Mechanisms of Treg Suppression: Still a Long Way to Go

regs work? This question has preoccupied and perplexed T cell biologists for nearly two decades (Tang and Bluestone, 2008; Vignali et al., 2008; Workman et al., 2009). As with all important questions, there are some issues where there is a general consensus and others where there is still considerable disagreement. I would venture that most would agree with the following four basic tenets related to T reg function. First, T regs are a critical peripheral tolerance mechanism that maintains immune homeostasis and prevents widespread autoimmunity (Ramsdell, 2003; Sakaguchi, 2003; Fontenot and Rudensky, 2005). Thus, determining how T regs work is a very important goal. Second, T regs can suppress or modulate the function of a wide variety of cell populations, in diverse anatomical locations and in multiple disease situations (Tang and Bluestone, 2008). Third, T regs have an extensive arsenal and can utilize multiple contact-dependent and contact-independent mechanisms (Shevach et al., 2006; Vignali et al., 2008). This may be important because not all cell populations will be sensitive to all T reg mechanisms. Fourth, in addition to natural, thymusderived T regs , there are several induced T reg (iT reg ) populations that can be generated in vitro or directly by T regs via one of several inhibitory cytokines (TGFβ, IL-10, IL-35; Shevach, 2006; Collison and Vignali, 2008). The mechanism of suppression used by these populations seems less complicated with each appearing to depend on one key inhibitory cytokine. Thus our discussion here will focus on natural T regs . However, I would argue that we know far less than we think we do about natural T reg function and there is probably more contention than consensus. It is likely that continued, extensive analysis, and unique tools and approaches are likely to be required before a clear picture emerges. Some of these issues can be encapsulated around three key questions that pertain to T reg function. Which mechanisms are most important? This remains a contentious issue with each mechanism having its set of protagonists and antagonists. The mechanisms utilized by natural T regs that have probably been examined and discussed the most are inhibitory cytokines (TGFβ, IL-10, IL-35), inhibitory receptors (CTLA4, LAG-3), cytotoxicity (Granzyme/Perforin) and metabolic disruption (IL-2 deprivation-mediated apoptosis, adenosine; Shevach et al., 2006; Tang and Bluestone, 2008; Vignali et al., 2008). Of course there may be several mechanisms that are important, with each contributing significantly in different disease scenarios, anatomical locations or against diverse cell types. Whether this is reality or appeasement remains to be resolved. Before one determines if a mechanism is important, one would first need to obtain convincing in vivo evidence that a particular mechanism has a clearly definable physiologic impact. In my view, too many strong conclusions have been derived from exclusively or predominantly in vitro studies. Even when in vivo experiments have been performed, they are either limited to one model system or have potential, inherent weaknesses or caveats. It is hard to define what is “sufficient” but it seems reasonable to propose that data should be derived from multiple in vivo models (at least three) using (1) mice harboring conditionally (and ideally temporally) deleted alleles and (2) in vivo blockade/neutralization with a specific monoclonal antibody. Indeed, one could argue that no proposed T reg mechanism has been extensively assessed in vivo thus far. Although we have championed the potential importance of IL-35 using five in vivo model systems with neutralizing antibodies and mutant T cell populations targeting the cytokine and its receptor (Collison et al., 2007, 2010, 2012), I would be the first to admit that more remains to be determined regarding the physiological importance of IL-35. So for mechanisms that have not reached this bar, further analysis is clearly required. Of course a major challenge is that some of these mechanisms may utilize molecules that also contribute to T reg development or homeostasis. Thus it may be very difficult, or even impossible, to divorce their roles in T reg function versus development/homeostasis. Furthermore, some mechanisms may be hard to target without affecting other cellular processes. The current debate regarding the importance of one proposed T reg mechanism illustrates many of these issues. T regs express high levels of the high affinity IL-2 receptor (CD25; Sakaguchi et al., 1995). It was initially proposed that T regs may act as a “sink” absorbing IL-2 from the local environment, thereby depriving recently stimulated T cells from the IL-2 required to initiate proliferation and subsequent differentiation. Subsequent analysis of mice lacking the capacity to make IL-2 or lacking CD25 expression revealed that IL-2 plays a critical role in maintaining peripheral T reg homeostasis (D’Cruz and Klein, 2005; Fontenot et al., 2005). It was suggested that these data refuted the idea that T regs act as an IL-2 “sink,” although T reg function in vivo was not directly examined (Maloy and Powrie, 2005). More recently, it has been suggested that IL-2 deprivation-mediated apoptosis, facilitated by high CD25 expression, is a prominent mechanism of suppression used by T regs (Pandiyan et al., 2007). These conclusions were supported predominantly by the observation that T cells targeted by T regs die by apoptosis in in vitro assays but are resistant if they lack Bim, a pro-apoMechanisms of Treg suppression: still a long way to go