Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy

Experimental allergic encephalomyelitis (EAE) is a paralytic autoimmune disease induced in susceptible animals by active immunization with myelin basic protein (MBP) or by passive transfer of MBP-specific T helper (TH) lymphocytes. We have analyzed the T cell receptor genes of 33 clonally distinct TH cells specific for a nonapeptide of MBP inducing EAE in B10.PL (H-2u) mice. All 33 TH cells used two alpha variable gene segments (V alpha 2.3, 61%; V alpha 4.2, 39%), the same alpha joining gene segment (J alpha 39), and two V beta and J beta gene segments (V beta 8.2-J beta 2.6, 79%; V beta 13-J beta 2.2, 21%). The anti-V beta 8 monoclonal antibody F23.1 was found to block completely recognition of the nonapeptide by V beta 8 TH cells in vitro and to reduce significantly the susceptibility of B10.PL mice to peptide-induced EAE.     

T cell specificity for class II (I-A) and the encephalitogenic N-terminal epitope of the autoantigen myelin basic protein

The role of class II restriction in T cell recognition of an epitope of the autoantigen myelin basic protein (MBP) has been investigated. Encephalitogenic PL/J(H-2u) and (PL/J X SJL/J(H-2s))F1 ((PLSJ)F1) clones, isolated after immunization with intact MBP, recognize the N-terminal 11 amino acid residues of MBP in association with I-Au class II molecules. The synthetic peptide MBP 1-11 has been tested in vivo for induction of EAE. Clinical and histological EAE occurs in PL/J and (PLSJ)F1 mice but not SJL/J. The class II restriction of T cells primed with MBP 1-11 has been examined in primary cultures in vitro. Similar to encephalitogenic T cell clones, isolated after continuous selection in vitro, the population of MBP 1-11-specific proliferative PL/J and (PLSJ)F1 T cells, recognize this epitope in association with I-Au class II molecules. Not all MBP-specific T cell clones which are restricted to I-Au class II molecules cause autoimmune encephalomyelitis. The specificity of these non-encephalitogenic clones has been examined in this report. These clones also recognize MBP 1-11. Thus recognition of an encephalitogenic T cell epitope is not sufficient for induction of EAE.     

Major histocompatibility complex-linked control of the murine immune response to myelin basic protein

Recent experiments have shown that different regions of myelin basic protein (MBP) are encephalitogenic for different inbred strains of mice. It was therefore of interest to determine whether the immune response to MBP was MHC associated, and if so, what subregion controlled this response. Because PL/J and A/J mice were good responders to mouse MBP and C57Bl/10SN were not, B10.PL(73NS) and B10.A mice were immunized with mouse MBP under conditions designed to induce EAE. These strains were found to be highly susceptible. Intra-H-2 recombinant mice were then assessed for susceptibility. B10.A(4R) and B10.MBR were susceptible, whereas B10.A(5R) were resistant. Thus, EAE induced by purified MBP is under the control of the MHC, and the response maps to the I-A subregion. Production of IL 2 in vitro by T cells from MBP-primed mice in the presence of antigen and adherent cells was blocked by monoclonal antibody to the I-A, but not the I-E, subregion. When the specificity of the encephalitogenic response was tested, peptide 1-37 was active in B10.PL(73NS) and B10.A mice, whereas peptide 89-169 was active in A.SW, SWR, and B10.T(6R) strains. Serum from mice immunized with MBP peptides was assayed for antibody content. PL, B10.PL, and B10.A mice made a good antibody response to peptides 1-37 and 43-88 but were nonresponsive to peptide 89-169. SJL, A.SW, SWR, and B10.T(6R) mice responded well to peptide 89-169 but were poorly responsive to peptides 1-37 and 43-88.     

Unusual pattern of surface marker expression on peripheral lymphocytes from aged humans suggestive of a population of less differentiated cells

SJL/J, PL/J, and (SJL/J x PL/J)F1 mice were immunized with bovine, guinea pig, mouse, or rat myelin basic proteins (MBP) in adjuvant containing Mycobacterium tuberculosis H37Ra. Twenty-four and 72 hr later, Bordetella pertussis vaccine was given i.v. All MBP tested induced experimental allergic encephalomyelitis (EAE) in SJL/J and F1 mice; however, bovine MBP was inactive in PL/J mice. Each strain was immunized in a similar manner with peptic peptides, residues 1-37, 43-88, and 89-169 of guinea pig MBP. In contrast to the SJL/J strain, which has been shown to recognize a major encephalitogenic determinant in peptide 89-169, PL/J and F1 mice responded primarily to an encephalitogenic determinant within peptide 1-37. Analysis of antibody levels showed that SJL/J mice made no antibody to peptide 1-37, although anti-peptide 89-169 antibodies were consistently found. Conversely, PL/J mice responded well to peptide 1-37, but only an occasional animal made a significant response to peptide 89-169. (SJL/J x PL/J)F1 mice were more susceptible to EAE than either parental strain, as shown by the percentage of animals showing neurologic signs and by clinical severity.     

CD28 costimulation is crucial for the development of spontaneous autoimmune encephalomyelitis

Multiple sclerosis (MS) is a severe central nervous system disease. Experimental autoimmune encephalomyelitis (EAE) mimics MS in mice. We report that spontaneous development of EAE in RAG-1-deficient mice transgenic for a myelin basic protein (MBP)-specific TCR (TgMBP+/RAG-1-/-) requires expression of the T cell costimulatory molecule CD28. Surprisingly, T cells from CD28-/-TgMBP+/RAG-1-/- mice proliferate and produce IL-2 in response to MBP1-17 peptide in vitro, excluding clonal anergy as the mechanism of CD28-regulated pathogenesis. Proliferation of autoaggressive T cells was dependent on the concentration of the MBP peptide, as was the development of MBP-induced EAE in CD28-deficient PL/J mice. These results provide the first genetic evidence that CD28 costimulation is crucial for MBP-specific T cell activation in vivo and the initiation of spontaneous EAE.     

Expression of the tyrosine phosphatase SRC homology 2 domain-containing protein tyrosine phosphatase 1 determines T cell activation threshold and severity of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4 Th1-mediated inflammatory demyelinating disorder of the CNS and a well-established animal model for multiple sclerosis. Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) is a cytosolic tyrosine phosphatase that is involved in regulating the T cell activation cascade from signals initiated through the TCR. To study the role of SHP-1 in EAE pathogenesis, we immunized B10.PL mice heterozygous for deletion of the SHP-1 gene (me(v+/-)) and B10.PL wild-type mice with the immunodominant epitope of myelin basic protein (MBP Ac1-11). T cell proliferation and IFN-gamma production were significantly increased in me(v+/-) mice after immunization with MBP Ac1-11. The frequency of MBP Ac1-11-specific CD4 T cells, analyzed by staining with fluorescently labeled tetramers (MBP1-11[4Y]: I-A(u) complexes), was increased in the draining lymph node cells of me(v+/-) mice compared with wild-type mice. In addition, me(v+/-) mice developed a more severe course of EAE with epitope spreading to proteolipid protein peptide 43-64. Finally, expansion of MBP Ac1-11-specific T cells in response to Ag was enhanced in me(v+/-) T cells, particularly at lower Ag concentrations. These data demonstrate that the level of SHP-1 plays an important role in regulating the activation threshold of autoreactive T cells.     

Kinetics and specificity of T and B cell responses in relapsing experimental allergic encephalomyelitis

T and B cell responses to myelin basic protein (MBP) and its relevant peptide fragments were examined throughout the course of MBP-induced relapsing experimental allergic encephalomyelitis (REAE) in (SJL X PL)F1 mice. T cell reactivity, measured by the antigen-driven proliferation of lymph node T cells in vitro, was directed predominantly against the encephalitogenic MBP-P2 peptide (amino acids 1 to 37) at all stages of disease. Levels of responsiveness did not correlate with disease expression, but declined over time to a relapse level that was four- to sixfold lower than that observed during peak acute stage reactivity. Relapse responses were further distinguished by the detection of host I-E restrictions on Lyt-1+ T cell recognition of P2, P2 recognition by acute-stage T cells occurring solely in the context of host I-A molecules. These data imply an increase in the heterogeneity of relapse T cell responses to MBP to include clones restricted by additional class II glycoproteins. A role for additional CNS autoantigens in the stimulation of relapse T cells is also considered. Serum antibody responses to MBP or the P2 fragment fluctuated randomly throughout R-EAE when total antibody activity (IgM plus IgG) was measured. However, analysis of individual isotypes of IgG immunoglobulins revealed an apparent correlation between peak antigen-binding activity and disease expression which may reflect either an effector or regulatory role for humoral immunity in recurrent EAE. Patterns of early antibody reactivity also distinguished F1 mice that developed or failed to develop disease signs after immunization, the latter exhibiting a consistent drop in antigen-binding activity 4 to 5 days before the usual onset of acute-stage paralysis. The results are considered with regard to possible mechanisms of chronic disease regulation in an environment of functional T cell suppression.     

Encephalitogenic T cells in the B10.PL model of experimental allergic encephalomyelitis (EAE) are of the Th-1 lymphokine subtype

T helper cells reactive to myelin basic protein are clearly implicated in the pathogenesis of murine EAE. We have developed a T cell line, BML-1 that (1) is reactive to the encephalitogenic amino terminal nonapeptide (1-9NAC) of MBP, (2) is I-Au restricted, and (3) induces relapsing EAE in B10.PL (H-2u) mice. Measurement of the lymphokine profile of BML-1 revealed secretion of IL-2, interferon-gamma and lymphotoxin but not IL-4. This profile is consistent with the Th1/DTH subtype. Coculture of BML-1 with MBP-primed B cells shows that BML-1 does not provide significant helper function in vitro. In addition, BML-1 secretion of interferon-gamma was found to inhibit LPS-induced anti-MBP antibody responses. This suggested that anti-MBP antibodies may not be necessary for induction of EAE. Sera from mice, in which severe disease was induced with the 1-9NAC peptide and Bordetella pertussis, showed no development of serum antibodies to MBP. These data show that MBP-reactive Th cells of the Th-1/DTH subtype can induce EAE and do not provide Th function for anti-MBP responses and that serum anti-MBP antibodies are not found in peptide 1-9NAC-induced disease. T cell lines specific for encephalitogenic epitopes and characterized for lymphokine secretion will provide a useful tool for understanding the role of T cells in the induction of EAE.     

Location of a new encephalitogenic epitope (residues 43 to 64) in proteolipid protein that induces relapsing experimental autoimmune encephalomyelitis in PL/J and (SJL x PL)F1 mice.

Synthetic peptides of proteolipid protein (PLP) were screened for their ability to induce experimental autoimmune encephalomyelitis (EAE) in SJL/J, PL/J, and (SJL x PL)F1 mice, and T cell lines were selected by stimulation of lymph node cells with PLP peptides. PLP 141-151 was found to be less encephalitogenic in SJL/J mice than PLP 139-151, due to deletion of two amino acids from the amino-terminal end. PLP 139-151 immunization induced relapsing EAE in SJL/J and F1 mice but not PL/J mice. In contrast, PLP 43-64 induced relapsing EAE in PL/J and F1 mice but not SJL/J mice. F1 T cell lines specific for either PLP 43-64 or PLP 139-151 adoptively transferred demyelinating EAE to naive F1 recipients. Haplotypes H-2s and H-2u appear to be immunologically co-dominant in F1 mice in the PLP EAE system, which differs from the H-2u dominance in F1 mice in the myelin basic protein EAE system. The identification of a PLP peptide that is encephalitogenic in PL/J mice, in addition to the previous demonstration of PLP peptides that are encephalitogenic for SWR mice (PLP 103-116) and SJL/J mice (PLP 139-151), lends support to a role for PLP as a target Ag in autoimmune demyelinating diseases.     

Influence of the H-2u haplotype on immune function in F1 hybrid mice. I. Antigen presentation

Previously, we reported that myelin basic protein (MBP) peptide 1-37 contains an encephalitogenic epitope for PL/J mice, and MBP peptide 89-169 is encephalitogenic for SJL/J mice. (SJPL)F1 hybrid mice do not respond to immunization with these peptides in a co-dominant manner because the encephalitogenic response to peptide 1-37 dominates. To examine this phenomenon more closely, we tested the ability of MBP-primed parental or F1 T cells to respond to MBP or MBP peptides in the context of PL, SJL, or F1 antigen-presenting cells (APC). It was found that the F1 T cells responded to either the protein or the peptides when these were presented in the context of F1 or PL APC. However, F1 T cells would not respond to MBP in the context of SJL APC, although the latter cells were functionally intact. This effect was not antigen-specific because SJL APC would not present ovalbumin or PPD to primed F1 T cells. F1 T cells from mice immune to the strongly antigenic bacterium Listeria monocytogenes responded to bacterial antigens presented by SJL APC, although at a significantly lower level compared to the results obtained when these antigens were presented by F1 or PL APC. This finding implied that unbalanced antigen presentation was a quantitative rather than a qualitative phenomenon. When F1 hybrid mice from other strain combinations were tested, a similar effect was observed whenever one of the parental strains was PL/J. This effect was mapped to the MHC in MHC-congenic B10 mice.     

Monomeric recombinant TCR ligand reduces relapse rate and severity of experimental autoimmune encephalomyelitis in SJL/J mice through cytokine switch

Our previous studies demonstrated that oligomeric recombinant TCR ligands (RTL) can treat clinical signs of experimental autoimmune encephalomyelitis (EAE) and induce long-term T cell tolerance against encephalitogenic peptides. In the current study, we produced a monomeric I-A(s)/PLP 139-151 peptide construct (RTL401) suitable for use in SJL/J mice that develop relapsing disease after injection of PLP 139-151 peptide in CFA. RTL401 given i.v. or s.c. but not empty RTL400 or free PLP 139-151 peptide prevented relapses and significantly reduced clinical severity of EAE induced by PLP 139-151 peptide in SJL/J or (C57BL/6 x SJL)F(1) mice, but did not inhibit EAE induced by PLP 178-191 or MBP 84-104 peptides in SJL/J mice, or MOG 35-55 peptide in (C57BL/6 x SJL/J)F(1) mice. RTL treatment of EAE caused stable or enhanced T cell proliferation and secretion of IL-10 in the periphery, but reduced secretion of inflammatory cytokines and chemokines. In CNS, there was a modest reduction of inflammatory cells, reduced expression of very late activation Ag-4, lymphocyte function-associated Ag-1, and inflammatory cytokines, chemokines, and chemokine receptors, but enhanced expression of Th2-related factors, IL-10, TGF-beta3, and CCR3. These results suggest that monomeric RTL therapy induces a cytokine switch that curbs the encephalitogenic potential of PLP 139-151-specific T cells without fully preventing their entry into CNS, wherein they reduce the severity of inflammation. This mechanism differs from that observed using oligomeric RTL therapy in other EAE models. These results strongly support the clinical application of this novel class of peptide/MHC class II constructs in patients with multiple sclerosis who have focused T cell responses to known encephalitogenic myelin peptides.     

Genetic control of the development of experimental allergic encephalomyelitis in rats. Separation of MHC and non-MHC gene effects

Experimental allergic encephalomyelitis (EAE)-susceptible Lew and EAE-resistant Brown Norway (BN) rats and the corresponding MHC congenic strains were examined for their ability to develop clinical and histologic EAE. The ability of T cells from these animals to proliferate in vitro in response to whole guinea pig (GP) myelin basic protein (MBP), rat MBP, and to the major encephalitogenic peptide of GP MBP 66-88 (GP 68-88) was also assessed. We found that Lewis (Lew) was highly susceptible and showed good T cell responses to GP, MBP, rat MBP, and GP 68-88. Lew.1N (BN MHC on Lew background) and BN were not susceptible and T cells from these strains showed significant responses to GP MBP, but not to rat MBP or GP 68-88. Although BN.B1 (Lew MHC on BN background) was not susceptible to actively induced EAE, MBP-specific Lew T cells could transfer severe disease to BN.B1. BN.B1 T cells showed responses to GP-MBP, rat MBP, and GP 68-88 and, when transferred to naive BN.B1 or Lew, induced only mild clinical EAE in both strains. Increasing the number of T cells from BN.B1 had no effect on the severity of clinical symptoms in either recipient, suggesting some deficiency in the T cell repertoire that is necessary for induction of severe EAE. These results suggest that 1) the T cell response to rat MBP and GP68-88 (but not to sites other than 68-88 in GP MBP) is necessary for susceptibility to EAE; 2) the ability to respond to both rat MBP and GP 68-88 is determined by the MHC gene products on APC; and 3) given a permissive MHC, the T cell response that results in EAE is influenced by non-MHC genes.     

Characterization of the antigen specificity and TCR repertoire,and TCR-based DNA vaccine therapy in myelin basic protein-induced autoimmune encephalomyelitis in DA rats

Like Lewis rats, DA rats are an experimental autoimmune encephalomyelitis (EAE)-susceptible strain and develop severe EAE upon immunization with myelin basic protein (MBP). However, there are several differences between the two strains. In the present study we induced acute EAE in DA rats by immunization with MBP and MBP peptides and examined the Ag specificity and TCR repertoire of encephalitogenic T cells. It was found that although immunization with MBP and a peptide corresponding to its 62-75 sequence (MBP(62-75)) induced clinical EAE, the responses of lymph node T cells isolated from MBP-immunized rats to MBP(62-75) was marginal, indicating that this peptide contains major encephalitogenic, but not immunodominant, epitopes. The TCR analysis by CDR3 spectratyping of spinal cord T cells revealed that Vbeta10 and Vbeta15 spectratype expansion was always found in MBP(62-75)-immunized symptomatic rats. On the basis of these findings, we examined the encephalitogenicity of Vbeta10- and Vbeta15-positive T cells. First, the adoptive transfer experiments revealed that Vbeta10-positive T line cells derived from MBP(62-75)-immunized rats induced clinical EAE in recipients. Second, administration of DNA vaccines encoding Vbeta10 and Vbeta15, alone or in combination, ameliorated MBP(62-75)-induced EAE. Collectively, it was strongly suggested that Vbeta10- and Vbeta15-positive T cells are encephalitogenic. Analyses of the Ag specificity and T cell repertoire of pathogenic T cells performed in this study provide useful information for designing specific immunotherapies against autoimmune diseases.     

Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice

PLP is the major protein constituent of central nervous system myelin. We have previously shown that SJL/J (H-2s) mice develop an acute form of EAE after immunization with PLP. The purpose of the present study was to identify an encephalitogenic determinant of PLP for SJL mice. We immunized SJL/J mice with a synthetic peptide identical to residues 130-147 QAHSLERVCHCLGKWLGH of murine PLP, a sequence having an amphipathic alpha-helical conformation. Although it did not induce disease, an overlapping peptide containing residues 139-154 HCLGKWLGHPDKFVGI was encephalitogenic. Immunization with this peptide induced severe clinical and histologic EAE in 3 of 20 mice. T cell enriched ILN cells from these mice responded specifically (3H-thymidine incorporation) to this peptide as well as to shorter analogues of this domain containing serine in place of cysteine at residues 138 and 140. Immunization with the serine-substituted PLP peptides 137-151 VSHSLGKWLGHPDKF and 139-151 HSLGKWLGHPDKF induced severe, acute EAE in 4 of 9 and 15 of 15 SJL mice, respectively, and their T cell enriched ILN cells responded not only to the analogues, but also to the native PLP sequence 139-154. These results indicate that residues 139-151 of murine PLP is an encephalitogenic determinant for SJL mice. Furthermore, like the PLP encephalitogenic domain for SWR (H-2q) mice, this determinant is also a T cell epitope with a coding sequence at the end of an exon.     

Identification of encephalitogenic V beta-4-bearing T cells in SJL mice. Further evidence for the V region disease hypothesis?

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by T cells bearing TCR of restricted heterogeneity. Thus, in the murine PL strain, V beta-8.2 is used by 80% of the encephalitogenic T cells. This observation has led to the successful prevention and reversal of EAE by the in vivo use of mAb directed to these restricted gene products. In SJL mice, the V beta-17a gene product has been shown to be used by approximately 50% of encephalitogenic T cells subsequent to immunization with a myelin basic protein (MBP)-derived peptide. However, the other V beta genes used by encephalitogenic T cells in SJL EAE have remained uncharacterized. We now report, for the first time, the beta-chain-encoding DNA sequence of two encephalitogenic, MBP-reactive, SJL-derived T cell clones. These clones which are specific for H-2s and the carboxyl-terminus (amino acid 92-103) of MBP, use TCR encoded by V beta-4. In addition, we demonstrate that the transfer of EAE by a heterogenous SJL-derived encephalitogenic T cell line can be prevented using an anti-V beta-4 antibody in vivo. V beta-4 usage has been previously described in a H-2u/MBP amino-terminus-reactive encephalitogenic T cell. The present findings may thus further support the "V region-disease" hypothesis.     

Oligodendrocyte-specific protein peptides induce experimental autoimmune encephalomyelitis in SJL/J mice.

Oligodendrocyte-specific protein (OSP) is a recently isolated and cloned, 207-aa, hydrophobic, four-transmembrane protein found in CNS myelin. It represents approximately 7% of total myelin protein. The OSP cDNA sequence has no significant homology with previously reported genes, but the predicted protein structure suggests that OSP is a CNS homologue of peripheral myelin protein-22. We previously reported the presence of anti-OSP Abs in the cerebrospinal fluid of relapsing-remitting multiple sclerosis (MS) patients, but not control patient groups. In this study, we tested the ability of a panel of 20-mer peptides with 10-aa overlaps, representing the sequence of murine OSP, to induce experimental autoimmune encephalomyelitis (EAE), an animal model for MS. SJL mice challenged with murine OSP peptides 52-71, 82-101, 102-121, 142-161, 182-201, and 192-207 exhibited clinical EAE. OSP:52-71 elicited severe relapsing-remitting EAE in some individuals. All other encephalitogenic peptides elicited, at most, a loss of tail tonicity from which the mice most often completely recovered. Mononuclear cell infiltrates and focal demyelination characteristic of EAE were evident. T cell proliferative responses were seen with all encephalitogenic peptides except 142-161 and 182-201. OSP peptides 72-91 and 132-151 did not cause clinical EAE, but did elicit robust proliferative responses. B10.PL and PL/J mice challenged with the same OSP peptide doses as SJL mice did not exhibit clinical EAE. These results in the SJL EAE model, together with the results from MS patient clinical samples, make OSP a promising candidate for autoantigenic involvement in MS.     

The myelin-associated oligodendrocytic basic protein region MOBP15-36 encompasses the immunodominant major encephalitogenic epitope(s) for SJL/J mice and predicted epitope(s) for multiple sclerosis-associated HLA-DRB1*1501

Autoimmune response to the myelin-associated oligodendrocytic basic protein (MOBP), a CNS-specific myelin constituent, was recently suggested to play a role in the pathogenesis of multiple sclerosis (MS). The pathogenic autoimmune response to MOBP and the associated pathology in the CNS have not yet been fully investigated. In this study, we have characterized the clinical manifestations, pathology, T cell epitope-specificity, and TCRs associated with experimental autoimmune encephalomyelitis (EAE) induced in SJL/J mice with recombinant mouse MOBP (long isoform, 170 aa). Analysis of encephalitogenic MOBP-reactive T cells for reactivity to overlapping MOBP peptides defined MOBP15-36 as their major immunodominant epitope. Accordingly, MOBP15-36 was demonstrated to be the major encephalitogenic MOBP epitope for SJL/J mice, inducing severe/chronic clinical EAE associated with intense perivascular and parenchymal infiltrations, widespread demyelination, axonal loss, and remarkable optic neuritis. Molecular modeling of the interaction of I-A(s) with MOBP15-36, together with analysis of the MOBP15-36-specific T cell response to truncated peptides, suggests MOBP20-28 as the core sequence for I-A(s)-restricted recognition of the encephalitogenic region MOBP15-36. Although highly focused in their epitope specificity, the encephalitogenic MOBP-reactive T cells displayed a widespread usage of TCR Vbeta genes. These results would therefore favor epitope-directed, rather than TCR-targeted, approaches to therapy of MOBP-associated pathogenic autoimmunity. Localization by molecular modeling of a potential HLA-DRB1*1501-associated MOBP epitope within the encephalitogenic MOBP15-36 sequence suggests the potential relevance of T cell reactivity against MOBP15-36 to MS. The reactivity to MOBP15-36 detected in MS shown here and in another study further emphasizes the potential significance of this epitope for MS.     

Experimental allergic encephalomyelitis mediated by cloned T cells specific for a synthetic peptide of myelin proteolipid protein. Fine specificity and T cell receptor V beta usage.

Proteolipid protein (PLP) is the major protein of central nervous system myelin. SJL (H-2s) mice immunized with a synthetic peptide corresponding to PLP residues 139-151 develop acute EAE. In this study, 6 IAs-restricted, CD4+, TCR alpha beta-bearing T cell clones were derived from SJL/J mice after immunization with this synthetic peptide. The clones responded in in vitro proliferative assays to the whole PLP molecule and to PLP peptide 139-151, but not to irrelevant Ag. They also responded to truncated and overlapping forms of the peptide but five distinct reactivity patterns were observed using these peptides. A panel of anti-TCR V beta mAb and TCR V beta-specific cDNA probes were used to determine the TCR V beta usage of the clones. Five clones were found to use four different V beta (V beta 2, V beta 6, V beta 10, or V beta 17a), whereas the V beta on the sixth clone could not be identified. Five of the clones induced EAE of varying severity upon adoptive transfer into naive syngeneic mice or mice pretreated with irradiation and pertussis and one clone was nonencephalitogenic. The Ag-specific proliferative response of all but the nonencephalitogenic clone could be blocked by an anti-CD4 mAb. Thus, the clones showed differences in their fine specifity, TCR V beta usage, sensitivity to antibody blocking, and encephalitogenic potency. These data demonstrate that the T cell response to the encephalitogenic PLP peptide 139-151 is heterogeneous.     

T cell recognition of distinct peptide:I-Au conformers in murine experimental autoimmune encephalomyelitis

We have used T cells bearing TCRs that are closely related in sequence as probes to detect conformational variants of peptide-MHC complexes in murine experimental autoimmune encephalomyelitis in H-2(u) mice. The N-terminal epitope of myelin basic protein (MBP) is immunodominant in this model. Our studies have primarily focused on T cell recognition of a position 4 analog of this peptide (MBP1-9[4Y]) complexed with I-A(u). Using site-directed mutagenesis, we have mapped the functionally important complementarity determining region residues of the 1934.4 TCR Valpha domain. One of the resulting mutants (Tyr(95) to alanine in CDR3alpha, Y95A) has interesting properties: relative to the parent wild-type TCR, this mutant poorly recognizes Ag complexes generated by pulsing professional APCs (PL-8 cells) with MBP1-9[4Y] while retaining recognition of MBP1-9[4Y]-pulsed unconventional APCs or insect cell-expressed complexes of I-A(u) containing tethered MBP1-9[4Y]. Insect cell expression of recombinant I-A(u) with covalently tethered class II-associated invariant chain peptide or other peptides which bind relatively weakly, followed by proteolytic cleavage of the peptide linker and replacement by MBP1-9[4Y] in vitro, results in complexes that resemble peptide-pulsed PL-8 cells. Therefore, the distinct conformers can be produced in recombinant form. T cells that can distinguish these two conformers can also be generated by the immunization of H-2(u) mice, indicating that differential recognition of the conformers is observed for responding T cells in vivo. These studies have relevance to understanding the molecular details of T cell recognition in murine experimental autoimmune encephalomyelitis. They are also of particular importance for the effective use of multimeric peptide-MHC complexes to characterize the properties of Ag-specific T cells.