The Balance Players of the Adaptive Immune System

It was the recognition of suppressor T cells, now called regulatory T cells (Treg), pioneered by Dr. Sakaguchi that highlighted the importance of regulatory cells in the maintenance of immunologic self-tolerance and immune homeostasis (1, 2). However, although a lot of attention has been given regulatory suppressive T cells, new findings suggest that regulatory T cells may also have effector capabilities. We recently reported that the immune system has established a mechanism to counteract the variety of immune-suppressive feedback signals: self-reactive, proinflammatory T cells that target immunosuppressive cells. Thus, we identified self-reactive T cells that recognize human leukocyte antigen (HLA)–restricted epitopes derived from proteins, including indoleamine 2,3-dioxygenase (IDO; refs. 3–7) and PD-L1 (8–12), expressed at inflammation sites in regulatory immune cells. Because these T cells can directly react against regulatory immune cells, such cells were termed antiregulatory T cells (anti-Tregs; ref. 13). Anti-Tregs may directly suppress the function of regulatory immune cells within immune regulatory networks as well as assisting the adaptive immune response by secreting proinflammatory cytokines at inflammation sites.

To keep the immune balance, regulatory immune cells, for example, Tregs, different dendritic cell subtypes, myeloid-derived suppressor cells, and M2 macrophages suppress or terminate immune responses. This regulatory arm secures the unresponsiveness or tolerance to self-antigens. Regulatory immune cells suppress immunity through a number of different cellular and extracellular factors. In contrast, specific anti-Tregs recognizing HLA-restricted derived epitopes, which are generated from intracellular degraded antigens, are able to directly eliminate regulatory immune cells (Fig. 1; ref. 3). In addition, anti-Tregs can boost local immune activation by the secretion of effector cytokines. Thus, anti-Tregs may function as specific first responder helper cells at the site of inflammation. It must be assumed that anti-Tregs themselves are hampered by the suppressive effects of their targets. Thus, in immune regulatory networks, anti-Tregs may suppress the function of other regulatory immune cells and vice versa. Hence, under normal physiologic conditions equilibrium between immune activation and suppression may be necessary to maintain immune homeostasis. The role of self-reactive effector and suppressor cells in immune-regulatory networks may thus be miscellaneous. Professional antigen-presenting cells highly express proteins such as PD-L1 and IDO, which are induced by interferons expressed at inflammation sites. In fact, circulating IDO- or PD-L1 specific anti-Tregs are present in healthy donors, although detection was not as frequent as detection in patients with cancer (3, 6, 9, 10). We have further verified that interferons expand populations of IDO-specific T cells by demonstrating that known IDO inducers (e.g., IFNγ) lead to expansion of IDO-specific T cells among human PBMCs without additional stimulation (3). Likewise, we recently found that subcutaneous IFNγ injections in C57 mice expand populations of PD-L1–specific T cells. If the mice were sacrificed 1 week after IFNγ injections, the spleens of the IFNγ-treated mice showed a strong PD-L1–specific T-cell response. Similarly, 3 days of treating C57 mice with the allergen DNFB led to an influx (or expansion) of PD-L1-specific T cells at the inflammation site (in prep.). Furthermore, it has been described that the very common cytomegalovirus (CMV) induces IDO expression in vivo, which has been suggested to confer an advantage to CMV-infected cells, allowing them to escape T-cell responses (14). Notably, we found that the presence of IDO-specific T-cell responses in the periphery was associated with the CMV T-cell response (6). It has long been thought that self-reactive T cells harboring T-cell receptors with high affinity to a target/human leukocyte antigen complex undergo clonal deletion to maintain self-tolerance. However, Yu and colleagues (15) recently demonstrated that clonal deletion prunes the T-cell repertoire but does not eliminate self-reactive T-cell clones. Hence, self-peptide–specific CD8 T cells were present in similar frequencies as those specific for non-self-antigens in the blood of healthy humans. These self-reactive T cells were significantly anergic compared with foreign-specific T cells; however, they could be activated by strong activation signals. These self-reactive T cells may escape thymic selection not only to participate in the fight against pathogens directly but also to provide the immune system with yet another layer of immune regulators as anti-Tregs that may contribute to immune homeostasis. Thus, both suppressive as well as effector regulatory cells participate in the complex network of cells that control the immune system and, consequently, together function as the Balance Players of the Adaptive Immune System (Fig. 1).

Many of the immune regulatory mechanisms considered helpful in autoimmune settings are used by tumors to suppress immune responses toward malignant cells in cancerous settings. Hence, various immune-tolerance mechanisms are exploited by cancer cells to achieve immune escape, which becomes more pronounced with disease progression. Thus, cancer cells as well as other regulatory immune cells [e.g., tumor-associated dendritic cells and myeloid-derived suppressor cells (MDSC)] express checkpoint inhibitors (e.g., PD-L1), inhibitory cytokines as well as metabolic enzymes (e.g., IDO) that restrain the antitumor activity of antitumor-specific T cells in the tumor microenvironment. In recent years, our growing knowledge of the factors responsible for protecting cancer cells from immune destruction has led to the development of novel, immune-based, anticancer treatment modalities (16, 17). Indeed, impressive clinical responses have been achieved by characterizing inhibitory T-cell pathways and targeting them with monoclonal antibodies against specific membrane proteins (18). The activation of anti-Tregs, e.g., by vaccination, may offer a novel very to directly target immune inhibitory pathways in the tumor microenvironment, modulate immune regulation, and potentially altering tolerance to tumor antigens. Thus, if successfully targeted, a therapeutic vaccination approach to activate anti-Tregs can, like the other approaches that target immune suppression (by checkpoint inhibition or small molecule inhibitors that target immunosuppressive molecules), contribute to antitumor immunity by relieving the immune suppression and thereby potentiating effective antitumor T-cell responses. However, unlike other approaches, it could also lead to epitope spreading of the potential target cells and immunologic memory (19), because anti-Tregs directly kill their target cells.

The first clinical testing of IDO vaccinations was performed in patients with NSCLC (NCT01543464; ref. 20). Furthermore, two additional trials have recently started to evaluate the safety of a vaccine targeting PD-L1–specific T cells in multiple myeloma (NCT03042793) and a combination vaccine that targets both IDO- and PD-L1–specific T cells with Nivolumab in metastatic melanoma (NCT03047928). Finally, industry-sponsored clinical trials in combination with anti-PD1 antibodies are being initiated in patients with NSCLC (www.iobiotech.com). In all of these trials the vaccinations were well tolerated by all patients with no severe toxicity. In the NSCLC study 2 patients received the vaccinations for 5 years (ESMO 2017, manuscript in preparation). Anti-Tregs are as described naturally present in vivo without vaccinations and both PD-L1 and IDO are induced by interferons as a counter-response to the inflammatory response. This provides a mechanism that ensures immune homeostasis, which keep anti-Tregs in check—therefore, the risk of triggering autoimmune-related adverse events by vaccination is minimal. This is confirmed in pre-clinical models as mice vaccinated with IDO or PD-L1 have shown no sign of toxicity (unpublished data), which supports the safety data from the clinical studies conducted so far.

Because immune-suppressive cells might antagonize the desired effects of therapeutic cancer vaccines, the addition of anti-Treg antigens would consequently be easily implementable and highly synergistic. Indeed, in a pre-clinical study, co-stimulation of anti–PD-L1 T cells augments T-cell response to a dendritic cell (DC) vaccine (21). In addition to PD-L1 and IDO we and others have identified pro-inflammatory self-reactive T cells that recognized HLA-restricted epitopes derived from other proteins normally expressed by regulatory immune cells—For example, tryptophan 2,3-dioxygenase (TDO; ref. 22), C-C motif chemokine 22 (23), forkhead box P3 (Foxp3; refs. 24, 25) and more recently programmed death-ligand 2 (PD-L2; ref. 26), and Arginase (27). Especially, the latter is highly interesting to activate in a therapeutic setting, because arginase-expressing myeloid cells contributes to an immunosuppressive tumor microenvironment that prevents effector lymphocyte proliferation (28). Specific targeting of arginase-expressing myeloid cells (e.g., neutrophils, TAMs, and MDSC) could potentially induce T-cell infiltration at the tumor site. The fact that Th1 inflammation signals expand IDO- and PD-L1–specific T cells suggests that combination of arginase with IDO and/or PD-L1–based vaccines may work synergistically. In this situation, arginase vaccination could induce Th1 inflammation at tumor sites where regulatory myeloid cells otherwise prevent infiltration of lymphocytes. This would, in turn, induce IDO and PD-L1, enabling further targeting by PD-L1- and/or IDO-specific T cells. The combination of vaccine epitopes like arginase, PDL1, and IDO would thus be highly beneficial and easy to implement in a clinical setting.

Immune system suppression plays a major role in cancer progression, with major mechanisms of tumor immune escape (29, 30). In a subset of patients with various cancer types, durable therapeutic responses can be generated via immune checkpoint blockade using antibodies against cytotoxic-T-lymphocyte–associated protein 4 (CTLA4) or PD1/programmed cell death 1 ligand 1 (PD-L1). However, checkpoint inhibitors are only efficient in a small fraction of patients with cancer. MDSCs and tumor-associated macrophages (TAM) play important roles in tumor immune evasion, and their accumulation in the tumor bed restrict the accumulation of T cells within the vicinity of cancer cells. Therefore, these suppressive cells constitute a major reason for the limited efficacy of checkpoint blockade in cancer treatment. The novel understanding of anti-Tregs may lead to a translatable strategy for improving the efficacy of checkpoint blockade through the activation of specific T cells that react to regulatory cells (including tumor cells) at the tumor site, thereby inducing local inflammation. We hypothesize that an anti-Treg–activating vaccine would attract T cells into the tumor, thereby inducing Th1 inflammation, which would further induce PD-L1 expression in cancer and immune cells, generating targets more susceptible to anti-PD1/PDL1 immunotherapy. Indeed, this was confirmed in pre-clinical models as anti-PD1 and IDO vaccination show synergy in both C57 and Balb-C mice (in preparation). Combinatorial therapy with an anti-Treg–based vaccine and checkpoint blockade could be effective in a much wider population of cancer patients.

The role of anti-Tregs in immune reactions supports a rationale for the targeting of these T cells in cancer immunotherapy. An obvious risk for such an approach, potential long-term toxicity due to vaccine-induced autoimmune mechanisms, appears to be minimal, illustrated both in mouse in vivo studies and in the human safety trials conducted thus far. Investigations to address some of the most clinically relevant questions are ongoing in preclinical as well as clinical studies, especially elucidating the potential benefits of combination strategies with other therapeutic modalities.

M.H. Andersen is a board member, scientific director, and has ownership interest (including patents) in IO Biotech.

The funders did not have a role in the writing of the article or the decision to submit the article for publication.

This work was supported by the Danish Cancer Society, the Danish Council for Independent Research, and Herlev Hospital.