Targeting ROS-sensing Nrf2 potentiates anti-tumor immunity of intratumoral CD8+ T and CAR-T cells.

Cytotoxic T lymphocytes (CTLs) play a crucial role in cancer rejection. However, CTLs encounter dysfunction and exhaustion in the immunosuppressive tumor microenvironment (TME). Although the reactive oxygen species (ROS)-rich TME attenuates CTL function, the underlying molecular mechanism remains poorly understood. The nuclear factor erythroid 2-related 2 (Nrf2) is the ROS-responsible factor implicated in increasing susceptibility to cancer progression. Therefore, we examined how Nrf2 is involved in anti-tumor responses of CD8+ T and chimeric antigen receptor (CAR) T cells in the ROS-rich TME. Here, we demonstrated that tumor growth in Nrf2-/- mice was significantly controlled and was reversed by T cell depletion and further confirmed that Nrf2 deficiency in T cells promotes anti-tumor responses using an adoptive transfer model of antigen-specific CD8+ T cells. Nrf2-deficient CTLs are resistant to ROS, and their effector functions are sustained in the TME. Furthermore, Nrf2 knockdown in human CAR-T cells enhanced the survival and function of intratumoral CAR-T cells in a solid tumor xenograft model and effectively controlled tumor growth. ROS-sensing Nrf2 inhibits the anti-tumor T cell responses, indicating that Nrf2 may be a potential target for T cell immunotherapy strategies against solid tumors.

NFAT1 and NFκB regulates expression of the common γ-chain cytokine receptor in activated T cells.

INTRODUCTION: Cytokines of the common γ chain (γc) family are critical for the development, differentiation, and survival of T lineage cells. Cytokines play key roles in immunodeficiencies, autoimmune diseases, allergies, and cancer. Although γc is considered an assistant receptor to transmit cytokine signals and is an indispensable receptor in the immune system, its regulatory mechanism is not yet well understood. OBJECTIVE: This study focused on the molecular mechanisms that γc expression in T cells is regulated under T cell receptor (TCR) stimulation. METHODS: The γc expression in TCR-stimulated T cells was determined by flow cytometry, western blot and quantitative RT-PCR. The regulatory mechanism of γc expression in activated T cells was examined by promoter-luciferase assay and chromatin immunoprecipitation assays. NFAT1 and NFκB deficient cells generated using CRISPR-Cas9 and specific inhibitors were used to examine their role in regulation of γc expression. Specific binding motif was confirmed by γc promotor mutant cells generated using CRISPR-Cas9. IL-7TgγcTg mice were used to examine regulatory role of γc in cytokine signaling. RESULTS: We found that activated T cells significantly upregulated γc expression, wherein NFAT1 and NFκB were key in transcriptional upregulation via T cell receptor stimulation. Also, we identified the functional binding site of the γc promoter and the synergistic effect of NFAT1 and NFκB in the regulation of γc expression. Increased γc expression inhibited IL-7 signaling and rescued lymphoproliferative disorder in an IL-7Tg animal model, providing novel insights into T cell homeostasis. CONCLUSION: Our results indicate functional cooperation between NFAT1 and NFκB in upregulating γc expression in activated T cells. As γc expression also regulates γc cytokine responsiveness, our study suggests that γc expression should be considered as one of the regulators in γc cytokine signaling and the development of T cell immunotherapies. Video Abstract.

Defects in aminoacyl-tRNA synthetase cause partial B and T cell immunodeficiency.

Aminoacyl-tRNA synthetases (ARSs) are emerging as important regulators in various immune diseases; however, their roles in immune cells remain unclear. In this study, using alanyl-tRNA synthetase (AARS) mutant (sti) mice with neurodegenerative disorder, we investigated the effect of translational fidelity in immune cells. Dysfunctional AARS caused disorders in immune cell responses and cellularity. The impairment was caused by dampened TCR signaling than cytokine signaling. Therefore, sti mutant inhibits TCR signaling, impeding T cell survival and responses. B cell numbers were decreased in sti mice. Despite low B cell cellularity, serum IgM, IgA, and IgE levels were higher in sti mice than in wild-type mice. Misacylation of ARS and the consequent translational infidelity induce disturbances in signaling pathways critical for immune cell survival and responses. Our findings provide a novel mechanism by which translational fidelity might play a critical role in cellular and humoral immune responses.

The Abundance and Availability of Cytokine Receptor IL-2Rß (CD122) Constrain the Lymphopenia-Induced Homeostatic Proliferation of Naive CD4 T Cells.

Lymphopenia-induced homeostatic proliferation (LIP) is a critical mechanism for restoring T cell immunity upon lymphodepleting insults or infections. LIP is primarily driven by homeostatic cytokines, such as IL-7 and IL-15, but not all T cells respond with the same efficiency to homeostatic proliferative cues. Although CD8 T cells vigorously proliferate under lymphopenic conditions, naive CD4 T cells are substantially impaired in their response to homeostatic cytokines, and they fail to fully expand. In this study, we show that the availability of IL-2Rß (CD122), which is a receptor subunit shared by IL-2 and IL-15, affects both the cytokine responsiveness and the LIP of naive CD4 T cells in the mouse. The enumeration of surface IL-2Rß molecules on murine naive CD4 and naive CD8 T cells revealed a 5-fold difference in IL-2Rß abundance. Notably, it was the limited availability of IL-2Rß that impaired CD4 T cell responsiveness to IL-15 and suppressed their LIP. As such, forced IL-2Rß expression on CD4 T cells by transgenesis bestowed IL-15 responsiveness onto naive CD4 T cells, which thus acquired the ability to undergo robust LIP. Collectively, these results identify IL-2Rß availability as a new regulatory mechanism to control cytokine responsiveness and the homeostatic proliferation of murine CD4 T cells.

Soluble γc receptor attenuates anti-tumor responses of CD8+ T cells in T cell immunotherapy.

Previous studies have shown that soluble common γ-chain (sγc) modulates CD4+ T cell immunity with antagonistic functions in γc cytokine signaling. However, the role of sγc in functional properties of effector CD8+ T cells has not been fully defined. In this study, we report a new mechanism by which the anti-tumor activity of mouse CD8+ T cells is suppressed in sγc of their own producing. While sγc significantly inhibits cytotoxicity of CD8+ T cells, blocking sγc production by genetic modification leads to potentiated effector function of CD8+ T cells, establishing persistent CD8+ T cells. This is due to the modulation of IL-2 and IL-15 signaling, which is required for expansion and survival of CD8+ T cells as well as for optimal cytotoxic activity. More efficient management of tumor growth was achieved by an adoptive transfer of sγc-deficient CD8+ T cells than that of wild-type or sγc-overexpressing CD8+ T cells. Blocking of IL-2 and IL-15 signaling by sγc attenuates the capacity of CD8+ T cells to mount an optimal response to the tumor, with both quantitative and qualitative effects on antigen-specific CD8+ T cells. These results could have a critical implication for the generation and survival of optimal effector T cells for adoptive immunotherapy of cancer.

The Potential Role of a Soluble γ-Chain Cytokine Receptor as a Regulator of IL-7-Induced Lymphoproliferative Disorders.

IL-7 is an essential, nonredundant growth factor for T and B cell generation and maintenance. While IL-7 deficiency results in lymphopenia, overexpression of IL-7 can cause neoplasia in experimental models. IL-7's involvement in neoplasia has been appreciated through studies of IL-7 transgenic (Tg) mice models and human lymphoma patients. Since we recently found that a soluble form of the common γ-chain (γc) cytokine receptor (sγc) antagonistically regulates IL-7 signaling, IL-7 and sγc double-Tg mice were generated to investigate the effects of sγc overexpression in IL-7-mediated lymphoproliferative disorders (LPDs). The overexpression of sγc prevents IL-7Tg-induced abnormal increase of LN cell numbers and the development of splenomegaly, resulting in striking amelioration of mortality and disease development. These results suggest that modification of γc cytokine responsiveness by sγc molecules might control various γc cytokine-associated hematologic malignancy, and also provide an alternative view to approach antitumor therapy.

IL-7 Induces an Epitope Masking of γc Protein in IL-7 Receptor Signaling Complex.

IL-7 signaling via IL-7Rα and common γ-chain (γc) is necessary for the development and homeostasis of T cells. Although the delicate mechanism in which IL-7Rα downregulation allows the homeostasis of T cell with limited IL-7 has been well known, the exact mechanism behind the interaction between IL-7Rα and γc in the absence or presence of IL-7 remains unclear. Additionally, we are still uncertain as to how only IL-7Rα is separately downregulated by the binding of IL-7 from the IL-7Rα/γc complex. We demonstrate here that 4G3, TUGm2, and 3E12 epitope masking of γc protein are induced in the presence of IL-7, indicating that the epitope alteration is induced by IL-7 binding to the preassembled receptor core. Moreover, the epitope masking of γc protein is inversely correlated with the expression of IL-7Rα upon IL-7 binding, implying that the structural alteration of γc might be involved in the regulation of IL-7Rα expression. The conformational change in γc upon IL-7 binding may contribute not only to forming the functional IL-7 signaling complex but also to optimally regulating the expression of IL-7Rα.

Molecular classification of basal cell carcinoma of skin by gene expression profiling.

Non-melanoma skin cancers (NMSC) including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are more common kinds of skin cancer. Although these tumors share common pathological and clinical features, their similarity and heterogeneity at molecular levels are not fully elaborated yet. Here, by performing comparative analysis of gene expression profiling of BCC, SCC, and normal skin tissues, we could classify the BCC into three subtypes of classical, SCC-like, and normal-like BCCs. Functional enrichment and pathway analyses revealed the molecular characteristics of each subtype. The classical BCC showed the enriched expression and transcription signature with the activation of Wnt and Hedgehog signaling pathways, which were well known key features of BCC. By contrast, the SCC-like BCC was enriched with immune-response genes and oxidative stress-related genes. Network analysis revealed the PLAU/PLAUR as a key regulator of SCC-like BCC. The normal-like BCC showed prominent activation of metabolic processes particularly the fatty acid metabolism. The existence of these molecular subtypes could be validated in an independent dataset, which demonstrated the three subgroups of BCC with distinct functional enrichment. In conclusion, we suggest a novel molecular classification of BCC providing insights on the heterogeneous progression of BCC.

The role of gut microbiota in T cell immunity and immune mediated disorders.

Gut microbiota was only considered as a commensal organism that aids in digestion, but recent studies revealed that the microbiome play a critical role in both physiological and pathological immune system. The gut microbiome composition is altered by environmental factors such as diet and hygiene, and the alteration affects immune cells, especially T cells. Advanced genomic techniques in microbiome research defined that specific microbes regulate T cell responses and the pathogenesis of immune-mediated disorders. Here, we review features of specific microbes-T cell crosstalk and relationship between the microbes and immunopathogenesis of diseases including in cancers, autoimmune disorders and allergic inflammations. We also discuss the limitations of current experimental animal models, cutting-edge developments and current challenges to overcome in the field, and the possibility of considering gut microbiome in the development of new drug.

Pharmacogenetic distribution of warfarin and its clinical significance in Korean patients during initial anticoagulation therapy.

During warfarin treatment, determining the optimal dose and maintaining the target PT-INR are challenging. Increasing evidence supports the theory that genotypic polymorphisms influence an individual's warfarin dose requirement. In this study, we evaluated allele frequencies and effects of CYP2C9 and VKORC1 on warfarin response during initial anticoagulation therapy in Korean patients. We enrolled patients who had initiated warfarin therapy and undergone PT-INR testing at least three times within the first month of anticoagulation therapy. All the participating patients were tested for the detection of CYP2C9*3 (c.1075A>C) and VKORC1-1639G>A. A melting-curve analysis after real-time PCR was performed using CYP2C9*3 and VK1639 genotyping kits (Idaho Technology, US). A total of 37 patients were enrolled in this study. CYP2C9*1/*1 (87%) and VKORC1-1639AA genotypes (89%) were predominant in Korea. The CYP2C9*3 and VKORC1-1639G alleles were found in five (13%) and four patients (11%), respectively. Patients with the CYP2C9*3 allele received a lower warfarin dose (P = 0.018) and tended to show more rapid PT-INR increase than CYP2C9*1/*1 genotype. Patients with the VKORC1-1639G allele nonsignificantly received higher warfarin dose than those without. The CYP2C9*3 and VKORC1-1639G alleles influenced warfarin response during the first month of anticoagulation therapy. Considering these results, CYP2C9 and VKORC1 genotyping can be an useful tool to estimate initial warfarin dose and frequency of PT-INR monitoring during the first month of anticoagulation therapy.

Innovative CAR-T Cell Therapy for Solid Tumor; Current Duel between CAR-T Spear and Tumor Shield.

Novel engineered T cells containing chimeric antigen receptors (CAR-T cells) that combine the benefits of antigen recognition and T cell response have been developed, and their effect in the anti-tumor immunotherapy of patients with relapsed/refractory leukemia has been dramatic. Thus, CAR-T cell immunotherapy is rapidly emerging as a new therapy. However, it has limitations that prevent consistency in therapeutic effects in solid tumors, which accounts for over 90% of all cancer patients. Here, we review the literature regarding various obstacles to CAR-T cell immunotherapy for solid tumors, including those that cause CAR-T cell dysfunction in the immunosuppressive tumor microenvironment, such as reactive oxygen species, pH, O2, immunosuppressive cells, cytokines, and metabolites, as well as those that impair cell trafficking into the tumor microenvironment. Next-generation CAR-T cell therapy is currently undergoing clinical trials to overcome these challenges. Therefore, novel approaches to address the challenges faced by CAR-T cell immunotherapy in solid tumors are also discussed here.

Aquatic Exercise at Thermoneutral Water Temperature Enhances Antitumor Immune Responses.

Despite the broad rehabilitative potential of aquatic exercises, the relationship between aquatic exercise and the immune system has not been fully elucidated to date. In particular, there are few specific and delicate immunological approaches to the effect of water temperature on immunity. Thus, we examined the effect of water temperature on immunity during aquatic exercise. The animal tumor model was adopted to examine the impact of aquatic exercise at thermoneutral temperature (TT; 29°C) on immunity compared with aquatic exercise at body temperature (BT; 36°C). Tumor-bearing mice were made to swim in TT water or in BT water for 3 wk and immune cells and their functional activity were analyzed using FACS. Tumor growth was significantly suppressed in mice that exercised in TT than in BT water. The tumor control correlated with the increased number of NK (2-fold), γδT cells (2.5-fold), NKT (2.5-fold), and cytotoxic CD8+ T cells (1.6-fold), which play a critical role in anti-tumor immune responses. Furthermore, the functional activity was dramatically improved in the TT group, showing enhanced production of IFNγ in CD8+ T cells compared with the BT group. This study demonstrates that aquatic exercise in TT water may improve protective immune responses more effectively than in BT water. Although the effects of water temperature on immune function need further verification in humans, this study suggests that water temperature in human hydrotherapy may be important for improving immune function.

Enzymatic Hydrolysis of Egg White Protein Exerts a Hypotensive Effect in Spontaneously Hypertensive Rats.

This study was conducted to investigate the hypotensive effect of egg white protein (EWP) hydrolysate (EWH) in spontaneously hypertensive rats (SHRs). The hydrolysis of EWP was effectively performed with a combination of 0.5% bromelain and 1% papain at 50°C for 60 min. The resulting hydrolysate did not elicit an allergic reaction as confirmed by human mast cell activation test. The systolic and diastolic blood pressures of the SHRs fed the EWH diet were observed to be significantly or numerically lower than those of the other groups during the experimental period of 28 d. EWH treatment significantly (p<0.05) upregulated the nitric oxide levels in hCMEC/D3 cells and the plasma of the SHRs compared to those in the control. Moreover, EWH ingestion significantly (p<0.01) reduced the plasma angiotensin II level of the SHRs compared with that in the control. In conclusion, beyond its basic nutritional value, EWH prevents and manages hypertension, and thus can be an invaluable resource for functional food development.

Specific Inhibition of Soluble γc Receptor Attenuates Collagen-Induced Arthritis by Modulating the Inflammatory T Cell Responses.

IL-17 produced by Th17 cells has been implicated in the pathogenesis of rheumatoid arthritis (RA). It is important to prevent the differentiation of Th17 cells in RA. Homodimeric soluble γc (sγc) impairs IL-2 signaling and enhances Th17 differentiation. Thus, we aimed to block the functions of sγc by inhibiting the formation of homodimeric sγc. The homodimeric form of sγc was strikingly disturbed by sγc-binding DNA aptamer. Moreover, the aptamer effectively inhibited Th17 cell differentiation and restored IL-2 and IL-15 signaling impaired by sγc with evidences of increased survival of T cells. sγc was highly expressed in SF of RA patients and increased in established CIA mice. The therapeutic effect of PEG-aptamer was tested in CIA model and its treatment alleviated arthritis pathogenesis with impaired differentiation of pathogenic Th17, NKT1, and NKT17 cells in inflamed joint. Homodimeric sγc has pathogenic roles to exacerbate RA progression with differentiation of local Th17, NKT1, and NKT17 cells. Therefore, sγc is suggested as target of a therapeutic strategy for RA.

Soluble common gamma chain exacerbates COPD progress through the regulation of inflammatory T cell response in mice.

Cigarette smoking (CS) is a major cause of considerable morbidity and mortality by inducing lung cancer and COPD. COPD, a smoking-related disorder, is closely related to the alteration of immune system and inflammatory processes that are specifically mediated by T cells. Soluble common gamma chain (sγc) has recently been identified as a critical regulator of the development and differentiation of T cells. We examined the effects of sγc in a cigarette smoke extract (CSE) mouse model. The sγc level in CSE mice serum is significantly downregulated, and the cellularity of lymph node (LN) is systemically reduced in the CSE group. Overexpression of sγc enhances the cellularity and IFNγ production of CD8 T cells in LN and also enhances Th1 and Th17 differentiation of CD4 T cells in the respiratory tract. Mechanistically, the downregulation of sγc expression mediated by CSE is required to prevent excessive inflammatory T cell responses. Therefore, our data suggest that sγc may be one of the target molecules for the control of immunopathogenic progresses in COPD.

Soluble γc cytokine receptor suppresses IL-15 signaling and impairs iNKT cell development in the thymus.

The soluble γc protein (sγc) is a naturally occurring splice isoform of the γc cytokine receptor that is produced by activated T cells and inhibits γc cytokine signaling. Here we show that sγc expression is also highly upregulated in immature CD4+CD8+ thymocytes but then downregulated in mature thymocytes. These results indicate a developmentally controlled mechanism for sγc expression and suggest a potential role for sγc in regulating T cell development in the thymus. Indeed, sγc overexpression resulted in significantly reduced thymocyte numbers and diminished expansion of immature thymocytes, concordant to its role in suppressing signaling by IL-7, a critical γc cytokine in early thymopoiesis. Notably, sγc overexpression also impaired generation of iNKT cells, resulting in reduced iNKT cell percentages and numbers in the thymus. iNKT cell development requires IL-15, and we found that sγc interfered with IL-15 signaling to suppress iNKT cell generation in the thymus. Thus, sγc represents a new mechanism to control cytokine availability during T cell development that constrains mature T cell production and specifically iNKT cell generation in the thymus.

Tumor suppressive effect of PARP1 and FOXO3A in gastric cancers and its clinical implications.

Poly (ADP-ribose) polymerase1 (PARP1) has been reported as a possible target for chemotherapy in many cancer types. However, its action mechanisms and clinical implications for gastric cancer survival are not yet fully understood. Here, we investigated the effect of PARP1 inhibition in the growth of gastric cancer cells. PARP1 inhibition by Olaparib or PARP1 siRNA could significantly attenuate growth and colony formation of gastric cancer cells, and which were mediated through induction of G2/M cell cycle arrest but not apoptosis. FOXO3A expression was induced by PARP1 inhibition, suggesting that FOXO3A might be one of downstream target of the PARP1 effect on gastric cancer cell growth. In addition, by performing tissue microarrays on the 166 cases of gastric cancer patients, we could observe that the expression status of PARP1 and FOXO3A were significantly associated with overall survival (OS) and relapse-free survival (RFS). Strikingly, combined expression status of PARP1 and FOXO3A showed better prediction for patient's clinical outcomes. The patient group with PARP1+/FOXO3A- expression had the worst prognosis while the patient group with PARP1-/FOXO3A+ had the most favorable prognosis (OS: P = 6.0 × 10(-9), RFS: P = 2.2 × 10(-8)). In conclusion, we suggest that PARP1 and FOXO3A play critical roles in gastric cancer progression, and might have therapeutic and/or diagnostic potential in clinic.

Genetic polymorphisms and plasma levels of tissue factor and tissue factor pathway inhibitor in venous thromboembolism.

Tissue factor (TF) and tissue factor pathway inhibitor (TFPI) play important roles in coagulation. The aim of this study was to investigate the distributions of TF and TFPI polymorphisms in Koreans and to analyze the association of these genetic polymorphisms with plasma levels and development of venous thromboembolism (VTE). The polymorphisms TF 5466 A > G, TF -603 A > G, TFPI -287 T > C and TFPI -33 T > C were investigated in 40 Korean VTE patients and 40 age-matched and sex-matched controls by real-time PCR followed by melting curve analysis and DNA sequence analysis. Plasma levels of TF and TFPI were measured by enzyme-linked immunosorbent assay. The G allele of TF 5466 was not detected, and allelic frequencies of TF -603 G, TFPI -287 C and TFPI -33 C were 27.5, 67.5 and 16.2%, respectively. The distributions of TF and TFPI polymorphisms were not different between patients and controls. The presence of TF -603 G allele was correlated with low plasma TF levels (P = 0.029). Mean plasma TFPI levels were similar between TFPI genotypic groups. Although not statistically significant, plasma TF and TFPI levels were higher in patients than controls. The distributions of TF and TFPI polymorphisms in Koreans were considerably different from whites, suggesting ethnic variations. The TF -603 A > G polymorphism was significantly correlated with decreased plasma TF levels. Neither genetic polymorphisms in TF and TFPI nor their plasma levels seem to act as direct risk factors for VTE.