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Friday, November 6, 2020



We have frequently reiterated the premise that no selfrespecting organism would permit the operation of an expanding enterprise such as a proliferating Tcell 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 Tcells 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 Tcells would have to compete for access to DCs with over­ whelming numbers of previously activated Tcells, with inevitable disastrous consequences for immunological fitness. For these reasons, our highly adapted immune systems have evolved ways of maintaining healthy competition between Tcells, which is achieved through downregulating immune responses upon clearance of a pathogen, along with culling of the majority of recently expanded Tcells. This is also necessary because the immune compartment is of a relatively finite size and cannot accommodate an infinite number of lymphocytes.

Damping down Tcell responses occurs via a number of mechanisms, some of which operate at the level of the activated Tcell itself, while others operate via additional Tcell subsets (regulatory Tcells) that use a variety of strategies to rein in Tcell responses, some of which are directed at the Tcell while others are directed at DCs. Regulatory Tcells will be discussed at length in Chapter 8, so here we will focus primarily on molecules present on activated Tcells that serve as “off switches” for such Tcells. Such molecules represent important immunological checkpoints, helping to keep Tcell responses within certain limits.


Downregulation of T‐cell responses

Figure 7.18 Downregulation of Tcell 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 Tcell receptor (TCR) complex and CD28 on the Tcell, respectively. (b) Antigen presentation to a previously activated Tcell that is bearing surface CTLA4 (a CD28related molecule that can also interact with B7 ligands) can lead to Tcell unresponsiveness owing to inhibitory signals delivered through CTLA4 costimulation (see main text for further details). (c) Whereas naive Tcells bearing surface Fas receptor are typically resistant to ligation of this receptor, activated Tcells 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 caspase8 within the FasR complex. Active caspase8 the propagates a cascade of further caspase activation events to kill the cell via apoptosis.

Signals routed through CTLA4 downregulate Tcell responses

Cytotoxic Tlymphocyte antigen4 (CTLA4) is structurally related to CD28 and also binds B7 (CD80/CD86) ligands. However, whereas CD28–B7 interactions are costimulatory, CTLA4B7 interactions act in an opposite fashion and contribute to the termination of TCR signaling (Figure 7.18). Whereas CD28 is constitutively expressed on Tcells, CTLA4 is not found on the resting cell but is rapidly upregulated within 3–4 hours following TCR/CD28induced activation. CTLA4 has a 10 to 20fold 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 CTLA4 suppresses Tcell 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 CTLA4 on Tcell activation. One mechanism is by simple competition with CD28 for binding of CD80/CD86 molecules on the DC. Another is through recruitment of SHP1 and SHP2 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. CTLA4 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 Tcells require CTLA4 expression to be induced after antigen engagement, Tregs constitutively express this receptor and this appears to play an important role in Tregmediated immune suppression. Tregs can use CTLA4 to bind CD80/CD86 on APCs, promoting transendocytosis and removal of B7 ligands from the APC cell surface, thereby downregulating immune responses. While this cellextrinsic function of CTLA4 is becoming widely recognized, it should be mentioned that Tregs also suppress immune responses in CTLA4 independent ways (as will be discussed in Chapter 8). Irrespective of its mechanism of action, CTLA4 is undoubtedly critical for keeping Tcells under control and in this regard is also important for preventing responses to self antigen. CTLA4deficient mice display a profound hyperproliferative disorder and die within 3 weeks of birth as a result of massive tissue infiltration and organ destruction by Tcells.

PD1 also represents an important Tcell checkpoint molecule

Another potent Tcell inhibitory receptor, programmed death 1 (PD1), 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 CTLinhibitory PD1 ligands on their surface. Similar to CTLA4, PD1 also belongs to the CD28 family of coreceptors, and mediates its inhibitory effect subsequent to antigen binding through recruitment of the phosphatase SHP2, which dephosphorylates and inactivates proximal signaling adaptors such as ZAP70 in Tcells and Syk in Bcells. Prior to antigen stimulation, Tcell PD1 expression is upregulated then triggered by either of its two receptors: PDL1, which is expressed mainly on nonlymphoid cells, and PDL2, expressed on APCs.

Thus, like CTLA4, PD1 is involved in the suppression of Tcelldriven immune responses. Unlike CTLA4 however, deficiency of which leads to fatal autoimmune disease in mice, loss of PD1 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 PD1 and CTLA4 function seems to reflect a propensity for PD1 activation to drive responses only in PD1expressing cells (cell intrinsic responses), whereas CTLA4 responses are more farreaching, not only through intrinsic processes but also through cell extrinsic Tcelldriven CTLA4mediated down­ regulation of CD28 on APCs and effector Tcells.

Importantly, PDL1 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 PDL1 on their surface to block CTLmediated killing. Indeed, preclinical animal studies using blocking antibodies directed against either PD1 or PDL1 have shown promising effects in restimulating the Tcellmediated immune response to promote tumor regression. Many PD1/PDL1 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 antiPD1 drug MK3475 in patients with advanced melanoma. Because PD1 action is primarily cell intrinsic, immuneassociated sideeffects with PD1blocking therapies have been considerably less severe than with CTLA4inhibitory therapies, which have also proved successful in the clinic. Therapies designed at re-stimulating Tcellmediated 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 longlived 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 Tcell activation and these include the Cbl family of proteins: cCbl, Cblb, and Cblc. 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 ubiquitinproteasome pathway. The ζ chain of the CD3 coreceptor complex has been identified as a target for cCblmediated ubiquitination and this can result in internalization and degradation of the TCR complex. Thus, cCbl proteins may raise the threshold for TCRinduced signals through destabilizing this complex. Mice doubly deficient in cCbl and Cblb (which appear to exert somewhat redundant functions) exhibit hyperresponsiveness to TCRinduced signals, resulting in excessive proliferation and cytokine production in naive as well as differentiated effector Tcells; 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 Tcells. Whereas TCR complexes are normally internalized and degraded after stimulation via cognate peptideMHC complexes (an event which contributes to the termination of TCR signals), TCR complexes fail to be internalized in cCbl/Cblbdeficient cells, leading to greatly extended TCR signaling and runaway Tcell 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 costimulation for proper Tcell activation. Surprisingly, mice deficient in Cblb lose the normal requirement for CD28 costimulation (i.e., signal 2) for Tcell proliferation; such cells make large amounts of IL2 and proliferate vigorously in response to TCR stimulation alone. This implies that Cblb plays a major role in maintaining the requirement for signal 2 for activation of naive Tcells. 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 Cblb in wildtype cells. Thus, for effective Vav activation, signals 1 and 2 are normally required. However, in the absence of Cblb, a sufficient amount of Vav activation is achieved through TCR stimulation alone, bypassing the need for CD28 costimulation.

Tcell death occurs through stimulation of membrane Fas receptors

Another important way of standing down Tcells from active duty is to kill such cells through programmed cell death (Figure 7.18). Naive Tcells, as well as recently activated Tcells,  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 caspase8 that is activated as a result of stimulation through the Fas receptor. However, upon several rounds of stimulation, experienced Tcells 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.

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