Classical selection theories suggest that antigen expression in the thymus should lead to central deletion of T cells encountering their cognate antigen. However, there are several possible explanations for the survival of MS2-3C8 Tg TCR T cells. Golli-MBP, the isoform of MBP expressed in the thymus, does not contain MBP 111–129, and therefore deletion would not necessarily be expected (24
). Even if classical MBP were to enter the thymus from the periphery and be presented by thymic APCs, the low binding affinity of MBP 111–129 to DRB1*0401 (16
) may allow their escape from deletion. This rationale might also explain the survival of Tg TCR T cells in other myelin protein–specific TCR Tg mice, humanized or not (21
). Except for MBP 83–99, each of these myelin peptides is either a poor MHC binder or not expressed in the thymus, e.g., MOG 35–55 and PLP 139–154 (29
). However, we observed a marked reduction in MS2-3C8 Tg T cells when the mice were crossed to Rag-1−/−
mice due to negative selection or lack of positive selection. Analysis of thymocyte cellularity and CD5 expression on CD4+
double positive thymocytes suggested the reduction is caused by thymic negative selection (unpublished data). Moreover, Tg T cells that escaped thymic deletion may have expressed endogenous mouse TCR together with the Tg α/β TCR heterodimer; thus, dual TCR expression on the Tg T cells may have led to their survival.
In MS2-3C8 TCR–HLA-DRB1*0401 Tg mice, Tg T cells were not activated in vivo ( D) and mice did not develop spontaneous EAE (unpublished data). Since the MS2-3C8 human TCC is a CD4+ T cell, we investigated the pathogenic characteristics of CD4+ Tg T cells. To eliminate the influence of CD8+ and DN Tg T cells on the induction of EAE, we isolated CD4+ Tg T cells from spleen cells and transferred CD4+ Th1 Tg cells into irradiated HLA-DRB1*0401 Tg mice. For comparison with the MS2-3C8 TCR, we generated HD4-1C2 TCR Tg mice from a human T cell clone which does not cross-react to the mouse epitope. We show that MS2-3C8 Tg CD4+ Th1 cells induced EAE, whereas HD4-1C2 Tg CD4+ Th1 cells did not, indicating that EAE induction is mediated by MS2-3C8 TCR recognition of the murine MBP 111–129–HLA-DRB1*0401 complex. This was confirmed using MS2-3C8 TCR–Rag-1−/− Tg T cells which lack endogenous mouse TCRs ( and ). Since the CD8+ Tg T cells expressed CD28 and produced high amounts of IFN-γ in response to MBP 111–129, we examined the pathogenic potential of CD8+ Tg T cells by adoptive transfer into irradiated HLA-DRB1*0401–IA−/− Tg mice. We did not observe any signs of EAE in animals receiving 10–20 million CD8+ Tg T cells, nor did the cotransfer of 10 million CD8+ Tg T cells at a 1:1 ratio with CD4+ Tg T cells suppress or worsen passive EAE (unpublished data). Active EAE could not be induced despite natural processing of MBP 111–129 and the stimulation of Tg T cells in vivo (unpublished data). Peripheral mechanisms of tolerance were not apparent as the explanation for ineffective active disease induction, since Tg T cells isolated from the spleen or lymph node proliferated in response to MBP 111–129, unaffected by addition of IL-2 (not depicted). Other mechanisms involved in active EAE suppression are currently under investigation.
MS presents with substantial clinical and pathologic heterogeneity (1
). Previous data from genetic studies on HLA-DR associations with MS (4
) and EAE experiments in animals with different MHC class II backgrounds and different encephalitogens (2
) suggest the clinical phenotype of MS–EAE is in part caused by the expression of different HLA-DR–MHC class II alleles and the T cell response to specific myelin peptides. Since the HLA-DR2 haplotype is strongly associated with MS, and HLA-DRB1*1501 and HLA-DRB5*0101 genes are coexpressed in humans carrying the HLA-DR2 allele, HLA-DRB1*1501-restricted MBP 83–99–specific TCR Tg mice were generated. The Tg mice developed spontaneous ascending paralysis (21
). Recently, we have developed a HLA-DRB5*0101-restricted MBP 83–99–specific human TCR Tg mouse (TL3A6 Tg mice). The TL3A6 Tg Th1 cells induced severe ascending paralysis, although they do not induce lingual paralysis in spite of weight loss (unpublished data). In accordance with the clinical symptoms, the lesions were predominantly observed in the spinal cord, and less inflammatory cells infiltrated into the cranial nerves (unpublished data). Therefore, lingual paralysis associated with the inflammation of brainstem and cranial nerves was a unique feature of MS2-3C8 Tg mice.
The patient from whom the MS2-3C8 TCC was derived did not suffer from chronic persisting dysphagia; however, the patient did present two episodes, one consisting of disturbances in tongue movement impairing swallowing of fluids and speech articulation and a second episode consisting of tongue numbness and dysgeusia (). These episodes were comprised between the two time points at which MS2-3C8 TCRs were isolated. We are well aware that the relationship between MBP 111–129–specific T cell responses (or the presence of a specific TCR) and the manifestation of clinical signs and symptoms should be interpreted with caution; however, the phenotypic characteristics of this particular EAE model and the similarities in the patient's clinical history are intriguing. Interestingly, ~30-40% of MS patients develop dysphagia (31
), albeit typically late in disease.
Clearly, several factors can contribute to the clinical manifestations of EAE in any one particular model, perhaps the two greatest contributors being the genetic background and the immunizing myelin antigen or the target autoantigen. It has been reported that the antigenic epitope specificity of myelin antigens influences the phenotype of EAE and location of the lesions. In Balb/c (IAd
) mice, IAd
-restricted MBP 59–76–specific T cells induce classical EAE with characteristic inflammation and demyelination of the CNS (34
). In contrast, IEd
-restricted MBP 151–168–specific T cells induce inflammation and demyelination of preferentially PNS myelin including nerves in the hind leg and spinal roots. The authors suggested two possibilities to explain their result. The first is that the distinct inflammation sites may be attributable to qualitative differences in MBP isoform composition in the CNS and PNS. The MBP 151–168 epitope, which is encoded by exons 6 and 7, exists only in the 18.5- and 21-kD MBP isoforms, whereas the MBP 59–76 epitope exists in all of the 14-, 17-, 18.5-, and 21-kD isoforms. The minimal epitope of MBP recognized by the MS2-3C8 TCR, MBP 116–123 (16
), is encoded by exons 5 and 6. Therefore, similar to the MBP 151–168 epitope recognition described above, only the 18.5- and 21-kD isoforms are recognized by MS2-3C8 Tg T cells. Although the quantitative distribution of these isoforms is unknown at the present, the unique EAE phenotype inducible by MS2-3C8 Tg T cells may be caused by the distinct distribution of MBP isoforms in the CNS and PNS. Second, the authors suggested that high versus low affinity interactions in the complex of MBP–IA or –IE molecules may generate distinct downstream signaling events after TCR engagement. Interestingly, MBP 59–76 strongly binds to IAd
molecules, whereas MBP 151–168 weakly binds to IEd
molecules. Such differences are likely to affect the expression of adhesion and costimulatory molecules involved in transmigration through perivascular endothelial cells of either the blood-brain or blood-nerve barrier, and thus influence factors key to T cell encephalitogenicity. This hypothesis was supported by EAE experiments in CH3 congenic mice where immunization of H2q
congenic C3H mice with PLP 103–116 induces typical ascending EAE. In contrast, immunization of H2p
congenic C3H mice with the same antigen induces atypical EAE, ataxia, and imbalance without any symptoms of ascending paralysis (35
). The binding affinity of PLP 103–116 to IAp
was lower than that to IAq
, suggesting that antigen binding affinity of myelin antigens to MHC class II molecules could influence the phenotype of EAE. Moreover, signals through TCR and costimulatory molecules could lead to the expression of adhesion molecules and chemokine production that are involved in the infiltration of pathogenic T cells. EAE experiments that used CD28-deficient mice supported this hypothesis. When the CD28-deficient mice were immunized with MOG 35–55, inflammatory cells predominantly infiltrated into the leptomeninges of the brain although immunization of wild-type mice with the same antigen induced typical ascending paralysis with the infiltrates in the parenchyma of the CNS (36
). Similarly, the poor binding of MBP 111–129 to HLA-DRB1*0401 molecules and the signaling events subsequent to TCR engagement may shape the unique EAE phenotype exhibited by the MS2-3C8 Tg TCR.
Properties of individual TCR, the pattern of migration of individual T cell clones, or the localization/abundance of antigens are all likely to come into play, as was suggested in the recent report of MOG Tg TCR mice. T cells specific for MOG35-55 gave rise to spontaneous optic neuritis, most often in the absence of typical EAE symptoms (37
). Moreover, in each model of EAE the overwhelming presentation of symptoms considered to be typical may mask those symptoms occurring less frequently, such as those described here. In this regard, TCR transgenic mice may be valuable to glean antigen/MHC-specific pathology from the otherwise heterogeneous presentation of disease that is observed in other models. With respect to the simultaneous occurrence of cranial nerve deficits and the typical sensorimotor signs, there is precedence in MS patients who not uncommonly develop trigeminal neuralgia or more rarely involvement of other cranial nerves.
Although it is difficult to extrapolate to human disease clinical findings from experimental animal models, our results show that inflammatory responses of MS2-3C8 Tg T cells directed against MBP 111–129 can induce unique clinical and pathological features of damage to the nervous system that are distinct from previously described Tg models (21
) based on TCR recognizing different MBP peptide–MHC complexes. Such correlations between specificity and restriction of myelin-specific T cells may provide important insights into the immunopathological heterogeneity of MS. Clearly, humanized Tg mice expressing a single TCR are biased and may not represent all clinical features of the MS patient from whom the Tg TCR had been derived. However, humanized TCR Tg mouse models could be an exemplary way to dissect the phenotypic heterogeneity linked to a T cell response against defined myelin peptides in the context of a disease-associated HLA-DR allele in the development of MS.