Clonotypic heterogeneity of lewis rat T cells specific for the encephalitogenic 68–86 region of myelin basic protein☆
Abstract
Experimental autoimmune encephalomyelitis was induced in a Lewis rat by sensitization with synthetic peptide GP68-86, representing the 68–86 sequence of guinea pig myelin basic protein (GPMBP). To delineate T cell determinants of GP68-86, lymph node cells from this rat were activated in culture with GP68-86 and were fused with cells of the mouse thymoma BW5147. The resultant hybrids were cloned by limiting dilution and screened for GP68-86-evoked secretion of IL 2 in the presence of rat splenocytes. Twelve T cell hybrids derived in this manner were tested for reactivity to different heterologous species of MBP as well as to substituted or truncated analogs of GP68-86. The hybrids generally exhibited potent reactivity to GPMBP but differed markedly in their reactivity to autologous rat MBP (RMBP). A few exceptional hybrids exhibited crossreactivity with peptides in which native serine75 or serine80 residues of GPMBP were substituted with either alanine75 (A75) or proline80 (P80) residues. These cross-reactive hybrids also possessed high levels of anti-RMBP reactivity. The remaining hybrids were unresponsive to the A75 and P80 substituted peptides and, with one exception, had relatively low levels of anti-RMBP reactivity. Unique reactivity patterns were also revealed by hybrid responses to peptides having modified C-terminal 84–86 residues. In summary, the contrasting fine specificities of different hybrids indicated that several distinct clones of T cells mediate the immune response of Lewis rats against the 68–86 region of GPMBP. Furthermore, heterogeneity in the hybrid response to “self” RMBP may reflect substantial differences in encephalitogenic potency of the T cell clones from which these hybrids were derived.
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Cited by (21)
Regulation of ocular inflammation - What experimental and human studies have taught us
2001, Progress in Retinal and Eye ResearchStudy of models of ocular autoimmunity and of autoimmune uveitis in humans has lead to a shift in the perceived nature of immune privilege from one based on anatomical isolation of the eye to a more dynamic, active process of immune tolerance. Using a variety of available models, the basis for this dynamic process of immune regulation is reviewed. The protective role of humoral immunity, the co-stimulatory function of B cells in EAU as well as the influence of cytokines within the inflammatory cascade are outlined. Modulation of the immune response and in particular the possible role of macrophages is explored. Within the current paradyme, a major effector cell is the CD4+ lymphocyte. Its maturation into a Th1 or Th2 phenotype process appears dependent on a number of exogenous factors, which while genetically determined can be manipulated prior to disease onset. Activation of CD4+ cells is dependent on presentation of immunoreactive peptide fragments. These fragments are well characterized in the Lewis rat for S-Ag and interphotoreceptor retinoid binding protein (IRBP). Mapping of the immunoreactivity to S-Ag has been recently completed in uveitis patients. An overlap with certain determinants identified in experimental models has been observed, in at least 2 disease entities. However, the response profile is not fixed in time and is subject to determinant spread. Future studies will be aimed at identifying with more detail immunologic triggers of inflammation in patients, and at better defining the interplay between effector and regulatory pathways both in the eye and in the systemic circulation.
Selection of anti-myelin basic protein T-cell lines in the Lewis rat: V- β 8.2 dominance and conserved complementarity-determining-region-3 motifs are dependent on serine at position 78 of myelin basic protein
2000, Journal of NeuroimmunologyIn the Lewis rat, the dominant T cell repertoire to myelin basic protein (MBP) is directed to the peptide 71–87 and the T cell receptors of pathogenic T cells are of the Vβ 8.2 genotype with short CDR3 sequences having a characteristic motif. However, this paradigm has been reached through analysis of long-term encephalitogenic lines and clones. We initiated the present study to examine the process of selection of the TCR Vβ 8.2 and characteristic CDR3 motifs upon immunization with guinea-pig MBP, and rat or guinea-pig 71–87 peptides. We found that the dominance of Vβ 8.2 developed progressively over 4–6 in vitro stimulations. Following immunization with rat 70–86, which differs from the guinea-pig peptide in one amino acid at position 78, the dominance of Vβ 8.2 and the characteristic CDR sequences are not seen. Thus, Vβ 8.2 dominance and specific CDR3 TCR motifs are seen with heterologous GpMBP but not with self rat MBP.
Immunohistochemical studies of mesolimbic dopaminergic neurons in Fischer 344 and Lewis rats
1996, Brain ResearchPrevious work has shown that the inbred Lewis and Fischer 344 rat strains differ in several behavioral measures related to mesolimbic dopamine function. Moreover, the ventral tegmental area (VTA) of the Lewis rat has been shown to contain higher levels of tyrosine hydroxylase compared to that of the Fischer rat by blot immunolabeling procedures. To investigate structural correlates of this biochemical difference, an immunohistochemical study of VTA dopaminergic neurons in these two strains was undertaken. Results show that the density and total number of tyrosine hydroxylase-positive neurons in the VTA of the Lewis rat is about 50% of that found in the Fischer rat. In contrast, examination of the substantia nigra in the same sections revealed no differences in the density and number of tyrosine hydroxylase-positive cells between these strains. Fischer-Lewis strain differences were also evident for cholecystokinin immunoreactivity in the VTA, with much lower levels seen in the Lewis rat, consistent with the known colocalization of this neuropeptide in many VTA dopamine neurons. The finding of 50%v fewer tyrosine hydroxylase-positive neurons in the VTA of the Lewis rat, along with our earlier results showing 45% higher levels of tyrosine hydroxylase by blot immunolabeling, would suggest much higher levels of tyrosine hydroxylase per VTA neuron in this strain. However, no obvious strain difference in the cellular intensity of tyrosine hydroxylase immunoreactivity could be detected by immunohistochemistry. Finally, the density of VTA dopamine neurons was assessed in 1-week-old Fischer and Lewis rats. In contrast to the results obtained for adult animals, no difference in the number of tyrosine hydroxylase-positive neurons was apparent in these young animals, indicating that the Fischer-Lewis strain difference in VTA dopamine neurons appears later in postnatal development. These anatomical findings shed new light on the differences in the mesolimbic dopamine system between Fischer and Lewis rats that may contribute to the behavioral differences exhibited by these animals.
T-helper lymphocytes specific for myelin basic protein: Activation-induced refractoriness of il-2 production pathways augments an anti-cd4-mediated proliferative deficit
1994, Cellular ImmunologyCloned and uncloned lines of encephalitogenic rat T cells produce IL-2 when activated with myelin basic protein (MBP) in the presence of irradiated splenocytes (SPL). Although these T cells use IL-2 as a primary mediator or autocrine growth, regulatory mechanisms controlling production of IL-2 have yet to be fully defined. This study shows that T cells reactivated within ∼7 days of a prior activation were refractory to the reinduction of MBP-stimulated IL-2 production. In contrast, T cells rested for >7 days regained the ability to produce optimal levels of IL-2 during activation with MBP. Cultures containing both activated and resting T cells responded to MBP by producing levels of IL-2 that were similar to those obtained from control cultures of resting T cells. The lack of IL-2 production during this refractory phase was associated with lowered responsiveness to MBP in proliferative assays as evidenced by right-shifted dose-response curves. However, this refractory phase did not affect the magnitude of responses elicited by optimal concentrations of MBP. The dissociation of proliferation from IL-2 production suggested parallel pathways of autocrine growth. Indeed, anti-MBP-proliferative responses were mediated by two distinct mechanisms distinguished by differential susceptibility to the anti-CD4 mAb W3/25. The W3/25-sensitive proliferation was desensitized in chronically activated T cells as well as in T cells activated once in the presence of the anti-CD4 mAb W3/25. Conversely, MBP responsiveness of W3/25-insensitive proliferation was unchanged by both chronic activation and by a prior activation in the presence of W3/25. In cultures of T cells recently activated by MBP in the presence of W3/25, the use of nonirradiated SPL rather than irradiated SPL reversed W3/25-mediated tolerance but did not restore MBP-stimulated IL-2 production. In summary, this study reveals mechanisms whereby the engagement of TcR and CD4 negatively regulates subsequent responsiveness of IL-2 production pathways and thereby impairs restimulation of IL-2-dependent proliferation by MBP-specific T-helper cells.
Parallel costimulatory pathways promote myelin basic protein-stimulated proliferation of encephalitogenic rat t cells
1994, Cellular ImmunologyActivation pathways responsible for myelin basic protein (MBP)-stimulated proliferation of encephalitogenic T cells were studied by derivation of new monoclonal antibodies against rat T cell surface proteins. These monoclonal antibodies were derived by immunization of Balb/c mice with THYB-2 T cell hybrids or with the GP2.E5 line of encephalitogenic T-helper cells.The LRTCI mAb inhibited MBP-stimulated IL-2 production by THYB-2 hybrids but not by THYB-1 hybrids and did not inhibit the alternative response of MBP-induced growth inhibition by either hybrid subset. Although LRTCI labeled virtually all rat leukocytes, it selectively inhibited proliferative responses to T cell mitogens but not to B cell mitogens. LRTC1 inhibited MBP-stimulated IL-2 production by GP2.E5 T cells but did not effectively block MBP-stimulated proliferation. Rather, LRTC1 acted in synergy with a second mAb (LRTC2) to effectively inhibit MBP-stimulated proliferation by GP2.E5 T cells. The observation that LRTC1 did not exhibit synergy with a third biologically active mAb (LRTC3) supported the hypothesis that LRTC1 and LRTC2 represented a specific combination of synergistic mAb. In contrast to the inhibitory activity on GP2.E5 T cells, LRTC1 and LRTC2 synergistically stimulated antigen-independent IL-2 production by the THYB-1 hybrid LSS-A1. These results support the hypothesis that GP2.E5 T cells respond to parallel costimulatory pathways that are respectively inhibited by LRTC1 and LRTC2 mAb, Furthermore, these synergistic mAb exhibited inhibitory or stimulatory activities that may be diagnostic of distinct T-helper cell subsets. These novel mAb may thereby facilitate studies of costimulatory pathways and T-helper cell subsets in the pathogenesis of autoimmune disease.
Differential recognition of sequences within the encephalitogenic region of myelin basic protein capable of eliciting cell-mediated immune responses in experimental autoimmune encephalomyelitis
1993, Journal of NeuroimmunologyThe fine specificity of myelin basic protein (MBP) epitopes capable of eliciting in vivo delayed-type hypersensitivity responses in Lewis rats with experimental autoimmune encephalomyelitis (EAE) was compared to those eliciting in vitro antigen-specific T cell proliferation and augmentation of disease transfer. Utilizing a panel of synthetic peptides with sequences representing the 68–86 region of guinea pig (GP-) or bovine myelin basic protein (B-MBP), animals were primed with one species of peptide and subsequently challenged with either the same peptide or peptides with truncations or substitutions representative of the other species of MBP. In regard to minimal length sequences capable of eliciting delayed-type hypersensitivity (DTH), rats primed with GP-MBP and complete Freund's adjuvant (CFA) exhibited a hierarchical pattern of responsiveness to challenge with a series of truncated peptides, ranking as follows: GP-68–86 > GP-72–86 > GP-68–84 > > GP-75–86 = no activity. This response pattern corresponds to that previously reported for T cell proliferation and activation for disease transfer. Furthermore, a comparison of these T cell-mediated immune parameters, as elicited by the substituted peptides, revealed the response patterns of DTH reactivity to be similar to that previously described for in vitro T cell proliferation with significant DTH responses generated only by the peptide species for which the animal was primed. In contrast, a cross-reactive pattern of recognition was observed in cells mediating disease transfer, with all four 68–86 sequences capable of augmenting activation for adoptive transfer of disease, regardless of the peptide species for which the animal was primed. The differential antigen recognition patterns observed for these EAE-associated immune responses supports the hypothesis that multiple TH cell subsets are involved in disease pathogenesis.
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This investigation was supported by NIH Grants AI-19273 and NS 06262, by a Postdoctoral Fellowship from the National Multiple Sclerosis Society (Grant FG 758-A-1), and by The Mulvihill Family Foundation in Memory of Rosemary Mulvihill Speth.
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Dr. Mark D. Mannie is a Postdoctoral Fellow of the National Multiple Sclerosis Society.
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Dr. Philip Y. Paterson is a Javits Neuroscience Investigator.