CCR 20^th Anniversary Commentary: From Regulatory T Cells to Checkpoint Monoclonal Antibodies—Immuno-oncology Advances Clinical Cancer Research

In 2011, Hanahan and Weinberg (1) elaborated on their initial “hallmark of cancer” concept by adding novel key aspects of cancer biology. The increasing knowledge at that time about the bidirectional interaction and manipulation of cancer and immune cells led the authors to include as part of their revised concept the ability of malignant tumors to escape from an efficient immune cell attack, referred to as “cancer immune evasion” (1). For decades, it had been assumed that tumors are able to shape the immune system, thereby preventing their active elimination. More than a century earlier, such visionaries as Paul Ehrlich had envisioned that “magic (i.e., immunologic) bullets” might be able to overcome tolerance to cancer. The description of murine tumors that could be eliminated upon depletion of so-called regulatory T cells (Treg), reported toward the end of the 20th century, provided the first preclinical evidence that CD25⁺ Treg function as cellular immune checkpoints (2). It was then understood that Treg might provide a potential target for cancer immunotherapy, reigniting the hope that cancer vaccination could be resuscitated after years of frustrating research (almost exclusively producing negative results at that time).

We and others set the stage in 2003 for the strategic development of Treg-targeting attempts with the aim of improving cancer treatment in humans, as we were able to demonstrate that patients suffering from untreated solid tumors have an expanded circulating blood Treg pool (3). In subsequent years, various groups, including our own, provided further evidence that Treg accumulate in the tumor microenvironment (4). Many tumor immunologists envisioned that Treg expansion and its negative prognostic impact represented a general phenomenon seen in all types of malignancies that might in turn allow development of strategies for Treg depletion as a reinforcement in cancer patients to reactivate their paralyzed immune response against the malignant tumor cells.

It became increasingly evident that the situation was much more complex, as we came to understand the unique identity of Treg and their role as the “bad guys” in the cancer field. Although some studies linked the high number of Treg in the tumor microenvironment in many tumor types (e.g., ovarian, gastric, breast, and renal cancers) with inferior survival, other authors demonstrated only a limited role of Treg abundance in the tumor microenvironment or in the peripheral blood for clinical outcome. In contrast, in some lymphoproliferative diseases, such as follicular lymphoma, increased numbers of Treg have been linked to an improved clinical course. This may, at least in part, be due to the physiologic role of Treg as suppressors of normal B-cell function. Although the hypothesis that Treg negatively regulate B cell-derived malignant cells is appealing, it has never been formally proven and the field of Treg/B-cell–derived malignancy interaction remains largely unstudied. In either case, the inhomogeneous data on the prognostic role of Treg in cancer highlighted that a general “Treg depletion” strategy may not be applicable to all malignancies. Moreover, intense research in the field of inflammation-driven malignant transformation suggested that in this particular situation, Treg may even protect the organism from malignancies, as they restrain the inflammatory process driving malignant transformation (5).

Finally, a systematic problem of immune-activating strategies is the inherent risk of triggering excessive autoimmunity. This concern is underscored by the seminal work of Sakaguchi and colleagues (6) in the mid-1990s, who first identified the role of CD4⁺CD25⁺ Treg in a mouse model inducing systemic autoimmunity by CD25⁺ depletion (comprising in the steady state mainly Treg). Conversely, two other groups of researchers reported their clinical observations from patients with a genetic mutation and subsequent inactivation of the Treg-specific transcription factor FoxP3, leading to a polyglandular autoimmune syndrome (IPEX-syndrome; refs. 7, 8). This may be a potential concern of translation of Treg-ablation approaches into the clinic, given that highly effective and systemically active Treg-depletion strategies are available. Although no specific Treg marker has been identified to date, various research groups have reported their results with preclinical models and in early clinical trials on Treg-depleting agents (reviewed in ref. 9). For example, low doses of cyclophosphamide have been shown to preferentially eliminate Treg and thus contribute to an improved response to vaccines in human clinical trials. An alternative approach that has already been translated into the clinic is the application of a diphtheria toxin–coupled IL2 (denileukin difitox), which binds to CD25-expressing Treg and after internalization depletes the Treg. Results from a clinical trial evaluating the use of a dendritic cell vaccination suggested that prior diphitox application enhances the clinical efficacy of this agent via Treg depletion; however, the toxicity of this construct is clinically significant, and until now no additional clinical evidence supports the broader applicability of this approach. Another translational approach is mAb-mediated targeting of CD25 by daclizumab, which is able to reprogram Treg into Th1 cells by downmodulating FoxP3 expression, and thereby inducing loss of suppressive activity paralleled by induction of IFNγ production. Recent data in humans show that anti-CCR4 mAb depletes Treg in cancer patients and boosts the induction of tumor-antigen (NY-ESO-1)–specific CD8⁺ T cells (10). This strategy is interesting, as it may also interfere with the recruitment process of Treg into the cancer microenvironment.

Another strategy that has been explored is targeting of signaling pathways critically involved in the regulation of Treg-mediated tolerance of tumors. One example of this approach is inactivation of the PI3K subunit p110Delta in Treg, which was shown to unleash CD8⁺ cytotoxic T cells and induce tumor regression in mice (11). Thus, inhibitors of the Delta subunit of the PI3K can break tumor-induced immune tolerance. However, targeting of cellular signaling pathways to functionally inactivate Treg as well as surface molecules to deplete them is not specific to Treg, as Treg-specific target molecules are still not identified. Moreover, other regulatory T-cell populations (i.e., induced Treg) also express the “Treg-specific” transcription factor FoxP3, in addition to other Treg markers. Thus, until now, naturally occurring Treg have not been thought to have a unique marker expression pattern. In our original article in 2003 (3), we therefore characterized the cells by functional testing as well, including proliferation and suppression assays, which still represents the gold standard for appropriate Treg identification and characterization. However, intense research during recent years has also highlighted that expression of CD3, CD4, CD25, CTLA-4, and CD39, as well as the absence of CD127 in humans, in combination with the intracellular positivity for FoxP3, approximates the ideal phenotype of naturally occurring Treg. Ki67, Helios, and CD45RA may also help to define the status of Treg as active or naïve. Novel (and clinically not yet evaluated) target structures may be the folate-receptor 4 or LAG-3, among many others. Also noteworthy is that Treg express CTLA-4 (which we also demonstrated in our initial work; ref. 3), which is targeted by the checkpoint antibody ipilimumab. Expression of CTLA-4 on Treg also explains Fc-receptor–mediated elimination of intratumoral Treg in murine cancer models after application of CTLA-4–binding antibodies (12). Another important and known checkpoint interaction is that of PD-1/PD-L1 (programmed death 1/programmed death ligand 1). This interaction is critical for CD4⁺ T-cell plasticity within the tumor microenvironment, as it supports differentiation of Th1 from Treg (13). In turn, blocking the PD-1/PD-L1 interaction drives a Th1 signature in cancer. These findings may, at least in part, explain the immune-activating effects induced by ipilimumab and PD-1/PD-L1–blocking agents, such as nivolumab.

The intense research in the field of cellular immune-suppression mechanisms highlighted in this commentary provides a detailed definition of the functional role of Treg in cancer biology and also supports advances in other research areas, including characterization of the ways in which T-cell activation in health, autoimmunity, and cancer is (dys)-regulated. These findings have also supported the development of checkpoint antibodies blocking CTLA-4 and PD1/PD-L1, which has dramatically changed the therapeutic landscape in types of cancer that have made only limited advances for decades (e.g., metastatic melanoma and lung cancer). We have a great deal to learn about how these therapeutics change the Treg compartment in various cancer subtypes, especially whether additional targeting of Treg in combination with immune-activating antibodies targeting checkpoint molecules may further increase the efficacy of these innovative immunologic therapeutics.

In summary, although many aspects of the role of Treg in cancer remain controversial, we are impressed by the rapid expansion of knowledge in the field of cancer immunology and how this knowledge has finally been translated into the clinic with the approval of the checkpoint mAbs, as described above. These advances led the editors of the journal Science to name cancer immunotherapy as the major scientific breakthrough of 2013 (14). The long-term remission rates seen in some of these patients fuel the hope that one day we may be able to reactivate the immune system of individual cancer patients in a more specific manner through manipulation of Treg biology. To achieve this goal, however, we must obtain a more detailed understanding of molecular mechanisms in individual tumor types that allow discrimination between the “immunosuppressive” Treg (promoting tumor escape) and the “anti-inflammatory” Treg (restricting chronic inflammation favoring malignant transformation) as well as the molecular mechanisms governing Treg recruitment and function in cancer.

No potential conflicts of interest were disclosed.

Conception and design: D. Wolf, A.M. Wolf

Writing, review, and/or revision of the manuscript: D. Wolf, A.M. Wolf