Sustained AhR activity programs memory fate of early effector CD8+ T cells.

Identification of mechanisms that program early effector T cells to either terminal effector T (Teff) or memory T (Tm) cells has important implications for protective immunity against infections and cancers. Here, we show that the cytosolic transcription factor aryl hydrocarbon receptor (AhR) is used by early Teff cells to program memory fate. Upon antigen engagement, AhR is rapidly up-regulated via reactive oxygen species signaling in early CD8+ Teff cells, which does not affect the effector response, but is required for memory formation. Mechanistically, activated CD8+ T cells up-regulate HIF-1α to compete with AhR for HIF-1ß, leading to the loss of AhR activity in HIF-1αhigh short-lived effector cells, but sustained in HIF-1αlow memory precursor effector cells (MPECs) with the help of autocrine IL-2. AhR then licenses CD8+ MPECs in a quiescent state for memory formation. These findings partially resolve the long-standing issue of how Teff cells are regulated to differentiate into memory cells.

Ammonia detoxification promotes CD8+ T cell memory development by urea and citrulline cycles.

Amino acid metabolism is essential for cell survival, while the byproduct ammonia is toxic and can injure cellular longevity. Here we show that CD8+ memory T (TM) cells mobilize the carbamoyl phosphate (CP) metabolic pathway to clear ammonia, thus promoting memory development. CD8+ TM cells use ß-hydroxybutyrylation to upregulate CP synthetase 1 and trigger the CP metabolic cascade to form arginine in the cytosol. This cytosolic arginine is then translocated into the mitochondria where it is split by arginase 2 to urea and ornithine. Cytosolic arginine is also converted to nitric oxide and citrulline by nitric oxide synthases. Thus, both the urea and citrulline cycles are employed by CD8+ T cells to clear ammonia and enable memory development. This ammonia clearance machinery might be targeted to improve T cell-based cancer immunotherapies.

TCR activation directly stimulates PYGB-dependent glycogenolysis to fuel the early recall response in CD8+ memory T cells.

Glycolysis facilitates the rapid recall response of CD8+ memory T (Tm) cells. However, it remains unclear whether Tm cells uptake exogenous glucose or mobilize endogenous sugar to fuel glycolysis. Here, we show that intracellular glycogen rather than extracellular glucose acts as the major carbon source for the early recall response. Following antigenic stimulation, Tm cells exhibit high glycogen phosphorylase (brain form, PYGB) activity, leading to glycogenolysis and release of glucose-6-phosphate (G6P). Elevated G6P mainly flows to glycolysis but is also partially channeled to the pentose phosphate pathway, which maintains the antioxidant capacity necessary for later recall stages. Mechanistically, TCR signaling directly induces phosphorylation of PYGB by LCK-ZAP70. Functionally, the glycogenolysis-fueled early recall response of CD8+ Tm cells accelerates the clearance of OVA-Listeria monocytogenes in an infected mouse model. Thus, we uncover a specific dependency on glycogen for the initial activation of memory T cells, which may have therapeutic implications for adaptive immunity.

Role of caprin-1 in carcinogenesis.

RNA-binding proteins serve an essential role in post-transcriptional gene regulation. Cytoplasmic activation/proliferation-associated protein-1 (caprin-1) is an RNA-binding protein that participates in the regulation of cell cycle control-associated genes. Caprin-1 acts alone or in combination with other RNA-binding proteins, such as RasGAP SH3-domain-binding protein 1 and fragile X mental retardation protein. In the tumorigenesis process, caprin-1 primarily functions by activating cell proliferation and upregulating the expression of immune checkpoint proteins. Through the formation of stress granules, caprin-1 is also involved in the process by which tumor cells adapt to adverse conditions, which contributes to radiation and chemotherapy resistance. Given its role in various clinical malignancies, caprin-1 holds the potential to be used as a biomarker and a target for the development of novel therapeutics. The present review describes this newly identified putative oncogenic protein and its possible impact on tumorigenesis.

The application of mRNA-based gene transfer in mesenchymal stem cell-mediated cytotoxicity of glioma cells.

Since the tumor-oriented homing capacity of mesenchymal stem cells (MSCs) was discovered, MSCs have attracted great interest in the research field of cancer therapy mainly focused on their use as carries for anticancer agents. Differing from DNA-based vectors, the use of mRNA-based antituor gene delivery benefits from readily transfection and mutagenesis-free. However, it is essential to verify if mRNA transfection interferes with MSCs' tropism and their antitumor properties. TRAIL- and PTEN-mRNAs were synthesized and studied in an in vitro model of MSC-mediated indirect co-culture with DBTRG human glioma cells. The expression of TRAIL and PTEN in transfected MSCs was verified by immunoblotting analysis, and the migration ability of MSCs after anticancer gene transfection was demonstrated using transwell co-cultures. The viability of DBTRG cells was determined with bioluminescence, live/dead staining and real time cell analyzer. An in vivo model of DBTRG cell-derived xenografted tumors was used to verify the antitumor effects of TRAIL- and PTEN-engineered MSCs. With regard to the effect of mRNA transfection on MSCs' migration toward glioma cells, an enhanced migration rate was observed with MSCs transfected with all tested mRNAs compared to non-transfected MSCs (p<0.05). TRAIL- and PTEN-mRNA-induced cytotoxicity of DBTRG glioma cells was proportionally correlated with the ratio of conditioned medium from transfected MSCs. A synergistic action of TRAIL and PTEN was demonstrated in the current co-culture model. The immunoblotting analysis revealed the apoptotic nature of the cells death in the present study. The growth of the xenografted tumors was significantly inhibited by the application of MSCPTEN or MSCTRAIL/PTEN on day 14 and MSCTRAIL on day 28 (p<0.05). The results suggested that anticancer gene-bearing mRNAs synthesized in vitro are capable of being applied for MSC-mediated anticancer modality. This study provides an experimental base for further clinical anticancer studies using synthesized mRNAs.

Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy.

Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable.

Differential expression and therapeutic efficacy of microRNA-346 in diabetic nephropathy mice.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, in which the SMAD signaling pathway plays an important role. The aim of the present study was to identify differentially expressed microRNAs (miRNAs) during the progression of DN and to investigate a selected miRNA in relation to SMAD3/4 and its therapeutic efficacy. The miRNA microarray was used to identify differentially expressed miRNAs in db/db DN mice. Reverse transcription-quantitative polymerase chain reaction and immunoblot analyses were used to detect SMAD3/4 expression. The development of DN in the db/db mice was demonstrated by glucose dysregulation and typical morphological changes in the kidney. miRNA-346 (miR-346) was identified as one of the differentially expressed miRNAs. The expression of SMAD3/4 was significantly attenuated by miR-346 administration and the therapeutic effects of miR-346 were observed in the DN mouse models. miR-346 was identified as a negative regulator of SMAD3/4. SMAD3/4 was upregulated in the renal tissue of db/db mice. The administration of miR-346 attenuated the SMAD3/4 expression in renal tissue and ameliorated the renal function and glomerular histology in DN mice. This study paves the way for clinical studies of miR-346 in DN.

Cancer cell-oriented migration of mesenchymal stem cells engineered with an anticancer gene (PTEN): an imaging demonstration.

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered to hold great potential as ideal carriers for the delivery of anticancer agents since the discovery of their tumor tropism. This study was performed to demonstrate the effects of phosphatase and tensin homolog (PTEN) engineering on MSCs' capacity for cancer cell-oriented migration. METHODS: MSCs were engineered with a PTEN-bearing plasmid and the expression was confirmed with Western blotting. A human glioma cell line (DBTRG) was used as the target cell; DBTRG cell-oriented migration of MSCs was monitored with a micro speed photographic system. RESULTS: The expression of transfected PTEN in MSCs was identified by immunoblotting analysis and confirmed with cell viability assessment of target cells. The DBTRG cell-oriented migration of PTEN-engineered MSCs was demonstrated by a real-time dynamic monitoring system, and a phagocytosis-like action of MSCs was also observed. CONCLUSION: MSCs maintained their capacity for cancer cell-directed migration after they were engineered with anticancer genes. This study provides the first direct evidence of MSCs' tropism post-anticancer gene engineering.

Intravital biobank and personalized cancer therapy: the correlation with omics.

Biobanks have played a decisive role in all aspects of the field of cancer, including pathogenesis, diagnosis, prognosis and treatment. The significance of cancer biobanks is epitomized through the appropriate application of various "-omic" techniques (omics). The mutually motivated relationship between biobanks and omics has intensified the development of cancer research. Human cancer tissues that are maintained in intravital biobanks (or living tissue banks) retain native tumor microenvironment, tissue architecture, hormone responsiveness and cell-to-cell signalling properties. Intravital biobanks replicate the structural complexity and heterogeneity of human cancers, making them an ideal platform for preclinical studies. The application of omics with intravital biobanks renders them more active, which makes it possible for the cancer-related evaluations to be dynamically monitored on a real-time basis. Integrating intravital biobank and modern omics will provide a useful tool for the discovery and development of new drugs or novel therapeutic strategies. More importantly, intravital biobanks may play an essential role in the creation of meaningful patient-tailored therapies as for personalized medicine.

Microparticles released by Listeria monocytogenes-infected macrophages are required for dendritic cell-elicited protective immunity.

Interplay between macrophages and dendritic cells in the processing and presentation of bacterial antigens for T-cell immune responses remains poorly understood. Using a Listeria monocytogenes (Lm) infection model, we demonstrate that dendritic cells (DCs) require the support of macrophages to elicit protective immunity against Lm infection. DCs themselves were inefficient at taking up Lm but capable of taking up microparticles (MPs) released by Lm-infected macrophages. These MPs transferred Lm antigens to DCs, allowing DCs to present Lm antigen to effector T cells. MP-mediated Lm antigen transfer required MHC class I participation, since MHC class I deficiency in macrophages resulted in a significant reduction of T-cell activation. Moreover, the vaccination of mice with MPs from Lm-infected macrophages produced strong protective immunity against Lm infection. We here identify an intrinsic antigen transfer program between macrophages and DCs during Lm infection, and emphasize that macrophages also play an essential role in DC-elicited Lm-specific T-cell responses.

Mast cell: insight into remodeling a tumor microenvironment.

Mast cells are of paramount importance to allergies, pathogen immune responses during infections, and angiogenesis, as well as innate and adaptive immune regulations. Beyond all these roles, mast cells are now more and more being recognized as modulators of tumor microenvironment. Notwithstanding mounting evidences of mast cell accumulation in tumors, their exact role in tumor microenvironment is still incompletely understood. In this review, we discuss the significant role of mast cells in the remodeling of tumor microenvironment by either releasing various factors after activation or interacting with other cells within tumor and, as a result, the possible role of mast cell in cancer invasion and metastasis. We also discuss recent findings that mast cells actively release microparticles, which account for the transfer of membrane-type receptor signal and regulatory molecules such as microRNAs to tumor cells and immune cells. These findings on mast cells provide further insights into the complexity of tumor microenvironment remodeling.

Potentiation of Th17 cytokines in aging process contributes to the development of colitis.

Th17 cells, which produce IL-17 and IL-22, promote autoimmunity in mice and have been implicated in the pathogenesis of autoimmune/inflammatory diseases in humans. However, the Th17 immune response in the aging process is still not clear. In the present study, we found that the induction of IL-17-producing CD4(+) T cells was significantly increased in aged individuals compared with young healthy ones. The mRNA expression of IL-17, IL-17F, IL-22, and RORC2 was also significantly increased in aged people. Similar to humans, Th17 cells as well as mRNAs encoding IL-17, IL-22 and RORγt were dramatically elevated in naïve T cells from aged mouse compared to young ones. In addition, CD44 positive IL-17-producing CD4(+) T cells were significantly higher in aged mice, suggesting that memory T cells are an important source of IL-17 production. Furthermore, the percentage of IL-17-producing CD4(+) T cells generated in co-culture with dendritic cells from either aged or young mice did not show significant differences, suggesting that dendritic cells do not play a primary role in the elevation of Th17 cytokines in aged mouse cells. Importantly, transfer of CD4(+)CD45Rb(hi) cells from aged mice induced more severe colitis in RAG(-/-) mice compared to cells from young mice, Taken together, these results suggest that Th17 immune responses are elevated in aging humans and mice and may contribute to the increased development of inflammatory disorders in the elderly.

Depletion of regulatory T cells facilitates growth of established tumors: a mechanism involving the regulation of myeloid-derived suppressor cells by lipoxin A4.

Regulatory T cells (Tregs) are thought to facilitate tumor development by suppressing protective antitumor immune responses. However, recent clinical and laboratory studies show that Tregs are a favorable element against cancer. In this study, we provide evidence that Tregs have both promoting and inhibiting effects on tumors, depending on the stage of tumor development. By using 0.5 mg cyclophosphamide, we constructed a murine liver cancer model in which Tregs were continuously and selectively depleted. Under such conditions, we found that tumor growth was inhibited at early stages but accelerated later on. Analysis of the tumor microenvironment disclosed that long-term Treg depletion by 0.5 mg cyclophosphamide treatment induced Gr-1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs). Ablation of MDSCs by anti-Gr-1 Ab blocked Treg depletion-induced promotion of tumor growth. Furthermore, lipoxygenases 5 and 12, two enzymes participating in the biosynthesis of the lipid anti-inflammatory mediator lipoxin A(4), were upregulated or downregulated by Treg depletion or adoptive transfer. Correspondingly, the levels of lipoxin A(4) were increased or decreased. Lipoxin A(4) thus regulated the induction of MDSCs in response to Treg depletion. These findings suggest that Tregs may play different roles at different stages of tumor growth: promoting early and inhibiting late tumor growth. Our study also suggests that the interplay among Tregs, MDSCs, and lipoxin A(4) tunes the regulation of tumor-associated inflammation.

Microparticles mediate enzyme transfer from platelets to mast cells: a new pathway for lipoxin A4 biosynthesis.

The inflammation-resolving lipid mediator lipoxin A4 (LXA4), which is derived from arachidonic acid in the context of inflammation, can be generated physiologically in vivo. However, the mechanism of physiologic formation of LXA4 remains elusive. In this report, we provide evidence that platelet-derived microparticles contain lipoxygenase 12 (12-LO) protein and act as a mediator in transferring 12-LO to mast cells, leading to the production of LXA4 by mast cells. Absence of either leukotriene, the precursor for LXA4, in mast cells or 12-LO in microparticles abolished LXA4 production. Using a mouse model, we demonstrated that platelet-derived microparticles were taken up by peritoneal mast cells in vivo and triggered LXA4 production. We also found that similar to LXA4, platelet-derived microparticles attenuated LPS- or dextran sulfate sodium-induced inflammation by regulating inflammatory cytokines. Together, these data suggest a critical role of platetlet-derived microparticles as a signal mediator, at least in LXA4 production, resulting in significant immunoregulatory consequences.

Selective depletion of CD4+CD25+Foxp3+ regulatory T cells by low-dose cyclophosphamide is explained by reduced intracellular ATP levels.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells have been shown to play important roles in mediating cancer development. Although cyclophosphamide (CY) has shown promise as a drug to selectively target Treg cells with low-dose in vivo, the underlying molecular mechanism remains unclear. In this report, we provide evidence that ATP, the energy molecule and signal element, accounts for the selective depletion of Treg cells by low-dose CY. Relative to conventional T cells or other cell types, ATP levels were much lower in Treg cells. This was due to Treg cells that downregulate one microRNA, miR-142-3p, and upregulate ecto-nucleoside triphosphate diphosphohydrolase CD39. The transfection of miR-142-3p or the blockade of CD39 could increase intracellular ATP levels of Treg cells, consequently decreasing the sensitivity of Treg cells to low-dose CY. On the other hand, the transfection of miR-142-3p inhibitor or the addition of soluble CD39 to the cultured CD4(+)CD25(-) T cells resulted in the decrease of intracellular ATP levels and increase of sensitivity of conventional T cells to low-dose CY. Furthermore, we found that the low levels of ATP attenuated the synthesis of glutathione, leading to the decrease of CY detoxification, thus increasing the sensitivity of Treg cells to low-dose CY. Therefore, we here identify a molecular pathway through which low-dose CY selectively ablates Treg cells. Our findings also imply that low levels of ATP are probably related to Treg cell function.

CD4+FOXP3+ regulatory T cell depletion by low-dose cyclophosphamide prevents recurrence in patients with large condylomata acuminata after laser therapy.

Condylomata acuminata (CA) caused by human papillomavirus (HPV) is a common sexually transmitted disease with half a million new cases diagnosed in the United States per year and the annual increase in incidence in China. Recurrence is a major challenge for CA treatment. Recently, we demonstrated that FOXP3(+) regulatory T (Treg) cells mediate the immunosuppression in large genital warts. Here, we further report that low-dose cyclophosphamide (CY), a conventional chemotherapy drug, can effectively prevent the recurrence of large CA in clinical patients after laser therapy. Surprisingly, although 9 out of 52 patients recur six weeks after the combination treatment, the re-administration of low-dose CY alone completely eliminates most recurred lesions. We provide evidence that low-dose CY not only depletes patients' Treg cells and enhances function of HPV-specific T cells and NK cells in the periphery, but also ameliorates the immune milieu of the lesion site, leading to the elimination of remnant viruses. These findings have important clinical significance, and potentially lead to a therapeutic breakthrough for the treatment of CA.

Hepatitis B virus protein X-induced expression of the CXC chemokine IP-10 is mediated through activation of NF-kappaB and increases migration of leukocytes.

Interferon-gamma inducible protein 10 (IP-10) involves inflammatory cell recruitment and cellular immune damage during virus infection. Although an increase of the peripheral IP-10 level is known in HBV-infected patients, the molecular basis of HBV infection inducing IP-10 expression has remained elusive. In the present study, we demonstrate that hepatitis B virus protein X (HBx) increases IP-10 expression in a dose-dependent manner. Transfection of the HBx-expressing vector into HepG2 cells results in nuclear translocation of NF-kappaB, which directly binds the promoter of IP-10 at positions from -122 to -113, thus facilitating transcription. The addition of the NF-kappaB inhibitor blocks the effect of HBx on IP-10 induction. In parallel, increase of NF-kappaB subunits p65 and p50 in HepG2 cells also augments IP-10 expression. Furthermore, we show that HBx induces activation of NF-kappaB through the TRAF2/TAK1 signaling pathway, leading to up-regulation of IP-10 expression. As a consequence, up-regulation of IP-10 may mediate the migration of peripheral blood leukocytes in a NF-kappaB-dependent manner. In conclusion, we report a novel molecular mechanism of HBV infection inducing IP-10 expression, which involves viral protein HBx affecting NF-kappaB pathway, leading to transactivation of the IP-10 promoter. Our study provides insight into the migration of leukocytes in response to HBV infection, thus causing immune pathological injury of liver.

Mast cells mobilize myeloid-derived suppressor cells and Treg cells in tumor microenvironment via IL-17 pathway in murine hepatocarcinoma model.

Tumor immunosuppression is commonly braided with chronic inflammation during tumor development. However, the relationship between immunosuppression and inflammation in tumor microenvironment is still unclear. We have demonstrated that mast cells are accumulated and exacerbate the inflammation and immunosuppression in tumor microenvironment via SCF/c-kit signaling pathway. Here, we further elucidate the underlying mechanism, which involves both myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our data showed that mast cells mobilized the infiltration of MDSCs to tumor and induced the production of IL-17 by MDSCs; MDSCs-derived IL-17 indirectly attracted Treg cells, enhanced their suppressor function, and induced the IL-9 production by Treg cells; in turn, IL-9 strengthened the survival and protumor effect of mast cells in tumor microenvironment. Our findings disclose a closed loop among mast cells, MDSCs and Treg cells in tumor microenvironment, which provides a new insight into the paralleled developments of inflammation and immunosuppression in tumor microenvironment. Based on these findings, we propose that targeting tumor inflammation might be a potential strategy to reverse the immunosuppression of tumor microenvironment, thus facilitating cancer immunotherapy.

Rapamycin inhibits lung metastasis of B16 melanoma cells through down-regulating alphav integrin expression and up-regulating apoptosis signaling.

Currently available data indicate the potential application of rapamycin and its analogues in the clinic as anticancer therapeutic agents through inhibiting tumor cell growth and tumor angiogenesis. However, whether rapamycin can directly suppress tumor metastasis remains unclear. In the present study, we demonstrated that rapamycin treatment results in reduced formation of metastatic nodules in the lung by B16 cells. This is due to two mechanisms. First, the expression of alphav integrin is down-regulated by rapamycin treatment, and subsequently, the phosphorylation of focal adhesion kinase (FAK) is reduced. Second, rapamycin promotes apoptosis by up-regulating the proapoptotic molecules Bid and Bax and down-regulating Bcl-xL. Blocking the apoptosis pathway by pan-caspase inhibitor zVAD partially reversed the suppression of rapamycin in B16 metastasis. Interestingly, rapamycin up-regulates Bax and Bid in B16 cells via the S6K1 pathway and down-regulates the expression of alphav integrin via other pathway(s). In addition, our data showed that autophagy was not involved in the mechanisms of rapamycin-mediated metastasis suppression. Our findings demonstrate a potential anti-metastatic effect of rapamycin via down-regulating alphav integrin expression and up-regulating apoptosis signaling, suggesting that rapamycin might be worthy of clinical evaluation as an antimetastatic agent.

Regulation of HIF-1alpha and VEGF by miR-20b tunes tumor cells to adapt to the alteration of oxygen concentration.

The regulation of HIF-1alpha is considered to be realized by pVHL-mediated ubiquitin-26S proteasome pathway at a post-transcriptional level. The discovery of a class of small noncoding RNAs, called microRNAs, implies alternative mechanism of regulation of HIF-1alpha. Here, we show that miR-20b plays an important role in fine-tuning the adaptation of tumor cells to oxygen concentration. The inhibition of miR-20b increased the protein levels of HIF-1alpha and VEGF in normoxic tumor cells; the increase of miR-20b in hypoxic tumor cells, nevertheless, decreased the protein levels of HIF-1alpha and VEGF. By using luciferase reporter vector system, we confirmed that miR-20b directly targeted the 3'UTR of Hif1a and Vegfa. On the other hand, the forced overexpression of HIF-1alpha in normoxic tumor cells downregulated miR-20b expression. However, HIF-1alpha knockdown in hypoxic tumor cells caused the increase of miR-20b. The differential expression of miR-20b has important biological significance in tumor cells, either enhancing the growth or favoring the survival of tumor cells upon the oxygen supply. Thus, we identify a novel molecular regulation mechanism through which miR-20b regulates HIF-1alpha and VEGF and is regulated by HIF-1alpha so to keep tumor cells adapting to different oxygen concentrations.