Targeting T regulatory (Treg) cells in immunotherapy-resistant cancers
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Abstract
Primary or secondary (i.e., acquired) resistance is a common occurrence in cancer patients and is often associated with high numbers of T regulatory (Treg) cells (CD4+CD25+FOXP3+). The approval of ipilimumab and the development of similar pharmacological agents targeting cell surface proteins on Treg cells demonstrates that such intervention may overcome resistance in cancer patients. Hence, the clinical development and subsequent approval of Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) targeting agents can serve as a prototype for similar agents. Such new agents aspire to be highly specific and have a reduced toxicity profile while increasing effector T cell function or effector T/T regulatory (Teff/Treg) ratio. While clinical development with large molecules has shown the greatest advancement, small molecule inhibitors that target immunomodulation are increasingly entering early clinical investigation. These new small molecule inhibitors often target specific intracellular signaling pathways [e.g., phosphoinositide-3-kinase delta (PI3K-δ)] that play an important role in regulating the function of Treg cells. This review will summarize the lessons currently applied to develop novel clinical agents that target Treg cells.
Keywords
INTRODUCTION
Immunotherapy with immune checkpoint inhibitors (ICI) has become the backbone of several treatment regimens for cancer and has resulted in unprecedented benefits for patients[1]. Notwithstanding this progress, many patients eventually experience disease progression while undergoing treatment with ICI, and the mechanisms of the underlying resistance remain elusive[2]. One important contributor to such resistance is the immunosuppressive tumor microenvironment[3-5]. Based on the state and quality of immune cells, the tumor microenvironment has been classified as immune-inflamed, immune-excluded, and immune-deserted[6,7]. A second classification incorporates the role of cancer-associated fibrosis to describe the response to ICI[8,9]. A third classification integrates the role of epithelial-mesenchymal transition (EMT) as a key factor for resistance to ICI[10]. T regulatory (Treg) cells emerge as key contributors of resistance to ICI and are included in each of the three above-mentioned classifications, primarily in immune-excluded or immune-enriched fibrosis conditions [Figure 1]. Considering that Treg cells play an important function in tissue homeostasis, responses to infections, and the control of autoimmunity, their involvement in immune-excluded or immune-enriched fibrosis conditions is perhaps expected[11]. Furthermore, Treg cells are no longer recognized as a single group of T cells, but instead consist of different subgroups with varied immunosuppressive properties against which distinct inhibitors can be developed[12]. This review will discuss the advances in drug development of large and small molecule agents to overcome Treg cell-mediated resistance to ICI.
Figure 1. Main Mechanisms of Resistance (primary or secondary) to ICI. There are three different classifications or models summarizing the main mechanisms of resistance to ICI. The first classification (blue triangles) describes the response to ICI in relationship to markers of EMT[10]: the more tumors show a status of EMT, the lesser they respond to ICI. The second classification associates the degree and type of fibrosis with responses to ICI (grey boxes)[8]: response to ICI is generally observed in conditions with immune-enriched fibrotic and non-fibrotic conditions. By contrast, immune-depleted or fibrotic conditions are not responsive to ICI. The third classification is based on the presence of specific immune cells or markers (red boxes)[5,6]: responses to ICI are commonly observed in patients with immune-inflamed conditions (characterized by a high CD8+/Treg cell ratio, B cells and TLS-rich tissues); conversely, responses are reduced in immune-excluded conditions (characterized by high vascular stroma content with fibrosis, chemokines, such as CCL, CCL2, CCL5, CCL13, CCL22, or cytokines TGF-β). Limited or no responses to ICI are observed in patients with an immune-deserted tumor microenvironment (lacking T cell priming, exhibiting tolerance, and displaying CAF-related markers). While Treg cells (green box) can be found in each of these conditions, their highest quantity and functional role are observed in either immune-excluded conditions or in immune-enriched fibrotic tissues. ICI: Immune checkpoint inhibitor; EMT: epithelial-mesenchymal transition; TLS: tertiary lymphoid structure; CCL: chemokine c-c-motif ligand; TGF-β: transforming growth factor beta; CAF: cancer-associated fibrosis.
BIOLOGY AND CHARACTERIZATION OF Treg CELLS
Early discovery of Treg cell biology
Originally described as T suppressor cells[13-16], Treg cells play a specific role in different phases of immune responses[17]. Treg cells were first identified as a subset of CD4+ T cells by their cell surface expression of CD25 (alpha chain of the IL-2 receptor) and consequently labeled as CD4+CD25+ Treg cells[18]. Functionally,
Ontogeny of Treg cells [Figure 2]
Figure 2. Characterization of Treg cells and subsets: CD4+ T cells egress from the thymus and differentiate in blood and tumor tissue. Depending on the degree of CD45RA and FOXP3 expression, CD4+ are defined as nTreg cells. CD4+ or nTreg cells egress into the periphery, where either cell population is subsequently altered and selected for different types of Treg cells. Based on the “selection model”, CD4+ naïve cells are selected to transition into iTreg cells, differing in their functional status as “Tr1 cells” or Th3 cells. FOXP3: Forkhead box protein P3; nTreg: natural T regulatory cells; iTreg: induced Treg cells; Tr1 cells: type 1 Treg cells; Th3 cells: T helper 3 cells.
Treg cells were defined by their anatomical site of differentiation and the detection of the Forkhead box protein P3 (FOXP3)[21]: (1) natural Treg cells (nTreg) are Treg cells that develop in the thymus and subsequently migrate to the periphery[22]; (2) induced Treg cells (iTreg) are those that evolve from naïve CD4+FOXP3- T cells upon stimulation in the periphery[21,23]. Unfortunately, Treg cells induced in vitro were also labeled as iTreg (i.e., inducible Treg). This has led to some confusion regarding the nomenclature of Treg cells. Therefore, the 3rd International Conference on regulatory T cells[24] has recommended the following nomenclature to resolve the existing confusion:
1. Thymus-derived Treg cells (tTreg) - in lieu of nTreg.
2. Peripherally-derived Treg cells (pTreg - i.e., FOXP3+ Treg cells that differentiate in the periphery) - in lieu of induced or adaptive Treg cells.
3. In vitro-iTreg - i.e., to differentiate Treg cells derived in vitro studies from those investigated during in vivo studies.
The above-mentioned classifications of Treg cells are based on ontogeny studies and two models are used to describe the generation of Treg cells. The first model is called “instructive model”. According to the “instructive model”, T cells are being “instructed” after T cell receptor (TCR) selection in the thymus. Intermediate TCR stimulation (in contrast to negative and positive selection) leads to the intracellular gene expression of FOXP3, which subsequently determines the generation of Treg cells. The second model is called “selection model”. According to this model, Treg cells are being “selected” rather than “instructed” from a pool of pre-formed T cells. According to this model, FOXP3 gene expression is independent of the strength of TCR stimulation and further assumes the presence of FOXP3- and FOXP3+ T cells in the thymus. Upon exposure to self-antigens, the FOXP3+ T cells are resistant to negative selection and form the majority of Treg cells[25].
Independent of the thymus, which is a key organ for the development of Treg cells, secondary lymphoid organs also appear to play a prominent role in generating CD4+FOXP3+ T cells from CD4+FOXP3- T cells[26]. Such pTreg cells can originate from sub-immunogenic stimuli, non-inflammatory conditions, long-lasting or chronic infections, and inflammation. Furthermore, they are frequently present in various cancers where they contribute to an immunosuppressive environment[27-30].
Classification of Treg cells
In general, CD4+CD25+ Treg cells are characterized by FOXP3[28-31]. Additionally, low expression of the IL-7 receptor alpha chain (CD127) on the cell surface of Treg cells often coincides with the intracellular presence of FOXP3[32]. Therefore, some classifications use the low expression of CD127 as an alternative marker to FOXP3, recognizing that this may not reflect the entire Treg cell population[33]. Using a composite of intracellular and cell surface proteins, four major subsets of CD4+ T cells, from which Treg cells are derived, are classified as non-Treg, naïve Treg, effector Treg and tumor-associated effector Treg cells [Table 1]. Each subset is further characterized by additional surface markers[31,34].
Two different classifications of Treg cells
| Classification of Treg cells[31] | |||
| Treg cells subsets | Phenotype markers | Characteristics | |
| Non Treg | CD45RA- CD4+CD25+FOXP3low CTLA-4+PD-1+ | No suppressive activity | |
| Naïve Treg | CD45RA+ CD4+CD25+FOXP3low CTLA-4lowPD-1- | Weak suppressive activity Differentiate into effector Treg cells | |
| Effector Treg | CD45RA- CD4+CD25++FOXP3++ CTLA-4++PD-1+ GITR+LAG3+CD127- | Strong suppressive activity Prone to apoptosis | |
| Tumor Effector Treg | CD45RA- CD4+CD25++FOXP3++ CTLA-4+++PD-1++ GITR++LAG3++CD127- | High activation and proliferation | |
| Classification of Treg cells based on the concept of “fractions (Fr)”[34,35] | |||
| Fraction | Classification | Definition/Phenotype | Characteristics |
| Fr 1 (= naïve or resting) | rTreg | CD45RA+ CD4+CD25lowFOXP3low CTLA-4lowCD127low/-Ki67- | Derived from the thymus Weak suppressive activity Proliferation and differentiation into effector Tregs by TCR stimulation |
| Fr 2 (= effector or activated) | eTreg | CD45RA- CD4+CD25hiFOXP3hi CTLA-4hi, PD-1+, ICOS+, GITR+, OX40+, CD15s+, CCR4+, CCR8+, IL-10+, TGF-β+ | Terminal differentiation status Strong suppressive activity Prone to apoptosis Tend to increase in peripheral blood with aging |
| Fr 3 (= non-Treg cells) | Non-Treg | CD45RA- CD4+CD25lowFOXP3low IL-2+, IFN-γ+, IL-17+ | Heterogenous population No suppressive activity |
Another nomenclature defines Treg cells as “fractions” [Table 1][34,35]. This nomenclature also takes into consideration elements of functionality. Each Treg cell fraction has distinct functions depending on the type of organ and anatomical location within the organ[36] [Table 1].
Some authors have preferred to define Treg cells based on their function. For example, “type 1 Treg cells” (Tr1) and T Helper (Th)3 cells are Treg cells that produce immunosuppressive factors[23,37]. In contrast to the
Molecular mechanisms generating Treg cells and their function [Figure 3]
Figure 3. General Concept of Developing Drugs Blocking Activity of Treg cells: In general, there are three main compartments enriched in Treg cells, which are currently being targeted with drugs: (1) Extracellularly by blocking Ligands (white background), such as IL-2. Alternatively, blocking specific receptors on Treg cells, e.g., CTLA-4, CCR4, with monoclonal antibodies, such as ipilimumab or mogamulizumab, can arrest the activity of Treg cells; (2) Intracellularly (red background), signaling pathways can be blocked with small molecule inhibitors, e.g., targeting PI3K-δ; (3) Transcription, gene modification is targeted with different pharmacological agents, such as antisense oligonucleotides, molecular glue, and small molecules. These pharmacological interventions are mainly in non-clinical or early clinical investigations. They target a variety of factors, of which HELIOS and FOXP3 are perhaps the most unique to Treg cells.
As highlighted above, FOXP3 is an important intracellular transcription factor determining the fate of
In addition to intracellular transcription factors and the interaction with TCR, chemokines such as C-C motif chemokine ligand (CCL22) can induce the formation of Treg cells[46]. CCL22, secreted by dendritic cells (DC) and macrophages, engages with its receptor C-C chemokine receptor (CCR4), which is predominantly expressed on Treg cells[47]. Blocking this CCL22/CCR4 axis and consequently removing Treg cells leads to anti-tumor immune responses[48]. Recent studies further show that FOXP3 is required to increase the expression of CCR4 on Treg cells[49]. This co-regulation underscores that soluble and molecular events determine the fate of Treg cells.
Epiregulation
The function or the generation of Treg cells can also be influenced by mechanisms of epiregulation[50]. In murine models, complement factors determined the methylation of the FOXP3 in Treg cells. Since complement is part of the innate immune system, epigenetic regulation of Treg cells appears to occur early during an immune response. Hence, interventions of blocking complement activation may have an impact on the generation of Treg cells.
Immunosuppressive function of Treg cells
The classifications of Treg cells can be based on functional studies for all Treg cells or their subsets. Generally, Treg cells exert their suppressive function in three ways: (1) soluble factors; (2) inhibitory receptors; (3) competition for activation or growth factors[51]. In recent years, the list of such mechanisms has expanded, and the following examples for each mechanism are presented to illustrate the basis for novel anti-cancer therapies targeting Treg cells.
1. Soluble Factors: IL-10 is secreted by Treg cells and is one of the key cytokines contributing to immune suppression in cancer[52]. IL-10 also acts on Treg cells themselves by expanding their number and increasing CTLA-4 expression[53]. TGF-β signaling is another cytokine that is associated with immunosuppression by Treg cells[54,55]. Like IL-10, TGF-β signaling can also induce Treg cells[56]. Its significance might surpass that of IL-10 in the function of Treg cells, as it also inhibits the differentiation and function of Th1 and Th2 cells. TGF-β signaling promotes the differentiation of Th17 and Th9 cells, differentiation of tissue-resident memory CD8+ T cells, generation of natural killer (NK) cells, and other tissue-resident cells, e.g., γδ T cells, innate lymphoid cells, and gut intraepithelial lymphocytes[57]. Given the tissue distribution of TGF-β signaling proteins and its feedback loop on Treg cells, it may be one factor contributing to the tissue-dependent functionality of Treg cells [Table 2].
Phenotype characteristics of Treg cells based on tissue distribution highlights the plasticity of Treg cells
| Tissue | Treg cell phenotype and function |
| Brain | IL-10, IL-33, IL-35, ST2, CTLA-4, TGF-β, IDO, 5-HT7, AREG |
| Lung | COX-2, PGE2, TGF-β, AREG, IL-33, CD103, PHD, HIFα |
| Liver | IL-10, IL-35, CTLA-4, TGF-β, SCFAs, AREG, RA, IDO1, COX2, PGE2, GITR, LAG3, ICOS, CD39/CD73, ST2 |
| Adrenal gland | β1-adrenergic receptors, Glucocorticoid receptor α |
| Lymph node | IDO, TGF-β, CTLA-4, ICOS, CXCR5, IL-2, CD28, CD103 |
| Skin | IL-10, TGF-β, GITR, CTLA-4, Jag1, IDO, OX40+, ARG2, CCR4, CCR6, CLA |
| Bone | CD39/CD73, RANK, PGE3, TGF-β, IDO, HIF1α, CXCR4 |
2. Inhibitory Receptors: Perhaps the most recognized inhibitory receptor expressed on Treg cells is the CTLA-4[35,58]. Because of its role in competing with CD28 for the co-stimulatory molecules CD80 (B7.1) and CD86 (B7.2) on antigen presenting cells (APCs), CTLA-4 can induce cell cycle arrest, inhibit the production of IL-2, and down-regulate ligands needed for the activation of T effector cells. Hence, it was termed an immune checkpoint inhibitor (ICI) and this critical discovery was recognized through the Nobel Prize awarded to James Allison and Tasuku Honjo[59]. This observation led to the discovery of similar receptors with inhibitory function, such as CD73[60,61]. The expression of CD73 in conjunction with TGF-β signaling contributes to a significant increase in Treg cells and renders ICI therapies ineffective.
3. Competition for Growth Factors: Interleukin-2 (IL-2) is not only produced by activated CD4+ and CD8+
Overall, these few examples demonstrate that Treg cell function can be induced and maintained by a variety of factors. Hence, activating or blocking these functions is relevant to therapeutic drug development. To appropriately assess the responses to therapies directed against Treg cells, it is necessary to detect and monitor the Treg cells in either tumor tissue or peripheral blood. This assumes that most Treg cells are selected in the periphery and that, regardless of their ontogeny, they share similar mechanisms of action.
METHODS TO MEASURE Treg CELLS
There are several methods to determine Treg cells in cancer patients. Multiparametric cellular flow cytometry (FC) was historically used to evaluate the Treg cells and their subsets[70,71]. Even today, the main advantage of flow cytometry is the quick turn-around time (i.e., generally within hours), and thus can be used to monitor Treg cells before and after novel treatments. An alternative tool to monitor Treg cells is mass cytometry[72,73]. Mass cytometry has a reduced risk of signal spill-over, thus improving background noise, and is a highly dimensional method to assess several complex markers simultaneously. The disadvantage of mass cytometry lies in the longer turn-around time, destruction of the specimen at the end of the examination, and the subsequent bioinformatic analyses of high-volume data[74]. The power of mass cytometry to measure small subsets of immune cells in blood is exemplified in an ongoing clinical study with the phosphoinositide-3-kinase delta (PI3K-δ) inhibitor roginolisib (IOA-244). In this study, mass cytometry detected a reduction in blood Treg cells across dose cohorts, which was only marginally detected with standard FC[75].
In tumor specimens, standard immunohistochemistry has also provided early insights into changes in
Transcriptomics provides another high-dimensional approach to assess Treg cells along with other changes in the tumor or blood[81]. Gene expression profiles can describe the Treg cells along with other immune cells using whole tissue extracts[82]. Under such conditions, the anatomical structure is lost for the benefit of detecting low signal events. A modification of this technique is single-cell transcriptomics approaches, which have revealed new functions of Treg cells[83]. Using this technology, the destruction of the tumor specimen is kept to a minimum while the detection of cellular events is increased. The disadvantage of this technology primarily lies in the processing and evaluation of high-volume data, which leads to long turn-around times.
Like Transcriptomics, Proteomics is a collection of high-dimensional data of proteins either within tumor tissue or proteins shed from tumors to the blood[84,85]. Thus, a wide range of secreted proteins can be evaluated, including chemokines (e.g., CCL22) or cytokines (e.g., IL-2, TGF-β) associated with Treg cells[86]. For drug development, Proteomics offers a broad discovery tool to study the effect of novel agents. From this discovery platform, specific diagnostic tools can also be developed, such as companion diagnostics or laboratory developed tests.
In vivo imaging has been used to describe the dynamics of Treg cells in animals[87]. While such studies in animals have shown important insights into T cell regulation in the presence of CTLA-4 inhibition, there are no such specific imaging tools available for appropriate clinical investigation. The most advanced imaging tool uses CD8-labeled PET imaging and reveals significant heterogeneity in CD8+ T cell distribution during immunotherapy in patients[88]. Therefore, to date, such imaging tools still need to prove their value to guide the drug development of novel agents.
While there are no regulatory-approved tests for assessing Treg cells or their function, FC is the most widely used laboratory test in clinical studies. In contrast to tissue-based tests, Treg cells in the blood can be monitored longitudinally either alone or in comparison to other blood-based immune cells.
Treg CELLS DURING IMMUNOTHERAPY AND THEIR ROLE IN RESISTANCE
Background
Treg cells play an important role in tissue homeostasis and co-regulation of other immune cell subsets[89]. In the following section, the role of Treg cells during immunotherapy will be reviewed and their potential as either prognostic (i.e., relevant to the disease progression and independent of therapies) or predictive (i.e., in assessing possible response to therapies) biomarkers[90].
Baseline levels of Treg cells in malignancies and their potential role as prognostic marker [Table 3]
Examples of malignancies with elevated Treg cells associated with treatment resistance
| Indication | Number of patients | Method and panel | Clinical observation | Ref. |
| Treg cells at baseline | ||||
| Pan-cancer | 15,512 | Meta-analysis of studies assessing FOXP3 in tumor tissue and OS | Influence factors for prognosis included tumor location, molecular subtype, tumor stage For most solid tumors, Treg cells correlated with poor OS | Shang et al. 2015[91] |
| Endometrial cancer | 82 | Flow cytometry using CD4+CD25+CD127- | Baseline associated with treatment resistance | Li et al. 2019[92] |
| Endometrial cancer | 275 | IHC with FOXP3 Flow cytometry using CD4+CD25+CD127- | Tumor tissue enriched for Treg cells at baseline and associated with poor OS Endometrial cancer cells expanded CD4+CD25+CD127- cells ex vivo | Kolben et al. 2022[93] |
| Breast cancer | 164 | Flow cytometry using CD4+CD25+FOXP3+ | High Treg cells in tumor tissue and draining lymph nodes associated with invasiveness Associated with CCL5 and increased expression of CCR5 on Treg cells | Qiu et al. 2022[94] |
| NSCLC | 64 | IHC CD3 and FOXP3 | High Treg cells in tumor tissue of patients with stage I are at risk of relapse | Petersen et al. 2006[95] |
| NSCLC | 28 | Peripheral blood and flow cytometry using CD4+CD25+ | CD4+CD25+ is higher compared to healthy subjects Increased CD8+CD28- lymphocytes | Karagöz et al. 2010[96] |
| NSCLC | 23 | Peripheral blood and flow cytometry using CD4+CD25+FOXP3+ | Treg cells elevated compared to healthy subjects Treg cells increase depending on the stage of NSCLC High intracellular CTLA-4 expression | Erfani et al. 2012[97] |
| NSCLC | 36 | Peripheral blood and flow cytometry using CD4+CD25+FOXP3+ | Treg cells elevated compared to healthy subjects Treg cells were negatively correlated with serum IL-17 | Hu et al. 2018[98] |
| NSCLC | 26 | Peripheral blood and flow cytometry using CD4+CD25+FOXP3+ | Treg cells elevated compared to healthy subjects Correlation of Th17 cells with Treg cells High levels of TGF-β, IL-17, IL-23 | Li et al. 2014[99] |
| NSCLC | 49 | Peripheral blood and flow cytometry using CD4+CD25+FOXP3+ | Treg cells increase depending on the stage of NSCLC Treg cells decreased after surgery | Chen et al. 2014[100] |
| NSCLC | 156 | Peripheral blood and flow cytometry using CD4+CD25+FOXP3+ | Treg cells produce TGF-β and IL-10 Naïve Treg cells elevated and correlated with poor outcome High frequency of terminal Treg cells correlated with improved outcome | Kotsakis et al. 2016[101] |
| NSCLC (EGFR mutation) | 323 (164 with EGFR mutation) | IHC for FOXP3 (clone 236A/E7) | Significant High FOXP3 expression in EGFR mutation-positive NSCLC Association with poor survival | Luo et al. 2021[102] |
| NSCLC (EGFR mutation) | 19 (6 EGFR-mutated and 13 EGFR-wildtype) | Flow cytometry with CD45RA-FOXP3+CD4+ (=Fraction 2) | EGFR mutation is non-inflamed (no presence of CD8+ T cells) High presence of Treg cells EGFR mutation induces CCL22, which induces Treg cells | Sugiyama et al. 2020[103] |
| Treg cells response during treatment (possible predictive value) | ||||
| Cutaneous melanoma | 40 | Flow cytometry using CD4+CD25highCD127-Foxp3+ | High baseline levels Reduction after 3 consecutive doses of ipilimumab Enrichment of CD39+HELIOS+ Treg cells | Bjoern et al. 2016[104] |
| Cutaneous melanoma | 32 | Flow cytometry using CD4+CD25+CD127-PD-1+ | Reduction after nivolumab or pembrolizumab treatment observed in patients responding to PD-1 inhibitors No reduction observed in patients with no response | Gambichler et al. 2020[105] |
| NSCLC | 31 | IHC using FOXP3 for tumor tissue and flow cytometry using CD4+CD25+ FOXP3+ for blood | Neo-adjuvant treatment with cetuximab/docetaxel/cisplatin showed a correlation of reduction in Treg cells and response Treg cells at diagnosis did not predict clinical response with therapy | Pircher et al.[106] |
| NSCLC | 132 | Flow cytometry using CD4+CD25+CD45RA-FOXP3+ | High Treg cells and TGF-β1 levels after 1 week of treatment with PD-1 inhibitors are associated with increased OS High Treg cells at baseline associated with longer OS and PFS | Koh et al. 2020[107] |
| NSCLC | 27 | IHC and mass cytometry T cell subsets | Ratio of PD1+ on CD8+/PD1+ on Treg cells was predictive of outcomes Ratio was predictive in other tumor types as well, i.e., gastric cancer and melanoma | Kumagai et al. 2022[108] |
| Renal cell carcinoma | 43 | Flow cytometry using CD4+CD25+CD127-FOXP3+ | Treatment with nivolumab reduced Treg cells only in responders when assessed after 3 months Inhibition with CXCR4 antagonist blocked Treg cell function in vitro Treatment with nivolumab reduced Treg cells only in responders when assessed after 3 months | Santagata et al. 2020[109] |
| Uveal melanoma | 9 | Mass cytometry using CD4+CD25+CD127- | Treg cells reduced within 3 months, while CD8+ and NK cells increased | Di Giacomo et al. 2022[110] |
The prognostic value of Treg cells was examined by a systematic meta-analysis using data from 76 articles, which included 17 different types of cancers and 15,512 cancer cases[91]. This study evaluated Treg cells as part of tumor-infiltrating lymphocytes (TILs). High numbers of Treg cells were associated with shorter overall survival (OS) in most tumor types (e.g., cervical, renal, melanoma, and breast cancer), but were associated with longer OS in colorectal, head and neck, and esophageal cancer. The main parameters that influenced the prognostic value included tumor location, stage of disease, and molecular subtype.
In addition to this meta-analysis, studies assessed the prognostic role of Treg cells in specific tumor types and a few important examples are described below.
In Non-small Cell Lung Cancer (NSCLC), the frequency of Treg cells in peripheral blood increases with the stage of NSCLC[96,97]. In 156 NSCLC patients, naïve Treg cells and not terminal Treg cells were correlated with poor outcomes[101]. These naïve Treg cells produced TGF-β and IL-10, indicating an immunosuppressive function. A study in the perioperative setting also found that Treg cells in peripheral blood increased with the stage of disease[100]. This increase in Treg cells was independent of histology such as squamous and adenocarcinoma. The postoperative Treg cell frequency was not reduced to levels comparable to healthy subjects, suggesting that the immunosuppressive condition remained intact after surgery. Therefore, some investigators proposed to use the presence of Treg cells in tumor tissue to assess the risk for relapse. For example, the Treg/TIL Combination Risk Index identified that patients with Stage I NSCLC and a high count of Treg cells were at risk of relapsing[95].
While another study also reported that Treg cells increased with the stage of NSCLC, it found that serum levels of IL-17 and not IL-10 were negatively correlated with Treg cells[98]. Gene expression of IL17 in lymphocytes was correlated with numbers of circulating Treg, suggesting that IL-17 is being produced by lymphocytes[99]. Thus, serum levels of immunomodulatory factors may not always reflect the function of
In 275 tumor specimens from patients with endometrial cancer, high FOXP3 expression was correlated with poor OS[93]. A similar observation was reported for patients with primary breast cancer, where Treg cells and CCL5 were co-expressed with standard prognostic markers for breast cancer[94]. The authors postulated that CCL5 engages the CCR5 on Treg cells and subsequently induces the production of TGF-β[94]. Like the CCL5/CCR5 axis, the chemokine receptor CCR8 (its ligand being CCL1) also plays a critical role in upregulating genes of intra-tumoral Treg cells as observed in patients with breast, colorectal, and lung cancer[112,113]. In each of these tumor types, the expression of CCR8 correlated with Treg cell signature and was associated with poor prognosis[114].
Oncogenic driver mutations are associated with a tumor microenvironment rich in immunosuppressive mediators and Treg cells. For instance, Epidermal Growth Factor Receptor (EGFR) mutations in NSCLC are associated with high levels of Treg cells[102]. The microenvironment of patients with EGFR-mutated NSCLC is immune-suppressed, as indicated by tissue expression of FOXP3 and PD-L1[102]. Furthermore, high numbers of Fraction 2 Treg cells, low numbers of CD8+ T cells (i.e., non-inflamed condition), and high levels of CCL22 (the main ligand for CCR4) are observed in EGFR-mutated NSCLC patients[103]. This immunosuppressive state was reversed during combination treatment of EGFR inhibitors and PD-1 monoclonal antibodies, leading to a reprogramming of the immune subsets, and consequently overcoming the resistance. Kirsten Rat Sarcoma Virus (KRAS) mutated tumors are also associated with high numbers of Treg cells, for example, in KRAS-mutated colorectal cancers[115]. KRAS-mutated tumors produce the immune suppressive mediators IL-10 and TGF-β1 and thus drive a phenotype switch from naïve to Treg cells[116]. Because of these observations in EGFR- and KRAS-mutated tumors, it is possible that other mutations are associated with similar immunosuppressive mediators and Treg cells[117].
In contrast to solid tumors, lymphoma patients may harbor four functionally distinct Treg cell groups: (1) Suppressor Treg cells: similar to solid tumors, this group of Treg cells is immunosuppressive; (2) Malignant
While the above-mentioned examples show how Treg cells are associated with survival, it remains unclear whether the presence of Treg cells is merely an epiphenomenon or a key driver of immune suppression in cancer patients. Therefore, changes in Treg cells after clinically meaningful responses to therapies may help to recognize where Treg cells are key drivers of tumor progression.
Treg cells as potential drivers of tumor progression and their potential role as predictive biomarkers [Table 3]
Studies of immunotherapy and other anti-cancer treatments were selected to determine whether Treg cells are potentially related to treatment outcomes, either as a negative or positive predictive marker[120]. For example, patients with hyperprogression during immunotherapy have elevated Treg cells, which is associated with treatment failure[121,122]. In such patients, Treg cells expand and copious amounts of immune suppressive cytokines (e.g., TGF-β1, IL-10) are secreted. Furthermore, Treg cells upregulate PD-1 expression during
Treg cell dynamics are not always associated with poor outcomes. For example, PD-L1-treated patients with NSCLC had high frequencies of circulating Treg cells one week after therapy. These levels were correlated with a high response rate, longer progression-free survival, and overall survival[107]. At the same time, TGF-β levels were elevated and associated with a favorable response to anti-PD-1 immunotherapy. A second study in patients with cutaneous melanoma also reported an association of high levels of Treg cells with improved outcomes after adjuvant PD-1-based therapies[124]. Several reasons may explain this difference between
In hematologic malignancies, Treg cells play a role in the regulation of bone marrow progenitor cells, in controlling the development of malignant clones (e.g., either by transcriptional changes in the malignant B- or T cell), and in influencing the immune cell composition. Some examples are used to illustrate the complexity of targeting Treg cells in hematologic malignancies. Patients with chronic lymphocytic leukemia (CLL) and responding to PI3K inhibitors idelalisib or duvelisib show a reduction in Treg cells[125]. Interestingly, this reduction in Treg cells seemed to coincide with toxicities reminiscent of autoimmune toxicities observed in patients receiving ICI[126]. Therefore, treatments with oral PI3K-δ inhibitors have offered new insights into the role of Treg cells or their mediators, such as the underappreciated role of IL-17[127-129]. Whether this effect of PI3K-δ inhibitors is uniquely related to the reduction in Treg cells remains to be determined, because a reduction or inhibition of the function of Treg cells is not always associated with autoimmune toxicities. One example of Treg cell reduction without autoimmune toxicities is observed in patients receiving Janus kinase (JAK) 1/2 inhibitors in Primary Myelofibrosis (PMF). Patients who respond to the treatment with the JAK 1/2 inhibitor ruxolitinib show a decrease in Treg cells[130]. Interestingly, the highest frequency of Treg cells was observed in patients with the highest allele frequency of the JAK2 V617F mutation. Furthermore, long-term treatment with ruxolitinib was associated with disease control and reduction in Treg cells[131]. In contrast to the experience with CTLA-4 targeting agents and PI3K inhibitors, the reduction in Treg cells was not associated with autoimmune toxicities. There are at least two factors that may explain the autoimmune toxicities in patients treated with anti-CTLA-4 antibodies or PI3K-δ inhibitors, while they are absent in patients receiving agents while reducing Treg cells. First, common among both drug groups is the question about specificity and selectivity. For example, monoclonal antibodies with a modified Fc framework have an altered response and perhaps also a reduced autoimmune-toxicity profile[132,133]. Additionally, for the designated PI3K-δ inhibitors, such as idelalisib[134], parsaclisib[135] and duvelisib[136], the selectivity profile in humans is less clear. All known PI3K-δ inhibitors are not as selective as originally assumed with some important safety implications as recently evaluated[137]. Second, in addition to specificity or high selectivity, the immune competency of patients may play a role. For example, in patients with CLL, the B cell function is disrupted. Hence, it is possible that the reduction in Treg cells induces the elevation of cytotoxic Th17 T cells[125].
Examples of drugs targeting Treg cells and Treg cell-mediated resistance [Table 4]
Examples of drugs targeting Treg cells
| Drug/Intervention | Observation | Ref. |
| Large molecules | ||
| CTLA-4 targeting agents | Intra-tumoral Treg cells unchanged after ipilimumab or tremelimumab therapy In neo-adjuvant setting, ipilimumab transiently increased Treg cells In patients with metastatic melanoma, Treg cells are reduced after extended treatment time (> 3 months) | Sharma et al. 2019[138] Retseck et al. 2018[139] Bjoern et al. 2016[104] Patel et al. 2023[140] |
| PD1 targeting agents | Ratio of expression on Treg/Teff cells after immunotherapy potentially predicts response PD1+ Treg cells may be dysfunctional | Kumagai et al. 2020[108] Lowther et al. 2016[141] |
| CCR-4 targeting agents | Monoclonal antibody mogamulizumab (NCT02705105) showed limited activity (ORR or 10%) either as monotherapy or in combination with nivolumab Blood and tumor Treg show a reduction for patients with ORR | Hong et al. 2022[142] |
| CCR-8 targeting agents | Subpopulation of Treg cells express CCR-8 Blocking CCR-8 appears not to be associated with autoimmune adverse events in animal studies Monoclonal antibody GS-1811 in early phase clinical trials (NCT05007782) | Kidani et al. 2022[143] Weaver et al. 2022[144] |
| CD25 targeting agents | CD25 high-affinity subunit alpha Monoclonal antibody RO7296682 (RG6292) had no overt adverse events in animals RO7296682 in clinical trials (NCT04158583) | Solomon et al. 2020[145] |
| IL-2 targeting agents | Selective inhibition of trimeric and not dimeric CD25 leads to Treg cell reduction | Wyant et al. 2023[146] |
| CEACAM-5 targeting agents | CEACAM-5 and 6 is expressed on highly suppressive Treg cells NEO201 reduces Treg cells | Cole et al. 2023[147] |
| Small molecules | ||
| Chemotherapies | Low-dose cyclophosphamide and vaccines Low-dose cyclophosphamide in CRC Docetaxel in NSCLC Sunitinib in RCC | Le et al. 2012[148] Ghiringhelli et al. 2007[149] Scurr et al. 2017[150] Roselli et al. 2013[151] |
| STAT3 (FOXP3) inhibition | Treg cell reduction | Revenko et al. 2022[152] |
| ATP-competitive PI3K-δ inhibitors | Drug-related Grade 3/4 toxicities limiting continuous dosing and reducing potential efficacy Treg cell reduction in tumor tissue Chemokines inducing Treg cells reduced in lymphoma patients | Eschweiler et al. 2022[153] Tarantelli et al. 2021[154] |
| Non-ATP competitive PI3K-δ inhibitor roginolisib (IOA-244) | Low grade 3/4 toxicity with no requirement of drug modifications Safety in long-term treated uveal melanoma Reduction in Treg cells, increase in CD8+ T and NK cells | Di Giacomo et al. 2022[110] |
| JAK1/2 inhibitors | Reduction in Treg cells in patients with PMF responding to ruxolitinib | Massa et al. 2014[130] |
| CDK4/6 | Reduction in Treg cells and increase in Teff cells, with a greater reduction in patients with responses to therapy | Scirocchi et al. 2022[155] |
| BCL2 (e.g., Venetoclax) | Reduction in peripheral Treg cells and enhancement of immune cells | Kohlhapp et al. 2021[156] |
The success of the CTLA-4 targeting agents such as ipilimumab has provided important lessons for future drug development concepts. Herein, we review drug candidates with specific inhibition profiles for Treg cells. Furthermore, the novel agents intend to provide a greater benefit/risk profile. Drugs designed to increase the Treg cells, such as for improving transplantation outcomes, will not be reviewed.
The lessons from the drug development of such agents support the hypothesis that Treg cells are key players in the resistance mechanisms of immunotherapy[157]. This explains the increasing number of drug candidates targeting Treg cells with an aim to rebalance the overall immune cell compartment[12,158].
Large Molecules: Because of the preferential expression of CTLA-4 on Treg cells, CTLA-4 inhibitors, such as ipilimumab or tremelimumab, are perhaps the prototype of selective Treg cell inhibitors, although a reduction in Treg cells cannot always be detected[138-140,159]. Both ipilimumab and tremelimumab have received approvals for a wide range of indications and form the backbone of many standard treatments[160]. With a greater understanding of dose and dose schedule, the use of CTLA-4 targeting agents is evolving. For example, it appears that continuous dosing may not be required to achieve the full effect of CTLA-4 targeting agents[104,161,162]. This is best observed in the neo-adjuvant setting, where limited doses of ipilimumab have contributed to a greater disease-free survival and revolutionized treatment for high-risk melanoma patients[163].
In addition to the approved anti-CTLA-4 agents, the group of approved anti-PD-1 targeting agents, such as pembrolizumab and nivolumab, can reduce Treg cells. In contrast to CTLA-4, PD1 is not preferentially expressed on Treg cells. Therefore, the ratio of PD1 expressing Teff and Treg cells can be used as a monitor for response[108,164,165]. Whether the PD1+ Treg cells are functionally immunosuppressive or have reduced functional activity remains a topic of ongoing research[141,165]. Since both main groups of ICI (i.e., CTLA-4 and PD1 targeting agents) affect Treg cells, clinical studies evaluating Treg cells during ICI therapies may provide valuable information for the development of novel inhibitors of Treg cells.
The chemokine receptor CCR4 is expressed on Treg cells. For example, the monoclonal antibody against CCR4, mogamulizumab, is approved for relapsed or refractory mycosis fungoides (MF) or Sézary syndrome (SS)[166]. Although mogamulizumab achieved an ORR of 10% in a diverse population with solid tumors[142], Treg cells in tumor tissue and circulating blood were reduced in patients with tumor responses, while there were no changes or even increases in Treg cells for patients who progressed.
In addition to the above-mentioned approved monoclonal antibodies, there are several drug development candidates designed to target specific proteins on Treg cells. One such drug is GS-1811, a monoclonal antibody blocking CCR8 on Treg cells[143,144]. This antibody is designed to remove the highly immune suppressive Treg cells, which express CCR8. This approach of reducing a specific subset of Treg cells may address the toxicity concerns otherwise observed with the CTLA-4 targeting agents. Furthermore, it appears that the expression of CCR8 is highly restricted to tumor-infiltrating Treg cells[144].
Targeting CD25 on Treg cells is another selective approach to block Treg cells. RO7296682 (also known as RG6292), a monoclonal antibody designed to specifically block the CD25-mediated function on Treg cells, is currently under clinical investigation (NCT04158583)[145]. Due to its design, RO7296682 promises to be more selective and less toxic than prior anti-CD25 monoclonal antibodies, such as daclizumab or basiliximab. As with GS-1811, the anticipated benefit is the reduced toxicity profile compared to the approved CTLA-4 targeting monoclonal antibodies.
Early non-clinical and clinical development efforts are currently targeting the ligand of CD25. This approach relies on blocking IL-2 or modifying the binding of IL-2. Recent technologies can generate multivalent, asymmetric IL-2-Fc fusions with different binding properties (including variable forms to either block or activate Treg cells)[167]. A more traditional approach consists in the generation of specific IL-2 blocking antibodies, such as AU-007[146]. AU-007 binds to the CD25-binding epitope of IL-2, which prevents the interaction with the trimeric IL-2R expressed on Treg cells, while not affecting the dimer of the IL-2R on memory or naïve T and NK cells. Patients receiving AU-007 had a decrease in Treg cells, with an increase in CD8+ T cells. This approach may overcome the known drug resistance in triple-negative breast cancer, where CD25+ Treg cells are associated with resistance to immunotherapy[168].
The surface protein CD38 is present on a wide range of immune cells, including Treg cells. The reduction in Treg cells following dosing of the anti-CD38 monoclonal antibody isatuximab plus atezolizumab in patients with advanced solid tumors was evaluated[169]. Surprisingly, isatuximab plus atezolizumab was not associated with a reduction in Treg cells, although nearly all patients showed a reduction in CD38+ T cells. The low overall response rate, diverse patient population, and low immune cell population at baseline may explain the lack of detectable changes in Treg cells.
The carcinoembryonic antigen-related cell adhesion molecules (CEACAM)-5 and CEACAM-6 are expressed on tumor cells and Treg cells with a profound immunosuppressive function[170]. The monoclonal antibody NEO20, which targets CAECAM-5 and -6, reduced Treg cells only in patients with long-term stable disease (SD)[147]. Therefore, the observations from the early clinical trials with the anti-CD38 and anti-CEACAM-5 monoclonal antibodies suggest that factors other than selectivity are important in the design of novel Treg cell inhibitors.
Small Molecules: In addition to the large molecules, small molecules are being used to target signaling pathways uniquely or preferentially present in Treg cells. There is an increasing list of small molecules that have been associated with the regulation of Treg cells[171]. Perhaps the most common treatments associated with a reduction in Treg cells are chemotherapies, such as cyclophosphamide, either as a therapy alone or in combination with vaccines[148]. In particular, the low dose cyclophosphamide (50 mg twice a day for a
Chemotherapies are not sufficiently selective for Treg cells and their subsets. Hence, more specific inhibitors may target unique pathways of Treg cells, such as targeting FOXP3. Recently, a screen from different compounds found potential candidates that would directly degrade FOXP3, such as derivatives of gallic acid[173]. AZD8701 is an antisense oligonucleotide (ASO) blocking STAT3 and thus indirectly FOXP3[152]. During the Phase 1 study of AZD8701 in combination with durvalumab (NCT00637039), the FOXP3 expression was reduced with a concurrent reduction in Treg cells.
Following the drug development experience of large molecules targeting CCR4, small molecule inhibitors of CCR4 are being investigated in patients[174]. For example, CCR4-351 is a small molecule inhibitor of CCR4, which reduces Treg cells in animal and in vitro models[174]. CCR4 small molecule inhibitors block the migration of Treg cells and therefore keep Treg cells from entering the tumor microenvironment[175]. Despite a wide range of different CCR4 small molecule inhibitors, their clinical development has not led to an approved agent to this date[176].
Another approach is blocking signaling pathways downstream of T cell receptors or co-stimulatory molecules. One such pathway is the PI3K-δ signaling pathway[177]. By blocking PI3K-δ signaling, Treg cells show reduced proliferation and, in patients’ plasma, chemokines such as CCL2, CCL3, CCL5, and CCL22 are decreased[125,154]. In solid tumors, blocking PI3K-δ signaling modulated immune homeostasis and reinforced PD-1 blockade[178]. Based on this observation, the combination of pembrolizumab with parsaclisib (a designated PI3K-δ inhibitor) was investigated in patients who had progressed on prior immunotherapies[179]. Unlike the combination of pembrolizumab with the JAK1 inhibitor itacitinib, parsaclisib rebalanced the immune environment towards an interferon (IFN)-γ signature. Patients receiving the combination of parsaclisib and pembrolizumab also showed responses in both ICI-naïve and ICI therapy-resistant tumors (8/28 patients; 28%). Another designated PI3K-δ inhibitor, AMG-319, was investigated in patients with head and neck cancers[153]. In post-treatment biopsies, Treg cells were reduced only in patients who tolerated AMG-319 for approximately 2 weeks, and thus were able to complete their scheduled treatment period. The tumor responses were minor and transient, most likely because the treatment was relatively short. These adenosine triphosphate (ATP)-competitive and designated PI3K-δ inhibitors, such as AMG-319 or idelalisib, have limitations due to their toxicity profile in patients with solid malignancies[180]. By contrast, the non-ATP, allosteric modulator and highly selective PI3K-δ inhibitor, roginolisib (IOA-244), has a lower rate of severe toxicity, which allows for treatments lasting greater than 6 months[75,181,182]. This well-tolerated profile is associated with a reduction in Treg cells and a simultaneous increase of CD8+ T and NK cells[183]. In patients with metastatic uveal melanoma, these changes in immune cell composition were associated with longer-than-expected overall survival (median OS of 20.8 compared to historic OS of 7.8 months)[110]. Whether roginolisib has the potential to overcome resistance to immunotherapy or prevent disease hyperprogression will be the objective of future investigation.
“Molecular glue” compounds, which are derived from cyclosporin A and FK506, are an emerging class of agents for clinical investigation[184]. Targeting IKZF2 (the gene that encodes for the zinc finger protein HELIOS, a member of the Ikaros family of transcription factors), the novel glue degrader NVP-DKY709 (=DKY709) reduces tumor resident and circulating Treg cells[185]. Because HELIOS is uniquely expressed in a subset of Treg cells[39], this approach promises a selective depletion of Treg cells. DKY709 has been under clinical investigation in a Phase 1 study since 2019, either as a monotherapy or in combination with the PD1 inhibitors PDR001 (NCT03891953; accessed 3rd December 2023). Results on the biomarker responses are soon to be presented.
Reprogramming of Treg cells provides an additional approach to reduce or alter the function of
Lastly, there are a growing number of approved small molecules that seem to affect Treg cells, although they were not specifically designed to target Treg cell pathways. We will highlight a few examples to illustrate such underappreciated drugs and their potential as immunotherapeutics. CDK4/6 inhibitors can reduce Treg cells and improve immune responses in patients with breast cancer[155]. Similarly, breast cancer patients treated with trastuzumab, either alone or in combination with chemotherapy, showed a reduction in Treg cells[191]. The JAK1/2 inhibitor ruxolitinib is associated with a reduction in Treg cells in patients with primary myelofibrosis[130,131]. The FLT3 inhibitor midostaurin reduced Treg cells in PBMCs from patients with AML[192]. Whether this effect is mediated via Dendritic Cells is being investigated[193]. The BCL2 inhibitor venetoclax, alone and in combination with pembrolizumab, improves immune responses and is associated with the reduction in Treg cells in animal studies[156]. SRC inhibition represents another target for Treg cell modification. The SRC inhibitor dasatinib seems to reduce Treg cells and enhance immune responses in preclinical models[194]. While these aforementioned approved small molecule inhibitors do not specifically target signaling pathways in Treg cells, they seem to have clinical benefits associated with a reduction in
CONCLUSION
Lessons from the drug development of CTLA-4 inhibitors may provide valuable insights to successfully develop new therapies targeting Treg cells. The research on Treg cells has uncovered a T cell population with great plasticity. Despite their relatively small size, Treg cells play a critical role in modulating immune responses to tumors. Hence, for novel drugs to be successfully developed in the clinic, the appropriate methods to assess the function of Treg cells need to be evaluated alongside the standard measures of clinical benefit. The discovery of the precise pharmacologic platform (i.e., large or small molecule) that will deliver the greatest advantage is currently an exciting area of drug development.
DECLARATIONS
Authors’ contributions
Reviewed literature, and critically reviewed, analyzed and interpreted the current state-of-the-art data: Spiliopoulou P, Kaur P, Hammett T, Di Conza G, Lahn M
Availability of data and materials
Not applicable.
Financial support and sponsorship
Not applicable.
Conflicts of interest
Spiliopoulou P declared that there are no conflicts of interest. Kaur P, Hammett T, and Lahn M are full-time employees of iOnctura SA; Di Conza G and Lahn M holds stocks in iOnctura SA.
Ethical approval and consent to participate
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Consent for publication
Not applicable.
Copyright
© The Author(s) 2024.
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Spiliopoulou P, Kaur P, Hammett T, Di Conza G, Lahn M. Targeting T regulatory (Treg) cells in immunotherapy-resistant cancers. Cancer Drug Resist 2024;7:2. http://dx.doi.org/10.20517/cdr.2023.46
AMA Style
Spiliopoulou P, Kaur P, Hammett T, Di Conza G, Lahn M. Targeting T regulatory (Treg) cells in immunotherapy-resistant cancers. Cancer Drug Resistance. 2024; 7: 2. http://dx.doi.org/10.20517/cdr.2023.46
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Spiliopoulou, Pavlina, Paramjit Kaur, Tracey Hammett, Giusy Di Conza, Michael Lahn. 2024. "Targeting T regulatory (Treg) cells in immunotherapy-resistant cancers" Cancer Drug Resistance. 7: 2. http://dx.doi.org/10.20517/cdr.2023.46
ACS Style
Spiliopoulou, P.; Kaur P.; Hammett T.; Di Conza G.; Lahn M. Targeting T regulatory (Treg) cells in immunotherapy-resistant cancers. Cancer Drug Resist. 2024, 7, 2. http://dx.doi.org/10.20517/cdr.2023.46
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