Immunological synapse formation between T regulatory cells and cancer-associated fibroblasts promotes tumour development.

Cancer-associated fibroblasts (CAFs) have emerged as a dominant non-hematopoietic cell population in the tumour microenvironment, serving diverse functions in tumour progression. However, the mechanisms via which CAFs influence the anti-tumour immunity remain poorly understood. Here, using multiple tumour models and biopsies from cancer patients, we report that α-SMA+ CAFs can form immunological synapses with Foxp3+ regulatory T cells (Tregs) in tumours. Notably, α-SMA+ CAFs can phagocytose and process tumour antigens and exhibit a tolerogenic phenotype which instructs movement arrest, activation and proliferation in Tregs in an antigen-specific manner. Moreover, α-SMA+ CAFs display double-membrane structures resembling autophagosomes in their cytoplasm. Single-cell transcriptomic data showed an enrichment in autophagy and antigen processing/presentation pathways in α-SMA-expressing CAF clusters. Conditional knockout of Atg5 in α-SMA+ CAFs promoted inflammatory re-programming in CAFs, reduced Treg cell infiltration and attenuated tumour development. Overall, our findings reveal an immunosuppressive mechanism entailing the formation of synapses between α-SMA+ CAFs and Tregs in an autophagy-dependent manner.

One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16.

Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al.1.

Potent induction of trained immunity by Saccharomyces cerevisiae ß-glucans.

Candida albicans cell wall component ß-glucan has been extensively studied for its ability to induce epigenetic and functional reprogramming of innate immune cells, a process termed trained immunity. We show that a high-complexity blend of two individual ß-glucans from Saccharomyces cerevisiae possesses strong bioactivity, resulting in an enhanced trained innate immune response by human primary monocytes. The training required the Dectin-1/CR3, TLR4, and MMR receptors, as well as the Raf-1, Syk, and PI3K downstream signaling molecules. By activating multiple receptors and downstream signaling pathways, the components of this ß-glucan preparation are able to act synergistically, causing a robust secondary response upon an unrelated challenge. In in-vivo murine models of melanoma and bladder cell carcinoma, pre-treatment of mice with the ß-glucan preparation led to a significant reduction in tumor growth. These insights may aid in the development of future therapies based on ß-glucan structures that induce an effective trained immunity response.

Investigating the role of "Immature Myeloid Cells" as Drivers of Inflammation and Disease Persistence in Psoriatic Arthritis.

Background: Despite the development of treatments targeting T cell co-stimulation and cytokines TNF, IL-12/23, and IL-17, less than half of patients within clinical trials achieve high levels of clinical response. This fact, as well as the absence of prognostic biomarkers represents major unmet clinical needs that necessitate further investigation of the disease pathophysiology. Myeloid cells are critical components of PsA inflammatory mechanisms, being a highly prevalent immune population in biopsies of PsA target tissues, such as the skin and the synovium. Through their antigen-presenting capacity and their pro-angiogenic and pro-inflammatory properties myeloid cells could contribute to persistent inflammation in PsA leading to treatment-resistant disease. To this end, we have recently shown the expansion of monocytes in the blood of PsA patients compared to healthy subjects. Importantly, we have also identified an immature myeloid cell population in patients with highly active, refractory disease, indicating the presence of an "emergency myelopoiesis" process in PsA. Aim of the study: In this research protocol, we aim to decipher the pro-inflammatory "myeloid signature" in patients with active PsA and explore the role of immature myeloid cells in disease pathophysiology and their potential as prognostic biomarkers. Methods: To address this, we will isolate and analyse monocytes and immature myeloid cells from PsA patients -before and after a 6-month treatment course- focusing on differences between responders and non-responders. In this context, we will perform a thorough phenotypic and functional analysis of these cells, identify their expression signature in an already established whole blood RNA-seq dataset and investigate their presence in target tissues, such as the skin and synovial fluid. Anticipated benefits: This study will elucidate the role of myeloid cells in disease propagation by further defining the involvement of immature myeloid cells in PsA.

Decoding of translation-regulating entities reveals heterogeneous translation deficiency patterns in cellular senescence.

Cellular senescence constitutes a generally irreversible proliferation barrier, accompanied by macromolecular damage and metabolic rewiring. Several senescence types have been identified based on the initiating stimulus, such as replicative (RS), stress-induced (SIS) and oncogene-induced senescence (OIS). These senescence subtypes are heterogeneous and often develop subset-specific phenotypes. Reduced protein synthesis is considered a senescence hallmark, but whether this trait pertains to various senescence subtypes and if distinct molecular mechanisms are involved remain largely unknown. Here, we analyze large published or experimentally produced RNA-seq and Ribo-seq datasets to determine whether major translation-regulating entities such as ribosome stalling, the presence of uORFs/dORFs and IRES elements may differentially contribute to translation deficiency in senescence subsets. We show that translation-regulating mechanisms may not be directly relevant to RS, however uORFs are significantly enriched in SIS. Interestingly, ribosome stalling, uORF/dORF patterns and IRES elements comprise predominant mechanisms upon OIS, strongly correlating with Notch pathway activation. Our study provides for the first time evidence that major translation dysregulation mechanisms/patterns occur during cellular senescence, but at different rates depending on the stimulus type. The degree at which those mechanisms accumulate directly correlates with translation deficiency levels. Our thorough analysis contributes to elucidating crucial and so far unknown differences in the translation machinery between senescence subsets.

Topically Administered NOX4 Inhibitor, GLX7013114, Is Efficacious in Treating the Early Pathological Events of Diabetic Retinopathy.

NADPH oxidases (NOXs) are major players in generating reactive oxygen species (ROS) and are implicated in various neurodegenerative ocular pathologies. The aim of this study was to investigate the role of a NOX4 inhibitor (GLX7013114) in two in vivo, experimental streptozotocin (STZ) paradigms depicting the early events of diabetic retinopathy (DR). Animals in the diabetic treated group received GLX7013114 topically (20 µL/eye, 10 mg/mL, once daily) for 14 days (paradigm A: preventive) and 7 days (paradigm B: treated) at 48 h and 4 weeks after STZ injection, respectively. Several methodologies were used (immunohistochemistry, Western blot, real-time PCR, ELISA, pattern electroretinography [PERG]) to assess the diabetes-induced early events of DR, namely oxidative stress, neurodegeneration, and neuroinflammation, and the effect of GLX7013114 on the diabetic insults. GLX7013114, administered as eye drops (paradigms A and B), was beneficial in treating the oxidative nitrative stress, activation of caspase-3 and micro- and macroglia, and attenuation of neuronal markers. It also attenuated the diabetes-induced increase in vascular endothelial growth factor, Evans blue dye leakage, and proinflammatory cytokine (TNF-α protein, IL-1ß/IL-6 mRNA) levels. PERG amplitude values suggested that GLX7013114 protected retinal ganglion cell function (paradigm B). This study provides new findings regarding the pharmacological profile of the novel NOX4 inhibitor GLX7013114 as a promising therapeutic candidate for the treatment of the early stage of DR. ARTICLE HIGHLIGHTS: NADPH oxidases (NOXs) are implicated in the early pathological events of diabetic retinopathy (DR). The NOX4 inhibitor GLX7013114, topically administered, reduced oxidative damage and apoptosis in the rat streptozotocin model of DR. GLX7013114 protected retinal neurons and retinal ganglion cell function and reduced the expression of pro-inflammatory cytokines in the diabetic retina. GLX7013114 diminished the diabetes-induced increase in vascular endothelial growth factor levels and Evans blue dye leakage in retinal tissue. GLX7013114 exhibits neuroprotective, anti-inflammatory, and vasculoprotective properties that suggest it may have a role as a putative therapeutic for the early events of DR.

IL-6 Signaling Attenuates TNF-α Production by Plasmacytoid Dendritic Cells in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is characterized by autoimmune joint destruction with debilitating consequences. Despite treatment advancements with biologic therapies, a significant proportion of RA patients show an inadequate clinical response, and restoration of immune self-tolerance represents an unmet therapeutic need. We have previously described a tolerogenic phenotype of plasmacytoid dendritic cells (pDCs) in RA patients responding to anti-TNF-α agents. However, the molecular mechanisms involved in tolerogenic reprogramming of pDCs in RA remain elusive. In this study, guided by transcriptomic analysis of CD303+CD123+ pDCs from RA patients in remission, we revealed enhanced expression of IL-6R and its downstream signaling compared with healthy pDCs. Functional assessment demonstrated that IL-6R engagement resulted in marked reduction of TNF-α secretion by pDCs whereas intracellular TNF-α was significantly increased. Accordingly, pharmacologic inhibition of IL-6R signaling restored TNF-α secretion levels by pDCs. Mechanistic analysis demonstrated impaired activity and decreased lysosomal degradation of ADAM17 (a disintegrin and metalloproteinase 17) sheddase in pDCs, which is essential for TNF-α cleavage. Importantly, reduction of TNF-α secretion by IL-6-treated pDCs attenuated the inflammatory potential of RA patient-derived synovial fibroblasts. Collectively, these findings position pDCs as an important source of TNF-α in RA pathogenesis and unravel an anti-inflammatory mechanism of IL-6 by limiting the pDC-derived TNF-α secretion.

Combined - whole blood and skin fibroblasts- transcriptomic analysis in Psoriatic Arthritis reveals molecular signatures of activity, resistance and early response to treatment.

Background: An interplay between immune cells and resident skin and joint stromal cells is implicated in psoriatic arthritis (PsA), yet the mechanisms remain elusive with a paucity of molecular biomarkers for activity and response. Combined transcriptomic and immunophenotypic analysis of whole blood and skin fibroblasts could provide further insights. Methods: Whole blood RNA-seq was performed longitudinally in 30 subjects with PsA at the beginning, one and six months after treatment, with response defined at six months. As control groups, 10 healthy individuals and 10 subjects with rheumatoid arthritis (RA) were recruited combined with public datasets from patients with psoriasis (PsO) and systemic lupus erythematous (SLE). Differential expression analysis and weighted gene co-expression network analysis were performed to identify gene expression signatures, while deconvolution and flow cytometry to characterize the peripheral blood immune cell profile. In a subset of affected and healthy individuals, RNA-seq of skin fibroblasts was performed and subjected to CellChat analysis to identify the blood-skin fibroblast interaction network. Results: PsA demonstrated a distinct "activity" gene signature in the peripheral blood dominated by TNF- and IFN-driven inflammation, deregulated cholesterol and fatty acid metabolism and expansion of pro-inflammatory non-classical monocytes. Comparison with the blood transcriptome of RA, PsO, and SLE revealed a "PsA-specific signature" enriched in extracellular matrix remodeling. This was further supported by the skin fibroblast gene expression profile, displaying an activated, proliferating phenotype, and by skin-blood interactome analysis revealing interactions with circulating immune cells through WNT, PDGF and immune-related semaphorins. Of note, resistance to treatment was associated with upregulation of genes involved in TGFß signaling and angiogenesis and persistent increase of non-classical monocytes. Differentially expressed genes related to platelet activation and hippo signaling discriminated responders and non-responders as early as one month after treatment initiation. Conclusion: Transcriptome analysis of peripheral blood and skin fibroblasts in PsA reveals a distinct disease activity signature and supports the involvement of skin fibroblasts through their activation and interaction with circulating immune cells. Aberrant TGFß signaling and persistently increased non-classical monocytes characterize treatment-resistant PsA, with pro-inflammatory pathways related to platelet activation and Hippo signaling predicting early response to treatment.

Ablation of NLRP3 inflammasome rewires MDSC function and promotes tumor regression.

Myeloid-derived suppressor cells (MDSCs) are myeloid precursors that exert potent immunosuppressive properties in cancer. Despite the extensive knowledge on mechanisms implicated in mobilization, recruitment, and function of MDSCs, their therapeutic targeting remains an unmet need in cancer immunotherapy, suggesting that unappreciated mechanisms of MDSC-mediated suppression exist. Herein, we demonstrate an important role of NLRP3 inflammasome in the functional properties of MDSCs in tumor-bearing hosts. Specifically, Nlrp3-deficient mice exhibited reduced tumor growth compared to wild-type animals and induction of robust anti-tumor immunity, accompanied by re-wiring of the MDSC compartment. Interestingly, both monocytic (M-MDSCs) and granulocytic (G-MDSCs) subsets from Nlrp3-/- mice displayed impaired suppressive activity and demonstrated significant transcriptomic alterations supporting the loss-of-function and associated with metabolic re-programming. Finally, therapeutic targeting of NLRP3 inhibited tumor development and re-programmed the MDSC compartment. These findings propose that targeting NLRP3 in MDSCs could overcome tumor-induced tolerance and may provide new checkpoints of cancer immunotherapy.

Regulatory T Cells in Autoimmunity and Cancer: A Duplicitous Lifestyle.

Regulatory T (Treg) cells, possess a strategic role in the maintenance of immune homeostasis, and their function has been closely linked to development of diverse pathologies including autoimmunity and cancer. Comprehensive studies in various disease contexts revealed an increased plasticity as a characteristic of Treg cells. Although Treg cell plasticity comes in various flavors, the major categories enclose the loss of Foxp3 expression, which is the master regulator of Treg cell lineage, giving rise to "ex-Treg" cells and the "fragile" Treg cells in which FOXP3 expression is retained but accompanied by the engagement of an inflammatory program and attenuation of the suppressive activity. Treg cell plasticity possess a tremendous therapeutic potential either by inducing Treg cell de-stabilization to promote anti-tumor immunity, or re-enforcing Treg cell stability to attenuate chronic inflammation. Herein, we review the literature on the Treg cell plasticity with lessons learned in autoimmunity and cancer and discuss challenges and open questions with potential therapeutic implications.

Patrolling human SLE haematopoietic progenitors demonstrate enhanced extramedullary colonisation; implications for peripheral tissue injury.

Systemic lupus erythematosus (SLE) is an autoimmune disease where bone-marrow-derived haematopoietic cells have a key role in its pathogenesis with accumulating evidence suggesting an aberrant function of haematopoietic stem/progenitor cells (HSPCs). We examined whether patrolling HSPCs differ from bone-marrow HSPCs both in SLE and healthy individuals, and how they participate in peripheral tissue injury. By employing next-generation RNA sequencing, the transcriptomes of CD34+ HSPCs deriving from the bone marrow and those patrolling the bloodstream of both healthy and individuals with SLE were compared. Patrolling SLE and Healthy human HSPC kinetics were examined through their inoculation into humanised mice. Patrolling and bone-marrow HSPCs have distinct molecular signatures, while patrolling SLE HSPCs showed an enhanced extramedullary gene expression profile. Non-mobilised, SLE-derived circulating HSPCs demonstrated altered homing capacities. Xenotransplantation of circulating HSPCs in humanised mice showed that human peripheral blood HSPCs possess the ability for extramedullary organ colonisation to the kidneys. Circulating and bone marrow-derived HSPCs are distinct in steady and diseased states. Patrolling SLE CD34+ HSPCs are able to home at extramedullary sites such as the spleen and kidneys, potentially participating in peripheral tissue injury.

Decoding the Myeloid-Derived Suppressor Cells in Lymphoid Malignancies.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid precursors which emerged as a potent regulator of the immune system, exerting suppressive properties in diverse disease settings. In regards to cancer, MDSCs have an established role in solid tumors; however, their contribution to immune regulation during hematologic malignancies and particularly in lymphomas remains ill-defined. Herein focused on lymphoma, we discuss the literature on MDSC cells in all histologic types, and we also refer to lessons learned by animal models of lymphoma. Furthermore, we elaborate on future directions and unmet needs and challenges in the MDSC field related to lymphoma malignancies which may shed light on the complex nature of the immune system in malignancies.

Regulatory T-cell Transcriptomic Reprogramming Characterizes Adverse Events by Checkpoint Inhibitors in Solid Tumors.

Immune checkpoint inhibitors (ICI), which target immune regulatory pathways to unleash antitumor responses, have revolutionized cancer immunotherapy. Despite the remarkable success of ICI immunotherapy, a significant proportion of patients whose tumors respond to these treatments develop immune-related adverse events (irAE) resembling autoimmune diseases. Although the clinical spectrum of irAEs is well characterized, their successful management remains empiric. This is in part because the pathogenic mechanisms involved in the breakdown of peripheral tolerance and induction of irAEs remain elusive. Herein, we focused on regulatory T cells (Treg) in individuals with irAEs because these cells are vital for maintenance of peripheral tolerance, appear expanded in the peripheral blood of individuals with cancer, and abundantly express checkpoint molecules, hence representing direct targets of ICI immunotherapy. Our data demonstrate an intense transcriptomic reprogramming of CD4+CD25+CD127- Tregs in the blood of individuals with advanced metastatic melanoma who develop irAEs following ICI immunotherapy, with a characteristic inflammatory, apoptotic, and metabolic signature. This inflammatory signature was shared by Tregs from individuals with different types of cancer developing irAEs and individuals with autoimmune diseases. Our findings suggest that inflammatory Treg reprogramming is a feature of immunotherapy-induced irAEs, and this may facilitate translational approaches aiming to induce robust antitumor immunity without disturbing peripheral tolerance.

Measuring Suppressive Activity and Autophagy in Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSC) are potent suppressor cells that accumulate in tumor microenvironment and inhibit anti-tumor responses. Assessment of cell-autonomous MDSC responses allows the precise characterization of MDSCs in various disease settings and elucidates the underlying mechanisms of MDSC-mediated immune suppression. Here we describe a protocol for the isolation of tumor infiltrating or splenic MDSC, as well as their subpopulations, from melanoma-inoculated mice using Fluorescent Activated Cell Sorting (FACS). We further provide protocols for investigation of the autophagy pathway and ex vivo assessment of MDSC suppressive function using lymph node responder cells. These assays allow a comprehensive characterization of MDSC in murine experimental models.

Neutrophils as Orchestrators in Tumor Development and Metastasis Formation.

Several lines of clinical and experimental evidence suggest that immune cell plasticity is a central player in tumorigenesis, tumor progression, and metastasis formation. Neutrophils are able to promote or inhibit tumor growth. Through their interaction with tumor cells or their crosstalk with other immune cell subsets in the tumor microenvironment, they modulate tumor cell survival. Here, we summarize current knowledge with regards to the mechanisms that underlie neutrophil-mediated effects on tumor establishment and metastasis development. We also discuss the tumor-mediated effects on granulopoiesis and neutrophil precursors in the bone marrow and the involvement of neutrophils in anti-tumor therapeutic modalities.

BCG Vaccination Induces Long-Term Functional Reprogramming of Human Neutrophils.

The tuberculosis vaccine bacillus Calmette-Guérin (BCG) protects against some heterologous infections, probably via induction of non-specific innate immune memory in monocytes and natural killer (NK) cells, a process known as trained immunity. Recent studies have revealed that the induction of trained immunity is associated with a bias toward granulopoiesis in bone marrow hematopoietic progenitor cells, but it is unknown whether BCG vaccination also leads to functional reprogramming of mature neutrophils. Here, we show that BCG vaccination of healthy humans induces long-lasting changes in neutrophil phenotype, characterized by increased expression of activation markers and antimicrobial function. The enhanced function of human neutrophils persists for at least 3 months after vaccination and is associated with genome-wide epigenetic modifications in trimethylation at histone 3 lysine 4. Functional reprogramming of neutrophils by the induction of trained immunity might offer novel therapeutic strategies in clinical conditions that could benefit from modulation of neutrophil effector function.

Innate Immune Training of Granulopoiesis Promotes Anti-tumor Activity.

Trained innate immunity, induced via modulation of mature myeloid cells or their bone marrow progenitors, mediates sustained increased responsiveness to secondary challenges. Here, we investigated whether anti-tumor immunity can be enhanced through induction of trained immunity. Pre-treatment of mice with ß-glucan, a fungal-derived prototypical agonist of trained immunity, resulted in diminished tumor growth. The anti-tumor effect of ß-glucan-induced trained immunity was associated with transcriptomic and epigenetic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor phenotype; this process required type I interferon signaling irrespective of adaptive immunity in the host. Adoptive transfer of neutrophils from ß-glucan-trained mice to naive recipients suppressed tumor growth in the latter in a ROS-dependent manner. Moreover, the anti-tumor effect of ß-glucan-induced trained granulopoiesis was transmissible by bone marrow transplantation to recipient naive mice. Our findings identify a novel and therapeutically relevant anti-tumor facet of trained immunity involving appropriate rewiring of granulopoiesis.

An intrinsic role of IL-33 in Treg cell-mediated tumor immunoevasion.

Regulatory T (Treg) cells accumulate into tumors, hindering the success of cancer immunotherapy. Yet, therapeutic targeting of Treg cells shows limited efficacy or leads to autoimmunity. The molecular mechanisms that guide Treg cell stability in tumors remain elusive. In the present study, we identify a cell-intrinsic role of the alarmin interleukin (IL)-33 in the functional stability of Treg cells. Specifically, IL-33-deficient Treg cells demonstrated attenuated suppressive properties in vivo and facilitated tumor regression in a suppression of tumorigenicity 2 receptor (ST2) (IL-33 receptor)-independent fashion. On activation, Il33-/- Treg cells exhibited epigenetic re-programming with increased chromatin accessibility of the Ifng locus, leading to elevated interferon (IFN)-γ production in a nuclear factor (NF)-κB-T-bet-dependent manner. IFN-γ was essential for Treg cell defective function because its ablation restored Il33-/- Treg cell-suppressive properties. Importantly, genetic ablation of Il33 potentiated the therapeutic effect of immunotherapy. Our findings reveal a new and therapeutically important intrinsic role of IL-33 in Treg cell stability in cancer.

Transcriptome reprogramming and myeloid skewing in haematopoietic stem and progenitor cells in systemic lupus erythematosus.

OBJECTIVES: Haematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to both myeloid and lymphoid cell lineages. We reasoned that the aberrancies of immune cells in systemic lupus erythematosus (SLE) could be traced back to HSPCs. METHODS: A global gene expression map of bone marrow (BM)-derived HSPCs was completed by RNA sequencing followed by pathway and enrichment analysis. The cell cycle status and apoptosis status of HSPCs were assessed by flow cytometry, while DNA damage was assessed via immunofluorescence. RESULTS: Transcriptomic analysis of Lin-Sca-1+c-Kit+ haematopoietic progenitors from diseased lupus mice demonstrated a strong myeloid signature with expanded frequencies of common myeloid progenitors (CMPs)-but not of common lymphoid progenitors-reminiscent of a 'trained immunity' signature. CMP profiling revealed an intense transcriptome reprogramming with suppression of granulocytic regulators indicative of a differentiation arrest with downregulation trend of major regulators such as Cebpe, Cebpd and Csf3r, and disturbed myelopoiesis. Despite the differentiation arrest, frequencies of BM neutrophils were markedly increased in diseased mice, suggesting an alternative granulopoiesis pathway. In patients with SLE with severe disease, haematopoietic progenitor cells (CD34+) demonstrated enhanced proliferation, cell differentiation and transcriptional activation of cytokines and chemokines that drive differentiation towards myelopoiesis, thus mirroring the murine data. CONCLUSIONS: Aberrancies of immune cells in SLE can be traced back to the BM HSPCs. Priming of HSPCs and aberrant regulation of myelopoiesis may contribute to inflammation and risk of flare. TRIAL REGISTRATION NUMBER: 4948/19-07-2016.

Balancing cancer immunotherapy and immune-related adverse events: The emerging role of regulatory T cells.

Advances in our understanding οf tumor immunity have prompted a paradigm shift in oncology, with the emergence of immunotherapy, where therapeutic agents are used to target immune cells rather than cancer cells. A real breakthrough in the field of immunotherapy came with the use of immune checkpoint inhibitors (ICI), namely antagonistic antibodies that block key immune regulatory molecules (checkpoint molecules), such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein (PD-1) and its ligand PD-L1, that under physiologic conditions suppress T cell effector function. However, despite the enormous success, a significant proportion of patients do not respond, while responses are frequently accompanied by life-threatening autoimmune related adverse events (irAEs). A major impediment in the effectiveness of ICI immunotherapy is the tumoral resistance, which is dependent on the immunosuppressive nature of tumor microenvironment (TME). Regulatory T cells (Tregs) are among the most abundant suppressive cells in the TME and their presence has been correlated with tumor progression, invasiveness as well as metastasis. Tregs are characterized by the expression of the transcription factor Foxp3 and various mechanisms ranging from cell-to-cell contact to secretion of inhibitory molecules have been implicated in their function. Notably, Tregs amply express most of the checkpoint molecules such as CTLA4, PD1 and LAG3 and therefore represent a direct target of ICI immunotherapy. Taking into consideration the critical role of Tregs in maintenance of immune homeostasis and avoidance of autoimmunity it is plausible that targeting of Tregs by ICI immunotherapy results in the development of irAEs. Since the use of ICI becomes common, and new immune checkpoint molecules are currently under clinical trials for the treatment of cancer, the occurrence of irAEs is expected to dramatically rise. Herein we review the current literature focusing on the role of Tregs in cancer evolution, ICI response and development of irAEs. Unraveling the complex mechanisms that hinder the tumor immune surveillance and in particular how ICI immunotherapy imprint on Treg activities to promote cancer regression while avoid development of irAEs, will empower the design of novel immunotherapeutic modalities in cancer with increased efficacy and diminished adverse events.