Dysregulation of mucosal-associated invariant T cells correlates with altered placental microenvironment in preterm birth.

Preterm birth (PTB) is a major problem affecting perinatal health, directly increasing the mortality risk of mother and infant that often results from the breakdown of the maternal-fetal immune balance. Increasing evidence shows the essential role of mucosal-associated invariant T (MAIT) cells to balance antibacterial function and immune tolerance function during pregnancy. However, the phenotype and function of placental MAIT cells and their specific mechanisms in PTB remain unclear. Here, we report that MAIT cells in placentas from PTBs show increased activation levels and decreased IFN-γ secretion capacity compared with those from normal pregnancies. Moreover, our data indicate gravidity is a factor affecting placental MAIT cells during pregnancies. Multi-omics analysis indicated aberrant immune activation and abnormal increase of lipids and lipid-like metabolites in the PTB placental microenvironment. Moreover, the proportion and activation of MAIT cells were positively correlated with the abnormal increase of lipids and lipid-like metabolites. Together, our work revealed that abnormal activation and impaired function of MAIT cells may be related to abnormal elevation of lipids and lipid-like metabolites in PTB.

Mucosal-Associated Invariant T Cell Dysregulation Correlates With Conjugated Bilirubin Level in Chronic HBV Infection.

BACKGROUND AND AIMS: Mucosal-associated invariant T (MAIT) cells are nonconventional T cells restricted to major histocompatibility complex class I-related protein 1 (MR1). They are highly abundant in human liver and activated by T-cell receptor (TCR)-dependent and TCR-independent mechanisms to exhibit rapid, innate-like effector responses. However, the roles of MAIT cells in chronic HBV infection are still open for study. This study aims to test their antiviral potential and investigate their dynamic changes and regulating factors during chronic HBV infection. APPROACH AND RESULTS: Blood samples from 257 chronic HBV-infected patients were enrolled, and nontumor liver specimens were collected from 58 HBV-infected HCC patients. Combining cell-culture experiments and human data, we showed that MAIT cells had strong cytotoxicity against HBV-transfected hepatocytes in an MR1-dependent way. However, circulating and hepatic MAIT cells in HBV-infected patients decreased significantly compared to controls. Correlation analysis suggested that MAIT cell frequency was associated with disease progression and inversely correlated with serum-conjugated bilirubin level. In particular, conjugated bilirubin not only directly promoted MAIT cell activation and apoptosis, but also impaired TCR-induced proliferation and expansion of MAIT cells, which could be partially rescued by IL-2 in the absence of conjugated bilirubin. Despite that MAIT cells from patients with high conjugated bilirubin levels showed decreased cytokine-producing capacity, the increased TCR-dependent antiviral cytokine production suggested MAIT cells as an important guardian of chronic HBV with high conjugated bilirubin. CONCLUSIONS: We reveal the MR1-dependent, anti-HBV potential of MAIT cells and identify conjugated bilirubin as a major factor dysregulating its frequency and function in chronic HBV-infected patients, suggesting a therapeutic target for MAIT-cell-based immunity against chronic HBV infection.

Elevated Hepatic CD1d Levels Coincide with Invariant NKT Cell Defects in Chronic Hepatitis B Virus Infection.

Activation of invariant NKT (iNKT) cells manifests antiviral immune responses in vivo. However, clinical trials have failed to show consistent hepatitis B virus (HBV) DNA reduction postadministration of iNKT cell-specific agonist α-galactosylceramide (α-GalCer). In this study, we aimed to investigate HBV infection-related iNKT cell defects and explore iNKT cell-based therapeutic potential for chronic hepatitis B (CHB). Liver specimens from 30 HBV-infected hepatocellular carcinoma patients were collected for CD1d/hepatitis B surface Ag (HBsAg) staining and/or intrahepatic iNKT cell assay. Two hundred and six chronic HBV-infected patients (including 130 CHB patients) were enrolled in the study of circulating iNKT cell frequency and function. We found that liver and hepatoma tissue that positively stained for HBsAg had higher CD1d expression as compared with HBsAg negatively stained counterparts. The elevated CD1d expression in infected tissue is supposed to facilitate the iNKT cell-based antiviral effects locally. However, iNKT cell defects that related with disease progression suggested iNKT cells attenuated their effects during chronic HBV infection. The residual iNKT cells in CHB patients showed aberrant activation and hyporesponsiveness to α-GalCer. Exogenous IL-2 fully rescued α-GalCer-induced expansion of iNKT cells from CHB patients, and synergistic effects of IL-2 and IL-15 helped to recover the CD1d-dependent IFN-γ production. In conclusion, our results highlight the increased CD1d expression in HBV-infected liver and differential iNKT cell defects associated with disease progression during chronic HBV infection. The reversibility of iNKT cell defects suggests protective immune responses could be partially recovered in CHB.

Betulin from Hedyotis hedyotidea ameliorates concanavalin A-induced and T cell-mediated autoimmune hepatitis in mice.

Hedyotis hedyotidea has been used in traditional Chinese medicine for the treatment of autoimmune diseases. However, the mechanisms underlying for the effect remain unknown. We previously showed that, among 11 compounds extracted from H hedyotidea, betulin produced the strongest suppressive effect on T cell activation. Here, we examined the hepatoprotective effects of betulin against acute autoimmune hepatitis in mice and the mechanisms underlying the effects. Freshly isolated mouse splenocytes were stimulated with concanavalin A (Con A, 5 µg/mL) in the presence of betulin, the cell proliferation was assessed with CSFE-dilution assay. Mice were injected with betulin (10, 20 mg·kg-1·d-1, ip) for 3 d. One hour after the last injection, the mice were injected with Con A (15 mg/kg, iv) to induce acute hepatitis. Blood samples and liver tissues were harvested at 10 h after Con A injection, and serum transaminase levels and liver histopathology were detected; serum levels of proinflammatory cytokines, hepatic T lymphocyte ratios, and functional statuses of conventional T and NKT cells were also analyzed. Betulin (16 and 32 µmol/L) dose-dependently suppressed the proliferation of Con A-stimulated mouse splenocytes in vitro. In Con A-challenged mice, preinjection with betulin (20 mg·kg-1·d-1) significantly decreased the levels of proinflammatory cytokines IFN-γ, TNF-α and IL-6, and ameliorated liver injury. Furthermore, pretreatment with betulin (20 mg·kg-1·d-1) significantly inhibited the Con A-induced activation of NKT and conventional T cells, and decreased production of proinflammatory cytokines IFN-γ, TNF-α and IL-6 in these two cell populations. Betulin has immunomodulatory effect on overly activated conventional T and NKT cells and exerts hepatoprotective action in mouse autoimmune hepatitis. The findings provide evidence for the use of H hedyotidea and its constituent betulin in the treatment of autoimmune diseases.

Distinctly altered lipid components in hepatocellular carcinoma relate to impaired T cell-dependent antitumor immunity.

BACKGROUND AND AIMS: T cells are master effectors of anti-tumor immunity in cancer. Recent studies suggest that altered lipid metabolism imposed by the tumor microenvironment constrains anti-tumor immunity. However, the tumor-associated lipid species changes that dampen T cell ability to control tumor progression are not fully understood. Here, we plan to clarify the influences of distinctly altered lipid components in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) on T-cell function, aiming to seek lipid metabolic targets for improving T cell anti-tumor effects. METHODS: Tumor tissues and non-tumor liver from HCC patients were collected for RNA-sequencing, lipid profiling and T cell characterizing, followed by correlation analysis. Additionally, the effects of significantly changed lipid components on anti-tumor potential of T cells were tested by in vitro cell experiments and/or in vivo tumor inoculated model. RESULTS: Altered lipid metabolism coincides with impaired T cell response in HBV-related HCC. Characteristic lipid composition, significantly marked by accumulation of long-chain acylcarnitines (LCACs) and reduction of lysophosphatidylcholines (LPCs), are found in the tumor tissue. Notably, LCACs accumulated are associated with T cells exhaustion and deficient functionality, while LPCs correlate to anti-tumor effects of T cells. In particular, supplement of LPCs, including LPC (20:0) and LPC (22:0), directly promote the activation and IFN-γ secretion of T cells in vitro, and suppress tumor growth in vivo. CONCLUSIONS: Our study highlights the distinctly changed lipid components closely related to T cell dysregulation in HCC, and suggests a promising strategy by decreasing LCACs and increasing LPCs for anti-tumor immunotherapy.

A similarity in peptide cross-reactivity between alloantigen- and nominal antigen-induced CD8+ T cell responses in vitro.

Raising tumor-specific allorestricted T cells in vitro for adoptive transfusion is expected to circumvent host tumor tolerance. However, it has been assumed that alloreactive T cell clones activated in vitro ranges from peptide-specific with high avidity to peptide-degenerate with low avidity. In this study, we examined the peptide specificity and cross-reactivity of T cell responses in vitro to an allogeneic epitope and a nominal epitope with a modified co-culture of lymphocytes and autologous monocytes. After binding to the monocyte via the interaction of its Fc part and the cell surface IgG Fc receptor type I (FcγRI), a fusion protein consisting of the extracellular domains of HLA-A2 molecule and the Fc region of IgG1 (the dimer) introduced a single epitope into the co-culture. The dimer-coated monocytes stimulated the proliferation of autologous CD8(+) T cells after co-culturing. The CD8(+) T cell responses were self-HLA-restricted for HLA-A2-positive (HLA-A2+ve) samples and allo-HLA-restricted for HLA-A2-negative (HLA-A2-ve) samples, since the co-cultural bulks stained with HLA-A2 tetramers, human interferon-gamma (IFN-γ) production in response to T cell receptor (TCR) ligands, and cytotoxicity against a panel of target cells exhibited peptide-specific properties. Two HLA-A2-restricted peptides with sequence homology were included, allowing the comparison of cross-reactivity between allo-antigen- and nominal antigen-induced CD8(+) T cell responses. Interestingly, the allo- and self-HLA-restricted CD8(+) T cell responses were similar in the peptide cross-reactivity, although the allorestricted T cell response seemed, overall, more intensive and had higher binding affinity to specific tetramer. Our findings indicated the alloreactive T cells raised by the co-culture in vitro were as peptide specific and cross-reactive as the self-HLA-restricted ones.

Long-Chain Acylcarnitines Induce Senescence of Invariant Natural Killer T Cells in Hepatocellular Carcinoma.

CD1d-restricted invariant natural killer T (iNKT) cells actively patrol the liver and possess valuable antitumor potential. However, clinical trials evaluating administration of iNKT cell-specific agonist α-galactosylceramide (α-GalCer) have failed to achieve obvious tumor regression. Improving the efficacy of iNKT cell-based immunotherapy requires a better understanding of the factors restraining the clinical benefits. In the context of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), we found circulating and hepatic iNKT cells were hyperactivated but demonstrated imbalances in ratio and defective α-GalCer responsiveness. Exogenous IL2 helped to expand residual α-GalCer-responsive clones with reduced T-cell receptor diversity. However, transcriptome-wide analysis revealed activation of the senescence-associated secretory phenotype and dampened cytotoxicity in iNKT cells, weakening their immune surveillance capacity. The senescent status of iNKT cells from the patients was further illustrated by cell-cycle arrest, impaired telomere maintenance, perturbed calcium transport-related biological processes, and altered metabolism. Lipidomic profiling revealed the accumulation of long-chain acylcarnitines (LCAC) and aberrant lipid metabolism in HCC tissue. Exogenous LCACs, especially palmitoyl-carnitine and stearoyl-carnitine, inhibited iNKT cell expansion and promoted senescence. Collectively, our results provide deeper insights into iNKT cell dysregulation and identify a cell senescence-associated challenge for iNKT cell-based immunotherapy in HBV-related HCC. The mechanistic links between iNKT cell senescence and accumulated LCACs suggest new targets for anti-HCC immunotherapies. SIGNIFICANCE: Patients with HBV-related HCC exhibit a cell senescence-associated dysregulation of invariant natural killer cells that is related to altered lipid metabolism and accumulated LCACs in tumor tissue.

Increased Non-MAIT CD161+CD8+ T Cells Display Pathogenic Potential in Chronic HBV Infection.

BACKGROUND & AIMS: CD161-expressing CD8+ T cells consist of mucosal-associated invariant T cells with semi-invariant T-cell receptor (TCR) use and non-mucosal-associated invariant T CD161+CD8+ T cells with polyclonal TCR repertoire. Although CD161+CD8+ T cells are enriched in liver and embrace hepatitis B virus (HBV)-specific T cells in chronic hepatitis B (CHB) patients, their roles in disease progression remain poorly understood. This study aimed to decipher their profiling and dynamic changes during chronic HBV infection. METHODS: Blood samples from 257 CHB patients and nontumor liver specimens from 73 HBV-positive patients were analyzed for CD161+CD8+ T-cell characterization by flow cytometry, TCR repertoire determination, transcriptomic analyses, and cell experiments. RESULTS: CD161+CD8+ T cells were increased and hyperactivated in patients, while positive correlation between the CD161+CD8+ T-cell ratio and HBV-DNA level suggested this was insufficient to control HBV replication. The overlap of complementarity determining region 3 sequences supported the switch between CD161-CD8+ and CD161+CD8+ populations. Although CD161+CD8+ T cells were endowed with innateness phenotype and enhanced antiviral capacity, the population from patients had impaired type I cytokine production, and increased interleukin 17 and granzyme B secretion. The increased CD161+CD8+ T cells and their increased granzyme B secretion correlated positively with inflammation-associated liver injury. Hepatic CD161+CD8+ T cells showed neutrophil-related pathogenic potential because they had increased transcript signatures and proinflammatory cytokine production in neutrophil recruitment- and response-related pathways that changed consistently in the injured liver. CONCLUSIONS: Our results highlight the reduced antiviral potency but increased pathogenic potential of CD161+CD8+ T cells in CHB patients, supporting CD161 expression as a marker of pathogenic CD8+ T subset and the intervention target for liver injury.

Mucosal-associated invariant T cells reduce and display tissue-resident phenotype with elevated IL-17 producing capacity in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the important causes of cancer-related mortality worldwide. Previous studies have demonstrated the crucial roles of mucosal-associated invariant T (MAIT) cells in regulating tumor immunity, while their roles in NSCLC remain largely unknown. The aim of this study is to evaluate clinical relevance of MAIT cells in blood and tumor tissues of patients with NSCLC. Here, we find that there is no significant difference in the frequency of circulating MAIT cells between NSCLC patients and healthy donors. However, the MAIT-frequency is significantly declined in lung tumor tissues compared to their peri-tumor counterparts, which relates to Tumor-Node-Metastasis (TNM) stage. The MAIT-frequency in lung tumor tissues is higher in node-negative patients compared to node-positive patients. Furthermore, tumor-infiltrating MAIT cells display a tissue-resident effector-memory phenotype and exhibit upregulated levels of exhaustion markers. The percentage of tissue-resident cells in MAIT tends to be higher in tumor tissues than in peri-tumor tissues. In addition, the percentage of IL-17A+ MAIT cells is significantly higher in lung tumor tissues than that in peri-tumor tissues. In summary, our results indicate the possible detrimental role of MAIT cells in the development and progression of NSCLC.

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.

The effects of PDL-Ig on collagen-induced arthritis.

Programmed death ligand (PDL) is a new member of the B7 family of costimulatory molecules that specifically interacts with programmed death 1 (PD-1) expressed on activated T cells, B cells, and myeloid cells. Collagen II (CII)-induced arthritis (CIA) is an experimental model of arthritis that has been used to dissect the pathogenesis of human rheumatoid arthritis. In this study, we have investigated the effects of PDL-Ig on CIA. Administration of PDL-Ig significantly ameliorated the disease as assessed by clinical arthritis score and histology in the joints. Expression of proinflammatory cytokines, such as IL-17 and IL-23, in the serum was reduced by PDL-Ig treatment. These results showed a beneficial effect of PDL-Ig on CIA through anti-inflammatory actions and inhibition of cell proliferation in response to CII, suggesting that the PD-1-PDL pathway may be involved in the pathogenesis of CIA, and thus PDL-Ig may be a useful therapy for the improvement of human rheumatoid arthritis.

The Expansion and Cytotoxicity Detection of Human iNKT Cells.

Invariant natural killer T (iNKT) cell is a type of innate-like T cell subsets with both T and NK cell phenotype and functions. They recognize lipid antigens presented by CD1d molecules and can be specifically activated by alpha-galactosylceramide (α-GalCer) in vitro. After activation, iNKT cells expand efficiently and exert direct killing effects. Based on it, we mainly introduce the protocols of detection of human iNKT cell functions in vitro, including in vitro expansion and their cytotoxicity to tumor cells.

Activation and Functional Alteration of Mucosal-Associated Invariant T Cells in Adult Patients With Community-Acquired Pneumonia.

Community-acquired pneumonia (CAP) remains the significant infectious cause of morbidity and mortality worldwide. Although mucosal-associated invariant T cells (MAIT) play roles in the pathogenesis of children CAP and ICU-associated pneumonia, their roles in adult CAP are largely unexplored. In this study, we investigated the frequency, phenotype, and function of MAIT cells in peripheral blood and bronchoalveolar lavage fluid (BALF) of adult CAP patients. Our data indicate that MAIT-cell frequency is profoundly lower in the peripheral blood of CAP patients compared to that in healthy individuals. Furthermore, the circulatory MAIT cells express higher levels of CD69 and PD-1 compared to those in healthy individuals. In BALF of CAP patients, MAIT-cell frequency is higher and MAIT cells express higher levels of CD69 and PD-1 compared to their matched blood counterparts. Levels of IL-17A and IFN-γ are increased in BALF of CAP patients compared to those in BALF of patients with pulmonary small nodules. The IL-17A/IFN-γ ratio is significantly positively correlated with MAIT frequency in BALF of CAP patients, suggesting a pathogenic role of MAIT-17 cells in CAP. Of note, blood MAIT-cell frequency in CAP patients is strongly negatively correlated with high-sensitivity C-reactive protein (hsCRP) and neutrophil count percentage in blood. The ability of circulating MAIT cells in CAP patients to produce IFN-γ is significantly impaired compared to those in healthy individuals. In summary, our findings suggest the possible involvement of MAIT cells in the immunopathogenesis of adult CAP.

Viral specific cytotoxic T cells inhibit the growth of TfR-expressing tumor cells with antibody targeted viral peptide/HLA-A2 complex.

A fusion protein of single chain antibody (scFv) specific for transferrin receptor (TfR, CD71) and viral peptide/HLA-A2 complex was prepared in this study to redirect cytotoxic T cells (CTLs) of viral specificity to tumor cells by attaching the ligand of T cell receptor (TCR) to tumor cells via binding of TfR scFv to TfR. The results demonstrate that the fusion protein can attach the active virus-peptide/HLA-A2 complex to HLA class I-negative, TfR-expressing K562 cells through binding of TfR scFv to TfR, and mediate cytotoxicity of viral peptide-specific CTLs against K562 cells in vitro. In addition, the fusion protein can induce inhibition of solid tumor formation and improve survival time in tumor xenograft nude mouse with the injection of the sorted viral peptide-specific CTLs generated by co-culture of peripheral blood lymphocytes from HLA-A2 positive donors with inactivated T2 cells pulsed with the viral peptide.

Detection, Expansion, and Isolation of Human MAIT Cells.

Mucosal-associated invariant T (MAIT) cells are a novel subset of innate-like T cells that recognize vitamin B metabolites from a range of microbes presented by MHC class I-related molecules (MR1). The term mucosal-associated invariant T cells derives from the fact that MAIT cells are abundant in the liver and mucosal tissues, and human MAIT cells use a semi-invariant TCR Vα7.2 Jα33 paired with Vß2 or Vß13. Here, based on the interaction between MAIT cell and its ligand 5-OP-RU/MR1, we describe the protocols for identification, rapid expansion, and isolation of human MAIT cells.

Mitochondrial-targeted ubiquinone alleviates concanavalin A-induced hepatitis via immune modulation.

BACKGROUND: Despite knowledge regarding the effects of antioxidants in ameliorating oxidative damage, evidence concerning their effects on activated immune cells is lacking. Here, a concanavalin A (Con A)-induced hepatitis mouse model was used to investigate the protective effects and immune regulatory mechanisms of mitochondrial-targeted ubiquinone (MitoQ). METHODS: Wild-type (WT) and CD1d-knockout (CD1d-/-, NKT cell deficient) mice were pretreated with MitoQ and then intravenously injected with a sublethal dose of Con A. Serum transaminase and inflammatory cytokine levels were tested. Immune cell functions and AMPK/mTORC1 pathway activation in liver tissue were also evaluated. RESULTS: NKT cells were critical for extensive pro-inflammatory cytokine production and prolonged liver injury upon Con A challenge, while IFN-γ-producing non-NKT cells played an important role during the hyperacute phase. MitoQ treatment not only ameliorated NKT cell-independent hyperacute hepatitis within 12 h post Con A administration but also alleviated NKT cell-dependent extended liver injury at 24 h. The underlying mechanisms involved an inhibition of the heightened activation of iNKT cells and conventional T cells, suppression of the excessive production of IFN-γ, TNF-α and IL-6, and modulation of aberrant AMPK and mTORC1 pathways. CONCLUSION: MitoQ efficiently alleviates Con A-induced hepatitis through immune regulation, suggesting a new therapeutic approach for immune-mediated liver injury by targeting mitochondrial ROS.

Heterogeneity of antigen specificity between HLA-A*02:01 and other frequent Chinese HLA-A2 subtypes detected by a modified autologous lymphocyte-monocyte coculture.

HLA-A2 is the most common serological HLA type among all ethnic groups. Through advances in DNA typing, more than 800 subtypes of HLA-A2 have been identified, and the existence of heterogeneity of antigen specificity among the HLA-A2 subtypes has been suggested by retrospective analyses of allogeneic transplantation patients and by studies of antigen amino acid structure. However, prior to this study, the antigenicity of a given subtype or the mismatch extent between two given subtypes could not be studied in vitro. Here, we used a modified autologous lymphocyte-monocyte coculture method to reveal heterogeneity of antigen specificity among HLA-A2 subtypes. The coculture was set up with HLA-A2 (non-A*02:01) lymphocytes and monocytes, and the monocytes were coated with an HLA-A*02:01/IgG1-Fc fusion protein (dimer) by high-affinity binding of the IgG1-Fc to FcgRI. Lymphocyte proliferation following coculture indicated that HLA-A*02:01 showed antigenicity against the HLA-A2 (non-A*02:01) subtype. Among the most frequent HLA-A2 subtypes in the Chinese population (HLA-A*02:01, -A*02:03, -A*02:06 and -A*02:07), we identified significant -A*02:01 antigenicity for T cells from -A*02:03 or -A*02:06 but not -A*02:07 individuals. Our findings were consistent with retrospective studies of allograft patients with a limited number of involved subtypes, indicating that this modified coculture method provides a practical and reliable means to study the antigenicity of HLA allele subtypes in vitro.

PMA treated THP-1-derived-IL-6 promotes EMT of SW48 through STAT3/ERK-dependent activation of Wnt/ß-catenin signaling pathway.

Colon cancer is one of the most common digestive malignant tumors that leads to high mortality worldwide, and metastasis is the primary cause of cancer-related death. It is well accepted that the epithelial-mesenchymal transition (EMT) plays a key role in the process of metastasis. As a cytokine that macrophage secretes, IL-6 is involved in the progression of tumors, including the invasion and metastasis via kinds of signaling pathways. However, the mechanism of interactions between IL-6, macrophage, EMT and colon cancer is not fully understood. Increased CD68+ macrophages and IL-6 level were found in colon tumor as compared to normal colon tissue. Metastatic lymph node showed even more CD68+ macrophages and higher IL-6 level than the primary tumor. These results suggested that macrophages and IL-6 play an important role in EMT of colon cancer. In order to investigate the effect of macrophage and IL-6 on EMT of colon cancer, we cultured human colon carcinoma cell line SW48 with conditioned medium (CM) from PMA-stimulated monocyte THP-1 cells and tested for IL-6 dependent EMT pathways. Wound healing assay and Transwell assay were used to analyze cell migration and invasion. Results showed that CM-treated SW48 cells increased IL-6 production and displayed elevated capacity of migration and invasion compared to untreated cells. Increased expressions of EMT markers (N-cadherin, Vimentin and ß-catenin) and decreased expression of EMT marker(E-cadherin) were found in CM-treated SW48 cells by Western Blot. The addition of an anti-IL-6 antibody significantly inhibited the increase of EMT markers (Vimentin and ß-catenin) as well as cell migration and invasion, suggesting that IL-6 played a critical role in promoting EMT of CM-treated SW48 cells. In addition, we found that the levels of p-STAT3 and p-ERK increased in CM-treated SW48 compared to untreated cells, which can be reversed by AG490, an inhibitor of JAK. In the meantime, the suppression of JAK-associated signaling pathways caused a decrease of ß-catenin. In summary, our study suggested that macrophage-induced IL-6 promotes migration and invasion of colon cancer cell via Wnt/ß-catenin pathway in STAT3/ERK-dependent way.

Antigen-specific CD8+ memory stem T cells generated from human peripheral blood effectively eradicate allogeneic targets in mice.

BACKGROUND: As the implantation and long-term existence of tumor-specific T cells in host are the prerequisite for adoptive immunotherapy, memory stem T cells (TSCM) with self-renewal and differentiation capacity show the greatest potential to implant and long-term exhibit function in vivo, compared with other T cells of differentiation stages. Hence, tumor-specific TSCM have become potential candidate for adoptive T cell therapy of cancer. Here, we reported a protocol to generate allogeneic antigen-specific CD8+ TSCM cells from human PBLs. METHODS: To prepare allogeneic antigen-specific CD8+ TSCM, we used an LCL named E007 of defined HLA allotyping as simulator, a co-culture of E007 and allogeneic PBLs was carried out in the presence of differentiation inhibitor TWS119 for 7 days. Sorting of proliferation cells ensured the E007-specificity of the prepared TSCM cells. The sorted lymphocytes underwent further expansion by cytokines IL-7 and IL-15 for further 7 days, making the E007-specific CD8 + TSCM expanded in number. The stem cell and T memory cell properties of the prepared CD8+ TSCM were observed in NOD-SCID mice. RESULTS: Our protocol began with 1 × 107 PBLs and resulted in 2 × 107 E007-specific CD8+ TSCM cells in 2 weeks of preparation. The prepared TSCM cells exhibited a proliferative history and rapid differentiation into effector cells upon the E007 re-stimulation. Importantly, the prepared TSCM cells were able to exist long and reconstitute other T cell subsets in vivo, eradicating the E007 cells effectively after transferred into the LCL burden mice. CONCLUSIONS: This protocol was able to prepare allogeneic antigen-specific CD8+ TSCM cells from human PBLs. The prepared TSCM showed the properties of stem cells and T memory cells. This study provided a reference method for generation of antigen-specific TSCM for T cell adoptive immunotherapy.

Fas/FasL interaction mediates imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells.

The rapid antitumor cytokine production and direct cytotoxicity confer invariant NKT (iNKT) cells ideal candidates for cancer therapy. However, the therapeutic potential of iNKT cells in T-cell malignant diseases remains elusive, as antigen presentation by T cells (T-T presentation) has been suggested to induce hyporesponsiveness of iNKT cells. In this study, we found discrepancies in iNKT cell responses against two T cell-origin cell lines (Jurkat and Molt-4). Human iNKT cells exhibited more intensive cytotoxicity and less efficient cytokine production in response to Fas-bearing Jurkat cells than those to the Fas-negative tumor cells (Molt-4 and myeloid-derived K562). The imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells were CD1d-dependent and relied mostly on Fas/FasL interaction. The impairment in cytokine production could be overcome by Fas/FasL blocking antibodies and exogenous IL-2. Elevated CD1d levels as well as CD1d and Fas co-localization were found in T-cell lymphomas. However, defects in frequency and function of circulating iNKT cells were observed in the patients, which could be partly rescued by exogenous IL-2. Collectively, the Fas/FasL-dependent aberrant iNKT cell responses and the reversibility of the defects suggest the distinct iNKT cell manipulation in CD1d- and Fas-bearing T cell malignancies.