Oncolytic α-herpesvirus and myeloid-tropic cytomegalovirus cooperatively enhance systemic antitumor responses.

Oncolytic virotherapy aims to activate host antitumor immunity. In responsive tumors, intratumorally injected herpes simplex viruses (HSVs) have been shown to lyse tumor cells, resulting in local inflammation, enhanced tumor antigen presentation, and boosting of antitumor cytotoxic lymphocytes. In contrast to HSV, cytomegalovirus (CMV) is nonlytic and reprograms infected myeloid cells, limiting their antigen-presenting functions and protecting them from recognition by natural killer (NK) cells. Here, we show that when co-injected into mouse tumors with an oncolytic HSV, mouse CMV (mCMV) preferentially targeted tumor-associated myeloid cells, promoted the local release of proinflammatory cytokines, and enhanced systemic antitumor immune responses, leading to superior control of both injected and distant contralateral tumors. Deletion of mCMV genes m06, which degrades major histocompatibility complex class I (MHC class I), or m144, a viral MHC class I homolog that inhibits NK activation, was shown to diminish the antitumor activity of the HSV/mCMV combination. However, an mCMV recombinant lacking the m04 gene, which escorts MHC class I to the cell surface, showed superior HSV adjuvanticity. CMV is a potentially promising agent with which to reshape and enhance antitumor immune responses following oncolytic HSV therapy.

IL-33/ST2 as a potential target for tumor immunotherapy.

IL-33, a member of the IL-1 family, was initially reported to be expressed constitutively in the nucleus of tissue-lining and structural cells. However, upon tissue damage or injury, IL-33 can be released quickly to bind with its cognate receptor ST2 in response to wound healing and inflammation and act as a DAMP. As a key regulator of Th2 responses, IL-33/ST2 signal is primarily associated with immunity and immune-related disorders. In recent years, IL-33/ST2 signaling pathway has been reported to promote the development of cancer and remodel the tumor microenvironment by expanding immune suppressive cells such as myeloid-derived suppressor cells or regulatory T cells. However, its role remains controversial in some tumor settings. IL-33 could also promote effective infiltration of immune cells such as CD8+ T and NK cells, which act as antitumor. These dual effects may limit the clinical application to target this cytokine axis. Therefore, more comprehensive exploration and deeper understanding of IL-33 are required. In this review, we summarized the IL-33/ST2 axis versatile roles in the tumor microenvironment with a focus on the IL-33-target immune cells and downstream signaling pathways. We also discuss how the IL-33/ST2 axis could be used as a potential therapeutic target for cancer immunotherapy.

Inducing immunogenic cell death in immuno-oncological therapies.

Immunotherapy has revolutionized cancer treatment and substantially improved patient outcomes with respect to multiple types of tumors. However, most patients cannot benefit from such therapies, mainly due to the intrinsic low immunogenicity of cancer cells (CCs) that allows them to escape recognition by immune cells of the body. Immunogenic cell death (ICD), which is a form of regulated cell death, engages in a complex dialogue between dying CCs and immune cells in the tumor microenvironment (TME), ultimately evoking the damage-associated molecular pattern (DAMP) signals to activate tumor-specific immunity. The ICD inducers mediate the death of CCs and improve both antigenicity and adjuvanticity. At the same time, they reprogram TME with a "cold-warm-hot" immune status, ultimately amplifying and sustaining dendritic cell- and T cell-dependent innate sensing as well as the antitumor immune responses. In this review, we discuss how to stimulate ICD based upon the biological properties of CCs that have evolved under diverse stress conditions. Additionally, we highlight how this dynamic interaction contributes to priming tumor immunogenicity, thereby boosting anticancer immune responses. We believe that a deep understanding of these ICD processes will provide a framework for evaluating its vital role in cancer immunotherapy.

Endogenous IL-33 exerts CD8+ T cell antitumor responses overcoming pro-tumor effects by regulatory T cells in a colon carcinoma model.

Interleukin-33 (IL-33) is a nuclear-associated cytokine of the IL-1 family. IL-33 and its receptor ST2 axis exert conflicting anti-tumor and pro-tumor effects in various tumors. In this study, we examined the role of endogenously produced IL-33 in the colon-26 tumor model, in which involvement of the IL-33:ST2 pathway was negligible on the tumor side. We found that the generation of regulatory T cells (Tregs) and CD8+ T cells, and IFN-γ expression by both CD4+ and CD8+ T cells (T cell activation) were impaired in IL-33-deficient mice. Overall antitumor responses, assessed by tumor growth and IFN-γ expression by tumor-infiltrating CD8+ T cells, were also impaired, even after Treg adjustment prior to tumor inoculation. These results indicate that endogenous IL-33 augmented CD8+ T cell-mediated antitumor responses in this colon carcinoma model, with higher CD8+ T cell-infiltration and overcoming pro-tumor effects by increased Tregs. Exogenous application of IL-33 into the tumors did not enhance CD8+ T cell-mediated antitumor responses despite marked elevation of innate responses showing upregulation of proinflammatory cytokine/chemokine expression, neutrophil recruitment, and dendritic cell activation. Our results suggest a dual role for endogenous IL-33 in antitumor responses and suggest that the balance of CD8+ T cells:Tregs in the tumor microenvironment is one of key factors for estimating the contribution of IL-33-mediated antitumor responses. Therefore, the development of IL-33-based cancer immunotherapy may require a target cell-specific approach.

TIM-3 as a Target for Cancer Immunotherapy and Mechanisms of Action.

Cancer immunotherapy has produced impressive clinical results in recent years. Despite the success of the checkpoint blockade strategies targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death receptor 1 (PD-1), a large portion of cancer patients have not yet benefited from this novel therapy. T cell immunoglobulin and mucin domain 3 (TIM-3) has been shown to mediate immune tolerance in mouse models of infectious diseases, alloimmunity, autoimmunity, and tumor Immunity. Thus, targeting TIM-3 emerges as a promising approach for further improvement of current immunotherapy. Despite a large amount of experimental data showing an immune suppressive function of TIM-3 in vivo, the exact mechanisms are not well understood. To enable effective targeting of TIM-3 for tumor immunotherapy, further in-depth mechanistic studies are warranted. These studies will also provide much-needed insight for the rational design of novel combination therapy with other checkpoint blockers. In this review, we summarize key evidence supporting an immune regulatory role of TIM-3 and discuss possible mechanisms of action.

LXR-dependent and -independent effects of oxysterols on immunity and tumor growth.

Oxysterols are involved in maintaining cellular cholesterol levels. Recently, oxysterols have been demonstrated to modulate the function of immune cells and tumor growth. These effects can be dependent on the activation of the oxysterol-binding liver X receptors (LXRs) or, as recently demonstrated for T and B cells, DCs and neutrophils, can be independent of LXR activation. LXR-dependent oxysterol effects can be ascribed to the activation of LXRα, LXRß or LXRαß isoforms, which induces transcriptional activation or trans-repression of target genes. The prevalent activation of one isoform seems to be cell-, tissue-, or context-specific, as shown in some pathologic processes, i.e., infectious diseases, atherosclerosis, and autoimmunity. Oxysterol-LXR signaling has recently been shown to inhibit antitumor immune responses, as well as to modulate tumor cell growth. Here, we review the mechanisms that link oxysterols to tumor growth, and discuss possible networks at the basis of LXR-dependent and -independent oxysterol effects on immune cells and tumor development.

A Trojan Horse Delivery Vehicle Carrying siRNA Nanotherapeutics with Multiple Tumor Microenvironment Responsiveness Elicits Robust Antitumor Immune Responses In Situ via a "Self-Synergistic" Approach.

The potential of small interfering RNAs (siRNAs) in the treatment of malignant tumors has attracted increasing attention due to their inherent advantages. However, their therapeutic performance strongly depends on the efficiency of their cytoplasmic delivery in vivo by the delivery vehicle with good cellular permeability and histocompatibility. Herein, a polycationic carrier camouflaged with macrophage membrane (MPM) is constructed biomimetically, which is condensed from endogenous spermine monomers through diselenide bonds. The developed Trojan horse delivery vehicle has desirable compression efficacy for siRNA oligo against PD-L1 (siPDL1) as well as intracytoplasmic release properties derived from its sequential degradation triggered by redox microenvironment in tumor cells. Furthermore, the coloading of photosensitizer can mediate photodynamic therapy (PDT) accompanied by the generation of reactive oxygen species (ROS) upon light irradiation applied, which accelerated the degradation of the carrier as well as the release of cargoes while enhancing the PD-L1 blockage-mediated immunotherapy by inducing in-situ immunogenic cell death. Moreover, the synchronously delivered siPDL1 attenuated the ROS-induced increase in immunosuppressive PD-L1 expression, thereby effectively eliciting a robust antitumor immune response with a "self-synergistic" manner in the xenograft breast cancer mouse model.

Circulating T cell profiles associate with enterotype signatures underlying hematological malignancy relapses.

Early administration of azithromycin after allogeneic hematopoietic stem cell transplantation was shown to increase the relapse of hematological malignancies. To determine the impact of azithromycin on the post-transplant gut ecosystem and its influence on relapse, we characterized overtime gut bacteriome, virome, and metabolome of 55 patients treated with azithromycin or a placebo. We describe four enterotypes and the network of associated bacteriophage species and metabolic pathways. One enterotype associates with sustained remission. One taxon from Bacteroides specifically associates with relapse, while two from Bacteroides and Prevotella correlate with complete remission. These taxa are associated with lipid, pentose, and branched-chain amino acid metabolic pathways and several bacteriophage species. Enterotypes and taxa associate with exhausted T cells and the functional status of circulating immune cells. These results illustrate how an antibiotic influences a complex network of gut bacteria, viruses, and metabolites and may promote cancer relapse through modifications of immune cells.

A lymphatic route for a hyperbranched heteroglycan from Radix Astragali to trigger immune responses after oral dosing.

Gut barrier makes a huge research gap between in vivo and in vitro studies of orally bioactive polysaccharides: whether/how they contact the related cells in vivo. A hyperbranched heteroglycan RAP from Radix Astragali, exerting antitumor and immunomodulatory effects in vitro and in vivo, is right an example. Here, we determined first that RAP's antitumor activity is immune-dependent. Being undegraded and non-absorbing, RAP quickly entered Peyer's patches (PPs) in 1 h where it directly targeted follicle dendritic cells and initiated antitumor immune responses. RAP was further delivered to mesenteric lymph node, bone marrow, and tumor. By contrast, the control Dendrobium officinale polysaccharide did not enter PPs. These findings revealed a blood/microbiota-independent and selective lymphatic route for orally administrated RAP to directly contact immune cells and trigger antitumor immune responses. This route bridges the research gap between the in vitro and in vivo studies and might apply to many other bioactive polysaccharides.

Stimuli-responsive nano vehicle enhances cancer immunotherapy by coordinating mitochondria-targeted immunogenic cell death and PD-L1 blockade.

Induction of immunogenic cell death promotes antitumor immunity against cancer. However, majority of clinically-approved drugs are unable to elicit sufficient ICD. Here, our study revealed that mitochondria-targeted delivery of doxorubicin (DOX) massively amplified ICD via substantial generation of reactive oxygen species (ROS) after mitochondrial damage. The underlying mechanism behind increased ICD was further demonstrated to be ascribed to two pathways: (1) ROS elevated endoplasmic reticulum (ER) stress, leading to surface exposure of calreticulin; (2) ROS promoted release of various mitochondria-associated damage molecules including mitochondrial transcription factor A. Nevertheless, adaptive upregulation of PD-L1 was found after such ICD-inducing treatment. To overcome such immunosuppressive feedback, we developed a tumor stimuli-responsive nano vehicle to simultaneously exert mitochondrial targeted ICD induction and PD-L1 blockade. The nano vehicle was self-assembled from ICD-inducing copolymer and PD-L1 blocking copolymer, and possessed long-circulating property which contributed to better tumor accumulation and mitochondrial targeting. As a result, the nano vehicle remarkably activated antitumor immune responses and exhibited robust antitumor efficacy in both immunogenic and non-immunogenic tumor mouse models.

Oncogenic Role of HMGB1 as An Alarming in Robust Prediction of Immunotherapy Response in Colorectal Cancer.

OBJECTIVE: To assess the correlation between HMGB1 expression and the patient prognosis in a multicancer context. METHODS: The potential oncogenic role of HMGB1 was explored in forty tumors through the TCGA, GEO, and Oncomine datasets. We analyzed the clinical prognostic value and antitumor immunotherapy of HMGB1 in a multicancer context using GEO (GSE111636). RESULTS: High expression of HMGB1 is present in multicancer cases, and its low expression is closely associated with the prognostic survival of patients, in terms of both overall and disease-free survival in ACC and LUAD. Further investigation revealed that the high expression of gastric and lung cancer is closely associated with low risk and better prognosis of patients based on COX and Kaplan-Meier analysis of OS, FP and PPS. HMGB1 expression was found to be significantly correlated with cancer-associated fibroblast and CD8+ T cell infiltration in the TME. The analysis of GO functional annotation/KEGG pathways indicates that HMGB1 may regulate tumor immunity-related pathways, such as the tumor immunotherapy response in colorectal cancer. The function of four genes as hubs are confirmed by in vitro HMGB1 knockdown which led to inhibition of cell proliferation and metastasis in SW620 and SW480 cells. CONCLUSION: HMGB1 is a potential novel biomarker for improving clinical prognosis and antitumor immunotherapy efficacy. CDK1, HMGB2, SSRP1, and H2AFV may serve as key nodes for HMGB1 in colorectal cancer.

Angiotensin-converting enzyme 2 connects COVID-19 with cancer and cancer immunotherapy.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than two million deaths. Underlying diseases, including cancer, are high-risk factors for severe COVID-19 outcomes. Angiotensin-converting enzyme 2 (ACE2), as a SARS-CoV-2 host cell receptor, plays a crucial role in SARS-CoV-2 invading human cells. ACE2 also has significant associations with cancer. Recent studies showed that ACE2 was inversely correlated with the activities of multiple oncogenic pathways and tumor progression phenotypes, and was positively correlated with antitumor immune response and survival prognosis in diverse cancers, suggesting a potential protective role of ACE2 in cancer progression. Positive expression of ACE2 is also correlated with programmed death-ligand 1 (PD-L1) in cancer. The positive associations of ACE2 expression with antitumor immune signatures and PD-L1 expression indicate that ACE2 expression is a positive predictor for the response to immune checkpoint inhibitors (ICIs). This was evidenced in multiple cancer cohorts treated with ICIs. Thus, ACE2 may build potential connections between COVID-19 and cancer and cancer immunotherapy. The potential connections suggest that ACE2 inhibitors may not be a good option for treating COVID-19 patients with cancer, particularly in cancer patients who are receiving immunotherapy. Furthermore, the relationships between ACE2, COVID-19, and cancer are worth confirming by more experimental and clinical data, considering that many cancer patients are at high risk for COVID-19.

Liver X Receptors: Regulators of Cholesterol Metabolism, Inflammation, Autoimmunity, and Cancer.

The interplay between cellular stress and immune response can be variable and sometimes contradictory. The mechanisms by which stress-activated pathways regulate the inflammatory response to a pathogen, in autoimmunity or during cancer progression remain unclear in many aspects, despite our recent knowledge of the signalling and transcriptional pathways involved in these diseases. In this context, over the last decade many studies demonstrated that cholesterol metabolism is an important checkpoint for immune homeostasis and cancer progression. Indeed, cholesterol is actively metabolized and can regulate, through its mobilization and/or production of active derivatives, many aspects of immunity and inflammation. Moreover, accumulation of cholesterol has been described in cancer cells, indicating metabolic addiction. The nuclear receptors liver-X-receptors (LXRs) are important regulators of intracellular cholesterol and lipids homeostasis. They have also key regulatory roles in immune response, as they can regulate inflammation, innate and adaptive immunity. Moreover, activation of LXRs has been reported to affect the proliferation and survival of different cancer cell types that show altered metabolic pathways and accumulation of cholesterol. In this minireview we will give an overview of the recent understandings about the mechanisms through which LXRs regulate inflammation, autoimmunity, and cancer, and the therapeutic potential for future treatment of these diseases through modulation of cholesterol metabolism.

Fyn tyrosine kinase, a downstream target of receptor tyrosine kinases, modulates antiglioma immune responses.

BACKGROUND: High-grade gliomas are aggressive and immunosuppressive brain tumors. Molecular mechanisms that regulate the inhibitory immune tumor microenvironment (TME) and glioma progression remain poorly understood. Fyn tyrosine kinase is a downstream target of the oncogenic receptor tyrosine kinase pathway and is overexpressed in human gliomas. Fyn's role in vivo in glioma growth remains unknown. We investigated whether Fyn regulates glioma initiation, growth and invasion. METHODS: We evaluated the role of Fyn using genetically engineered mouse glioma models (GEMMs). We also generated Fyn knockdown stem cells to induce gliomas in immune-competent and immune-deficient mice (nonobese diabetic severe combined immunodeficient gamma mice [NSG], CD8-/-, CD4-/-). We analyzed molecular mechanism by RNA sequencing and bioinformatics analysis. Flow cytometry was used to characterize immune cellular infiltrates in the Fyn knockdown glioma TME. RESULTS: We demonstrate that Fyn knockdown in diverse immune-competent GEMMs of glioma reduced tumor progression and significantly increased survival. Gene ontology (GO) analysis of differentially expressed genes in wild-type versus Fyn knockdown gliomas showed enrichment of GOs related to immune reactivity. However, in NSG and CD8-/- and CD4-/- immune-deficient mice, Fyn knockdown gliomas failed to show differences in survival. These data suggest that the expression of Fyn in glioma cells reduces antiglioma immune activation. Examination of glioma immune infiltrates by flow cytometry displayed reduction in the amount and activity of immune suppressive myeloid derived cells in the Fyn glioma TME. CONCLUSIONS: Gliomas employ Fyn mediated mechanisms to enhance immune suppression and promote tumor progression. We propose that Fyn inhibition within glioma cells could improve the efficacy of antiglioma immunotherapies.

Penetrable Nanoplatform for "Cold" Tumor Immune Microenvironment Reeducation.

Lack of tumor-infiltration lymphocytes (TILs) and resistances by overexpressed immunosuppressive cells (principally, myeloid-derived suppressor cells (MDSCs)) in tumor milieu are two major challenges hindering the effectiveness of immunotherapy for "immune-cold" tumors. In addition, the natural physical barrier existing in solid cancer also limits deeper delivery of drugs. Here, a tumor-targeting and light-responsive-penetrable nanoplatform (Apt/PDGs/@pMOF) is developed to elicit intratumoral infiltration of cytotoxic T cells (CTLs) and reeducate immunosuppressive microenvironment simultaneously. In particular, porphyrinic metal-organic framework (pMOF)-based photodynamic therapy (PDT) induces tumor immunogenic cell death (ICD) to promote CTLs intratumoral infiltration and hot "immune-cold" tumor. Upon being triggered by PDT, the nearly 10 nm adsorbed drug-loaded dendrimer de-shields from the nanoplatform and spreads into the deeper tumor, eliminating MDSCs and reversing immunosuppression, eventually reinforcing immune response. Meanwhile, the designed nanoplatform also has a systemic MDSC inhibition effect and moderate improvement of overall antitumor immune responses, resulting in effective suppression of distal tumors within less significant immune-related adverse effects (irAEs) induced.

Cancer Epigenetics, Tumor Immunity, and Immunotherapy.

Epigenetic mechanisms, including DNA methylation, histone post-translational modifications, and chromatin structure regulation, are critical for the interactions between tumor and immune cells. Emerging evidence shows that tumors commonly hijack various epigenetic mechanisms to escape immune restriction. As a result, the pharmaceutical modulation of epigenetic regulators, including 'writers', 'readers', 'erasers', and 'remodelers', is able to normalize the impaired immunosurveillance and/or trigger antitumor immune responses. Thus, epigenetic targeting agents are attractive immunomodulatory drugs and will have major impacts on immuno-oncology. Here, we discuss epigenetic regulators of the cancer-immunity cycle and current advances in developing epigenetic therapies to boost anticancer immune responses, either alone or in combination with current immunotherapies.

IL-36ß Promotes CD8+ T Cell Activation and Antitumor Immune Responses by Activating mTORC1.

Cytokine-amplified functional CD8+ T cells ensure effective eradication of tumors. Interleukin 36α (IL-36α), IL-36ß, and IL-36γ share the same receptor complex, composed of the IL-36 receptor (IL-36R), and IL-1RAcP. Recently, we revealed that IL-36γ greatly promoted CD8+ T cell activation, contributing to antitumor immune responses. However, the underlying mechanism of IL-36-mediated CD8+ T cell activation remains understood. In the current study, we proved that IL-36ß had the same effect on CD8+ T cell as IL-36γ, and uncovered that IL-36ß significantly activated mammalian target of rapamycin complex 1 (mTORC1) of CD8+ T cells. When mTORC1 was inhibited by rapamycin, IL-36ß-stimulated CD8+ T cell activation and expansion was drastically downregulated. Further, we elucidated that IL-36ß-mediated mTORC1 activation was dependent on the pathway of phosphatidylinositol 3 kinase (PI3K)/Akt, IκB kinase (IKK) and myeloid differentiation factor 88 (MyD88). Inhibition of PI3K or IKK by inhibitor, or deficiency of MyD88, respectively, suppressed mTORC1 signal, causing arrest of CD8+ T cell activation. Additionally, it was validated that IL-36ß significantly promoted mTORC1 activation and antitumor function of CD8+ tumor-infiltrating lymphocytes (TILs) in vivo, resulting in inhibition of tumor growth and prolongation of survival of tumor-bearing mice. Taken together, we substantiated that IL-36ß could promote CD8+ T cell activation through activating mTORC1 dependent on PI3K/Akt, IKK and MyD88 pathways, leading to enhancement of antitumor immune responses, which laid the foundations for applying IL-36ß into tumor immunotherapy.

Tumor Microenvironment-Triggered Aggregated Magnetic Nanoparticles for Reinforced Image-Guided Immunogenic Chemotherapy.

Anticancer therapies, which can induce cell death and elevate antitumor immune response in the meantime, are considered as effective treatments for many types of cancers. Immunogenic cell death (ICD) induced by chemodrugs is a promising and typical strategy to achieve cell cytotoxicity and immunological enhancement together. However, due to the low level of ICD induction and less tumor-targeting accumulation, application of traditional ICD inducers is limited. Here, tumor-targeting core-shell magnetic nanoparticles (ETP-PtFeNP:α-enolase targeting peptide modified Pt-prodrug loaded Fe3O4 nanoparticles) are developed to reinforce ICD induction of loaded-oxaliplatin (IV) prodrug. After tumor-targeting accumulation and endocytosis, platinum (IV) complexes are activated by intracellular reductive elimination to yield and release the Pt (II) congener, oxaliplatin, leading to DNA lesions and reactive oxygen species (ROS) generation. Simultaneously, in-progress-released ferric ions elicit highly toxic ROS (·OH or ·OOH) burst and interfere with the intracytoplasmic redox balance (like endoplasmic reticulum stress), leading to ICD-associated immunogenicity enhancement and specific antitumor immune responses to kill the tumor cells synergistically. Meanwhile, the transverse relaxation rate R 2 of ETP-PtFeNP is remarkably increased by more than three times while triggered by reductant, suggesting ETP-PtFeNP a high-sensitivity T 2 contrast agent for magnetic resonance imaging.

Exploiting Uptake of Nanoparticles by Phagocytes for Cancer Treatment.

Many cancers including ovarian, pancreatic, colon, liver, and stomach cancers are largely confined to the peritoneal cavity. Peritoneal tumors are directly accessible by intraperitoneal injections. Previously we demonstrated that intraperitoneal injection of nanoparticles and subsequent ingestion by tumor-associated phagocytes can be used to either directly impact tumors or stimulate antitumor immune responses. Here we outline methods to specifically utilize iron oxide nanoparticles with the ID8-Defb29/Vegf-A murine ovarian cancer model and discuss the tendency of phagocytes to ingest nanoparticles and the potential of phagocytes to carry nanoparticles to tumors resulting in direct killing of tumor cells or stimulate antitumor immune responses in peritoneal cancers. This basic approach can be modified as needed for different types of tumors and nanoparticles.

The Different Functional Distribution of "Not Effector" T Cells (Treg/Tnull) in Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide, ranking as high as the second leading cause of cancer-related deaths in industrialized countries. Consistent with immunosurveillance theory, the immune system is crucial to protect the host from developing tumors, and the major players in tumoral immunity are effector T cells. Anyway, cancer cells develop strategies of immunoevasion influencing the cancer-specific lymphocyte priming, activation, and effector function. Therefore, the T cell subsets that mature during the stages of tumor growth, differently contribute to disease progression and/or regression. In our study, we analyzed the intra-tumoral and peripheral T cell subsets' distribution in 30 patients with CRC, in order to clarify their functional role toward cancer. We found that percentage of infiltrating effector T cells decreased in cancer tissue than in healthy mucosa and that the tumor microenvironment negatively influences the cytolytic activity of T lymphocytes reactive to cancer cells. Moreover, we found that the tumor tissue was infiltrated by a large amount of "not effector" T (neT) cells with a regulatory or an anergic profile, which are unable to kill cancer cells, may be contributing to the CRC promotion. The presence of neT cells was investigated also in the peripheral blood of CRC patients, demonstrating that the peripheral T regulatory cells can inhibit the proliferation of effector T cells, confirming their immunosuppressive properties. Finally, monitoring the changes in circulating neT cells' frequencies after the tumor removal, we confirmed the role of cancer in the modulation of immune system, in particular, in supporting a Tregs-mediated immunosuppression.