Lag3: From Bench to Bedside.

The introduction of immune checkpoint inhibitors represented a breakthrough treatment for metastatic melanoma, but the effect of these agents is not limited to a single cancer type. Promising results have been reported in various solid tumors, for example, lung cancer. The success of these drugs depends on the activation of tumor-infiltrating lymphocytes and primary and acquired resistance have been reported alongside a high rate of immune-related adverse events when agents targeting different immune checkpoints are given in combination. Numerous other targets have been investigated to overcome the resistance, improve the activity, and reduce the toxicity of checkpoint inhibitor therapy. Among these, the most promising is Lymphocyte-activation gene 3 (LAG-3), a transmembrane protein involved in cytokine release and inhibitory signaling in T cells. Preclinical data showed that LAG-3 is a negative regulator of both CD4+ T cell and CD8+ T cell and the activity on CD8+ T cell is independent of CD4+ activation. On the CD8+ T cell, LAG-3 activation abrogates the antigen presentation whereas on the CD4+ T cell, arrests the S phase of the cell cycle. The blockade of LAG-3 has been tested in several combination therapies, and recent clinical data showed a good safety profile and a synergistic effect with anti-PD-1, suggesting that this combination could become a standard treatment for metastatic melanoma. In this review, we report the available preclinical data and the new clinical data on LAG-3 blockade in different solid tumors, and we discuss LAG-3 as potential prognostic and predictive factor, together with possible future applications.

Acquired resistance to cancer immunotherapy: Role of tumor-mediated immunosuppression.

In the tumor microenvironment (TME), tumor cells are constantly evolving to reduce neoantigen generation and the mutational burden to escape the anti-tumor response. This will lower tumor reactivity to the adaptive immune response and give rise to tumor intrinsic factors, such as altered expression of immune regulatory molecules on tumor cells. Tumor-extrinsic factors, such as immunosuppressive cells, soluble suppressive molecules or inhibitory receptors expressed by immune cells will alter the composition and activity of tumor-infiltrating lymphocytes (TILs) (by increasing T regulatory cells:T effector cells ratio and inhibiting T effector cell function) and promote tumor growth and metastasis. Together, these factors limit the response rates and clinical outcomes to a particular cancer therapy. Within the TME, the cross-talks between immune and non-immune cells result in the generation of positive feedback loops, which augment immunosuppression and support tumor growth and survival (termed as tumor-mediated immunosuppression). Cancer immunotherapies, such as immune checkpoint inhibitors (ICIs) and adoptive cell transfer (ACT), have shown therapeutic efficacy in hematologic cancers and different types of solid tumors. However, achieving durable response rates in some cancer patients remains a challenge as a result of acquired resistance and tumor immune evasion. This could be driven by the cellular and molecular suppressive network within the TME or due to the loss of tumor antigens. In this review, we describe the contribution of the immunosuppressive cellular and molecular tumor network to the development of acquired resistance against cancer immunotherapies. We also discuss potential combined therapeutic strategies which could help to overcome such resistance against cancer immunotherapies, and to enhance anti-tumor immune responses and improve clinical outcomes in patients.

T-cell responses and therapies against SARS-CoV-2 infection.

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.

Metabolic reprogramming of T regulatory cells in the hypoxic tumor microenvironment.

Metabolic dysregulation in the hypoxic tumor microenvironment (TME) is considered as a hallmark of solid tumors, leading to changes in biosynthetic pathways favoring onset, survival and proliferation of malignant cells. Within the TME, hypoxic milieu favors metabolic reprogramming of tumor cells, which subsequently affects biological properties of tumor-infiltrating immune cells. T regulatory cells (Tregs), including both circulating and tissue-resident cells, are particularly susceptible to hypoxic metabolic signaling that can reprogram their biological and physicochemical properties. Furthermore, metabolic reprogramming modifies Tregs to utilize alternative substrates and undergo a plethora of metabolic events to meet their energy demands. Major impact of this metabolic reprogramming can result in differentiation, survival, excessive secretion of immunosuppressive cytokines and proliferation of Tregs within the TME, which in turn dampen anti-tumor immune responses. Studies on fine-tuning of Treg metabolism are challenging due to heterogenicity of tissue-resident Tregs and their dynamic functions. In this review, we highlight tumor intrinsic and extrinsic factors, which can influence Treg metabolism in the hypoxic TME. Moreover, we focus on metabolic reprogramming of Tregs that could unveil potential regulatory networks favoring tumorigenesis/progression, and provide novel insights, including inhibitors against acetyl-coA carboxylase 1 and transforming growth factor beta into targeting Treg metabolism for therapeutic benefits.

Transcriptome of CD8+ tumor-infiltrating T cells: a link between diabetes and colorectal cancer.

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.

Differential gene expression of tumor-infiltrating CD33+ myeloid cells in advanced- versus early-stage colorectal cancer.

Colorectal cancer (CRC) has high mortality rates, especially in patients with advanced disease stages, who often do not respond to therapy. The cellular components of the tumor microenvironment are essentially responsible for dictating disease progression and response to therapy. Expansion of different myeloid cell subsets in CRC tumors has been reported previously. However, tumor-infiltrating myeloid cells have both pro- and anti-tumor roles in disease progression. In this study, we performed transcriptomic profiling of cells of myeloid lineage (CD33+) from bulk CRC tumors at varying disease stages. We identified differentially expressed genes and pathways between CRC patients with advanced stage and early stages. We found that pro-angiogenic and hypoxia-related genes were upregulated, while genes related to immune and inflammatory responses were downregulated in CD33+ myeloid cells from patients with advanced stages, implying that immune cell recruitment and activation could be compromised in advanced disease stages. Moreover, we identified a unique "poor prognosis CD33+ gene signature" by aligning top upregulated and downregulated genes in tumor-infiltrating myeloid cells from our analyses with data from The Cancer Genome Atlas. Our results showed that this gene signature is an independent prognostic indicator for disease-specific survival in CRC patients, potentially reflecting its clinical importance.

Transcriptomic Profiling of Circulating HLA-DR- Myeloid Cells, Compared with HLA-DR+ Myeloid Antigen-presenting Cells.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells with potent immunosuppressive functions, which can inhibit the activation of immune responses under a steady-state condition and pathological conditions. We performed transcriptomic profiling of circulating CD33+HLA-DR+ myeloid antigen-presenting cells (APCs) and CD33+HLA-DR- myeloid cells (potentially MDSCs) in healthy individuals. We sorted both subpopulations from peripheral blood mononuclear cells (PBMCs) of 10 healthy donors and performed RNA sequencing (RNA-Seq). We found that several signaling pathways associated with the positive regulation of immune responses, such as antigen presentation/processing, FcγR-mediated phagocytosis and immune cell trafficking, phosphoinositide 3-kinase (PI3K)/Akt signaling, DC maturation, triggering receptor expressed on myeloid cells 1 (TREM1) signaling, nuclear factor of activated T cells (NFAT) and IL-8 signaling were downregulated in CD33+HLA-DR- myeloid cells. In contrast, pathways implicated in tumor suppression and anti-inflammation, including peroxisome proliferator-activated receptor (PPAR) and phosphatase and tensin homolog (PTEN), were upregulated in CD33+HLA-DR- myeloid cells. These data indicate that PPAR/PTEN axis could be upregulated in myeloid cells to keep the immune system in check in normal physiological conditions. Our data reveal some of the molecular and functional differences between CD33+HLA-DR+ APCs and CD33+HLA-DR- myeloid cells in a steady-state condition, reflecting the potential suppressive function of CD33+HLA-DR- myeloid cells to maintain immune tolerance. For future studies, the same methodological approach could be applied to perform transcriptomic profiling of myeloid subsets in pathological conditions.

Expression of immune checkpoints and T cell exhaustion markers in early and advanced stages of colorectal cancer.

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.

Risk factors for high-dose methotrexate associated acute kidney injury in patients with hematological malignancies.

High dose methotrexate (HDMTX)-induced acute kidney injury (AKI) is a well-known adverse event in hemato-oncology patients. Our purpose was to define factors and setup cut-offs that may help better identify patients at-risk for developing AKI following HDMTX. All consecutive patients who received MTX dose ≥1 g were retrospectively reviewed. We compared patients with or without renal toxicity. We used a logistic regression model to define baseline variables associated with AKI. Overall survival (OS) was estimated by the Kaplan-Meier method employing log-rank test. Between 2012 and 2017, 160 patients were included with a total of 265 courses. Indications included: primary central nervous system (CNS) lymphoma, CNS prophylaxis in other lymphoma types, acute lymphatic leukemia and others. Median age at diagnosis was 58 years (range, 18-84), 54% were males, median MTX dose was 1941 mg/m2 (range, 743-5442) and AKI developed in 9% of drug administrations (n = 24). In univariate analysis: age > 40, LDH > 380 units/L, eGFR < 112 mL/min, albumin <3.6 mg/dL at baseline and Charlson comorbidity index (CCI) were associated with AKI. In multivariable analysis, only LDH > 380 units/L (OR = 4.1, 95% confidence interval [CI] 1.04-20.9, P = .04) and albumin levels <3.6 g/dL (OR = 4.17, 95% CI 1.04-6.5, P = .04) remained significant. In patients with AKI, median drug elimination was longer (8 days vs 5 days). In 80% of cases, the creatinine levels returned to normal within 1 month. Yet, the median survival of patients who developed AKI was 37 months, compared to 145 months in patients without AKI (Log rank = 0.015). In conclusion, LDH > 380 units/L and albumin <3.6 g/dL were the strongest factors associated with AKI in patients receiving HDMTX. Although the rise in creatinine levels was almost uniformly reversible, AKI was associated with increased mortality rates.

CSF-1 in Inflammatory and Arthritic Pain Development.

Pain is one of the most debilitating symptoms in many diseases for which there is inadequate management and understanding. CSF-1, also known as M-CSF, acts via its receptor (CSF-1R, c-Fms) to regulate the development of the monocyte/macrophage lineage and to act locally in tissues to control macrophage numbers and function. It has been implicated in the control of neuropathic pain via a central action on microglia. We report in this study that systemic administration of a neutralizing anti-CSF-1R or CSF-1 mAb inhibits the development of inflammatory pain induced by zymosan, GM-CSF, and TNF in mice. This approach also prevented but did not ameliorate the development of arthritic pain and optimal disease driven by the three stimuli in mice, suggesting that CSF-1 may only be relevant when the driving inflammatory insults in tissues are acute and/or periodic. Systemic CSF-1 administration rapidly induced pain and enhanced the arthritis in an inflamed mouse joint, albeit via a different pathway(s) from that used by systemic GM-CSF and TNF. It is concluded that CSF-1 can function peripherally during the generation of inflammatory pain and hence may be a target for such pain and associated disease, including when the clinically important cytokines, TNF and GM-CSF, are involved. Our findings have ramifications for the selection and design of anti-CSF-1R/CSF-1 trials.

G-CSF Receptor Blockade Ameliorates Arthritic Pain and Disease.

G-CSF or CSF-3, originally defined as a regulator of granulocyte lineage development via its cell surface receptor (G-CSFR), can play a role in inflammation, and hence in many pathologies, due to its effects on mature lineage populations. Given this, and because pain is an extremely important arthritis symptom, the efficacy of an anti-G-CSFR mAb for arthritic pain and disease was compared with that of a neutrophil-depleting mAb, anti-Ly6G, in both adaptive and innate immune-mediated murine models. Pain and disease were ameliorated in Ag-induced arthritis, zymosan-induced arthritis, and methylated BSA/IL-1 arthritis by both prophylactic and therapeutic anti-G-CSFR mAb treatment, whereas only prophylactic anti-Ly6G mAb treatment was effective. Efficacy for pain and disease correlated with reduced joint neutrophil numbers and, importantly, benefits were noted without necessarily the concomitant reduction in circulating neutrophils. Anti-G-CSFR mAb also suppressed zymosan-induced inflammatory pain. A new G-CSF-driven (methylated BSA/G-CSF) arthritis model was established enabling us to demonstrate that pain was blocked by a cyclooxygenase-2 inhibitor, suggesting an indirect effect on neurons. Correspondingly, dorsal root ganglion neurons cultured in G-CSF failed to respond to G-CSF in vitro, and Csf3r gene expression could not be detected in dorsal root ganglion neurons by single-cell RT-PCR. These data suggest that G-CSFR/G-CSF targeting may be a safe therapeutic strategy for arthritis and other inflammatory conditions, particularly those in which pain is important, as well as for inflammatory pain per se.

Modelling aggressive prostate cancers of young men in immune-competent mice, driven by isogenic Trp53 alterations and Pten loss.

Understanding prostate cancer onset and progression in order to rationally treat this disease has been critically limited by a dire lack of relevant pre-clinical animal models. We have generated a set of genetically engineered mice that mimic human prostate cancer, initiated from the gland epithelia. We chose driver gene mutations that are specifically relevant to cancers of young men, where aggressive disease poses accentuated survival risks. An outstanding advantage of our models are their intact repertoires of immune cells. These mice provide invaluable insight into the importance of immune responses in prostate cancer and offer scope for studying treatments, including immunotherapies. Our prostate cancer models strongly support the role of tumour suppressor p53 in functioning to critically restrain the emergence of cancer pathways that drive cell cycle progression; alter metabolism and vasculature to fuel tumour growth; and mediate epithelial to mesenchymal-transition, as vital to invasion. Importantly, we also discovered that the type of p53 alteration dictates the specific immune cell profiles most significantly disrupted, in a temporal manner, with ramifications for disease progression. These new orthotopic mouse models demonstrate that each of the isogenic hotspot p53 amino acid mutations studied (R172H and R245W, the mouse equivalents of human R175H and R248W respectively), drive unique cellular changes affecting pathways of proliferation and immunity. Our findings support the hypothesis that individual p53 mutations confer their own particular oncogenic gain of function in prostate cancer.

Targeting MDM4 as a Novel Therapeutic Approach in Prostate Cancer Independent of p53 Status.

Metastatic prostate cancer is a lethal disease in patients incapable of responding to therapeutic interventions. Invasive prostate cancer spread is caused by failure of the normal anti-cancer defense systems that are controlled by the tumour suppressor protein, p53. Upon mutation, p53 malfunctions. Therapeutic strategies to directly re-empower the growth-restrictive capacities of p53 in cancers have largely been unsuccessful, frequently because of a failure to discriminate responses in diseased and healthy tissues. Our studies sought alternative prostate cancer drivers, intending to uncover new treatment targets. We discovered the oncogenic potency of MDM4 in prostate cancer cells, both in the presence and absence of p53 and also its mutation. We uncovered that sustained depletion of MDM4 is growth inhibitory in prostate cancer cells, involving either apoptosis or senescence, depending on the cell and genetic context. We identified that the potency of MDM4 targeting could be potentiated in prostate cancers with mutant p53 through the addition of a first-in-class small molecule drug that was selected as a p53 reactivator and has the capacity to elevate oxidative stress in cancer cells to drive their death.

Role of circular RNAs in colorectal tumor microenvironment.

Circular RNAs (circRNAs) are a class of endogenous noncoding RNA, which were previously considered as a byproduct of RNA splicing error. Numerous studies have demonstrated the altered expression of circRNAs in organ tissues during pathological conditions and their involvements in disease pathogenesis and progression, including cancers. In colorectal cancer (CRC), multiple circRNAs have been identified and characterized as "oncogenic", given their involvements in the downregulation of tumor suppressor genes and induction of tumor initiation, progression, invasion, and metastasis. Additionally, other circRNAs have been identified in CRC and characterized as "tumor suppressive" based on their ability of inhibiting the expression of oncogenic genes and suppressing tumor growth and proliferation. circRNAs could serve as potential diagnostic and prognostic biomarkers, and therapeutic targets or vectors to be utilized in cancer therapies. This review briefly describes the dynamic changes of the tumor microenvironment inducing immunosuppression and tumorigenesis, and outlines the biogenesis and characteristics of circRNAs and recent findings indicating their roles and functions in the CRC tumor microenvironment. It also discusses strategies and technologies, which could be employed in the future to overcome current cancer therapy challenges associated with circRNAs.

Transcriptome of Tumor-Infiltrating T Cells in Colorectal Cancer Patients Uncovered a Unique Gene Signature in CD4+ T Cells Associated with Poor Disease-Specific Survival.

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.

FoxP3+ T regulatory cells in cancer: Prognostic biomarkers and therapeutic targets.

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.

Exosomes: Biological Carriers and Promising Tools for Cancer Immunotherapy.

Exosomes are recognized as new therapeutic targets for cancer biomedicine and cancer immunotherapy [...].

Targeting TIM-3 in solid tumors: innovations in the preclinical and translational realm and therapeutic potential.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) have shown a great therapeutic efficacy in cancer patients. However, a significant proportion of cancer patients remain unresponsive or show limited response. T cell immunoglobulin and mucin-domain containing protein-3 (TIM-3) is a co-inhibitory receptor expressed on various cell types and is involved in the attenuation of immune responses. TIM-3 and its ligands are highly expressed in various solid malignancies and some studies have reported its association with worse disease outcomes. Thus, targeting TIM-3 could be a promising therapeutic approach to treat cancer patients. AREAS COVERED: This review describes the role of TIM-3 and its ligands in regulating anti-tumor immunity and their contribution to cancer progression. Moreover, this review focuses on the preclinical models and translational data from important studies published in PubMed till October 2020, which demonstrate the therapeutic benefits of targeting TIM-3 signaling. EXPERT OPINION: Despite the promising data obtained from targeting TIM-3 in preclinical models, precise mechanisms underlying the anti-tumor effects of TIM-3 inhibition are not fully elucidated. Therefore, mechanistic studies are required to provide better insights into the anti-tumor effects of targeting TIM-3, and clinical data are necessary to determine the safety profiles and therapeutic efficacy of TIM-3 inhibition in cancer patients.