Administration of PLP139-151 primes T cells distinct from those spontaneously responsive in vitro to this antigen

We examined the TCR repertoire used by naive SJL mice in their in vitro spontaneous response to proteolipid protein (PLP) 139-151 by Vbeta-Jbeta spectratyping and compared it to that used after immunization with the peptide. T cells from immunized mice use the public rearrangement Vbeta10-Jbeta1.1, but naive mice do not; in contrast, TCR CDR3-beta rearrangements of Vbeta18-Jbeta1.2 and Vbeta19-Jbeta1.2 consistently are associated with the spontaneous response. T cells involved in spontaneous and induced responses can each recognize PLP(139-151) presented in vivo, but its s.c. administration has different consequences for the two repertoires. Four days after immunization, T cells associated with spontaneous responsiveness appear in the draining lymph nodes but disappear by day 10 and never appear elsewhere. Simultaneously, Vbeta10-Jbeta1.1 T cells are likewise activated in the lymph nodes by day 4 and spread to the spleen by day 10. Eight- to 10-wk-old naive mice use a narrower repertoire of TCRs than do immunized age-matched mice. Induced Vbeta10-Jbeta1.1 T cells home to the CNS during experimental autoimmune encephalomyelitis, whereas we failed to detect Vbeta18-Jbeta1.2 and Vbeta19-Jbeta1.2 TCR rearrangements in the CNS. Thus, we observe that administration of PLP(139-151) primes a T cell repertoire distinct from the one responsible for spontaneous responsiveness. This "immunized" repertoire substitutes for the naive one and becomes dominant at the time of disease onset.     

Functional activation of myelin-specific T cells by virus-induced molecular mimicry

Molecular mimicry is the process by which T cells activated in response to determinants on an infecting microorganism cross-react with self epitopes, leading to an autoimmune disease. Normally, infection of SJL/J mice with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) results in a persistent CNS infection, leading to a chronic progressive, CD4(+) T cell-mediated demyelinating disease. Myelin damage is initiated by T cell responses to virus persisting in CNS APCs, and progressive demyelinating disease (50 days postinfection) is perpetuated by myelin epitope-specific CD4(+) T cells activated by epitope spreading. We developed an infectious model of molecular mimicry by inserting a sequence encompassing the immunodominant myelin epitope, proteolipid protein (PLP) 139-151, into the coding region of a nonpathogenic TMEV variant. PLP139-TMEV-infected mice developed a rapid onset paralytic inflammatory, demyelinating disease paralleled by the activation of PLP139-151-specific CD4(+) Th1 responses within 10-14 days postinfection. The current studies demonstrate that the early onset demyelinating disease induced by PLP139-TMEV is the direct result of autoreactive PLP139-151-specific CD4(+) T cell responses. PLP139-151-specific CD4(+) T cells from PLP139-TMEV-infected mice transferred demyelinating disease to naive recipients and PLP139-151-specific tolerance before infection prevented clinical disease. Finally, infection with the mimic virus at sites peripheral to the CNS induced early demyelinating disease, suggesting that the PLP139-151-specific CD4(+) T cells could be activated in the periphery and traffic to the CNS. Collectively, infection with PLP139-151 mimic encoding TMEV serves as an excellent model for molecular mimicry by inducing pathologic myelin-specific CD4(+) T cells via a natural virus infection.     

IL-10 plays an important role in the homeostatic regulation of the autoreactive repertoire in naive mice

We have previously shown that naive SJL (H-2(s)) mice, which are highly susceptible to myelin proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), have a very high frequency (1/20,000 CD4 T cells) of PLP(139-151)-reactive T cells in the naive repertoire. In this study, we examine the function of this endogenous PLP(139-151)-reactive repertoire in vivo and find that this repertoire encompasses the precursors of pathogenic T cells. Because SJL mice do not develop spontaneous EAE, we have explored the mechanisms that keep this autopathogenic repertoire in check and prevent the development of spontaneous autoimmunity. We crossed IL-4 and IL-10 deficiency onto the SJL background and analyzed the roles of these two immunoregulatory cytokines in regulating the size and effector function of the endogenous PLP(139-151)-reactive repertoire and development of autoimmune disease. We find that IL-10 is important in the homeostatic regulation of the endogenous PLP(139-151)-reactive repertoire in that it both limits the size of the repertoire and prevents development of effector autoaggressive T cells. SJL IL-10(-/-) mice with high numbers of PLP(139-151)-specific precursors in the repertoire did not develop spontaneous EAE, but when they were injected with pertussis toxin, they showed atypical clinical signs of EAE with small numbers of typical mononuclear cell infiltrates predominantly in the meninges. EAE could be inhibited by prior tolerization of the mice with soluble PLP(139-151) peptide. These findings indicate that IL-10 may contribute to the regulation of the endogenous autoimmune repertoire.     

Viral delivery of an epitope from Haemophilus influenzae induces central nervous system autoimmune disease by molecular mimicry

Multiple sclerosis (MS) is an autoimmune CNS demyelinating disease in which infection may be an important initiating factor. Pathogen-induced cross-activation of autoimmune T cells may occur by molecular mimicry. Infection with wild-type Theiler's murine encephalomyelitis virus induces a late-onset, progressive T cell-mediated demyelinating disease, similar to MS. To determine the potential of virus-induced autoimmunity by molecular mimicry, a nonpathogenic neurotropic Theiler's murine encephalomyelitis virus variant was engineered to encode a mimic peptide from protease IV of Haemophilus influenzae (HI), sharing 6 of 13 aa with the dominant encephalitogenic proteolipid protein (PLP) epitope PLP(139-151). Infection of SJL mice with the HI mimic-expressing virus induced a rapid-onset, nonprogressive paralytic disease characterized by potent activation of self-reactive PLP(139-151)-specific CD4(+) Th1 responses. In contrast, mice immunized with the HI mimic-peptide in CFA did not develop disease, associated with the failure to induce activation of PLP(139-151)-specific CD4(+) Th1 cells. However, preinfection with the mimic-expressing virus before mimic-peptide immunization led to severe disease. Therefore, infection with a mimic-expressing virus directly initiates organ-specific T cell-mediated autoimmunity, suggesting that pathogen-delivered innate immune signals may play a crucial role in triggering differentiation of pathogenic self-reactive responses. These results have important implications for explaining the pathogenesis of MS and other autoimmune diseases.     

Detection of autoreactive myelin proteolipid protein 139-151-specific T cells by using MHC II (IAs) tetramers

Detection of autoreactive T cells using MHC II tetramers is difficult because of the low affinity of their TCR. We have generated a class II tetramer using the IA(s) class II molecule combined with an autoantigenic peptide from myelin proteolipid protein (PLP; PLP(139-151)) and used it to analyze myelin PLP(139-151)-reactive T cells. Using monomers and multimerized complexes labeled with PE, we confirmed the specificity of the reagent by bioassay and flow cytometry. The IA(s) tetramers stimulated and stained the PLP(139-151)-specific 5B6 TCR transgenic T cells and a polyclonal cell line specific for PLP(139-151), but not a control T cell line specific for PLP(178-191). We used this reagent to optimize conditions to detect low affinity autoreactive T cells. We found that high pH ( approximately 8.0) and neuraminidase treatment enhances the staining capacity of PLP(139-151) tetramer without compromising specificity. Furthermore, we found that induction of calcium fluxing by tetramers in T cells may be used as a sensitive measure to detect autoreactive T cells with a low affinity. Taken together, the data show that the tetrameric reagent binds and stimulates PLP(139-151)-reactive T cells with specificity. This tetrameric reagent will be useful in studying the evolution of PLP(139-151)-specific repertoire in naive mice and its expansion during the autoimmune disease experimental autoimmune encephalomyelitis.     

Initiation and exacerbation of autoimmune demyelination of the central nervous system via virus-induced molecular mimicry: implications for the pathogenesis of multiple sclerosis

Epidemiological studies indicate that infectious agents are important in the pathogenesis of multiple sclerosis (MS). Our previous reports showed that the infection of SJL mice with a nonpathogenic variant of Theiler's murine encephalomyelitis virus (TMEV) engineered to express a naturally occurring Haemophilus influenzae-encoded molecular mimic (HI574-586) of an immunodominant self-myelin proteolipid protein epitope (PLP139-151) induced a rapid-onset demyelinating disease associated with the activation of PLP139-151-specific Th1 responses. The current results extend our previous findings in four critical respects. We show that disease initiation by the H. influenzae mimic is prevented by tolerance to the self PLP139-151 epitope, definitively proving the occurrence of infection-induced molecular mimicry. We demonstrate that the H. influenzae mimic epitope can be processed from the flanking sequences within the native mimic protein. We show that the H. influenzae mimic epitope only induces an immunopathologic self-reactive Th1 response and subsequent clinical disease in the context of the TMEV infection and not when administered in complete Freund's adjuvant, indicating that molecular mimicry-induced disease initiation requires virus-activated innate immune signals. Lastly, we show that the infection of SJL mice with TMEV expressing the H. influenzae mimic can exacerbate a previously established nonprogressive autoimmune disease of the central nervous system. Collectively, these findings illustrate the evolving mechanisms by which virus infections may contribute to both the initiation and exacerbation of autoimmune diseases, and they have important implications for MS pathogenesis.     

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.     

SJL Mice Infected with Acanthamoeba castellanii Develop Central Nervous System Autoimmunity through the Generation of Cross-Reactive T Cells for Myelin Antigens

We recently reported that Acanthamoeba castellanii (ACA), an opportunistic pathogen of the central nervous system (CNS) possesses mimicry epitopes for proteolipid protein (PLP) 139–151 and myelin basic protein 89–101, and that the epitopes induce experimental autoimmune encephalomyelitis (EAE) in SJL mice reminiscent of the diseases induced with their corresponding cognate peptides. We now demonstrate that mice infected with ACA also show the generation of cross-reactive T cells, predominantly for PLP 139–151, as evaluated by T cell proliferation and IA^s/dextramer staining. We verified that PLP 139–151-sensitized lymphocytes generated in infected mice contained a high proportion of T helper 1 cytokine-producing cells, and they can transfer disease to naïve animals. Likewise, the animals first primed with suboptimal dose of PLP 139–151 and later infected with ACA, developed EAE, suggesting that ACA infection can trigger CNS autoimmunity in the presence of preexisting repertoire of autoreactive T cells. Taken together, the data provide novel insights into the pathogenesis of Acanthamoeba infections, and the potential role of infectious agents with mimicry epitopes to self-antigens in the pathogenesis of CNS diseases such as multiple sclerosis.     

Synthetic Peptide dendrimers block the development and expression of experimental allergic encephalomyelitis

Multiple Ag peptides (MAPs) containing eight proteolipid protein (PLP)(139-151) peptides arranged around a dendrimeric branched lysine core were used to influence the expression and development of relapsing experimental allergic encephalomyelitis (EAE) in SJL mice. The PLP(139-151) MAPs were very efficient agents in preventing the development of clinical disease when administered after immunization with the PLP(139-151) monomeric encephalitogenic peptide in CFA. The treatment effect with these MAPs was peptide specific; irrelevant multimeric peptides such as guinea pig myelin basic protein GPBP(72-84) MAP (a dendrimeric octamer composed of the 72-84 peptide) and PLP(178-191) MAP (a dendrimeric octamer composed of the PLP(178-191) peptide) had no treatment effect on PLP(139-151)-induced EAE. PLP(139-151) MAP treatment initiated after clinical signs of paralysis also altered the subsequent course of EAE; it limited developing signs of paralysis and effectively limited the severity and number of disease relapses in MAP-treated mice over a 60-day observation period. PLP(139-151) MAP therapy initiated before disease onset acts to limit the numbers of Th17 and IFN-gamma-producing cells that enter into the CNS. However, Foxp3(+) cells entered the CNS in numbers equivalent for nontreated and PLP(139-151) MAP-treated animals. The net effect of PLP(139-151) MAP treatment dramatically increases the ratio of Foxp3(+) cells to Th17 and IFN-gamma-producing cells in the CNS of PLP(139-151) MAP-treated animals.     

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.     

Intrinsic and induced regulation of the age-associated onset of spontaneous experimental autoimmune encephalomyelitis

Multiple sclerosis is characterized by perivascular CNS infiltration of myelin-specific CD4(+) T cells and activated mononuclear cells. TCR transgenic mice on the SJL background specific for proteolipid protein (PLP)(139-151) develop a high incidence of spontaneous experimental autoimmune encephalomyelitis (sEAE). We examined the intrinsic mechanisms regulating onset and severity of sEAE. CD4(+) T cells isolated from the cervical lymph nodes, but not spleens, of diseased 5B6 transgenic mice are hyperactivated when compared with age-matched healthy mice and produce both IFN-gamma and IL-17, indicating that the cervical lymph node is the initial peripheral activation site. The age-associated development of sEAE correlates with a decline in both the functional capacity of natural regulatory T cells (nTregs) and in PLP(139-151)-induced IL-10 production and a concomitant increase in IL-17 production. Anti-CD25-induced inactivation of nTregs increased the incidence and severity of sEAE. Conversely, induction of peripheral tolerance via the i.v. injection of PLP(139-151)-pulsed, ethylcarbodiimide-fixed APCs (PLP(139-151)-SP) inhibited the development of clinical disease concomitant with increased production of IL-10 and conversion of Foxp3(+) Tregs from CD4(+)CD25(-) progenitors. These data indicate that heterogeneous populations of Tregs regulate onset of sEAE, and that induction of peripheral tolerance can be exploited to prevent/treat spontaneous autoimmune disease.     

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.     

Identification and characterization of a second encephalitogenic determinant of myelin proteolipid protein (residues 178-191) for SJL mice.

We previously described a synthetic peptide of myelin proteolipid protein (PLP), peptide 139-151, which induces experimental allergic encephalomyelitis in SJL/J (H-2s) mice. We have now identified an additional determinant, PLP residues 178-191, that is also a potent encephalitogen in this strain. When PLP peptide 178-191 was compared with peptide 139-151 on an equimolar basis, the day of onset of disease induced by PLP 178-191 was earlier, but the incidence, severity, and histologic features were indistinguishable. Lymph node cells from animals immunized with the whole PLP molecule responded to both PLP 178-191 and 139-151, suggesting immunologic codominance of the two epitopes. PLP 178-191 elicited stronger proliferative responses and this may relate to the earlier onset of disease induced with this peptide. Two CD4+, peptide-specific, I-A(s)-restricted T cell lines, selected by stimulation of lymph node cells with either PLP 178-191 or 139-151, were each encephalitogenic in naive syngeneic mice. The presence of multiple encephalitogenic codominant PLP epitopes within a single mouse strain emphasizes the complexity of the immune response to PLP and its potential as a target Ag in autoimmune demyelinating diseases.     

Fine specificity of CD4+ T cell responses to the dominant encephalitogenic PLP 139-151 peptide in SJL/J mice

PLP 139-151(S) is the major encephalitogenic epitope of PLP in the SJL/J mouse. CD4⁺ T cells specific for PLP 139-151(S) induce a relapsing-remitting form of EAE which is similar to the human demyelinating disease MS in both clinical course and histopathology. We are interested in events involved in activation of autoreactive T cells and how to specifically regulate these immune response to both prevent and treat ongoing demyelinating disease. In the current study, we examined the effect of both amino acid substitutions and deletions in the native PLP 139-151(S) peptide to identify which residues are critical for immunogenicity and encephalitogenicity. Conservative and nonconservative substitutions at position 145 diminished or completely destroyed the encephalitogenic potential of the peptide without affecting the ability to recall a proliferative response in lymph node T cells primed with the native PLP 139-151(S) peptide indicating an interesting dichotomy between ability to induce T cell proliferation and ability to induce active clinical disease. In addition, tryptophan at position 144 was identified as a critical TCR contact site as a peptide containing an alanine for tryptophan at this position (A144) primed a unique population of T cells which did not cross react with the native PLP 139-151(S). In addition, A144 was unable to stimulate PLP 139-151(S)-specific T cells in vitro or to induce active relapsing EAE in vivo. The significance of these results to the potential development of new strategies for preventing and treating T cell-mediated autoimmune diseases is discussed.Special issue dedicated to Dr. Majorie B. Lees.     

Mycobacterium bovis strain bacillus Calmette-Guérin-induced liver granulomas contain a diverse TCR repertoire, but a monoclonal T cell population is sufficient for protective granuloma formation

Granuloma formation is a form of delayed-type hypersensitivity requiring CD4(+) T cells. Granulomas control the growth and dissemination of pathogens, preventing host inflammation from harming surrounding tissues. Using a murine model of Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) infection we studied the extent of T cell heterogeneity present in liver granulomas. We demonstrate that the TCR repertoire of granuloma-infiltrating T cells is very diverse even at the single-granuloma level, suggesting that before granuloma closure, a large number of different T cells are recruited to the lesion. At the same time, the TCR repertoire is selected, because AND TCR transgenic T cells (Valpha11/Vbeta3 anti-pigeon cytochrome c) are preferentially excluded from granulomas of BCG-infected AND mice, and cells expressing secondary endemic Vbeta-chains are enriched among AND cells homing to granulomas. Next, we addressed whether TCR heterogeneity is required for effective granuloma formation. We infected 5CC7/recombinase-activating gene 2(-/-) mice with recombinant BCG that express pigeon cytochrome c peptide in a mycobacterial 19-kDa bacterial surface lipoprotein. A CD4(+) T cell with a single specificity in the absence of CD8(+) T cells is sufficient to form granulomas and adequately control bacteria. Our study shows that expanded monoclonal T cell populations can be protective in mycobacterial infection.     

neonatally primed lymph node, but not splenic T cells, display a Gly-Gly motif within the TCR beta-chain complementarity-determining region 3 that controls affinity and may affect lymphoid organ retention

Ig-proteolipid protein 1 (Ig-PLP1) is an Ig chimera expressing the encephalitogenic PLP1 peptide corresponding to amino acid residues 139-151 of PLP. Newborn mice given Ig-PLP1 in saline on the day of birth and challenged 7 wk later with PLP1 peptide in CFA develop an organ-specific neonatal immunity that confers resistance against experimental allergic encephalomyelitis. The T cell responses in these animals are comprised of Th2 cells in the lymph node and anergic Th1 lymphocytes in the spleen. Intriguingly, the anergic splenic T cells, although nonproliferative and unable to produce IFN-gamma or IL-4, secrete significant amounts of IL-2. Studies were performed to determine whether the two populations display any structural differences in the TCR H chain variable region that could contribute to the differential affinity and retention in different organs. Responsive Th2 lymph node T cells and anergic splenic lymphocytes were immortalized, and the structures of their TCR Vbeta were determined. The results show that Vbeta and Jbeta usage was random, but the CDR3 regions of the lymph node cells had a conserved Gly-Gly motif. Analysis of TCR affinity/avidity correlated the Gly-Gly motif with lower affinity and retention of the Th2 cells in the lymph node. Also, it is suggested that a higher TCR affinity may be a contributing factor for the development of the neonatal Th1 response in the spleen.     

Discrete T cell populations with specificity for a neo-self-antigen bear distinct imprints of tolerance

Mice expressing the Torpedo acetylcholine receptor alpha-chain as a neo-self-Ag exhibit a reduced frequency of T cells responding to the immunodominant epitope Talpha146-162 indicating a degree of tolerance. We characterized tolerance induction in these animals by analyzing the residual Talpha146-162-responsive T cell population and comparing it to that of nontransgenic littermates. Using CD4(high) sorting, we isolated the vast majority of Ag-reactive T cells from both strains of mice. Quantitative studies of the CD4(high) populations in transgenic mice following immunization with Talpha146-162 revealed a diminished expansion of cells expressing the canonical TCRBV6 but not other TCRBV gene segments when compared with nontransgenic littermates. In addition, CD4(high) cells from transgenic mice were functionally hyporesponsive to Talpha146-162 in terms of proliferation and cytokine secretion regardless of TCRBV gene segment use. TCR sequence analysis of transgenic Vbeta6(+)CD4(high) cells revealed a reduced frequency of cells expressing a conserved motif within the TCRbeta CDR3. Thus, the canonical Talpha146-162 responsive, Vbeta6(+) population demonstrates both quantitative and qualitative deficits that correlate with an altered TCR repertoire whereas the non-Vbeta6 population in transgenic mice exhibits only a reduction in peptide responsiveness, a qualitative defect. These data demonstrate that discrete autoreactive T cell populations with identical peptide/MHC specificity in Torpedo acetylcholine receptor-alpha-transgenic animals bear distinct tolerance imprints.     

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.     

The protective immune response to heat shock protein 60 of Histoplasma capsulatum is mediated by a subset of V beta 8.1/8.2+ T cells

Immunization with recombinant heat shock protein 60 (rHsp60) from Histoplasma capsulatum or a region of the protein designated fragment 3 (F3) confers protection from a subsequent challenge in mice. To determine the T cell repertoire involved in the response to Hsp60, T cell clones from C57BL/6 mice immunized with rHsp60 were generated and examined for Vbeta usage by flow cytometry and RT-PCR. Vbeta8.1/8.2(+) T cells were preferentially expanded; other clones bore Vbeta4, -6, or -11. When Vbeta8.1/8.2(+) cells were depleted in mice, Vbeta4(+) T cell clones were almost exclusively isolated. Measurement of cytokine production demonstrated that nine of 16 Vbeta8.1/8.2(+) clones were Th1, while only three of 13 non-Vbeta8.1/8.2(+) clones were Th1. In mice immunized with rHsp60, depletion of Vbeta8.1/8.2(+), but not Vbeta6(+) plus Vbeta7(+), T cells completely abolished the protective efficacy of Hsp60 to lethal and sublethal challenges. Examination of the TCR revealed that a subset of Vbeta8.1/2(+) clones that produced IFN-gamma and were reactive to F3 shared a common CDR3 sequence, DGGQG. Transfer of these T cell clones into TCR alpha/beta(-/-) or IFN-gamma(-/-) mice significantly improved survival, while transfer of other Vbeta8.1/8.2(+) clones that were F3 reactive but were Th2 or clones that were not reactive to F3 but were Th1 did not confer protection. These data indicate that a distinct subset of Vbeta8.1/8.2(+) T cells is crucial for the generation of a protective response to rHsp60.     

Autoantigen-responsive T cell clones demonstrate unfocused TCR cross-reactivity toward multiple related ligands: implications for autoimmunity

It has been suggested that the cross-reaction of a single T cell receptor with multiple different peptide ligands is a mechanism for maintaining a diverse yet compact immune repertoire. In the context of autoimmune disease it is important to understand how this property is balanced against the maintenance of self-tolerance. Specifically, whether the cross-reactivity inherent in the immune system is focused or unfocused will have important consequences for the development of autoimmune disease. If cross-reactivity is "focused," then in an immune response to a foreign antigen all T cell receptors that recognize the foreign antigen will cross-react with a specific autoantigenic peptide. However, if cross-reactivity is "unfocused," an immune response to a foreign antigen will result in the activation of a small number of self-reactive cells within a larger pool of cells specific for the foreign antigen. We have tested whether cross-reactivity is focused or unfocused by generating a panel of T cell clones that respond to two closely related ligands. W144 is an autoantigenic peptide of myelin proteolipid protein, PLP 139-151 (HSLGKWLGHPDKF), and Q144 is an altered peptide of PLP 139-151 bearing a glutamine for tryptophan substitution at position 144. The Q144-responsive clones have a broad degree of cross-reactivity with other position 144 substituted peptides. We find that despite their characteristic responses to Q144 and W144, the patterns of responses of these clones to other structurally related ligands are random, demonstrating that cross-reactivity is unfocused in the absence of selection. Maintaining a diverse range of cross-reactive interactions may limit nonspecific responses to autoantigens.