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The existence of various T regulatory cell (Treg) subsets in colorectal cancer (CRC) could play a variety of functions in the regulation of anti-cancer immunity. We studied correlations between CD4+ Treg subsets with the expression of immunological checkpoints on CD4+ T cells, including PD-1, TIM-3, LAG-3, and CTLA-4 in CRC patients with early and advanced TNM staging. Strong positive correlations were found between frequencies of FoxP3+ Tregs and FoxP3+Helios+ Tregs with frequencies of various immune checkpoint-expressing CD4+ T cells in the tumor microenvironment (TME). However, there were strong negative correlations between frequencies of FoxP3−Helios− T cells and these immune checkpoint-expressing CD4+ T cells. Specifically, in the TME, we found that the correlations between FoxP3+ Tregs, FoxP3+Helios+ Tregs, FoxP3+Helios− Tregs, and FoxP3−Helios− T cells with CD4+LAG-3+ T cells and CD4+CTLA-4+ T cells were higher in patients with early stages, suggesting the potential of these highly immunosuppressive cells in inhibiting inflammatory responses in the TME. However, the correlations between FoxP3+ Tregs, FoxP3+Helios+ Tregs, and FoxP3−Helios− T cells with CD4+TIM-3+ T cells were higher in patients with advanced stages. This is the first study to explore correlations of Treg subpopulations with immune checkpoint-expressing CD4+ T cells in CRC based on clinicopathological features of CRC patients. The findings of our study provide a justification for focusing on these cells that possess highly immunosuppressive features. Understanding the correlations between different immune checkpoints and Treg subsets in CRC patients has the potential to enhance our understanding of core mechanisms of Treg-mediated immunosuppression in cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: Colorectal cancer is one of the leading causes of global cancer-related mortality. Tumor-infiltrating effector immune cells play critical roles in tumor control, and their activity can dictate disease outcomes. In this study, we provide evidence of the associations between different CD8+ T cell subpopulations with disease-free survival (DFS) in CRC patients. We report associations between higher levels of certain circulating and tumor-infiltrating CD8+ T cell subsets and improved clinical outcomes in CRC patients. T cells in the tumor microenvironment (TME) have diverse roles in anti-tumor immunity, including orchestration of immune responses and anti-tumor cytotoxic attack. However, different T cell subsets may have opposing roles in tumor progression, especially in inflammation-related cancers such as colorectal cancer (CRC). In this study, we phenotypically characterized CD3+CD4- (CD8+) T cells in colorectal tumor tissues (TT), normal colon tissues (NT) and in circulation of CRC patients. We investigated the expression levels of key immune checkpoints (ICs) and Treg-related markers in CD8+ T cells. Importantly, we investigated associations between different tumor-infiltrating CD8+ T cell subpopulations and disease-free survival (DFS) in CRC patients. We found that FoxP3 expression and ICs including PD-1, CTLA-4, TIM-3, and LAG-3 were significantly increased in tumor-infiltrating CD8+ T cells compared with NT and peripheral blood. In the TME, we found that TIM-3 expression was significantly increased in patients with early stages and absent lymphovascular invasion (LVI) compared to patients with advanced stages and LVI. Importantly, we report that high levels of certain circulating CD8+ T cell subsets (TIM-3-expressing, FoxP3−Helios−TIM-3+ and FoxP3−Helios+TIM-3+ cells) in CRC patients were associated with better DFS. Moreover, in the TME, we report that elevated levels of CD25+ and TIM-3+ T cells, and FoxP3+Helios−TIM-3+ Tregs were associated with better DFS. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) has been raised as a pandemic disease since December 2019. Immunosuppressive cells including T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs) are key players in immunological tolerance and immunoregulation; however, they contribute to the pathogenesis of different diseases including infections. Tregs have been shown to impair the protective role of CD8+ T lymphocytes against viral infections. In COVID-19 patients, most studies reported reduction, while few other studies found elevation in Treg levels. Moreover, Tregs have a dual role, depending on the different stages of COVID-19 disease. At early stages of COVID-19, Tregs have a critical role in decreasing antiviral immune responses, and consequently reducing the viral clearance. On the other side, during late stages, Tregs reduce inflammation-induced organ damage. Therefore, inhibition of Tregs in early stages and their expansion in late stages have potentials to improve clinical outcomes. In viral infections, MDSC levels are highly increased, and they have the potential to suppress T cell proliferation and reduce viral clearance. Some subsets of MDSCs are expanded in the blood of COVID-19 patients; however, there is a controversy whether this expansion has pathogenic or protective effects in COVID-19 patients. In conclusion, further studies are required to investigate the role and function of immunosuppressive cells and their potentials as prognostic biomarkers and therapeutic targets in COVID-19 patients. [ABSTRACT FROM AUTHOR]

T regulatory cells (Tregs) play different roles in the regulation of anti-tumor immunity in colorectal cancer (CRC), depending on the presence of different Treg subsets. We investigated correlations between different CD4+ Treg/T cell subsets in CRC patients with immune checkpoint-expressing CD4+ T cells. Positive correlations were observed between levels of different immune checkpoint-expressing CD4+ T cells, including PD-1, TIM-3, LAG-3, and CTLA-4 with FoxP3+ Tregs, Helios+ T cells, FoxP3+Helios+ Tregs, and FoxP3+Helios− Tregs in the tumor microenvironment (TME). However, negative correlations were observed between levels of these immune checkpoint-expressing CD4+ T with FoxP3−Helios− T cells in the TME. These correlations in the TME highlight the role of cancer cells in the upregulation of IC-expressing Tregs. Additionally, positive correlations were observed between levels of FoxP3+ Tregs, Helios+ T cells, FoxP3+Helios+ Tregs, and FoxP3+Helios− Tregs and levels of CD4+CTLA-4+ T cells and CD4+PD-1+ T cells in peripheral blood mononuclear cells (PBMCs) and normal tissue-infiltrating lymphocytes (NILs). These observations suggest that CTLA-4 and PD-1 expressions on CD4+ T cell subsets are not induced only by the TME. This is the first study to investigate the correlations of different FoxP3+/−Helios+/− T cell subsets with immune checkpoint-expressing CD4+ T cells in CRC patients. Our data demonstrated strong correlations between FoxP3+/Helios+/− Tregs but not FoxP3−Helios+/− non-Tregs and multiple immune checkpoints, especially in the TME, providing a rationale for targeting these cells with highly immunosuppressive characteristics. Understanding the correlations between different immune checkpoints and Treg/T cell subsets in cancer patients could improve our knowledge of the underlying mechanisms of Treg-mediated immunosuppression in cancer. [ABSTRACT FROM AUTHOR]

There is an increased risk of colorectal cancer (CRC) development in patients with non-insulin-dependent type 2 diabetes. CD8+ T cells have been implicated in diabetes and are crucial for anti-tumor immunity. However, transcriptomic profiling for CD8+ T cells from CRC diabetic patients has not been explored. We performed RNA sequencing and compared transcriptomic profiles of CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) in CRC diabetic patients with CRC nondiabetic patients. We found that genes associated with ribogenesis, epigenetic regulations, oxidative phosphorylation and cell cycle arrest were upregulated in CD8+ TILs from diabetic patients, while genes associated with PI3K signaling pathway, cytokine response and response to lipids were downregulated. Among the significantly deregulated 1009 genes, 342 (186 upregulated and 156 downregulated) genes were selected based on their link to diabetes, and their associations with the presence of specific CRC pathological parameters were assessed using GDC TCGA colon database. The 186 upregulated genes were associated with the presence of colon polyps history (P = 0.0007) and lymphatic invasion (P = 0.0025). Moreover, CRC patients with high expression of the 186 genes were more likely to have poorer disease-specific survival (DSS) (Mantel–Cox log-rank P = 0.024) than those with low score. Our data provide novel insights into molecular pathways and biological functions, which could be altered in CD8+ TILs from CRC diabetic versus nondiabetic patients, and reveal candidate genes linked to diabetes, which could predict DSS and pathological parameters associated with CRC progression. However, further investigations using larger patient cohorts and functional studies are required to validate these findings. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID‐19) is caused by SARS‐CoV‐2, a novel coronavirus strain. Some studies suggest that COVID‐19 could be an immune‐related disease, and failure of effective immune responses in initial stages of viral infection could contribute to systemic inflammation and tissue damage, leading to worse disease outcomes. T cells can act as a double‐edge sword with both pro‐ and anti‐roles in the progression of COVID‐19. Thus, better understanding of their roles in immune responses to SARS‐CoV‐2 infection is crucial. T cells primarily react to the spike protein on the coronavirus to initiate antiviral immunity; however, T‐cell responses can be suboptimal, impaired or excessive in severe COVID‐19 patients. This review focuses on the multifaceted roles of T cells in COVID‐19 pathogenesis and rationalizes their significance in eliciting appropriate antiviral immune responses in COVID‐19 patients and unexposed individuals. In addition, we summarize the potential therapeutic approaches related to T cells to treat COVID‐19 patients. These include adoptive T‐cell therapies, vaccines activating T‐cell responses, recombinant cytokines, Th1 activators and Th17 blockers, and potential utilization of immune checkpoint inhibitors alone or in combination with anti‐inflammatory drugs to improve antiviral T‐cell responses against SARS‐CoV‐2. [ABSTRACT FROM AUTHOR]

T Regulatory cells (Tregs) can have both protective and pathological roles. They maintain immune homeostasis and inhibit immune responses in various diseases, including cancer. Proportions of Tregs in the peripheral blood of some cancer patients increase by approximately two-fold, compared to those in healthy individuals. Tregs contribute to cancer development and progression by suppressing T effector cell functions, thereby compromising tumor killing and promoting tumor growth. Highly immunosuppressive Tregs express upregulated levels of the transcription factor, Forkhead box protein P3 (FoxP3). Elevated levels of FoxP3+ Tregs within the tumor microenvironment (TME) showed a positive correlation with poor prognosis in various cancer patients. Despite the success of immunotherapy, including the use of immune checkpoint inhibitors, a significant proportion of patients show low response rates as a result of primary or acquired resistance against therapy. Some of the mechanisms which underlie the development of therapy resistance are associated with Treg suppressive function. In this review, we describe Treg contribution to cancer development/progression, and the mechanisms of Treg-mediated immunosuppression. We discuss the prognostic significance of FoxP3+ Tregs in different cancers and their potential use as prognostic biomarkers. We also describe potential therapeutic strategies to target Tregs in combination with other types of immunotherapies aiming to overcome tumor resistance and improve clinical outcomes in cancer patients. Overall, understanding the prognostic significance of FoxP3+ Tregs in various cancers and their contribution to therapy resistance could help in the development of more effective targeted therapeutic strategies to enhance the clinical outcomes in cancer patients. [ABSTRACT FROM AUTHOR]

Despite recent advances in colorectal cancer (CRC) treatment, a large proportion of patients show limited responses to therapies, especially in advanced stages. There is an urgent need to identify prognostic biomarkers and/or therapeutic targets in advanced stages, aiming to improve the efficacy of current treatments. We aimed to determine prognostic biomarkers in tumor tissue and circulation of CRC patients, with a special focus on T cell exhaustion markers. We found that mRNA levels of PD-1, TIM-3, CTLA-4, TIGIT, CD160, CD244, KLRG1, TOX2, TOX3, Ki-67, and PRDM1 were elevated in CRC tumor tissues. We also investigated differences in gene expression between early and advanced disease stages. We found that TOX and potentially TIM-3, CTLA-4, VISTA, TIGIT, KLRG1, TOX2, SIRT1, Ki-67, and Helios mRNA levels in tumor tissue were elevated in advanced disease stages, suggesting their potential roles in CRC progression. In contrast, PD-1 and CD160 levels in tumor tissue were downregulated in advanced stages. In the circulation of CRC patients, mRNA levels of PD-1, VISTA and LAG-3 were higher than those of healthy individuals. Moreover, in circulation, PD-1, CTLA-4 and TIGIT mRNA levels were reduced in advanced stages. Interestingly, levels of PD-1 in both tumor tissue and circulation were reduced in advanced stages, suggesting that targeting PD-1 in patients with advanced stages could be less effective. Altogether, these findings suggest some potential T cell exhaustion markers that could be utilized as prognostic biomarkers and/or therapeutic targets for CRC. However, further investigations and validations in larger cohorts are required to confirm these findings. [ABSTRACT FROM AUTHOR]

Lactate dehydrogenase C (LDHC) is an archetypical cancer testis antigen with limited expression in adult tissues and re-expression in tumors. This restricted expression pattern together with the important role of LDHC in cancer metabolism renders LDHC a potential target for immunotherapy. This study is the first to investigate the immunogenicity of LDHC using T cells from healthy individuals. LDHC-specific T cell responses were induced by in vitro stimulation with synthetic peptides, or by priming with autologous peptide-pulsed dendritic cells. We evaluated T cell activation by IFN-γ ELISpot and determined cytolytic activity of HLA-A*0201-restricted T cells in breast cancer cell co-cultures. In vitro T cell stimulation induced IFN-γ secretion in response to numerous LDHC-derived peptides. Analysis of HLA-A*0201 responses revealed a significant T cell activation after stimulation with peptide pools 2 (PP2) and 8 (PP8). The PP2- and PP8-specific T cells displayed cytolytic activity against breast cancer cells with endogenous LDHC expression within a HLA-A*0201 context. We identified peptides LDHC41−55 and LDHC288−303 from PP2 and PP8 to elicit a functional cellular immune response. More specifically, we found an increase in IFN-γ secretion by CD8 + T cells and cancer-cell-killing of HLA-A*0201/LDHC positive breast cancer cells by LDHC41−55- and LDHC288−303-induced T cells, albeit with a possible antigen recognition threshold. The majority of induced T cells displayed an effector memory phenotype. To conclude, our findings support the rationale to assess LDHC as a targetable cancer testis antigen for immunotherapy, and in particular the HLA-A*0201 restricted LDHC41–55 and LDHC288–303 peptides within LDHC. [ABSTRACT FROM AUTHOR]

Tumor-infiltrating lymphocytes (TILs) play indispensable roles in the progression and response to treatment of solid tumors. However, the prognostic significance of CD4+ TILs is not fully disclosed in cancers generally and in CRC in particular, mainly due to the existence of different functional subsets of CD4+ T cells. We performed transcriptomic profiling of CD4+ TILs isolated from CRC patients in order to identify differentially expressed genes and their functional pathways in early versus advanced disease stages. We found that in advanced stages, genes related to immune and inflammatory responses, in particular Th1-mediated immune response and cytotoxicity-mediated genes, were downregulated; while epigenetic-mediated silencing genes were upregulated. Interestingly, we identified genes, which were steadily upregulated or downregulated in CD4+ TILs with CRC progression from stage I to IV. Additionally, of the top 200 deregulated genes, 43 upregulated and 64 downregulated genes showed similar deregulation trends in the cancer genome atlas CRC dataset. From these 97 deregulated genes, we identified a "poor prognosis CD4 gene signature (ppCD4sig)". Patients with high ppCD4sig score showed shorter disease-specific survival (DSS) and progression-free interval (PFI). The ppCD4sig was an independent prognostic indicator for DSS (HR = 1.73, 95% CI 1.32–2.27, P = 0.0001) and PFI (HR = 1.75, 95% CI 1.3–2.35, P = 0.0016). Additionally, patients at advanced stages and at a younger age (<55 years) were more likely to have a high ppCD4sig score. Altogether, our data provide novel insights and a unique prognostic gene signature of CD4+ TILs in the CRC microenvironment. [ABSTRACT FROM AUTHOR]

Blockade of inhibitory immune checkpoints (ICs) is a promising therapeutic approach; however, it has shown limited success in some cancers including colorectal cancer (CRC). The tumor microenvironment (TME) is largely responsible for response to therapy, and its constituents may provide robust biomarkers for successful immunotherapeutic approaches. In this study, we performed phenotypical characterization and critical analyses of key inhibitory ICs and T regulatory cell (Treg)-related markers on CD4+ T cell subsets in CRC patients, and compared with normal colon tissues and peripheral blood from the same patients. We also investigated correlations between the levels of different CD4+ T cell subsets and the clinicopathologic features including disease stage and tumor budding. We found a significant increase in the levels of CD4+FoxP3+Helios+ T cells, which represent potentially highly immunosuppressive Tregs, in the CRC TME. Additionally, tumor-infiltrating CD4+ T cells upregulated programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), T cell immunoglobulin and mucin domain-3 (TIM-3) and lymphocyte-activation gene 3 (LAG-3). We also characterized the expression of PD-1, CTLA-4, TIM-3, and LAG-3 on different CD4+FoxP3−/+Helios−/+ T cell subsets. Interestingly, we found that CTLA-4, TIM-3, and LAG-3 were mainly co-expressed on FoxP3+Helios+ Tregs in the TME. Additionally, FoxP3high Tregs expressed higher levels of Helios, CTLA-4 and TIM-3 than FoxP3low T cells. These results highlight the significance of Tregs in the CRC TME and suggest that Tregs may hamper response to IC blockade in CRC patients, but effects of different IC inhibition regimes on Treg levels or activity warrants further investigations. We also found that CD4+CTLA-4+ T cells in circulation are increased in patients with advanced disease stage. This study simultaneously provides important insights into the differential levels of CD4+ T cell subpopulations and IC expression in CRC TME, compared to periphery and associations with clinicopathologic features, which could be used as potential biomarkers for CRC progression and response to therapy. [ABSTRACT FROM AUTHOR]

T Regulatory cells (Tregs) act as a double-edged sword by regulating immune homeostasis (protective role) and inhibiting immune responses in different disease settings (pathological role). They contribute to cancer development and progression by suppressing T effector cell (Teff) functions. Decreased ratios of intratumoral CD8+ T cells to Tregs have been associated with poor prognosis in most cancer types. Targeting immune checkpoints (ICs), such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death-1 (PD-1), by immune checkpoint inhibitors (ICIs) in cancer patients has been beneficial in inducing anti-tumor immune responses and improving clinical outcomes. However, response rates remain relatively low, ranging from 15 to 40% depending on cancer type. Additionally, a significant proportion of patients who initially demonstrates a clinical response can acquire resistance overtime. This acquired resistance could occur due to the emergence of compensatory mechanisms within the tumor microenvironment (TME) to evade the anti-tumor effects of ICIs. In this review, we describe the immunosuppressive role of Tregs in the TME, the effects of currently approved ICIs on Treg phenotype and function, and the mechanisms of acquired resistance to ICIs mediated by Tregs within the TME, such as the over-expression of ICs, the up-regulation of immunosuppressive molecules, and apoptotic Treg-induced immunosuppression. We also describe potential therapeutic strategies to target Tregs in combination with ICIs aiming to overcome such resistance and improve clinical outcomes. Elucidating the Treg-mediated acquired resistance mechanisms should benefit the designing of well-targeted therapeutic strategies to overcome resistance and maximize the therapeutic efficacy in cancer patients. [ABSTRACT FROM AUTHOR]

Immune evasion is a characteristic of most human malignancies and is induced via various mechanisms. Immunosuppressive cells, including myeloid‐derived suppressor cells (MDSC) and regulatory T cells (Treg), are key mediators in assisting tumors to escape immune surveillance. Expansion of MDSC, Treg and elevated levels of immune checkpoints (IC) are frequently detected in the tumor microenvironment and periphery of cancer patients. Various therapeutic agents have been shown to target MDSC and to block IC for inducing anti‐tumor immunity and reversal of tumor immune escape. Importantly, some recent studies have shown that MDSC targeting improves the efficacy of IC blockade in cancer therapy. However, there is a pressing need to improve our understanding of the distinct role of these cells to develop combination therapy that attacks tumor cells from all frontiers to improve cancer therapeutics. Herein, we discuss the role of MDSC in cancer progression, interactions with IC in the context of anti‐cancer immunity and the current therapeutic strategies to target MDSC and block IC in cancer. Expansion of MDSC, Treg and elevated expression levels of immune checkpoints (IC) are frequently detected in the tumor microenvironment and periphery of cancer patients. Recent studies have shown that MDSC targeting improves the efficacy of IC blockade in cancer therapy. Herein, we discuss the role of MDSC in cancer progression, interactions with IC and the current therapeutic strategies to target MDSC and block IC in cancer. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) can be antitumorigenic or pro-tumorigenic in colorectal cancer (CRC) depending on the presence of different Treg subsets with various immunosuppressive molecules. Some studies reported the phenotypic characteristics of tumor-infiltrating immune cells in CRC, but limited studies have focused on the co-expression of suppressive molecules on immune cells. The aim of this study was to characterize immune cells in the tumor microenvironment (TME), compared to paired adjacent non-tumor colon tissue of CRC patients. Additionally, we investigated co-expression of immunosuppressive molecules on different Treg subsets in the TME, normal colon tissue, and peripheral blood of CRC patients and healthy donors. In this preliminary study, we report that the majority of CD3+ T cells in the TME are CD4+ T cells with high co-expression of programmed death 1 (PD-1)/cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and PD-1/CD39 molecules. Levels of CD4+FoxP3+Helios+ Tregs were significantly increased in the TME. Furthermore, we observed increased levels of PD-1/CTLA-4 and PD-1/CD39 co-expressing cells within FoxP3+Helios+ and FoxP3+Helios− Treg subsets, indicative of their potent immunosuppressive potential. These results suggest synergistic associations between PD-1/CTLA-4 and PD-1/CD39 in dampening T-cell activation and function along with suppressing tumor-specific immune responses, suggesting that dual blockade of these molecules could be a more effective strategy for inducing antitumor immune responses in CRC. [ABSTRACT FROM AUTHOR]

Exhaustion of immune cells in COVID-19 remains a serious concern for infection management and therapeutic interventions. As reported, immune cells such as T effector cells (Teff), T regulatory cells (Tregs), natural killer cells (NKs), and antigen-presenting cells (APCs) exhibit uncontrolled functions in COVID-19. Unfortunately, the mechanisms that orchestrate immune cell functionality and virus interaction are still unknown. Recent studies linked adaptive immune cell exhaustion to underlying epigenetic mechanisms that regulate the epigenetic transcription of inhibitory immune checkpoint receptors (ICs). Further to that, the over-activation of T cells accompanied by the dysfunctionality of DCs and Tregs may enhance uncontrollable alveoli inflammation and cytokine storm in COVID-19. This might explain the reasons behind the failure of DC-based vaccines in inducing sufficient anti-viral responses. This review explains the processes behind the over-activation and exhaustion of innate and adaptive immune cells in COVID-19, which may contribute to developing novel immune intervention strategies. • Overexpression of ICs and mitochondrial impairment in T cells contribute to increasing the severity of COVID-19. • T cell Exhaustion is caused primarily by ICs overexpression, even in low cytokine-producing T cells. • Over-activation of T cells in COVID-19 may elicit epithelial cell apoptosis and cytokine storm initiation. • SARS-CoV-2 promotes ICs upregulation by increasing epigenetic chromatin accessibility and ICs transcription. • Interaction between SARS-CoV-2 ORF8/ORF6 proteins and MHCI/II promotes the exhaustion of cytotoxic immune cells. [ABSTRACT FROM AUTHOR]

Among the different immunotherapy approaches, immune checkpoint inhibition (ICI) therapy and chimeric antigen receptor (CAR) T cells have arguably shown the greatest potential. Keywords: CD4; immunotherapy; immune checkpoint inhibitor (ICI); cancer; T lymphocyte EN CD4 immunotherapy immune checkpoint inhibitor (ICI) cancer T lymphocyte 1 2 2 09/08/21 20210906 NES 210906 In the last 20 years, remarkable advances in the field of immunotherapy have led to new therapeutic options for patients with a wide range of cancer types. [Extracted from the article]

Abstract: Conversion of conventional T cells into T regulatory cells (Tregs) has been proposed as a potential mechanism for Treg expansion in cancer. However, this evidence is supported by in vitro or mouse model studies with no data from in vivo or human studies to support its role in enriching peripheral and tumor-infiltrating Tregs. Recent work has shown that induced FoxP3+ Tregs (iTregs) do not express Helios; an Ikaros family transcription factor. We analyzed peripheral blood samples from untreated renal cell carcinoma (RCC) patients and following interleukin (IL)-2 treatment for the expression of FoxP3 and Helios. Our work shows that expanded peripheral FoxP3+ Tregs in untreated RCC patients co-express Helios. Interestingly, IL-2 administration results in expansion of FoxP3+Helios+ natural Tregs (nTregs) significantly more than FoxP3+Helios− iTregs. Our work shows that the increased FoxP3+ Treg subpopulation in RCC patients co-express Helios, indicating that they could be derived from natural but not induced Tregs. [Copyright &y& Elsevier]

Regulatory T cells (Treg) are a sub-population of T cells that suppress self-reactivity and are implicated in immune tolerance towards malignant cells. Circulating Treg cells are increased in several cancers. In endometrial cancer Treg cells have been investigated only in tumour tissues and, in contrast to some other tumours, fewer Treg cells were reported in endometrial cancer compared with benign controls. Flow cytometry was used to determine the frequency of circulating Treg cells in women undergoing hysterectomy for either endometrial cancer (n = 24) or non- cancer-related conditions (n = 21). Circulating Treg cells were more abundant in women with cancer compared to those without (4.68% vs. 3.66%, p = 0.05, Mann-Whitney test). This relationship disappeared, however, when only data from post-menopausal women were included in the analysis. Mean Treg cell frequency was 4.65% in postmenopausal women with cancer (n = 23) and 4.73% in postmenopausal controls (n = 5) ( p = 0.9). In women without cancer we found that mean Treg cell frequency was higher in postmenopausal women (4.73%, n = 5) in comparison to premenopausal controls (3.33%, n = 16) ( p = 0.02). These results suggest that the increased proportion of Treg cells seen in endometrial cancer patients might be, at least in part, attributed to their postmenopausal status or age. [ABSTRACT FROM AUTHOR]

Abstract: Cryopreservation of peripheral blood mononuclear cells (PBMC) is essential for many clinical and research assays. Some studies reported consistent changes in PBMC phenotype following cryopreservation. We hypothesized that PBMC freezing may have a negative impact on estimation of the frequency of T regulatory cell (Treg). Treg levels were measured in 6 fresh PBMC samples isolated from 6 healthy donors and these levels were re-measured after freezing for three weeks. Herein, we report a significant reduction in Treg frequency in all samples following cryopreservation. [Copyright &y& Elsevier]

The human 5T4 (h5T4) oncofoetal antigen is expressed by a wide variety of human carcinomas including colorectal, ovarian, gastric and renal, but rarely on normal tissues. Its restricted expression on tumour tissues as well as its association with tumour progression and bad prognosis has driven the development of a MVA-based vaccine (TroVax) which has been tested in several early phase clinical trials and these studies have led to the start of a phase III trial in renal cell carcinoma patients. We have recently shown that CD8+ T cells recognizing h5T4 can be generated in the absence of CD4+ T cells from peripheral blood lymphocytes of human healthy individuals. We report the existence and expansion of human CD4+ T cells against h5T4 by stimulation with autologous monocyte-derived dendritic cells infected with a replication defective adenovirus encoding the h5T4 cDNA (Ad-h5T4). The h5T4-specific T-cell responses in normal individuals are enhanced by initial depletion of CD25+ cells (putative T regulatory cells) prior to the in vitro stimulation. We have identified a novel h5T4-derived 15-mer peptide recognized by CD4+ T cells in HLA-DR4 positive healthy individuals. Interestingly, CD4+ T cells spontaneously recognizing a different 5T4 epitope restricted by HLA-DR were identified in tumour-infiltrating lymphocytes isolated from a regressing renal cell carcinoma lung metastasis. Our data show that CD4+ T cells recognizing h5T4 can be expanded and detected in healthy individuals and a renal cell carcinoma patient. Such h5T4-specific CD4+ T cells boosted or induced by vaccination could act to modulate both cell or antibody mediated anti-tumour responses. [ABSTRACT FROM AUTHOR]

CD4+CD25+ regulatory T (Treg) cells are present in increased numbers in patients with advanced cancer and CD25+ T cell depletion potentiates tumour immunity in animal models. The aim of this study was to assess the feasibility and safety of adoptive transfer of CD25+ depleted autologous T cells in patients with advanced renal cell carcinoma and to examine resulting changes in lymphocyte subsets. Six patients with advanced renal cell carcinoma underwent leukapheresis followed by conditioning chemotherapy with cyclophosphamide and fludarabine. The autologous leukapheresis product was depleted of CD25+ cells using CliniMACS® System then re-infused into the patient. Efficient CD25+ depletion from all leukapheresis products was achieved and 0.55–5.87 × 107/kg CD3+ cells were re-infused. Chemotherapy related haematological toxicity was observed, but blood counts recovered in all patients allowing discharge after a mean inpatient stay of 21 days. One patient subsequently developed a rapidly progressive neurological syndrome. A transient reduction in CD25+ subset was noted in the peripheral blood of 5 out of 6 patients with evidence of increased T cell responses to PHA in 4 out of 6 patients. One patient showed increased specific proliferative responses to the tumour associated antigen h5T4 coinciding with the nadir of Treg cells. Given the transient nature of the reduction in CD25+ subset and the observed toxicity there is a need to explore further strategies to improve the safety and efficacy of this approach. Nevertheless, the results provide proof of concept in potentiation of tumour antigen T cell responses when Treg cell levels are depleted. [ABSTRACT FROM AUTHOR]

Regulatory T cells are important in maintaining immune homeostasis, mediating peripheral tolerance and preventing autoimmunity. Increased frequencies of CD4+CD25high T regulatory (TReg) cells have been documented in the peripheral blood of patients with several types of cancer consistent with a role in tumour escape from immunological control. We have investigated the presence of TReg cells systemically and in situ in previously untreated patients with renal cell carcinoma (RCC). We have shown that there is a significant increased frequency of CD4+CD25high T cells in RCC patients ( n = 49) compared to normal donors ( n = 38), respectively, 2.47% versus 1.50%; P < 0.0001. We confirmed these data using the FOXP3 marker of TReg cells in a subset of these patients and normal donors. The population of TReg cells identified showed the expected phenotype with CD4+CD25high population in both RCC patients and normal donors contained higher proportions of CD45RO and GITR than CD4+CD25−/low populations and exhibiting suppressive activity in an anti-CD3 and anti-CD28 induced proliferation assay. CD4+FOXP3+ T cells were detected in the tumour microenvironment by immunofluorescence and the numbers enumerated in lymphocytes recovered following enzymatic disaggregations of biopsies; their frequency was higher in the tumour than the peripheral blood of the same patients. The early follow up data show an association between higher peripheral blood regulatory T-cell count and adverse overall survival. These data confirm the increase of TReg cells in RCC patients and provide impetus to further investigate modulation of TReg activity in RCC patients as part of therapy. [ABSTRACT FROM AUTHOR]

Abstract: Modest work has been performed to improve the sensitivity of residual disease detection or investigate the contribution that the immune system makes in controlling metastatic tumor growth, in particular, the frequency and biological actions of peptide-specific CD8+ T lymphocytes in limiting metastatic disease and/or maintaining remission. Fifty-three peripheral blood samples from 32 prostate cancer (PC) patients were investigated for the presence of circulating prostate-specific antigen (PSA)-expressing cells (CPECs) using a highly sensitive and specific assay combining immunomagnetic epithelial cell enrichment with nested RT-PCR of PSA mRNA. Using HLA-A2 tetramer complexes, frequency of CD8+ T cells specific for PSA-derived peptides was determined. Additionally, serum concentrations of PSA and testosterone were measured. CPECs were detected in 26% of peripheral blood samples from PC patients. CD8+ T cells specific for PSA-derived peptides were detected at low frequency in HLA-A2-positive PC patients. The correlation between these PSA-specific CD8+ T cells and residual prostate tumor cells and clinical measures was investigated. Our data suggest that frequency of PSA-specific CD8+ T cells is correlated to CPECs, but not to serum PSA level. [Copyright &y& Elsevier]

Colorectal cancer (CRC) is influenced by infiltration of immune cell populations in the tumor microenvironment. While elevated levels of cytotoxic T cells are associated with improved prognosis, limited studies have reported associations between CD4+ T cells and disease outcomes. We recently performed transcriptomic profiling and comparative analyses of sorted CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) from bulk tumors of CRC patients with varying disease stages. In this study, we compared the transcriptomes of CD4+ with CD8+ TILs. Functional annotation pathway analyses revealed the downregulation of inflammatory response-related genes, while T cell activation and angiogenesis-related genes were upregulated in CD4+ TILs. The top 200 deregulated genes in CD4+ TILs were aligned with the cancer genome atlas (TCGA) CRC dataset to identify a unique gene signature associated with poor prognosis. Moreover, 69 upregulated and 20 downregulated genes showed similar trends of up/downregulation in the TCGA dataset and were used to calculate "poor prognosis score" (ppScore), which was significantly associated with disease-specific survival. High ppScore patients showed lower expression of Treg-, Th1-, and Th17-related genes, and higher expression of Th2-related genes. Our data highlight the significance of T cells within the TME and identify a unique candidate prognostic gene signature for CD4+ TILs in CRC patients. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibition is an effective anti-cancer therapeutic approach but has shown limited efficacy in treating colorectal cancer (CRC) patients. Importantly, immune constituents of the tumor microenvironment (TME) can influence therapy response and cancer progression. We investigated the expression of immune checkpoints (ICs) on lymphoid populations within the CRC TME and compared with cells from normal colon tissues using samples from 50 patients with varying disease stages. We found that the levels of B cells, T cells, and NK cells were similar, IC-expressing CD4+ and CD4+CD8+ double positive T cells were higher, while CD8+ T cells and CD4−CD8− double negative T cells were significantly lower in CRC tumors. Notably, patients with mismatch-repair deficiency/microsatellite instability-high tumors had higher levels of IC-expressing CD4+ and CD8+ T cells than patients with proficient MMR and microsatellite stable tumors. Lastly, The Cancer Genome Atlas Colon Adenocarcinoma datasets showed associations between low expression of selective genes and poorer progression-free interval. Our findings highlight differential expression of ICs on lymphoid cells in CRC tumors in the era of cancer immunotherapy, which at present is solely approved for anti-PD-1 therapy in patients with dMMR/MSI-H tumors. Further investigations into their functionality have potentials for deciphering resistance mechanisms to IC inhibition. [ABSTRACT FROM AUTHOR]

T cell immunoglobulin mucin-3 (TIM-3) is an immune checkpoint identified as one of the key players in regulating T-cell responses. Studies have shown that TIM-3 is upregulated in the tumor microenvironment (TME). However, the precise role of TIM-3 in colorectal cancer (CRC) TME is yet to be elucidated. We performed phenotypic and molecular characterization of TIM-3+ T cells in the TME and circulation of CRC patients by analyzing tumor tissues (TT, TILs), normal tissues (NT, NILs), and peripheral blood mononuclear cells (PBMC). TIM-3 was upregulated on both CD4+ and CD3+CD4− (CD8+) TILs. CD4+TIM-3+ TILs expressed higher levels of T regulatory cell (Tregs)-signature genes, including FoxP3 and Helios, compared with their TIM-3− counterparts. Transcriptomic and ingenuity pathway analyses showed that TIM-3 potentially activates inflammatory and tumor metastatic pathways. Moreover, NF-κB-mediated transcription factors were upregulated in CD4+TIM-3+ TILs, which could favor proliferation/invasion and induce inflammatory and T-cell exhaustion pathways. In addition, we found that CD4+TIM-3+ TILs potentially support tumor invasion and metastasis, compared with conventional CD4+CD25+ Tregs in the CRC TME. However, functional studies are warranted to support these findings. In conclusion, this study discloses some of the functional pathways of TIM-3+ TILs, which could improve their targeting in more specific therapeutic approaches in CRC patients. [ABSTRACT FROM AUTHOR]

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype, and it exhibits resistance to common breast cancer therapies. Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1 (PD-1) and its ligand, PD-L1, have been approved to treat various cancers. However, the therapeutic efficacy of targeting PD-1/PD-L1 axis in breast cancer is under clinical investigation. In addition, the mechanisms of action of drugs targeting PD-1 and PD-L1 have not been fully elucidated. In this study, we investigated the effect of human TNBC cell lines, MDA-MB-231 and MDA-MB-468, and the non-TNBC cell line, MCF-7, on the expression of immune checkpoints (ICs) on CD4+ T cell subsets, including regulatory T cells (Tregs), using a co-culture system. We also examined the effect of blocking PD-1 or PD-L1 separately and in combination on IC expression by CD4+ T cell subsets. We found that breast cancer cells upregulate the expression of ICs including PD-1, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) and lymphocyte activation gene-3 (LAG-3) in CD4+ T cell subsets. We also found that the co-blockade of PD-1 and PD-L1 further upregulates the co-expression of TIM-3 and LAG-3 on CD4+CD25+ T cells and CD4+CD25+FoxP3+Helios+ Tregs in the presence of TNBC cells, but not in non-TNBC cells. Our results indicate the emergence of compensatory inhibitory mechanisms, most likely mediated by Tregs and activated non-Tregs, which could lead to the development of TNBC resistance against PD-1/PD-L1 blockade. [ABSTRACT FROM AUTHOR]

A letter to the editor in response to an article "Expression of Helios in Peripherally Induced Foxp3+ Regulatory T-Cells" that was published in the previous issue of the journal.

Interactions between immune checkpoints (ICs) and their ligands negatively regulate T cell activation pathways involved in physiological immune responses against specific antigens. ICs and their ligands are frequently upregulated in the tumor microenvironment (TME) of various malignancies, and they represent significant barriers for induction of effective anti-tumor immune responses. Several IC inhibitors (ICIs) have been developed, with some currently in clinical trials and others have been approved for the treatment of different cancers. However, tumor cells are able to counteract the activity of ICIs and can commission additional inhibitory pathways via expression of other ICs/ligands within the TME. This review discusses the expression of various ICs/ligands in the TME and their impact on tumor immune evasion. Additionally, we discuss various regulatory mechanisms, including genetic and epigenetic, and other modulatory factors including hypoxia and the presence of immunosuppressive populations in the TME, which result in upregulation of ICs in various cancers. Moreover, we discuss the prognostic significance of ICs and their ligands, and the potential strategies to enhance treatment responses to ICIs. This review aims to advance our current knowledge on the role of ICs in the TME and the clinical benefits of targeting them. [ABSTRACT FROM AUTHOR]

• Boswellic acids have multiple beneficial pharmacological activities. • AKBA and β-BA have the ability to reduce proliferation and activation of T cells without cytotoxicity. • AKBA exhibits greater anti-proliferative effects and inhibits CD25 expression on T cells at a low concentration without affecting cell viability. • ABA has cytotoxic effects on T cells. Boswellic acids have been recognized as anti-inflammatory and immunomodulatory agents with potentials to control autoimmune and inflammatory diseases. However, their effects on T cell proliferation and activation are not fully elucidated. In this study, we investigated effects of individual compounds including β-Boswellic acids (β-BA), 11-keto-β-Boswellic acid (β-KBA), 3- O -acetyl β-Boswellic acids (β-ABA), and 3- O -acetyl-11-keto-β-Boswellic acid (β-AKBA) on human peripheral blood mononuclear cells (PBMCs) and their potential role in modulating immune responses. We showed that β-BA, KBA, and AKBA at a 0.025 µM concentration significantly reduced T cell proliferation without inducing cytotoxicity, however, ABA showed cytotoxic effects at this concentration. β-BA and KBA showed significantly reduced T cell proliferation at 0.05 µM concentration without cytotoxic effects. Interestingly, we found that AKBA at 0.025 µM concentration significantly reduced CD25 expression on both CD4+ and CD8+ T cells without cytotoxic effects. Additionally, β-BA reduced CD25 expression on both CD4+ and CD8+ T cells at 0.05 µM concentration with no cytotoxicity. In this study, we determined the optimum concentration of each of these compounds that have the potential to reduce T cell activation without cytotoxic effects. Our findings show that both β-BA and AKBA have the ability to inhibit T cell proliferation and activation without inducing cytotoxicity. Further investigations are required to fully understand the mechanisms underlying these effects and the potential therapeutic benefits of these compounds in different autoimmune and inflammatory settings. [ABSTRACT FROM AUTHOR]

• Tumor-infiltrating lymphocytes are key players in predicting prognoses of cancer patients. • High levels of PD1+ with TIGIT+ or PD-1+ with TIM-3+ CD8+ TILs are associated with improved DFS. • Combination of immune checkpoints on CD8+ TILs are more sensitive predictive biomarkers in CRC patients than single ones. Immune checkpoints (ICs) are highly expressed on tumor-infiltrating immune cells (TIICs) in different malignancies, including colorectal cancer (CRC). T cells play crucial roles in shaping CRC, and their presence in the tumor microenvironment (TME) has proven to be one of the best predictors of clinical outcomes. A crucial component of the immune system is cytotoxic CD8+ T cells (CTLs), which play decisive roles in the prognosis of CRC. In this study, we investigated associations of immune checkpoints expressed on tumor-infiltrating CD8+ T cells with disease-free survival (DFS) in 45 naïve-treatment CRC patients. First, we examined the associations of single ICs, and found that CRC patients with higher levels of T-cell immunoglobulin and ITIM-domain (TIGIT), T-cell immunoglobulin and mucin domain-3 (TIM-3) and programmed cell death-1 (PD-1) CD8+ T cells tended to have longer DFS. Interestingly, when PD-1 expression was combined with other ICs, there were more evident and stronger associations between higher levels of PD-1+ with TIGIT+ or PD-1+ with TIM-3+ tumor-infiltrating CD8+ T cells and longer DFS. Our findings for TIGIT were validated in The Cancer Genome Atlas (TCGA) CRC dataset. This study is the first to report on the association of co-expression of PD-1 with TIGIT and PD-1 with TIM-3 in CD8+ T cells and improved DFS in treatment-naïve CRC patients. This work highlights the significance of immune checkpoint expression on tumor-infiltrating CD8+ T cells as critical predictive biomarkers, especially when co-expression of different ICs is considered. [ABSTRACT FROM AUTHOR]

Abstract: Cytotoxic T Lymphocyte Antigen 4 (CTLA4) blockade has shown antitumor activity against common cancers. However, the exact mechanism of immune mediation by anti-CTLA4 remains to be elucidated. Further understanding of how CTLA4 blockade with tremelimumab mediates immune responses may allow a more effective selection of responsive patients. Our results show that tremelimumab enhanced the proliferative response of T effector cells (Teff) upon TCR stimulation, and abrogated Treg suppressive ability. In the presence of tremelimumab, frequencies of IL-2-secreting CD4+ T cells and IFN-γ-secreting CD4+ and CD8+ T cells were increased in response to polyclonal activation and tumor antigens. Importantly, Treg frequency was not reduced in the presence of tremelimumab, and expanded Tregs in cancer patients treated with tremelimumab expressed FoxP3 with no IL-2 release, confirming them as bona fide Tregs. Taken together, this data indicates that tremelimumab induces immune responses mainly by direct activation of Teff rather than by affecting Tregs. [ABSTRACT FROM AUTHOR]

Flow cytometry and fluorescence-activated cell sorting have become invaluable tools to analyze and isolate specific cell populations in a wide range of biomedical research and clinical applications. In countless approaches worldwide, scientists are using single cell analyses to better understand the significance and variation within different cellular populations, and fluorescence-activated cell sorting has become a major technique for cell isolation in both basic and clinical research. However, majority of available cell sorters are pressurized, droplet-based systems, which apply significant environmental pressure and shear stress to cells during sorting. Recently, the flow cytometry community has become increasingly aware about the potential negative effects this could have on sorted cells and the term "sorter induced cell stress" (SICS) has been proposed. However, up to date only a limited number of studies have investigated the effects of cell sorting on cell viability and function. Therefore, solid data on the effects of sheath pressure and nozzle size on survival and function of sorted cells are surprisingly rare. With this in mind, we sorted "CD4+" T-cells and "live" cells from human peripheral blood mononuclear cells (PBMCs) at different sort conditions and analyzed their quality before and after sorting in a series of assays. Here we present our findings in reference to cell viability and cell proliferation following sorting on different instruments (BD FACSAria III SORP and BD FACSJazz), utilizing different nozzle sizes (70 to 100 μm) and sheath pressure settings (20 to 70 psi). The results show no significant differences in cell viability and proliferation after the different tested sort conditions, but rather differences between individual experiments. These findings are evaluated and their potential significance in cell sorting experiments is discussed. • Analyzing the effect of sorter induced stress on cell viability and proliferation • Human PBMCs were sorted using different sorters and nozzles • Viability and proliferation of sorted cells were studied by 7AAD and CFSE • Sorting at different conditions did not reduce cell viability • Cell proliferation was not altered after different sort conditions for up to 7 days [ABSTRACT FROM AUTHOR]

T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) is an inhibitory immune checkpoint, which suppresses anti-tumor immune responses. TIM-3 expression on different immune cells in periphery and tumor microenvironment (TME) of colorectal cancer (CRC) patients has not been fully investigated. We found that TIM-3 was mainly expressed on monocytic myeloid cells (MMCs) and antigen-presenting cells (APCs) in circulation but was mainly expressed on T cells and APCs in the TME. Additionally, TIM-3− T cells co-expressed higher levels of PD-1 than TIM-3+ T cells in normal tissue. In contrast, TIM-3+ T cells in the TME showed significantly higher PD-1 expression. Interestingly, there was a trend towards increased levels of TIM-3+ APCs with disease stages; however, levels of TIM-3+ T cells were decreased with disease stages in the TME. This study shows the differential expression of TIM-3 on different immune cells in circulation and TME of CRC patients, and their associations with disease stages. • High TIM-3 expression in CRC warrants characterization on immune cell populations. • TIM-3 was mainly expressed on MMCs and APCs in blood, APCs and T cells in the TME. • Unlike normal tissue, TIM-3 was co-expressed with PD-1 in tumor-infiltrating T cells. • There was a trend towards increased levels of TIM-3+ APCs with disease stages. • Levels of TIM-3+ T cells were decreased with disease stages in the TME. [ABSTRACT FROM AUTHOR]

Cancer immune evasion is a major stumbling block in designing effective anticancer therapeutic strategies. Although considerable progress has been made in understanding how cancers evade destructive immunity, measures to counteract tumor escape have not kept pace. There are a number of factors that contribute to tumor persistence despite having a normal host immune system. Immune editing is one of the key aspects why tumors evade surveillance causing the tumors to lie dormant in patients for years through “equilibrium” and “senescence” before re-emerging. In addition, tumors exploit several immunological processes such as targeting the regulatory T cell function or their secretions, antigen presentation, modifying the production of immune suppressive mediators, tolerance and immune deviation. Besides these, tumor heterogeneity and metastasis also play a critical role in tumor growth. A number of potential targets like promoting Th1, NK cell, γδ T cell responses, inhibiting Treg functionality, induction of IL-12, use of drugs including phytochemicals have been designed to counter tumor progression with much success. Some natural agents and phytochemicals merit further study. For example, use of certain key polysaccharide components from mushrooms and plants have shown to possess therapeutic impact on tumor-imposed genetic instability, anti-growth signaling, replicative immortality, dysregulated metabolism etc. In this review, we will discuss the advances made toward understanding the basis of cancer immune evasion and summarize the efficacy of various therapeutic measures and targets that have been developed or are being investigated to enhance tumor rejection. [ABSTRACT FROM AUTHOR]