As argued above, it is difficult to reconcile with allosteric models of discrimination the ability of a pMHC ligand to be a non-agonist for a mature T cell but an agonist mediating positive selection for a developing thymocyte with the same TCR. Rather, these data imply that the relationship between the biophysical parameters of pMHC-TCR interaction and agonist activity is determined by the differentiation state of the T cell and that the discrimination threshold is actively altered during intrathymic development to give rise to this changing response to weak and strong TCR-binding pMHCs.
There are reasons to also consider whether post-thymic differentiation processes also modify the ligand discrimination properties of T cells. If it were perfect, thymic selection would secure the release of an “ideal” repertoire of T cells (T cells that can be activated by pathogen-derived pMHCs but are not overtly responsive to self-derived pMHCs). However, it is clear that negative selection is imperfect and models of peripheral tolerance emphasize the role of micro-environments (e.g. costimulatory molecules, cytokines) in lymph nodes and other secondary lymphatic organs in limiting potential auto-immune catastrophes resulting from T cell engagement with self ligands able to signal beyond the activation threshold of the mature T cell. Such T cells may receive a detectable signal upon engagement with some self pMHC ligands, but, in the absence of inflammation, this activation would lead to anergy or tolerance. A non-exclusive view of peripheral tolerance is that aside from the influence of cosignals on T cell fate in the periphery upon self-recognition, there is a cell-autonomous effect of self-pMHC engagement. Such recognition might alter the signaling machinery to biochemically ‘erase’ the potential of self ligands to generate an activating signal in the mature lymphocytes: this process is classically called ‘tuning’. We will review some of the experimental studies suggesting such behavior among T cells, discuss how our differential feedback model can shed some light on this process, and present a conjecture on the mechanism of tunability.
3.1 Tuning during T cell development
CD4+CD8+ thymocytes are responsive in terms of ERK phosphorylation to endogenous (self-derived) ligands and this activation is necessary to their full differentiation. After release to the periphery, the same T cells lack this overt biochemical self-responsiveness. In making such a comparison, we believe it is crucial to focus on early events in a T cell’s response towards a given spectrum of ligands (such as ERK1/2 phosphorylation or CD69 upregulation) when evaluating changes in TCR-signaling coupling during differentiation. This is because analysis of more functional outcomes (cytokine secretion, cytotoxicity, proliferation) are complicated by the potential effects of gene remodeling differences between the immature and mature cells and by changes in the cell’s capacity to perceive and respond to non-antigen-associated factors such as costimuli, cytokines, etc.
The simplest view of this ‘erasure’ of overt responsiveness to positively selecting ligands that do not also induce subsequent negative selection [55
] is that the maturing T cells increase the threshold necessary for activation in a global sense. Under such conditions, weak TCR binders such as the self pMHCs involved in positive selection, would never provide enough input to reach the new threshold, whereas foreign pMHC that are good receptor binders would do so when they are available at a high enough density on APC. However, it is clear that such a simple scheme demands that the T cell lose potential sensitivity for the foreign agonists as compared to the immature thymocytes. This makes little biological sense – such a view suggests that positive selection evolved in such a way as to decrease
the ability of the adaptive immune system to detect foreign antigens. As it turns out, such a sacrifice is not necessary nor does it occur. Several groups [57
] have clearly shown that as a T cell matures, it maintains the same sensitivity to foreign agonist ligands while at the same time completely losing the capacity to show a functional response to weak TCR binders. Furthermore, at the level of proximal TCR signaling, the agonists retain their ability to generate fully phosphorylated TCRζ ITAMs and induce ZAP70 activation, whereas the weak binders change the pattern and not just extent of early signaling, producing an excess of hypo-phosphorylated TCRζ and failing to activate ZAP70. These findings are not compatible with an across-the-board decrease in sensitivity of the TCR to transmit signals, but rather clearly indicate that the threshold for discrimination between ligands of distinct TCR binding capacity has been altered. Very recent data indicate that, as presumed but not directly tested in the earlier studies, this occurs without measurable change in the lifetimes of the pMHC interactions with the TCR [59
]. Given these results, one must conclude that maturation changes how ligand-engaged TCRs couple to the signaling apparatus, or perhaps more precisely, how the signaling machinery of the T cell process signals downstream of initial kinase activation.
What might these changes be? Stark et al.
] have pointed out that there are different TCR signaling networks yielding ERK1/2 activation (namely through RAS-GRDP and GRB2), with different characteristics (transient or sustained activation), so one could imagine how the same input (pMHC engagement by TCR) and the same signaling output (phosphorylated ERK1/2) might be connected through different signaling pathways. Consequently, the tunability of ligand discrimination in maturing T cells would correspond to a qualitative “rewiring” of the TCR signaling machinery. The central issue is how particular biochemical changes (for example, switching from Ras-GRDP in the thymus to GRB2 in peripheral T cells) actually effect the necessary functional changes (positive selection with self pMHC at the DP stage vs. self restriction in the naïve stage in the periphery). This theme will be elaborated below and specific biochemical changes suggested that might contribute to such tuning not of triggering threshold but of ligand discrimination in signal propagation both among thymocytes and peripheral T cells.
3.2 Tunability during T cell differentiation in the periphery
Recent experimental results suggest that T cells may modify their ligand discrimination threshold in the periphery. The direction of this change in some but not other studies appears consistent with theoretical considerations positing the generation of peripheral tolerance through reduced responsiveness to self induced by chronic exposure to self-ligands, without loss of useful sensitivity to foreign stimuli. This concept of tuning of T cell responsiveness in the periphery came from Grossman and colleagues [45
]. The core model (the tunable-adaptation threshold model or TAT) argues that long-term adaptation to tonic levels of self-induced TCR signaling desensitizes the receptor response just enough to prevent overt responses to this self-ligand landscape, while preserving the capacity of the T cells to respond to acute contact with more potent foreign ligands.
Experimental testing of the TAT model came through adoptive transfers of T cells into TAP−/−
mice with a defect in presentation of Class I pMHC [62
] or into mice with abolished expression of MHC-II [45
]: T cells, left in the latter pMHC-deficient environment for at least a week were shown to gain increased signaling and functional responsiveness towards foreign pMHC upon rechallenge. Quite in contrast to these reports, Stefanova et al.
] as well as the Davis group [3
] have shown that T cells can take advantage of endogenous ligands to boost T cells’ response towards agonist ligands.
It is likely that major differences in experimental settings account for these divergent results. In the two cases in which loss of self pMHC contact induced gain of responsiveness in T cells [45
], these cells had been kept in lymphopenic environments for substantial time periods during which proliferation and differentiation into an effector/memory cell state occurred. As compared to naïve cells, previously-activated cells of this type are known to be more responsive to antigen and to require less costimulation, consistent with what these two groups reported. In contrast, the experiments showing a positive contribution of self to T cell sensitivity or responsiveness [3
] were done under acute settings, involved cells from normal animals, or examined cells within a few hours of adoptive transfer rather than many days or weeks, that is, prior to lymphopenia-induced proliferation and changes in phenotype.
Thus, under normal circumstances, in animals with intact lymphoid compartments, self-ligands tend to make a net positive contribution to the responsiveness of naïve T cells, rather than blunting their response to maintain tolerance. This is not to say that a more subtle form of self-induced TAT does not occur under physiological circumstances: available data and the illustrations above for synergistic self-ligands () indicate that exposure to these pMHCs will induce a small amount of SHP-1 recruitment to the TCR pool, just sufficient to prevent effective responses by poor TCR binders. This is quite consistent with the TAT model. However, because of the ability of agonist-induced MAPK activation to override this low level of negative regulation, there is a net positive effect of TCR engagement by self pMHCs apparently due to pre-association of key signaling molecules with the receptor that enhances activation of MAPK pathway.
Another set of experiments has addressed the issue of tunable-activation threshold upon constant agonist pMHC stimulation. Singh et al.
established a model in which different levels of agonist ligands were chronically presented in vivo
: these ligands induced naïve T cells in lymphopenic environments to proliferate, then enter a stable state with a low turnover rate and decrease their responsiveness to the same pMHC ligand to an extent that depended on the level of chronic pMHC presentation [63
]. In particular, chronic stimulation with constant low levels of antigen left the cells more responsive (in terms of signaling, cytokine production or proliferative capabilities upon re-challenge) than did exposure to (four-fold) higher levels of the same antigen. In these circumstances, strong agonist ligands for naïve cells became partial agonists for cells chronically exposed to low amounts of agonist ligand in vivo
(such T cells showed a characteristic transient ERK response) whereas the same pMHC acted as a null/weak ligand for cells exposed chronically to higher amounts of agonist (showing a diminished ERK response). This sliding scale of pMHC responsiveness depending on the receptor occupancy history of the T cells matched the predictions of the Tunable-Activation Threshold model. Recent developments demonstrated the role of other T cells in adjusting the tuning process (through extrinsic regulatory functions) for chronically-activated T cells [64
]. Understanding quantitatively the underlying mechanisms of this tuning remains challenging, even more so because this adaptation of the TCR machinery does not apply for all functional outputs: responsiveness in terms of CD69 upregulation or actin polymerization was not modulated with chronic exposure to antigen [63
Recently, experimental work in our lab unraveled another manifestation of tuning in T cell activation, namely a transient hypersensitivity of T cells towards self-like ligands. Using naïve lymphocytes that have been activated ex vivo
for one day and rested after APC removal for five days, or activated ex vivo
and expanded for 5 days [4
], responsiveness towards sub-threshold ligands was tested. The 5C.C7 clone was shown to gain responsiveness towards MCC102G
(a non-agonist for naïve 5C.C7 T cells) and the OT-1 clone was shown to gain responsiveness towards EIINFEKL/H-2Kb
. This hypersensitivity of T cells was also shown to be transient, as memory-type T cells regained a spectrum of responsiveness similar to that of the initial naïve T cells. These observations emphasize that ligand discrimination is not set for T cells endowed with a given TCR, but rather can be tuned/rewired depending on the differentiation state.
The functional relevance as well as the underlying molecular mechanism for this transient hypersensitivity in T cells is still under analysis. One change contributing to this hypersensitivity is the down-regulation of SHP-1 phosphatase (the main component of TCR negative feedback unraveled by Stefanova et al.
]. Re-establishment of near resting state levels of SHP-1 through retroviral infection of T cells corrected the transient hypersensitivity towards non-agonist ligands, without affecting the T cell’s responsiveness towards agonist ligands (a prediction of our computer model [4
]). What triggers SHP-1 downregulation remains unknown, but is clearly worth investigating because of this phenomenon’s implications with respect to autoimmunity and therapeutic responses to self antigens such as in cancer treatment. More generally, the observation that the up/downregulation of a single key signaling component (SHP-1) can abrogate or allow self-responsiveness suggests the existence of master regulators in ligand discrimination.
3.3 Tunability and the differential feedback model
As discussed in the first part of this section, the existence of differential feedbacks in TCR signaling provides a major step forward in understanding ligand discrimination at the biochemical level. A computer simulation of these feedback effects illustrated how the threshold of pMHC-TCR lifetime in terms of activation of T cells can be set by the detailed properties of the kinetic competition between the positive and negative pathways. A consequence of this feature of TCR signal control is that modest up and downregulation of signaling protein levels in the feedback pathways can be anticipated to modulate in dramatic ways the threshold of ligand discrimination.
This leads us to propose a simple algorithm for enforcing positive/negative selection and peripheral tolerance that also optimizes ligand discrimination and foreign antigen sensitivity. First, low levels of a key inhibitory signaling component (e.g. SHP-1) or high levels of a key stimulatory signaling component (e.g. Lck) will endow DP thymocytes with responsiveness to endogenous ligands. Following initiation of positive selection through such signaling, increased expression of the inhibitory or decreased expression of the stimulatory enzymes can modify the effective discrimination threshold for effective signaling until responsiveness to endogenous ligands is lost, preventing negative selection by the pMHCs involved in positive selection and producing a self-recognizing but not overtly self-reactive T cell for release to the periphery. This is an optimal algorithm for TCR response tuning, at least at the conceptual level, as it allows any pMHC binding to the TCR more avidly than endogenous ligands to be stimulatory, offering the repertoire the widest range of foreign ligand sensitivity in the periphery. It is especially attractive because the loss of self-reactivity does not come at the expense of foreign ligand sensitivity, as would a more general blunting of the T cell signaling capacity of a cell. Such global blunting would prevent self-responses but also diminish sensitivity for low levels of foreign ligands, an undesirable outcome of the tuning process and one that is not seen experimentally [48
]. This algorithm easily accounts for the differential change in signaling properties of a given T cell undergoing maturation. Some experimental evidence is also consistent with this algorithm: SHP-1 has been shown to be low in DP thymocytes [65
]; Lck association with the TCR and with CD4 also is high in DP thymocytes [58
]. These two observations correlate well with hypersensitivity of DP thymocytes: quantitative analysis and computer modeling will further test this correlation and help identify critical kinetic components in the tuning of T cell ligand discrimination in the thymus.