressed. An alternative possibility is that these features of the TCR of MBP 68 – 86-reactive T cells are dictated by considerations of antigen selection throughout ontogeny both in the thymus and in the periphery – i.e., that such β chains are conformationally the most appropriate for triggering by an epitope of 68 – 86 complexed to class II RT1.B^l MHC molecules. We show here that active experimental allergic encephalomyelitis, while delayed in onset, occurs in heavily irradiated animals, but not in the absence of a thymus, a finding indicating that this autoimmune disease is caused by a T cell subpopulation derived from the post-irradiation adult thymus. These disease-causing T cells are heavily Vβ8.2⁺ , CDR3 AspSer⁺ and use few N region additions. We conclude that T cells with these TCR β chain features can be generated in the adult thymus and most likely reflect requirements imposed by antigen selection.     

The central nervous system environment controls effector CD4+ T cell cytokine profile in experimental allergic encephalomyelitis

In experimental allergic encephalomyelitis (EAE), CD4⁺ T cells infiltrate the central nervous system (CNS). We derived CD4⁺ T cell lines from SJL/J mice that were specific for encephalitogenic myelin basic protein (MBP) peptides and produced both Th1 and Th2 cytokines. These lines transferred EAE to naive mice. Peptide-specific cells re-isolated from the CNS only produced Th1 cytokines, whereas T cells in the lymph nodes produced both Th1 and Th2 cytokines. Mononuclear cells isolated from the CNS, the majority of which were microglia, presented antigen to and stimulated MBP-specific T cell lines in vitro. Although CNS antigen-presenting cells (APC) supported increased production of interferon (IFN)-γ mRNA by these T cells, there was no increase in the interleukin (IL)-4 signal, whereas splenic APC induced increases in both IFN-γ and IL-4. mRNA for IL-12 (p40 subunit) was up-regulated in both infiltrating macrophages and resident microglia from mice with EAE. We have thus shown that a Th1 cytokine bias within the CNS can be induced by CNS APC, and that IL-12 is up-regulated in microglial cells within the CNS of mice with EAE. Microglia may therefore control Th1 cytokine responses within the CNS.     

Tumor necrosis factor blockade in actively induced experimental autoimmune encephalomyelitis prevents clinical disease despite activated T cell infiltration to the central nervous system

Recently, we demonstrated that experimental autoimmune encephalomyelitis (EAE) in the rat, passively transferred using myelin basic protein (MBP)-reactive encephalitogenic CD4⁺ T cells, was preventable by administration of a p55-tumor necrosis factor-IgG fusion protein (TNFR-IgG). This was despite quantitatively and qualitatively normal movement of these MBP-specific T cells to the central nervous system (CNS). To extend these findings, the effect of TNFR-IgG on EAE actively induced by injection of MBP in complete Freund's adjuvant was examined. This form of EAE in the rat typically involves an acute, self-limiting neurological deficit, substantial CNS inflammation, but minimal demyelination. Here we show that administration of TNFR-IgG prior to onset of disease signs completely prevented the neurological deficit or markedly reduced its severity. This blockade of clinical disease was dissociated from weight loss which occurred at the same tempo and magnitude as in control rats exhibiting neurological signs of disease such as paralysis. The timing of TNF blockade was critical as established clinical disease was relatively refractory to TNFR-IgG treatment. Activated CD4⁺ T cells expressing normal or elevated levels of VLA4, major histocompatibility complex class II, MRC OX40 and CD25 were isolated from or immunohistochemically localized in the CNS of clinically healthy rats treated before disease onset. There was a reduction of the amount of other inflammatory leukocytes in the CNS of these treated animals but, more importantly, the activation state of inflammatory leukocytes, as well as that of microglia isolated from treated animals, was reduced. Thus, TNFR-IgG, when administered before disease onset, appears to act by inhibiting an effector function of activated T cells and possibly other inflammatory leukocytes necessary to bring about the neurological deficit. However, while TNF is a critically important cytokine for the early events leading to initiation of EAE, it is not a necessary factor in the acute neurological deficit characteristic of this form of EAE, once disease onset has occurred.     

Antigen-specific down-regulation of myelin basic protein-reactive T cells during spontaneous recovery from experimental autoimmune encephalomyelitis: further evidence of apoptotic deletion of autoreactive T cells in the central nervous system

Experimental autoimmune encephalomyelitis (EAE) was inducedin Lewis rats by the I.v. injection of 10⁷ cloned V_ß8.2⁺T cells specific for the 72–89 peptide of guinea pig myelinbasic protein(MBP). Some animals were injected simultaneouslywith 10⁷ cloned T cells specific for ovalbumin(OVA). Lymphocyteswere isolated from the spinal cord and from the peripheral lymphoidorgans of these rats and the frequencies of MBP-peptide-specificor OVA-specific proliferating cells were estimated by limitingdilution analysis at different times after cell transfer. Thefrequencies of cells specific for MBP_72–89 or OVA in thespinal cord were highest 5 days after cell transfer (MBP_72–89,1 in 1149; OVA, 1 in 1116). On day 7, when the rats were recovering,the frequency of cells specific for MBP_72–89 in the spinalcord fell dramatically to <1 in 10⁵, while that of OVA-specificcells decreased to a much lesser extent (1 in 7001). The frequenciesof MBP_72–89-specific cells in the peripheral lymphoidorgans during and after recovery were also much lower than thoseof OVA-specific cells. A similar pattern of down-regulationof the MBP-peptide-specific, but not the OVA-specific, T cellresponse was observed in the spleen and mesenteric lymph nodes(MLN) of rats 38 days after the active induction of EAE by immunizationwith equal amounts of MBP and OVA in adjuvants. In the passivelytransferred model, cells isolated from the spinal cord and MLNon day 7 did not regain responsiveness to MBP_72–89 afterincubation with high levels of IL-2, indicating that the unresponsivenesswas not due to T cell anergy. Thus this study demonstrates thatthere is a specific down-regulation of the MBP_72–89-specificT cell response during spontaneous recovery from EAE. This conclusionis consistent with our previous observation that V_ß8.2⁺T cells are selectively eliminated from the CNS by apoptosisduring recovery from EAE induced by the passive transfer ofV_ß8.2⁺ T cells reactive to this MBP peptide. In contrastto autoreactive T cells, the non-autoreactive T cells that accumulatein the CNS during EAE appear to recirculate to the peripherallymphoid organs.     

IL‐12‐polarized Th1 cells produce GM‐CSF and induce EAE independent of IL‐23

CD4⁺ T‐helper (Th) cells reactive against myelin antigens mediate the mouse model experimental autoimmune encephalomyelitis (EAE) and have been implicated in the pathogenesis of multiple sclerosis (MS). It is currently debated whether encephalitogenic Th cells are heterogeneous or arise from a single lineage. In the current study, we challenge the dogma that stimulation with the monokine IL‐23 is universally required for the acquisition of pathogenic properties by myelin‐reactive T cells. We show that IL‐12‐modulated Th1 cells readily produce IFN‐γ and GM‐CSF in the CNS of mice and induce a severe form of EAE via an IL‐23‐independent pathway. Th1‐mediated EAE is characterized by monocyte‐rich CNS infiltrates, elicits a strong proinflammatory cytokine response in the CNS, and is partially CCR2 dependent. Conversely, IL‐23‐modulated, stable Th17 cells induce EAE with a relatively mild course via an IL‐12‐independent pathway. These data provide definitive evidence that autoimmune disease can be driven by distinct CD4⁺ T‐helper‐cell subsets and polarizing factors.     

Fasudil mediates cell therapy of EAE by immunomodulating encephalomyelitic T cells and macrophages

Although Fasudil has shown therapeutic potential in EAE mice, the mechanism of action are still not fully understood. Here, we examined the immunomodulatory effect of Fasudil on encephalitogenic mononuclear cells (MNCs), and tested the therapeutic potential of Fasudil‐treated MNCs in active EAE. Fasudil inhibited expression of CCL20 on T cells and migration of T cells, decreased CD4⁺IFN‐γ⁺ and CD4⁺IL‐17⁺ T cells, but increased CD4⁺IL‐10⁺ and CD4⁺TGF‐β⁺ T cells. Fasudil reduced expression of CD16/32 and IL‐12, while elevating expression of CD206, CD23, and IL‐10. Fasudil also decreased levels of iNOS/NO, enhanced levels of Arg‐1, and inhibited the TLR‐4/NF‐κB signaling and TNF‐α, shifting M1 macrophage to M2 phenotype. These modulatory effects of Fasudil on T cells and macrophages were not altered by adding autoantigen MOG_35–55 to the culture, i.e., autoantigen‐independent. Further, we observed that, in vitro, Fasudil inhibited the capacity of encephalitogenic MNCs to adoptively transfer EAE and reduced TLR‐4/p‐NF‐κB/p65 and inflammatory cytokines in spinal cords. Importantly, Fasudil‐treated encephalitogenic MNCs exhibited therapeutic potential when injected into actively induced EAE mice. Together, our results not only provide evidence that Fasudil mediates the polarization of macrophages and the regulation of T cells, but also reveal a novel strategy for cell therapy in MS.     

Heterogeneity of rat encephalitogenic T cells elicited by variants of the myelin basic protein (68–86) peptide

By immunizing Lewis rats with myelin basic protein (MBP) peptide variants derived from the major encephalitogenic epitope of guinea pig (MBP(68–88) and then isolating encephalitogenic T cells from these animals, we demonstrated that the variant peptides do not elicit the same encephalitogenic T cell subsets as those induced by the wild-type peptide or by intact MBP. Rather, the pathogenic T cells differed in clonal composition as reflected by their heterogeneous responses to a panel of variant peptides and by their T cell receptor usage. Thus, molecules mimicking the MBP(68–88) autoantigen can elicit pathogenic T cell subsets without necessarily cross-reacting with T cells specific for the original autoantigen. This suggests that a more clonally diverse group of pathogenic T cells might be involved in EAE than has been apparent from studies with intact MBP or its unaltered peptides.     

Diagnosis and assessment of preclinical and clinical autoimmune encephalomyelitis using peripheral blood lymphocyte TCR

In organ-specific autoimmune diseases, T cells involved in the disease development bear a particular type of TCR and infiltrate the target organ predominantly. However, it is difficult to identify disease-inducing T cells in peripheral blood lymphocytes (PBL) because such T cells are very few in number in a large pool of unrelated T cells. In the present study, we demonstrate that CDR3 spectratyping can identify experimental autoimmune encephalomyelitis (EAE)-specific patterns (oligoclonal expansion of Vβ8.2 with the shortest CDR3) in PBL at the preclinical and clinical stages of acute EAE. Analysis of nucleotide and predicted amino acid sequences of Vβ8.2 CDR3 of spectratype-derived clones revealed that CASSDSSYEQYFGPG, which is one of the representative sequences of encephalitogenic T cell clones, constituted the predominant population in both PBL and spinal cord T cells. In chronic relapsing EAE, the EAE-specific spectratype pattern in PBL was observed during the 1st and 2nd attacks, but not at the remission and full recovery stage. These findings indicate that the spectratyping pattern in PBL reflects the disease activity of acute and chronic relapsing EAE. Thus, CDR3 spectratyping using PBL can be used for diagnosis and assessment of T cell-mediated autoimmune diseases and is applicable to human autoimmune diseases.     

Th1‐mediated experimental autoimmune encephalomyelitis is CXCR3 independent

Drugs that block leukocyte trafficking ameliorate multiple sclerosis (MS). Occurrences of opportunistic infection, however, highlight the need for novel drugs that modulate more restricted subsets of T cells. In this context, chemokines and their receptors are attractive therapeutic targets. CXCR3, a Th1‐associated chemokine receptor, is preferentially expressed on T cells that accumulate in MS lesions and central nervous system (CNS) infiltrates of mice with experimental autoimmune encephalomyelitis (EAE). Surprisingly, mice genetically deficient in either CXCR3 or CXCL10 succumb to EAE following active immunization with myelin antigens. EAE is mediated by a heterogeneous population of T cells in myelin‐immunized mice. Hence, disease might develop in the absence of CXCR3 secondary to the compensatory action of encephalitogenic CCR6⁺ Th17 cells. However, in the current study, we show for the first time that blockade or genetic deficiency of either CXCR3 or of its primary ligand has no impact on clinical EAE induced by the adoptive transfer of highly polarized Th1 effector cells. Our data illustrate the fact that, although highly targeted immunotherapies might have more favorable side effect profiles, they are also more likely to be rendered ineffective by inherent redundancies in chemokine and cytokine networks that arise at sites of neuroinflammation.     

T cell receptor Vβ repertoire in mice lacking endogenous mouse mammary tumor provirus

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) Vβ segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, ?7, ?8 and ?9) and 38CH (Mtv^?) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, ?7, ?8 or ?9. The TcR Vβ chain (TcR Vβ) usage in these mice was analyzed using monoclonal antibodies specific for TcR Vβ2,Vβ3, Vβ4,β5,Vβ6,Vβ7,Vβ8,Vβ11,Vβ12 and Vβ14 segments. Both Mtv-8⁺ mice and Mtv-9⁺ mice deleted TcR Vβ5⁺ and Vβ11⁺ T cells. Moreover, we also observed the deletion of TcR Vβ12⁺ cells by Mtv-8 and Mtv-9 products. Mtv-6⁺ and Mtv-7⁺ animals deleted TcR Vβ3⁺ and Vβ35⁺ cells, and TcR Vβ6⁺,Vβ7⁺ and Vβ8.1⁺ cells, respectively. Unexpectedly, TcR Vβ8.2⁺ cells were also deleted in some backcross mice expressing Mtv-7. TcR Vβ8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn → Asp) in position 19 on the TcR Vβ8.2 fragment. Reactivities of BALB.D2 TcR Vβ8.2 and 38CH TcR Vβ8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR Vβ2⁺, Vβ4⁺ and Vβ8⁺ CD4⁺ T cell subsets in Mtv-8⁺ and Mtv-9⁺ mice, and TcR Vβ4⁺ CD4⁺ T cells in Mtv-6⁺ and Mtv-7⁺ mice, when compared with the T cell repertoire of Mtv^? mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.     

T cell receptor antagonist peptides are highly effective inhibitors of experimental allergic encephalomyelitis

The feasibility of using T cell receptor (TcR) antagonist peptides to inhibit autoimmune disease has been examined. First, the fine antigenic structure of the I-A^s-restricted encephalitogenic determinant proteolipid protein (PLP) 139–151 has been analyzed. It was found that residues 145 and 148 were I-A^s anchor residues, and residue 144 appeared to be especially critical in T cell activation. Residues 142, 143, 146, and 147 were found to be crucial for activation of some, but not all, of the T cells studied. Next, good I-A^s-binding nonantigenic analogs were tested for TcR antagonism. Accordingly, several single substitution analogs were identified which could act as TcR antagonists. Moreover, when two such analogs were combined, the resulting TcR antagonist pool inhibited most of the PLP 139–151-specific T cell clones in vitro. When the efficacy of this TcR antagonist pool in inhibiting EAE induction in vivo was examined, it was found that the analog pool was a remarkably potent inhibitor of disease induction. The TcR antagonist pool was approximately 10-fold more potent than our best major histocompatibility complex blocker and was still capable of significant inhibition when injected in equimolar amounts with the encephalitogenic PLP 139–151 determinant.     

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.