Signalling mechanisms underlying priming and tolerance of T cells

The primary mission of the immune system is to defend against invading pathogens. The normal healthy body can distinguish self from non-self antigens. When a new antigen is encountered, such discriminatory capacity would generate a productive immune response against invasive pathogens or exert antigen-specific tolerance, the latter to prevent harmful immune responses against self-components or non-dangerous food or environmental antigens. Peripheral tolerance plays an important role in preventing T cells response to self or harmless antigens. A breakdown in tolerance within an individual can result in the development of a variety of autoimmune disorders. Full T cell activation requires at least two signals. The first one is provided by the TcR recognizing cognate peptides derived from antigen in the context of appropriate MHC molecules expressed by antigen presenting cells (APC). The second is mediated by “co-stimulation” via interaction of CD28 on the T cell with CD80/86 on the APC. The clonal anergy is induced when the TcR is ligated in the absence of co-stimulation, one of the proposed mechanisms of peripheral tolerance, describes a state of long lasting unresponsiveness to antigen, in the T cell. Despite widely studies in this area, however, the mechanisms of induction of anergy and the efficient markers for diagnosis of anergy are still not clear. One of the mechanisms which contributes to forming tolerance is anergy, which can be defined as defect in cellular proliferation and IL-2 production. Furthermore, GTPase Rap1 has been reported to inhibit the generation of pERK signals and to accumulate in tolerant cells. However, most of previous studies have done by biochemical assessment of signaling in T cell lines or clones upon polyclonal stimulations in vitro, and thus has generated some conflicting data. For solving this problem, our lab has developed the technique, laser scanning cytometry (LSC), for observation of responses in individual antigen-specific T cells within their environmental niche within primary or secondary lymphoid tissue. By LSC, it has reported that there are significant differences in the amplitude and cellular localization of phosphorylated ERK signals when naive and in vitro-primed and tolerized T cells respond to Ag. To further investigate the role of Rap1 by LSC, it revealed that counter regulation in Rap1 and phosphor-ERK expression during the maintenance phase of tolerance and priming of antigen-specific CD4+ T cells in vitro and in vivo. In T cells, the maintenance phase of anergy has been reported to reflect defective activation of transcription factor, such as c-Jun/c-Fos, that are involved in formation of the AP-1 complex, which is required for inducing transcription of the IL-2 gene and optimal activation and effector function of T cells. In turn, this appears to be determined by the lack of recruitment of the ERK, JNK and p38 MAPK signaling cascades. The small GTPase, Rap1, has long been implicated in such desensitisation of ERK, and the consequent reduced IL-2 production, observed in tolerised T cells. However, the most of these studies were processed with T cell lines or clones in vitro and as such are not necessarily representative of physiological responses of primary antigen-specific T cells. Consistent with the previous finding, we have extended these studies to investigate whether Rap1 plays a role in determining commitment to anergy and priming during induction and maintenance phases. As expected, analysis in the DNA synthesis during maintenance phase reported that the primed T cells exhibited a higher response than either naive or anergic T cells, whilst the anergic T cells displayed an even lower DNA synthesis than naive T cells undergoing a primary response. To further investigation in cytokine production of IL-2 and IFNγ at 24, 48 and 96 hour during the maintenance phase, consistent with previous studies, the primed T cells produced the highest levels of IL-2, relative to anergic cells with the lowest levels, at the first 24 hours after challenge with antigen. However, the IL-2 production from primed and anergic T cells both drop down from 48 hours and to very low level at 96 hours but accompanying with gradual increase of IFNγ production. This implicates both anergic and primed cells consumed IL-2 secreted in the early stage of maintenance phase for supporting following cellular differentiation. The assessment of cellular proliferation also indicates that both primed and anergic cells had undergone several rounds of division. Whereas the primed cells proliferated more and faster than anergic cells over the first two days, after that anergic cells were able to catch up with primed cells. Consistent with above proliferative responses, the primed T cells showed higher levels of ERK activation than anergic cells at day 1 but lower levels of ERK activation than anergic cells at day 3. Surprisingly, there is no difference in Rap1 activation between primed and anergic T cells during maintenance phase. The additional finding from cellular proliferation during maintenance phase revealed that both primed and anergic cells undergo clonal expansion during induction of priming and tolerance, which leads the further investigation in functional outcomes, MAPK signaling and mTOR pathways studies during induction phase. The primed cells exhibited higher levels of DNA synthesis than anergic cells at 48 hours whereas they had similar levels of DNA synthesis at 96 hours. The IL-2 and IFNγ production were only detectable within the first 48 hours but not 96 hour. Collaborating with the data from cellular proliferation indicates the IL-2 were consumed for promoting the cells survival and proliferation since both populations showed clear peaks representing differential numbers of cell division from day 2 (48 hour) onwards, whereas the primed cells proliferated more and faster than anergic cells during whole induction phase. Moreover, cyclic activation of ERK was seen in the primed T cells and at higher levels of activation than in the anergic population, which did not exhibit these kinetics in western blotting. Interestingly, the primed T cells exhibited slightly higher levels of Rap1 than anergic cells from 48 hour until 96 hour during induction phase. Consistent with data from in vitro, the proliferation response in mimicking physiological model also can be replicated. Additionally, the counter regulation in ERK and Rap1 activation also occurred during the induction of priming and tolerance, which is investigated by adenoviral gene transfer of Ad Rap1 S17N, an inactive mutant of Rap1. Furthermore, modulation of Rap1 expression with Ad Rap1 S17N in cells during induction of anergy, revealed that Rap1 activity acts to limit cellular proliferation and thus switching off Rap1 activity upregulates cellular proliferation to generate a phenotype more resembling priming of normal (or GFP-) T cells by antiCD3+anti-CD28, which showed higher proliferation that GFP- cells stimulated with anti-CD3 only. However, when these adenoviral transfer experiments were repeated in the more physiological model, the higher proliferation exhibited in anergic Ad Rap1 S17N transduced cells were not replicated, suggesting that the enhancing effect of Ad Rap1 S17N might be substituted by signals generated under these more physiological conditions. There did not appear any difference between anergic and primed cells in terms of ERK/Rap1 signalling during the induction phase and introduction of Ad Rap1 S17N did not modulate ERK activity in transduced cells treated with anti-CD3 or anti-CD3+anti-CD28, suggesting that Rap may target some other effector during the induction phase. To sum up these data, Rap signaling in anergy and priming as well as the use of the dominant negative construct suggested that Rap was not acting to suppress ERK activation during induction of anergy. The further investigation in the downstream targets, c-Myc, did not see any direct connection with ERK/Rap1 activation during induction of anergy and priming. Moreover, the primed T cells tend to skew to catabolic rather anabolic metabolic pathways, when compared to anergic T cells during the induction phase, as evidenced by the primed cells exhibiting upregulation and phosphorylation of AMPK and Raptor to inhibit mTORC1 funtion and in turn, lower levels of pp70 S6K. However, the expression of phosphorylated Rictor in anergic t cells was higher than that of primed T cells, indicating inhibition of mTORC2 in anergic T cells resulting in downregulation of AKT activation during this induction phase.