Generally, B10.RIII mice develop significant EAU after they are immunized with 100 µg IRBP161–180 along with pertussis toxin. In this study, to better delineate the difference of ocular inflammation between control and rapamycin-treated groups, we induced EAU with 50 µg/mouse IRBP161–180 in the absence of pertussis toxin. With a mild uveitis as a control, we were able to observe the maximal effect of rapamycin on EAU. Control mice developed peak EAU on day 14 in response to the lower dose of IRBP161–180, and the uveitis subsided on day 21. However, we have demonstrated that low dose rapamycin exacerbated and prolonged EAU. On day 21, we still observed a robust ocular inflammatory response and retinal damage. Coinciding with this finding, we found that daily treatment of low dose rapamycin increased the frequency of antigen-specific T cells in the mice with EAU. In our unpublished study, we also found that daily treatment of SJL mice with low dose rapamycin consistently augmented PLP178–191-induced experimental autoimmune encephalomyelitis. This further validated the effect of low dose rapamycin in an autoimmune response in a different disease setting. To our knowledge, this is the first study showing that low dose rapamycin enhances the autoimmune response in the setting of uveitis. The significance of this observation is twofold.
First, rapamycin is a potent immunosuppressant clinically used to treat transplant rejection and some autoimmune diseases. In light of this finding, suboptimal dosing of rapamycin could exert an immunological effect that is opposite to the desired therapeutic intention. For instance, graft rejection and inflammatory responses may be augmented if an inadequate dose of rapamycin is given to patients. In addition, it is well documented that genetic variations, pharmacokinetics, drug interaction and poor treatment compliance can all affect the serum level of rapamycin 
. Many patients with organ transplantation or autoimmune diseases commonly receive multiple medications, which can accelerate rapamycin metabolism. Thus, this experiment underscores the need to monitor the pharmacokinetics of rapamycin to ensure the intended therapeutic outcome. In the current clinical trial, it is worth noting that the uveitis patients receive rapamycin via local injection. The local application of rapamycin may achieve better therapeutic concentration in the eye and avoid hepatic biotransformation. Therefore, it would be interesting to compare the efficacy and safety profile between local and systemic therapies of rapamycin in uveitis.
In addition to the potential clinical significance, this study is immunologically intriguing. Although rapamycin is a well documented immune suppressant, we now report that low dose rapamycin can actually exacerbate autoimmune uveitis. This observation is paradoxical to the known effect of rapamycin. However, this finding is in keeping with recent publications suggesting this unexpected impact of low does rapamycin. A number of reports showed the development of interstitial pneumonitis in the absence of infection as well as chronic inflammation-related anemia after rapamycin treatment 
. Araki et al. have demonstrated that rapamycin promotes virus-specific memory CD8+ T cell immunity in both murine and macaque models 
. In this study, we found that depletion of CD8+ cells reduced rapamycin-enhanced EAU (). This suggests that CD8+ lymphocytes contribute to low dose rapamycin-augmented autoimmune response in EAU. In addition, Ford and colleagues report an enhancement of antigen-specific T cell response by rapamycin in the setting of Listeria monocytogenes
. Consistent with this finding, rapamycin has been shown to enhance IL-12 and IL-23 production induced by bacteria 
. Thus, low dose rapamycin could also augment autoantigen-driven inflammation. It is well documented that IL-12 promotes the development of Th1 cells, whereas IL-23 plays a critical role in Th17 differentiation. Here, we showed that low dose rapamycin augmented IFN-γ- but not IL-17-producing cells () in the context of EAU. It is unclear if low dose rapamycin treatment skews Th1 differentiation, or it is a phenomenon related to this specific model since IRBP-induced EAU displays a strong Th1 response 
. Thus, further study is needed.
Recent research in a lymphocytic choriomeningitis virus model has demonstrated that rapamycin treatment during the early T cell expansion phase increases the population of memory T lymphocytes, whereas rapamycin enhances the function of memory cells when they enter the contraction stage 
. In this study, we found that treating mice with low dose rapamycin during antigen sensitization (days 0–7) led to a severe and sustained EAU. Furthermore, the exacerbation of EAU by low dose rapamycin coincided with increased frequency of the T cells that specifically respond to IRBP. This suggests that low dose rapamycin may augment the T cell response to antigen priming, enhance effector T cell function and contribute to long-term survival. Despite the problematic effect of low dose rapamycin within the setting of autoimmunity, the effect of rapamycin on memory T cells could be beneficial to improve the long-term immune memory for host defense. Whether rapamycin might be used as an effective adjuvant to boost the efficacy of vaccination and immunotherapy against infections or cancers remains to be tested.
Presently, the molecular mechanism by which low dose rapamycin enhances T cell response remains to be fully elucidated. It is well documented that rapamycin inhibits mTOR through the interaction with FK506 binding protein 12 
. mTOR is a serine/threonine kinase that plays an important role in regulating the cellular response to external and nutritional cues 
. Using RNA-interference knockdown technique, Araki et al. have shown that rapamycin facilitates memory T cell differentiation via mTOR complex 1 pathway 
. It is thought that metabolic status exerts a pivotal impact on T cell programming. High metabolic state drives effector T cell development, whereas low metabolic cue is in favor of memory T cell differentiation 
. This notion is further supported by a recent study showing that suppression of fatty acid metabolism promotes a memory T cell response 
. Thus, inhibition of mTOR by low dose rapamycin may reset the metabolic switch, leading to more memory T cell development. However, high dose rapamycin also blocks mTOR activity. Yet it displays a potent T cell suppression effect. Thus, another mechanism is likely to be involved in the response that develops following low dose rapamycin. After antigen activation, T cells expand rapidly. During the subsequent contraction phase, a majority of effector T cells undergo apoptosis, and a few surviving lymphocytes develop into the memory lineage. A recent study showed that long-term rapamycin treatment can improve mouse longevity 
, suggesting that rapamycin may regulate the apoptosis process. In fact, our study showed that pre-treatment of rapamycin attenuated AICD and apoptotic molecule expression in antigen-activated lymphocytes. In the setting of autoimmune diseases, waves of naïve T cells will gain an access to antigens during different stages of the inflammatory process. Rapamycin-primed naïve lymphocytes could resist AICD after encountering an antigen. Thus, low dose rapamycin may prolong the life span of effector lymphocytes and facilitate the generation of memory T cells by its potential anti-apoptotic effect. However, we believe that inhibition of AICD is only a part of the mechanism by which low dose rapamycin augments the immune response. A recent study demonstrated that autophagy enhances immune response in part by releasing ATP to recruit dendritic cells, CD4+ and CD8+ lymphocytes 
. Since rapamycin inhibits mTOR, a negative regulator of autophagy 
, low dose rapamycin could exacerbate EAU by augmenting autophagy 
. Although treatment with rapamycin during different stages of EAU development resulted in a similar outcome of ocular inflammation (), our study showed that peripheral T cells from the mice treated with low dose rapamycin from day 1 to day 21 exhibited an enhanced IFN-γ response to ocular antigen re-stimulation (). This suggests that low dose rapamycin during other phases of EAU development may augment ocular inflammation through different immune cells and/or mediators.
In summary, our study reveals that low dose rapamycin enhances the autoimmune response in the setting of uveitis. This finding is clinically important as it raises the concern of an unintended consequence from low dose rapamycin and underscores the need to validate dosing and pharmacokinetics during rapamycin therapy. In addition, these data suggest that rapamycin may be used to help enhance a developing immune response, a premise requiring additional investigation in different model systems.