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CFSE-based proliferation assays and CD25 expression within different FoxP3 +/– Helios +/– T-cell subsets. A. Representative flow cytometric plots showing FoxP3 − Helios − , FoxP3 − Helios + , FoxP3 + Helios + and FoxP3 + Helios − T-cell subsets (first plot) and their proliferation (second plot) as measured by CFSE loss. B. Mean percentage ± SEM of cell proliferation of these different subsets in PBMCs isolated from 9 healthy donors. Flow cytometric plots and mean percentage ± SEM of CD25 expression in non-activated (C) and activated (D) FoxP3 +/− Helios +/− T-cell subsets in PBMCs isolated from 5 healthy donors.
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Regulatory T cells (Tregs) are key players of immune regulation/dysregulation both in physiological and pathophysiological settings. Despite significant advances in understanding Treg function, there is still a pressing need to define reliable and specific markers that can distinguish different Treg subpopulations. Herein we show for the first time...
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... allows the release of IL-2 from TCR-stimulated CD4 + T effector cells that can be used by cells expressing the high affinity IL-2R α chain (CD25). We found that FoxP3 + Helios − CD4 + Tregs proliferated significantly higher (59.2 ± 5.4%, Figure 5A & 5B) than all other subpopulations, which showed similar proliferation levels (32-35%, Figure 5B). Having less proliferation capability in response to TCR stimulation even in the presence of IL-2 secreted by effector cells confirms that FoxP3 + Helios + are more anergic, a characteristic of bona fide Tregs. ...
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... allows the release of IL-2 from TCR-stimulated CD4 + T effector cells that can be used by cells expressing the high affinity IL-2R α chain (CD25). We found that FoxP3 + Helios − CD4 + Tregs proliferated significantly higher (59.2 ± 5.4%, Figure 5A & 5B) than all other subpopulations, which showed similar proliferation levels (32-35%, Figure 5B). Having less proliferation capability in response to TCR stimulation even in the presence of IL-2 secreted by effector cells confirms that FoxP3 + Helios + are more anergic, a characteristic of bona fide Tregs. ...
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... measured levels of CD25 expression before and after TCR stimulation to determine the role of IL-2 in inducing proliferation of different subsets. As expected, without activation FoxP3 + Helios + and FoxP3 + Helios − Tregs showed constitutive expression of significantly higher levels of CD25 (75.2 ± 2.3% and 73.7 ± 8.2%, respectively; Figure 5C), compared with FoxP3 − Helios + and FoxP3 − Helios − subsets (19.4 ± 3.7% and 17.1 ± 5.3%, respectively; Figure 5C). Following TCR stimulation, CD25 was induced on FoxP3 − Helios + (52.2 ± 15.2%, Figure 5D), FoxP3 + Helios − (94.8 ± 2.8%, Figure 5D), and FoxP3 − Helios − (56.8 ± 12.0%, Figure 5D), but not on FoxP3 + Helios + Tregs, which showed similar levels of CD25 as per before stimulation (79.7 ± 8.5%, Figure 5D) levels of IL-10 + cells (6.4 ± 2.1%, Figure 4E), and did not secrete significant levels of effector cytokines. ...
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... measured levels of CD25 expression before and after TCR stimulation to determine the role of IL-2 in inducing proliferation of different subsets. As expected, without activation FoxP3 + Helios + and FoxP3 + Helios − Tregs showed constitutive expression of significantly higher levels of CD25 (75.2 ± 2.3% and 73.7 ± 8.2%, respectively; Figure 5C), compared with FoxP3 − Helios + and FoxP3 − Helios − subsets (19.4 ± 3.7% and 17.1 ± 5.3%, respectively; Figure 5C). Following TCR stimulation, CD25 was induced on FoxP3 − Helios + (52.2 ± 15.2%, Figure 5D), FoxP3 + Helios − (94.8 ± 2.8%, Figure 5D), and FoxP3 − Helios − (56.8 ± 12.0%, Figure 5D), but not on FoxP3 + Helios + Tregs, which showed similar levels of CD25 as per before stimulation (79.7 ± 8.5%, Figure 5D) levels of IL-10 + cells (6.4 ± 2.1%, Figure 4E), and did not secrete significant levels of effector cytokines. ...
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... expected, without activation FoxP3 + Helios + and FoxP3 + Helios − Tregs showed constitutive expression of significantly higher levels of CD25 (75.2 ± 2.3% and 73.7 ± 8.2%, respectively; Figure 5C), compared with FoxP3 − Helios + and FoxP3 − Helios − subsets (19.4 ± 3.7% and 17.1 ± 5.3%, respectively; Figure 5C). Following TCR stimulation, CD25 was induced on FoxP3 − Helios + (52.2 ± 15.2%, Figure 5D), FoxP3 + Helios − (94.8 ± 2.8%, Figure 5D), and FoxP3 − Helios − (56.8 ± 12.0%, Figure 5D), but not on FoxP3 + Helios + Tregs, which showed similar levels of CD25 as per before stimulation (79.7 ± 8.5%, Figure 5D) levels of IL-10 + cells (6.4 ± 2.1%, Figure 4E), and did not secrete significant levels of effector cytokines. In contrast, FoxP3 + Helios − T cells secreted effector cytokines, and contained the highest proportions of IFN-γ + cells. ...
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... expected, without activation FoxP3 + Helios + and FoxP3 + Helios − Tregs showed constitutive expression of significantly higher levels of CD25 (75.2 ± 2.3% and 73.7 ± 8.2%, respectively; Figure 5C), compared with FoxP3 − Helios + and FoxP3 − Helios − subsets (19.4 ± 3.7% and 17.1 ± 5.3%, respectively; Figure 5C). Following TCR stimulation, CD25 was induced on FoxP3 − Helios + (52.2 ± 15.2%, Figure 5D), FoxP3 + Helios − (94.8 ± 2.8%, Figure 5D), and FoxP3 − Helios − (56.8 ± 12.0%, Figure 5D), but not on FoxP3 + Helios + Tregs, which showed similar levels of CD25 as per before stimulation (79.7 ± 8.5%, Figure 5D) levels of IL-10 + cells (6.4 ± 2.1%, Figure 4E), and did not secrete significant levels of effector cytokines. In contrast, FoxP3 + Helios − T cells secreted effector cytokines, and contained the highest proportions of IFN-γ + cells. ...
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... expected, without activation FoxP3 + Helios + and FoxP3 + Helios − Tregs showed constitutive expression of significantly higher levels of CD25 (75.2 ± 2.3% and 73.7 ± 8.2%, respectively; Figure 5C), compared with FoxP3 − Helios + and FoxP3 − Helios − subsets (19.4 ± 3.7% and 17.1 ± 5.3%, respectively; Figure 5C). Following TCR stimulation, CD25 was induced on FoxP3 − Helios + (52.2 ± 15.2%, Figure 5D), FoxP3 + Helios − (94.8 ± 2.8%, Figure 5D), and FoxP3 − Helios − (56.8 ± 12.0%, Figure 5D), but not on FoxP3 + Helios + Tregs, which showed similar levels of CD25 as per before stimulation (79.7 ± 8.5%, Figure 5D) levels of IL-10 + cells (6.4 ± 2.1%, Figure 4E), and did not secrete significant levels of effector cytokines. In contrast, FoxP3 + Helios − T cells secreted effector cytokines, and contained the highest proportions of IFN-γ + cells. ...
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... expected, without activation FoxP3 + Helios + and FoxP3 + Helios − Tregs showed constitutive expression of significantly higher levels of CD25 (75.2 ± 2.3% and 73.7 ± 8.2%, respectively; Figure 5C), compared with FoxP3 − Helios + and FoxP3 − Helios − subsets (19.4 ± 3.7% and 17.1 ± 5.3%, respectively; Figure 5C). Following TCR stimulation, CD25 was induced on FoxP3 − Helios + (52.2 ± 15.2%, Figure 5D), FoxP3 + Helios − (94.8 ± 2.8%, Figure 5D), and FoxP3 − Helios − (56.8 ± 12.0%, Figure 5D), but not on FoxP3 + Helios + Tregs, which showed similar levels of CD25 as per before stimulation (79.7 ± 8.5%, Figure 5D) levels of IL-10 + cells (6.4 ± 2.1%, Figure 4E), and did not secrete significant levels of effector cytokines. In contrast, FoxP3 + Helios − T cells secreted effector cytokines, and contained the highest proportions of IFN-γ + cells. ...
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Citations
... 94,104 Additionally, the observation of mRNA cytokine profiles similar to Th3 represents the first description of a potential presence of Th3 cells in the decidua. 93 The main suppressive effects of Th3 Treg cells are mediated by TGF-β in a cell-contact independent manner 105,106 (Table 1). The Relationship with URSA CD4 + CD25 + Treg-deficient mouse model was established to demonstrate that allogeneic fetuses are consistently rejected without Treg cells. ...
Recurrent spontaneous abortion (RSA) is defined as two or more consecutive pregnancy failures, which brings tremendous stress to women of childbearing age and seriously affects family well-being. However, the reason in about 50% of cases remains unknown and is defined as unexplained recurrent spontaneous abortion (URSA). The immunological perspective in URSA has attracted widespread attention in recent years. The embryo is regarded as a semi-allogeneic graft to the mother. A successful pregnancy requires transition to an immune environment conducive to embryo survival at the maternal–fetal interface. As an important member of regulatory immunity, regulatory T (Treg) cells play a key role in regulating immune tolerance at the maternal–fetal interface. This review will focus on the phenotypic plasticity and lineage stability of Treg cells to illustrate its relationship with URSA.
... Surprisingly, analysis of Helios expression in tTreg revealed that tTregs from the control group had much lower expression of Helios than their counterparts from colon and mLN of I3AA-treated mice ( Fig 6H). As previously reported, Helios hi Tregs divide much more slowly than Helios lo Tregs (Elkord et al, 2015;Thornton et al, 2019). We conclude that I3AA induces impaired activation and proliferation of Treg, contributing to the functional defect of Tregs in the large intestine during Blastocystis infection. ...
The large intestine harbors microorganisms playing unique roles in host physiology. The beneficial or detrimental outcome of host-microbiome coexistence depends largely on the balance between regulators and responder intestinal CD4+ T cells. We found that ulcerative colitis-like changes in the large intestine after infection
with the protist Blastocystis ST7 in a mouse model are associated with reduction of anti-inflammatory Treg cells and simultaneous expansion of pro-inflammatory Th17 responders. These alterations in CD4+ T cells depended on the tryptophan metabolite indole-3-acetaldehyde (I3AA) produced by this single-cell eukaryote. I3AA reduced the Treg subset in vivo and iTreg development in vitro by modifying their sensing of TGFb, concomitantly affecting recognition of self-flora antigens by conventional CD4+ T cells. Parasite-derived I3AA also induces over-exuberant TCR signaling, manifested by increased CD69 expression and downregulation of co-inhibitor PD-1. We have thus identified a new mechanism dictating CD4+ fate decisions. The findings thus shine a new light on the ability of the protist microbiome and tryptophan metabolites, derived from them or other sources, to modulate the adaptive immune compartment, particularly in the context of gut inflammatory disorders.
... Helios was proposed as a specific marker for thymic-derived Treg cells (47) and the co-expression of Helios and Foxp3 was reported to increase the regulatory activity of human engineered regulatory T cells (74). Moreover, Helios expression has been linked to the control of several regulatory functions of Treg cells (46) and has been described as an exclusive marker of activated Treg cells expressing the markers GARP/LAP (75). We could confirm our findings obtained in the RNA-Seq experiments on the protein level in flow cytometric analyses demonstrating that BX-795-induced expression of the transcription factor Helios in a fraction of Th-IL-2 cells ( Figure S8). ...
Background
Treg cells have been shown to be an important part of immune-homeostasis and IL-2 which is produced upon T cell receptor (TCR)-dependent activation of T lymphocytes has been demonstrated to critically participate in Treg development.
Objective
To evaluate small molecule inhibitors (SMI) for the identification of novel IL-2/Treg enhancing compounds.
Materials and methods
We used TCR-dependent and allergen-specific cytokine secretion of human and mouse T cells, next generation messenger ribonucleic acid sequencing (RNA-Seq) and two different models of allergic airway inflammation to examine lead SMI-compounds.
Results
We show here that the reported 3-phosphoinositide dependent kinase-1 (PDK1) SMI BX-795 increased IL-2 in culture supernatants of Jurkat E6-1 T cells, human peripheral blood mononuclear cells (hPBMC) and allergen-specific mouse T cells upon TCR-dependent and allergen-specific stimulation while concomitantly inhibiting Th2 cytokine secretion. RNA-Seq revealed that the presence of BX-795 during allergen-specific activation of T cells induces a bona fide Treg cell type highly similar to iTreg but lacking Foxp3 expression. When applied in mugwort pollen and house dust mite extract-based models of airway inflammation, BX-795 significantly inhibited Th2 inflammation including expression of Th2 signature transcription factors and cytokines and influx into the lungs of type 2-associated inflammatory cells such as eosinophils.
Conclusions
BX-795 potently uncouples IL-2 production from Th2 inflammation and induces Th-IL-2 cells, which highly resemble induced (i)Tregs. Thus, BX-795 may be a useful new compound for the treatment of allergic diseases.
... For example, instead of a Foxp3, Helios has been explained as an activated GARP + /LAP + Tregs transcription factor. Also, Foxp3 short hairpin RNA may change GARP expression, but GARP short hairpin RNA does not affect the expression of Foxp3 in Tregs [40,41]. In addition, the same as Foxp3, signal transducer and activator of transcription 3 (STAT3) is one of the transcription factors which take part in GARP regulation through the Interleukin-6 (IL-6) signaling in CD4 + naive T cell [42]. ...
... They concluded that human lung cancer-related cell lines, H520 and A549, may induce high expression of GARP in Tregs. Also, the population of GARP + Foxp3 + Tregs was significantly higher than that of Foxp3-conventional T cells in the tumor tissue [40]. Additionally, PLGA/PEG Nano encapsulated Nasturtium Officinale extract was successful in inducing apoptosis in A549 cancer cell line [74]. ...
... Additionally, the GARP overexpression in stage I and II lung cancer without distant metastasis and lymphatic invasion is significantly higher than in stage III and IV lung cancer with distant metastasis and lymphatic invasion [40]. Therefore, according to these findings, GARP could be a cancer early-stage prognostic factor, and it must introduce as a novel predictive biomarker [40]. ...
T regulatory cells play a crucial role in antitumor immunity suppression. Glycoprotein-A repetitions predominant (GARP), transmembrane cell surface marker, is mostly expressed on Tregs and mediates intracellular organization of transforming growth factor-beta (TGF-β). The physiological role of GARP is immune system homeostasis, while it may cause tumor development by upregulating TGF-β secretion. Despite the vast application of anti- programmed cell death protein-1 (PD-1)/programmed death-ligand 1 (PD-L1) and anti-cytotoxic T-lymphocyte Antigen-4 (CTLA-4) antibodies in immunotherapy, anti-GARP antibodies have the advantage of better response in patients who has resistance to anti-PD-1/PD-L1. Furthermore, simultaneous administration of anti-GARP antibody and anti-PD-1/PD-L1 antibody is much more effective than anti-PD-1/PD-L1 alone. It is worth mentioning that the GARP–mTGF-β complex is more potent than secretory TGF-β to induce T helper 17 cells differentiation in HIV + patients. On the other hand, TGF-β is an effective cytokine in cancer development, and some microRNAs could control its secretion by regulating GARP. In the present review, some information is provided about the undeniable role of GARP in cancer progression and its probable importance as a novel prognostic biomarker. Anti-GARP antibodies are also suggested for cancer immunotherapy.
... Foxp3 is crucial for the development and maintenance of the suppressive Treg lineage. However, it was reported that Helios should be a better marker of activated Tregs expressing Glycoprotein A repetitions predominant (GARP/LRRC32)/LAP than Foxp3 [52]. LAP and TGF-β form inactive complexes known as latent TGF-β complexes on the surface of T cells. ...
... These complexes can be cleaved to release active TGF-β [53]. GARP plays a critical role in the formation and expression of latent TGF-β complexes at the cell surface by anchoring the complexes to the cell membrane [52]. These new markers all have the characteristic of being located on the cell membrane, making them more valuable than Foxp3 in translational medicine. ...
Atherosclerosis, which is characterized by chronic inflammation in the arterial wall, is driven by immune cells and cytokines. Recent evidence indicated that interleukin (IL)-27 showed pleiotropic properties in immune diseases. However, precise mechanisms of IL-27, especially in atherosclerosis remains unknown. In our research, we examined the influence of the administration of IL-27 and an anti-IL-27p28 antibody (anti-IL-27p28-Ab) on both the initiation and the progression of atherosclerosis. In the groups (both the initiation and the progression) receiving recombinant IL-27 administration, the formation of atherosclerotic plaques was suspended, and the percentage of regulatory T cells (LAP+ or Foxp3+) in the spleen and peripheral blood was increased. Meanwhile, the number of T helper 1 (Th1) and T helper 17 (Th17) cells was decreased. In the peripheral blood plasma, TGF-β and IL-10 expression were increased, while the levels of IFN-γ and IL-17 were reduced. As for lesions, the mRNA expression of Foxp3, TGF-β, and IL-10 was increased, while that of IFN-γ and IL-17 was reduced. In the anti-IL-27p28 antibody groups, we obtained opposite results. We also observed that DCs treated with IL-27 display a tolerogenic phenotype and that IL-27–treated tolerogenic DCs (tDCs) are likely to play a protective role during atherosclerosis. Our study indicates that IL-27 or adoptive transfer of IL-27 loaded tDCs may be a new therapeutic approach in atherosclerosis.
... 146,147 However, Helios expression can be upregulated by both nTregs and iTregs. [148][149][150][151][152] Similarly, Nrp-1 expression is found on Tregs even when the capacity to develop nTregs or iTregs is impaired. 149 Given their complex program and dynamic phenotype, the use of surface markers to study Tregs requires careful consideration. ...
... These cells are distinct from helios − FoxP3 + CD4 T cells which are largely non-suppressive. 150 172 In Tregs, activation of the PD-1/PD-L1 axis phosphorylates SHP2 which inhibits the phosphorylation of ZAP70 and AKT, thus suppressing TCR and CD28 downstream signaling which are essential to Treg function. 173 As an essential immune checkpoint, the PD-1/PD-L1 axis modulates tolerance, tumor development, and response to infections. ...
Inflammation is a biological process that dynamically alters the surrounding microenvironment, including participating immune cells. As a well‐protected organ surrounded by specialized barriers and with immune privilege properties, the central nervous system (CNS) tightly regulates immune responses. Yet in neuroinflammatory conditions, pathogenic immunity can disrupt CNS structure and function. T cells in particular play a key role in promoting and restricting neuroinflammatory responses, while the inflamed CNS microenvironment can influence and reshape T cell function and identity. Still, the contraction of aberrant T cell responses within the CNS is not well understood. Using autoimmunity as a model, here we address the contribution of CD4 T helper (Th) cell subsets in promoting neuropathology and disease. To address the mechanisms antagonizing neuroinflammation, we focus on the control of the immune response by regulatory T cells (Tregs) and describe the counteracting processes that preserve their identity under inflammatory challenges. Finally, given the influence of the local microenvironment on immune regulation, we address how CNS‐intrinsic signals reshape T cell function to mitigate abnormal immune T cell responses.
... The expression and function of Helios and Eos in Treg cells from patients with RA have been studied recently. Helios has been suggested as a marker for functional Treg cells in patients with RA [37,38]. Moreover, Helios enhances the function of induced Treg cells from RA patients in cooperation with FoxP3 [39]. ...
Regulatory T (Treg) cells play an important role in immune tolerance and contribute to the prevention of autoimmune diseases, including rheumatoid arthritis (RA). The differentiation, function and stability of Treg cells is controlled by members of the Ikaros zinc finger transcription factor family. In this study, we aimed to reveal how the expression of Ikaros transcription factors is affected by disease activity in RA. Therefore, we analyzed the ex vivo expression of Ikaros, Helios, Aiolos and Eos in Treg cells, Th17 cells and Th1 cells from RA patients by flow cytometry. We found significantly reduced expression of Helios, Aiolos and Eos in Treg cells from RA patients as compared to healthy controls. Moreover, Helios and Aiolos levels correlated with disease activity, as assessed by DAS28-CRP. In addition, Ikaros, Helios and Aiolos were significantly downregulated in Th1 cells from RA patients, while no difference between healthy individuals and RA was observed in Th17 cells. In summary, Helios and Aiolos expression in Treg cells correlates with disease activity and the expression levels of Ikaros transcription factors are diminished in Treg cells from RA patients. This observation could explain the reduced stability of Treg cells in RA.
... Interestingly, GARP is not expressed by resting CD4 + Foxp3 + Treg, but it is upregulated upon Treg activation. In comparison, CD4 + Helios + Foxp3cells upregulate the expression of GARP/LAP upon TCR stimulation, supporting that Foxp3 and GARP are not regulated by each other (37). ...
... Another protein that interacts with GARP, lysosomal associated transmembrane 4B (LAPTM4B), was identified in a yeast two hybrid assay (37). LAPTM4B expression increased upon Treg activation, and it directly decreased the surface expression of GARP and the secretion of LTGF-b1. ...
... In this context, the link between GARP and Foxp3 remains elusive. On the one hand, several publications support the idea that GARP and Foxp3 expression is independent of one and other (8,36,37,46). On the other hand, it has been described that Treg specific transcription of LRRC32 is Foxp3 mediated, resulting from the synergistic interaction of Foxp3 with NFAT (5). ...
Regulatory T cells (Treg) play a critical role in immune homeostasis by suppressing several aspects of the immune response. Herein, Glycoprotein A repetitions predominant (GARP), the docking receptor for latent transforming growth factor (LTGF-β), which promotes its activation, plays a crucial role in maintaining Treg mediated immune tolerance. After activation, Treg uniquely express GARP on their surfaces. Due to its location and function, GARP may represent an important target for immunotherapeutic approaches, including the inhibition of Treg suppression in cancer or the enhancement of suppression in autoimmunity. In the present review, we will clarify the cellular and molecular regulation of GARP expression not only in human Treg but also in other cells present in the tumor microenvironment. We will also examine the overall roles of GARP in the regulation of the immune system. Furthermore, we will explore potential applications of GARP as a predictive and therapeutic biomarker as well as the targeting of GARP itself in immunotherapeutic approaches.
... Classification of FoxP3 + Tregs into subsets helps to investigate Treg cell differentiation in both normal and disease conditions, as well as to alter immune responses by modulating specific FoxP3 + Treg subpopulations [35]. Our group has also recently proposed that FoxP3 + Helios + Tregs constitute a functional subset of Tregs with higher suppressive characteristics [36]. Tumor tissues in CRC patients were characterized by high levels of Helios + Tregs compared to PBMCs and normal colon tissues [24,37], suggesting their potential roles in CRC progression [12]. ...
... Classification of FoxP3 + Tregs into subsets helps to investigate Treg cell differentiation in both normal and disease conditions, as well as to alter immune responses by modulating specific FoxP3 + Treg subpopulations [35]. Our group has also recently proposed that FoxP3 + Helios + Tregs constitute a functional subset of Tregs with higher suppressive characteristics [36]. Tumor tissues in CRC patients were characterized by high levels of Helios + Tregs compared to PBMCs and normal colon tissues [24,37], suggesting their potential roles in CRC progression [12]. ...
There are different subsets of T regulatory cells (Tregs), orchestrating critical roles in the regulation of anti-tumor immunity in colorectal cancer (CRC). In this study, we report that a high frequency of circulating CD4 + FoxP3 + Tregs was associated with poorer disease-free survival (DFS), while their higher frequencies in tumor-infiltrating CD4 + Tregs was associated with better DFS. We further investigated such associations with four Tregs/T cells expressing or lacking FoxP3 and Helios (FoxP3 ± Helios ±). For the first time, we report that a high frequency of circulating CD4 + FoxP3 + Helios + Tregs was associated with poorer DFS, while a high frequency of tumor-infiltrating CD4 + FoxP3 − Helios − T cells was associated with poorer DFS. In the four FoxP3 ± Helios ± T cell subsets expressing any of the immune checkpoints (ICs) investigated, we found that a high frequency of CD4 + FoxP3 + Helios − PD-1 + Tregs in circulation was associated with worse DFS. We also found that high frequencies of FoxP3 + Helios + CTLA-4 + Tregs, FoxP3 + Helios − CTLA-4 + Tregs, and FoxP3 − Helios + CTLA-4 + CD4 + T cells in circulation were associated with worse DFS. In contrast, high frequencies of CD4 + TIM-3 + T cells, FoxP3 + Helios + TIM-3 + Tregs, and FoxP3 − Helios + TIM-3 + CD4 + T cells in circulation were associated with longer DFS. Our data show that certain CD4 + Treg/T cell subsets could serve as independent predictive biomarkers in CRC patients. Identification of the exact subpopulations contributing to clinical outcomes is critical for prognoses and therapeutic targeting.
