For the first time, the effects of age on immune tolerance have been examined in a well-defined model, a transgenic mouse in which the TAChR α chain is expressed primarily in muscle. This model is ideal since it can be used to assess both B-cell and T-cell tolerance. In addition, the T-cell fine specificity of the TAChR response has been mapped to a single determinant and tolerance to this T-cell epitope in the young transgenic model was previously demonstrated (21
). However, it was possible that this fine specificity would be altered with age. We reasoned that a reduced frequency of dominantly responding clones due to a decline in receptor diversity (10
) might reveal reactivities against other less dominant TAChR epitopes. Alternatively, mechanisms that regulate the fine specificity of the T-cell responses in young animals might be altered in the old. For example, epitope spreading, reported in induced T-cell tolerance to specific antigens (36
) and in the progression of T-cell responses (37
), could conceivably be increased with age. However, this was not observed in the AChR response, the immunodominance of p146-162 was well maintained with age in TAChR-immunized nontransgenic mice (). This result is in agreement with a study of T-cell responses to hen egg lyzozyme in 2 and 20 month old mice; no change in the fine specificity of the peptide response was seen when measured as the frequency of IFN-γ producing T cells (38
). Our results suggest that factors controlling T-cell fine specificity of the TAChR response are still intact in 20 month old mice and verify the importance of addressing tolerance to p146-162 in the old transgenic mice.
In view of the reported age-associated alterations in T-cell development and function (10
), the maintenance of T-cell tolerance to the AChR in old mice might not have been expected. Nonetheless, tolerance was evidenced by diminished T-cell proliferative responses to p146-162 as well as other epitopes of the α chain extracellular domain and in low antibody titers to the TAChR α chain. Moreover, another measure of tolerance in young TAChR α transgenic mice, the reduction in frequency of high avidity CD4high
antigen-specific cells (28
), was observed in the old transgenic mice as well. Thus, in seeming contradiction to the paradigm that reactions to self-antigens increase with age (1
), we found that both T-cell and B-cell tolerance to the AChR neo-self antigen were maintained.
Given this persistence of tolerance in the old transgenic mice, it appears that mechanisms thought to promote the loss of T-cell tolerance with age are not applicable in this model. For example, it has been proposed that thymic involution would compromise negative selection, possibly resulting in an increase in the escape of autoreactive T cells to the periphery (40
). However, this was clearly not seen in our model or alternatively, peripheral mechanisms of tolerance remained active and effectively eliminated any escapees. It also has been suggested that age-related loss of T-cell tolerance could be due to T-cell signaling alterations that impinge upon apoptotic mechanisms or that promote anergy of Th2-type regulatory cells (reviewed in 39
). Alterations in T-cell regulation are of special interest since differences in foxp3+
) or function (42
) have been reported in thymus and blood from patients with MG. Patients in these studies were mostly young (41
). However, in one study reporting diminished suppressive capacity of Tregs
from MG patients (42
), twelve of twenty-one individuals exhibited disease onset after the age of 50 years. Thus, it is conceivable that alterations in Treg
number and/or function could be a contributing factor in late-onset MG, as well as early-onset MG.
Significantly, the effects of age on Treg
profiles in healthy humans and mice have been addressed and could have implications for late-onset MG. While age-related alterations in certain Treg
functions were seen in some studies (15
), suppressive capabilities were generally maintained (15
). In addition, the frequencies of Tregs
are widely reported to be increased in old individuals (15
). In support of this finding, we found that the Treg
frequency within the CD4+
T-cell population from old unimmunized TAChR α transgenic mice was significantly elevated over that of the young transgenic mice and this enhanced frequency was also evident in CD4+
T cells derived from p146-162 immunized mice. While it is far from clear whether the increased proportion of Tregs
in the old transgenic mice helps to maintain tolerance to the TAChR studied here, this is an important area for future investigation.
Importantly, our results indicating that tolerance to the neo-self AChR is not broken in the old mice, raise a more basic question. In the face of this tolerance maintenance, how is tolerance disturbed in autoimmune diseases where symptoms are first seen in old age? One possible explanation for loss of tolerance in late-onset MG is that in some individuals, deficiencies in T-cell regulation are present at a young age but are not sufficient to result in clinical manifestations of autoimmunity. Thus, autoimmune challenges may be held in check although autoimmune memory T cells are generated. Upon re-exposure to the self antigen in old age, due to age-related tissue damage or exposure to mimicking foreign antigens, “recall” autoimmune memory cells established at a young age would respond vigorously and rapidly. T-cell tolerance, already weak in these individuals, would be “broken” by the strong memory response. In support of this hypothesis, in B-cell studies, Nobrega et al. have demonstrated that although auto-antibody levels were higher in old C57BL/6 mice, the autoantigen recognition profile was the same in young and old (45
). Moreover, our own work has shown that autoreactive B cells first established by immunization of young mice could be recalled in old age to give a vigorous antibody response and produce autoimmune MG (22
). Thus, similar to autoreactive B cells, it may be that certain self-reactive T cells in the elderly reflect specificities first established in youth.