Targeting Tregs in Tumors.

This story began as an idea inspired by the observations of others and ended up with a drug in the clinic through articles in Nature, Cell, and Nature Immunology, and a biotech startup along the way. If you cast your mind back to the 1990s, you will recall that suppressor T cells had gone out of favor due to an inability to isolate and characterize them, essentially at the molecular level. People did not even like to use the ‘‘S’’ word! A renaissance gave birth to ‘‘regulatory T cells’’ with the seminal discovery by Sakaguchi and others (1995), published in Journal of Immunology in 1995, that CD25 (the interleukin [IL]-2a receptor) marked a small population of CD4+ T cells with potent suppressive activity. There was a lot of activity in the early days with people trying to understand what regulatory T cells (Tregs) were and how they functioned. There were 2 key articles from the groups of Sakaguchi and colleagues (Takahashi and others, 1998) and Thornton and Shevach (1998) in 1998 in which they performed some critical experiments using transwell plates. These are like regular 96-well plates but include a transwell insert where you can put cells in a top or bottom compartment that are separated by a semipermeable membrane. With small pores you could get diffusion of cytokines and various agents, but you did not get transfer of cells. The key question immunologists were asking was whether Tregs needed contact to mediate suppression? If you put effector T cells and Tregs in a well, the effector cell proliferation is prevented by the suppressor cell. But when they put Tregs in the top compartment and effector cells in the bottom compartment, there was no longer suppression. This gave birth to the term ‘‘contact-dependent suppression. ’’ The whole Treg field came to this paradigm thinking that Tregs had to have physical contact with effector cells to mediate suppression. This term stuck for some time, but over the next 10 years or so, more analysis of Treg function gave rise to the realization that these cells can and do suppress through soluble factors, specifically, inhibitory cytokines. Probably the first article of note was in 1999 by Powrie and colleagues (Asseman and others, 1999) who found that Tregs make a lot of IL-10, which constitutes a key mechanism of suppression. She had a whole series of terrific articles particularly focusing on the importance of IL-10 in colitis and other models, highlighting, among other things, the critical role of IL-10 in Treg function. Then, of course, there was a whole series of studies showing that transforming growth factor beta (TGFb) is also an important mechanism of Treg suppression. The emergence of cytokines as important mediators of Treg function created a bit of a quandary, because if cytokines are important, then that would infer that contact was not so critical. Then we came onto the scene, so to speak, with our discovery of the inhibitory cytokine IL-35 (Ye and others, 2021). We were looking for other soluble mediators of Treg suppression and discovered IL-35, a heterodimeric IL-12 family member, which was published in Nature in 2007 (Collison and others, 2007). This got us back to the issue of how can inhibitory cytokines (IL-10, IL-35, and TGFb) be so important for Treg function if the latter require contact to mediate suppression? We mulled over this for some time as it seemed like a contradiction. Then we had an idea. What if it is not the suppression that is contact dependent, but rather the augmentation or boosting of Treg function that was contact dependent. A fantastic postdoc in my laboratory, Lauren Collison, who was first author on the IL-35 Nature article (Collison and others, 2007), took up the challenge to address this conundrum. She performed a series of experiments, which led to a really critical article published in The Journal of Immunology in 2009 (Collison and others, 2009). If contact between effector cells and Tregs was required for boosting, then we should see more inhibitory cytokine production from Treg stimulated in the presence of effector T cells versus their absence. Indeed, we saw that these boosted Tregs were making a lot more IL-10 and IL-35 (Collison and others, 2009). We then asked, what if we went back to the transwell experiments of 1998 and put effector cells in the top compartment with Tregs; would that be sufficient to boost their functionality and mediate transwell suppression? We first reproduced the previous observations of Shimon and Ethan that, indeed, we had no Treg suppression across a transwell with Tregs in the top compartment and effector cells in the bottom compartment. However, when we added effector cells with Tregs in the top compartment, now we could see great suppression of the effector cells in the bottom compartment (Collison and others, 2009). How could that happen if the Tregs are not actually contacting them? This gave rise to the notion that these effector cells in the top well were boosting Treg