DAMPING T‐CELL ENTHUSIASM
We have frequently reiterated the premise that no self‐respecting organism would permit the operation of an expanding enterprise such as a proliferating T‐cell population without some sensible controlling mechanisms. There are some similarities here with regulations governing corporate takeovers in the business world, where it has been deemed prudent to ensure that no single enterprise is permitted to completely dominate the marketplace. Such monopoly practices, if allowed to occur in an unregulated way, would eventually eliminate all competition. Not a good thing for diversity or overall fitness.
In a similar vein, in order to preserve immunological diversity and the capacity to rapidly respond to new challenges of an infectious nature, it is necessary to ensure that T‐cells specific for particular epitopes are not allowed to proliferate indefinitely and ultimately dominate the immune compartment. This would inevitably reduce the probability that responses to freshly encountered antigens would ever get off the ground, as naive T‐cells would have to compete for access to DCs with over whelming numbers of previously activated T‐cells, with inevitable disastrous consequences for immunological fitness. For these reasons, our highly adapted immune systems have evolved ways of maintaining healthy competition between T‐cells, which is achieved through downregulating immune responses upon clearance of a pathogen, along with culling of the majority of recently expanded T‐cells. This is also necessary because the immune compartment is of a relatively finite size and cannot accommodate an infinite number of lymphocytes.
Damping down T‐cell responses occurs via a number of mechanisms, some of which operate at the level of the activated T‐cell itself, while others operate via additional T‐cell subsets (regulatory T‐cells) that use a variety of strategies to rein in T‐cell responses, some of which are directed at the T‐cell while others are directed at DCs. Regulatory T‐cells will be discussed at length in Chapter 8, so here we will focus primarily on molecules present on activated T‐cells that serve as “off switches” for such T‐cells. Such molecules represent important immunological checkpoints, helping to keep T‐cell responses within certain limits.
Figure 7.18 Downregulation of T‐cell responses. (a) Antigen presentation by a mature dendritic cell (DC) provides effective antigenic stimulation via peptide–MHC (signal 1) and B7 ligands (signal 2) that engage the T‐cell receptor (TCR) complex and CD28 on the T‐cell, respectively. (b) Antigen presentation to a previously activated T‐cell that is bearing surface CTLA‐4 (a CD28‐related molecule that can also interact with B7 ligands) can lead to T‐cell unresponsiveness owing to inhibitory signals delivered through CTLA‐4 co‐stimulation (see main text for further details). (c) Whereas naive T‐cells bearing surface Fas receptor are typically resistant to ligation of this receptor, activated T‐cells acquire sensitivity to Fas receptor (FasR) engagement within a week or so of activation. Engagement of FasR on susceptible cells results in activation of the programmed cell death machinery as a result of recruitment and activation of caspase‐8 within the FasR complex. Active caspase‐8 the propagates a cascade of further caspase activation events to kill the cell via apoptosis.
Signals routed through CTLA‐4 downregulate T‐cell responses
Cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) is structurally related to CD28 and also binds B7 (CD80/CD86) ligands. However, whereas CD28–B7 interactions are co‐stimulatory, CTLA‐4–B7 interactions act in an opposite fashion and contribute to the termination of TCR signaling (Figure 7.18). Whereas CD28 is constitutively expressed on T‐cells, CTLA‐4 is not found on the resting cell but is rapidly upregulated within 3–4 hours following TCR/CD28‐induced activation. CTLA‐4 has a 10‐ to 20‐fold higher affinity for both B7.1 and B7.2 and can therefore compete favorably with CD28 for binding to the latter even when present at relatively low concentrations. The mechanism by which CTLA‐4 suppresses T‐cell activation has been the subject of lively debate, as this receptor appears to recruit a similar repertoire of proteins (such as PI3K) to its intracellular tail as CD28 does. A number of mechanisms have been proposed to account for the inhibitory effect of CTLA‐4 on T‐cell activation. One mechanism is by simple competition with CD28 for binding of CD80/CD86 molecules on the DC. Another is through recruitment of SHP‐1 and SHP‐2 protein tyrosine phosphatases to the TCR complex, which may contribute to the termination of TCR signals by dephosphorylating proteins that are required for TCR signal propagation. CTLA‐4 may also antagonize the recruitment ofthe TCR complex to lipid rafts, which is where many of the signaling proteins that propagate TCR signals reside.
Although conventional T‐cells require CTLA‐4 expression to be induced after antigen engagement, Tregs constitutively express this receptor and this appears to play an important role in Treg‐mediated immune suppression. Tregs can use CTLA‐4 to bind CD80/CD86 on APCs, promoting trans‐endocytosis and removal of B7 ligands from the APC cell surface, thereby downregulating immune responses. While this cell‐extrinsic function of CTLA‐4 is becoming widely recognized, it should be mentioned that Tregs also suppress immune responses in CTLA‐4 independent ways (as will be discussed in Chapter 8). Irrespective of its mechanism of action, CTLA‐4 is undoubtedly critical for keeping T‐cells under control and in this regard is also important for preventing responses to self antigen. CTLA‐4‐deficient mice display a profound hyperproliferative disorder and die within 3 weeks of birth as a result of massive tissue infiltration and organ destruction by T‐cells.
PD‐1 also represents an important T‐cell checkpoint molecule
Another potent T‐cell inhibitory receptor, programmed death 1 (PD‐1), is currently creating quite a stir because of the emerging clinical success of antitumor therapies that seek to block its action and reactivate the immune response against tumors expressing CTL‐inhibitory PD‐1 ligands on their surface. Similar to CTLA‐4, PD‐1 also belongs to the CD28 family of co‐receptors, and mediates its inhibitory effect subsequent to antigen binding through recruitment of the phosphatase SHP‐2, which dephosphorylates and inactivates proximal signaling adaptors such as ZAP‐70 in T‐cells and Syk in B‐cells. Prior to antigen stimulation, T‐cell PD‐1 expression is upregulated then triggered by either of its two receptors: PD‐L1, which is expressed mainly on nonlymphoid cells, and PD‐L2, expressed on APCs.
Thus, like CTLA‐4, PD‐1 is involved in the suppression of T‐cell‐driven immune responses. Unlike CTLA‐4 however, deficiency of which leads to fatal autoimmune disease in mice, loss of PD‐1 has a less drastic outcome, resulting in the development of a range of different autoimmune diseases depending on the genetic background of the mice. This difference between PD‐1 and CTLA‐4 function seems to reflect a propensity for PD‐1 activation to drive responses only in PD‐1‐expressing cells (cell intrinsic responses), whereas CTLA‐4 responses are more far‐reaching, not only through intrinsic processes but also through cell extrinsic T‐cell‐driven CTLA‐4‐mediated down regulation of CD28 on APCs and effector T‐cells.
Importantly, PD‐L1 is expressed at significantly high levels on many tumor types, which is correlated with poor clinical prognosis. This indicates that tumor cells may aggressively express PD‐L1 on their surface to block CTL‐mediated killing. Indeed, preclinical animal studies using blocking antibodies directed against either PD‐1 or PD‐L1 have shown promising effects in re‐stimulating the T‐cell‐mediated immune response to promote tumor regression. Many PD‐1/PD‐L1 blocking therapies are now in advanced phase clinical trials and have shown impressive clinical responses in multiple tumor types, including a 38% response rate by the anti‐PD‐1 drug MK‐3475 in patients with advanced melanoma. Because PD‐1 action is primarily cell intrinsic, immune‐associated side‐effects with PD‐1‐blocking therapies have been considerably less severe than with CTLA‐4‐inhibitory therapies, which have also proved successful in the clinic. Therapies designed at re-stimulating T‐cell‐mediated antitumor immunity are particularly desirable, as activating the adaptive immune system to target tumors not only offers an exquisite layer of precision, because of the generation of highly specific antigen receptors against tumor antigens, but also generates long‐lived memory, which may significantly lesson the chances of tumor relapse.
Cbl family ubiquitin ligases restrain TCR signals
A number of other molecules have been identified that may be involved in reigning in T‐cell activation and these include the Cbl family of proteins: c‐Cbl, Cbl‐b, and Cbl‐c. Membersof the Cbl family are protein ubiquitin ligases that can catalyze the degradation of proteins through attaching polyubiquitin chains to such molecules, thereby targeting them for destruction via the ubiquitin‐proteasome pathway. The ζ chain of the CD3 co‐receptor complex has been identified as a target for c‐Cbl‐mediated ubiquitination and this can result in internalization and degradation of the TCR complex. Thus, c‐Cbl proteins may raise the threshold for TCR‐induced signals through destabilizing this complex. Mice doubly deficient in c‐Cbl and Cbl‐b (which appear to exert somewhat redundant functions) exhibit hyperresponsiveness to TCR‐induced signals, resulting in excessive proliferation and cytokine production in naive as well as differentiated effector T‐cells; such mice die from autoimmune disease as a consequence. This appears to be due to a defect in downmodulation of the TCR complex in activated T‐cells. Whereas TCR complexes are normally internalized and degraded after stimulation via cognate peptide–MHC complexes (an event which contributes to the termination of TCR signals), TCR complexes fail to be internalized in c‐Cbl/Cbl‐b‐deficient cells, leading to greatly extended TCR signaling and runaway T‐cell expansion.
Cbl family proteins can also exert their influence on TCR signaling in other ways and may have an especially important role in maintaining the requirement for CD28 co‐stimulation for proper T‐cell activation. Surprisingly, mice deficient in Cbl‐b lose the normal requirement for CD28 co‐stimulation (i.e., signal 2) for T‐cell proliferation; such cells make large amounts of IL‐2 and proliferate vigorously in response to TCR stimulation alone. This implies that Cbl‐b plays a major role in maintaining the requirement for signal 2 for activation of naive T‐cells. Although it is not yet clear exactly how this operates, activation of Vav, which occurs downstream of TCR as well as CD28 receptor stimulation, appears to be suppressed by Cbl‐b in wild‐type cells. Thus, for effective Vav activation, signals 1 and 2 are normally required. However, in the absence of Cbl‐b, a sufficient amount of Vav activation is achieved through TCR stimulation alone, bypassing the need for CD28 co‐stimulation.
T‐cell death occurs through stimulation of membrane Fas receptors
Another important way of standing down T‐cells from active duty is to kill such cells through programmed cell death (Figure 7.18). Naive T‐cells, as well as recently activated T‐cells, express the membrane Fas (CD95) receptor, but are insensitive to stimulation via this receptor as these cells contain an endogenous inhibitor (FLIP) of the proximal signaling molecule caspase‐8 that is activated as a result of stimulation through the Fas receptor. However, upon several rounds of stimulation, experienced T‐cells become sensitive to stimulation via their Fas receptors, most likely owing to loss of FLIP expression, and this situation results in apoptosis of these cells. Mice defective in expression of either Fas or FasL manifest a lymphoproliferative syndrome that results in autoimmune disease due to a failure to cull recently expanded lymphocytes.