The transcription factor TCF-1 initiates the differentiation of T(FH) cells during acute viral infection.

Induction of the transcriptional repressor Bcl-6 in CD4(+) T cells is critical for the differentiation of follicular helper T cells (T(FH) cells), which are essential for B cell-mediated immunity. In contrast, the transcription factor Blimp1 (encoded by Prdm1) inhibits T(FH) differentiation by antagonizing Bcl-6. Here we found that the transcription factor TCF-1 was essential for both the initiation of T(FH) differentiation and the effector function of differentiated T(FH) cells during acute viral infection. Mechanistically, TCF-1 bound directly to the Bcl6 promoter and Prdm1 5' regulatory regions, which promoted Bcl-6 expression but repressed Blimp1 expression. TCF-1-null T(FH) cells upregulated genes associated with non-T(FH) cell lineages. Thus, TCF-1 functions as an important hub upstream of the Bcl-6-Blimp1 axis to initiate and secure the differentiation of T(FH) cells during acute viral infection.

Cutting Edge: Transcription Factor BCL6 Is Required for the Generation, but Not Maintenance, of Memory CD8+ T Cells in Acute Viral Infection.

The differentiation of memory CD8+ T cells is critical to the long-term cellular immunity. The transcription factor BCL6 has been reportedly important for the generation and maintenance of memory CD8+ T cells; however, using the newly established BCL6 conditional knockout mouse model, we demonstrate that BCL6 is dispensable for the maintenance of established memory CD8+ T cell pool, although BCL6 is still required for the generation of CD8+ memory precursors upon acute viral infection. In addition, BCL6 promotes the expression of TCF-1 via directly binding to the Tcf7 (gene symbol for TCF-1) allele in CD8+ memory precursors and forced expression of TCF-1 restores the generation of BCL6-deficient memory precursors. Thus, our findings clarify that BCL6 is dispensable for the maintenance of memory CD8+ T cells, but functions as an important upstream of TCF-1 to regulate the generation of memory precursors in acute viral infection.

The characteristic expression of B7-H3 and B7-H4 in liver biopsies from patients with HBV-related acute-on-chronic liver failure.

Hepatitis B virus (HBV) is a major public health problem, and HBV-related acute-on-chronic liver failure (HBV-ACLF) has an extremely poor prognosis due to a lack of effective treatments. B7-H3 and B7-H4 are two novel members of the B7 superfamily that are actively involved in regulating the pathogenesis of infectious diseases. However, the intrahepatic expression of both members in HBV-ACLF patients has yet to be described. In this study, we analyzed the expression of B7-H3 and B7-H4 in HBV-ACLF biopsies by immunohistochemistry. Our results showed that both members were observed in all HBV-ACLF samples, and their expression was chiefly observed on infiltrating inflammatory cells and the damaged bile ducts. Immunofluorescence double staining showed that B7-H4 was expressed chiefly on CD3(+) T cells, CD68(+) macrophages, CK-18(+) bile ducts, and CD31(+) endothelial cells, while B7-H3 was found on all cell types detected. The expression of the programmed death (PD)-1 ligands, PD-L1 and PD-L2, was also detected in these liver tissues and they were found to be co-expressed with B7-H3 and B7-H4. These results suggest that the B7-family signaling is most likely to affect the pathogenesis of this disease, and a clear understanding of their functional roles may further elucidate the disease process.

The GTPase Rab3b/3c-positive recycling vesicles are involved in cross-presentation in dendritic cells.

Antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. In this study, we examined the effect of siRNA-mediated knockdown of 57 Rab GTPases, the key regulators of membrane trafficking, on antigen cross-presentation. Twelve Rab GTPases were identified to be associated with antigen cross-presentation, and Rab3b/3c was indicated to be colocalized with MHC class I molecules at perinuclear tubular structure. Tracing with fluorescence protein-tagged beta(2)-microglobulin demonstrated that the MHC class I molecules were internalized from the plasma membrane to Rab3b/3c-positive compartments, which were also colocalized with the internalized transferrin. Moreover, depletion of Rab3b/3c strongly reduced the fast phase recycling rate of transferrin receptors. Furthermore, the Rab3b/3c-positive compartments were colocalized with a fraction of Rab27a at a juxtaposition of phagosomes. Together, these data demonstrate that Rab3b/3c-positive recycling vesicles are involved in and may constitute one of the recycling compartments in exogenous antigen cross-presentation.

MnO2-melittin nanoparticles serve as an effective anti-tumor immunotherapy by enhancing systemic immune response.

Cancer vaccines are viewed as a promising immunotherapy to eradicate malignant tumors and aim to elicit the patients' own tumor-specific immune response against tumor cells. However, few cancer vaccines have been applied due to the low immunogenicity of antigen and invalidation of adjuvant. Herein, we designed a tumor microenvironment (TME) responsive MnO2-melittin nanoparticles (M-M NPs). The M-M NPs consumed glutathione and produced •OH via Fenton-like reaction in the mimic TME, specifically caused tumor cell death in vitro, activated cGAS-STING pathway in vitro and promoted the maturation of antigen-presenting cells in vitro and in vivo to elicit systemic anti-tumor immune response including the augmentation of tumor-specific T cells and more productions of pro-inflammatory cytokines and chemokines, which all were stronger than MnO2 NPs and melittin. The anti-tumor effects of M-M NPs were evaluated in three subcutaneous tumor models and the B16-F10 lung metastasis model and the tumor growth and lung metastasis were more obviously inhibited in the M-M NPs treated mice, compared with MnO2 NPs and melittin treatments. More importantly, only M-M NPs promoted the MHC-I cross-dressing by dendritic cells to prime tumor-specific CD8+ T cells and remarkably suppressed the growth of left tumors if express cognate antigen while treating on the right in the bilateral tumor model. Our findings proposed a strategy to enhance the cancer vaccine efficiency which showed great therapeutic effect on tumor immunotherapy.

Liver X receptor ß is required for the survival of single-positive thymocytes by regulating IL-7Rα expression.

Liver X receptors (LXRs) are known as key transcription factors in lipid metabolism and have been reported to play an important role in T-cell proliferation. However, whether LXRs play a role in thymocyte development remains largely unknown. Here, we demonstrated that LXRß deficiency caused a reduction in single-positive (SP) thymocytes, whereas the transitions from the double-negative to SP stage were normal. Meanwhile, LXRß-null SP thymocytes exhibited increased apoptosis and impairment of the IL-7Rα-Bcl2 axis. In addition, the LXR agonist T0901317 promoted the survival of SP thymocytes with enhanced IL-7Rα expression in wild-type mice but not in LXRß-deficient mice. Mechanistically, LXRß positively regulated the expression of IL-7Rα via direct binding to the Il7r allele in SP thymocytes, and forced expression of IL-7Rα or Bcl2 restored the survival of LXRß-defective SP thymocytes. Thus, our results indicate that LXRß functions as an important transcription factor upstream of IL-7Rα to promote the survival of SP thymocytes.

A haplotype in STAT4 gene associated with rheumatoid arthritis in Caucasians is not associated in the Han Chinese population, but with the presence of rheumatoid factor.

OBJECTIVE: Several studies have shown that a haplotype (rs11889341, rs7574865, rs8179673 and rs10181656) in STAT4 is associated with the development of RA in Caucasian, Korean and Japanese populations. The aim of this study was to investigate the effect of STAT4 on susceptibility to RA in the Han Chinese population. METHODS: Unrelated 750 RA patients and individually matched 750 healthy controls were genotyped for single nucleotide polymorphism (SNP), rs11889341, in STAT4, which tags the susceptibility haplotype. Association analyses were performed on the whole data set and on sex subsets. Significant relationships were determined between clinical variables and rs11889341 for each disease subtype in the studied groups. RESULTS: There was no evidence of a significant association between rs11889341 and RA. The heterozygous CT genotype was associated with RA in female group [P = 0.027; odds ratio (OR) 1.31; 95% CI 1.03, 1.65]. No association was found in male group and in any subsets of RA based on sex, RF and anti-cyclic citrullinated peptide (anti-CCP) antibody. However, in the Han Chinese population with RA disease, we observed a significantly decreased frequency of the minor T allele and TT genotype in the RF-positive subgroup compared with RF-negative subgroup (T allele: P = 0.024; OR 0.73; 95% CI 0.56, 0.95; TT genotype P = 0.013; OR 0.49; 95% CI 0.28, 0.86). CONCLUSION: The STAT4 RA-susceptibility haplotype identified in other previously reported populations has not been replicated in the Han Chinese population with individually matched case-control study design. It is associated only with the presence of RF in the Han Chinese population with RA disease.

Intrahepatic PD-1/PD-L1 up-regulation closely correlates with inflammation and virus replication in patients with chronic HBV infection.

Chronic hepatitis B was characterized by fluctuant immune response to infected hepatocytes resulting in hepatic inflammation and virus persistence. Recently, Programmed Death-1 (PD-1) and its ligand PD-L1 have been demonstrated to play an essential role in balancing antiviral immunity and inflammation in the livers of acute hepatitis B patients, significantly influencing disease outcome. PD-1 up-regulation in peripheral T cells is associated with immune dysfunction in chronic hepatitis B patients. However, the effect of PD-1/PD-L1 on hepatic damage and chronic infective status is still unknown in patients with chronic HBV infection. Here, we report up-regulation of PD-1 and PD-L1 in liver biopsies from 32 chronic HBV patients compared to 4 healthy donors. PD-1/PD-L1 up-regulation was significantly associated with hepatic inflammation and ALT elevation. Moreover, appropriate up-regulation but not overexpression of PD-L1 in the active phase of chronic hepatitis B as well as lower expression of PD-L1 in the inactive phase in liver residential antigen presenting cells (including Kupffer cells and sinusoidal endothelial cells) may contribute to viral inhibition. Our data suggest that the intrahepatic interaction of PD-1 and PD-L1 might play an important role in balancing the immune response to HBV and immune-mediated liver damage in chronic HBV infection.

Ceria nanoparticles promoted the cytotoxic activity of CD8+ T cells by activating NF-κB signaling.

Cytotoxic CD8+ T cells (CTLs) are crucial for controlling intracellular pathogens as well as cancer. However, how to promote the cytotoxic activity of CTL cells in vitro and in vivo remains largely unknown. On the other hand, ceria nanoparticles (CNPs) are widely used in biomedical fields, but the role of CNPs in CTL cells is still unclear. In this study, we found that the activated antigen-specific (P14) and nonspecific CD8+ T cells with CNP treatment both produced more cytokines, including interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α), and released more effector molecules, such as granzyme B and perforin, and then exhibited higher killing activity of P14 cells in vitro and stronger viral clearance capacity of CTL cells in vivo. Mechanistically, the activated P14 cells with CNP treatment inhibited the production of reactive oxygen species, and therefore promoted the activity of NF-κB signaling. Importantly, while the P14 cells were simultaneously treated by IMD-0354, a specific inhibitor of NF-κB signaling, the increases of IL-2 and TNF-α productions and granzyme B and perforin releases were remedied, and the P14 cells eventually exhibited the natural killing activity in vitro. Thus, our results demonstrated that CNP treatment promoted the cytotoxic activity of CTL cells and provide new ideas in the usage of CNPs and fascinating pharmacological potentials for clinical application, especially cancer immunotherapy.

The Kinase Complex mTOR Complex 2 Promotes the Follicular Migration and Functional Maturation of Differentiated Follicular Helper CD4+ T Cells During Viral Infection.

Follicular helper CD4+ T (TFH) cells are critical for optimal B-cell-mediated humoral immunity by initiating, fueling, and sustaining germinal center reactions. The differentiation of TFH cells relies on multiple intrinsic and extrinsic factors; however, the details by which these factors are integrated to coordinate TFH differentiation are largely unknown. In this study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) viral infection, we demonstrate that mTOR complex 2 (mTORC2) kinase integrates TCR signaling and ICOS-mediated co-stimulation to promote late differentiation and functional maturation of virus-specific TFH cells. Specifically, mTORC2 functions to maintain TFH lineage specifications, including phenotypes, migratory characteristics, and functional properties. Thus, our results highlight the importance of mTORC2 in guarding TFH phenotypic and functional maturation.

Sinomenine promotes differentiation but impedes maturation and co-stimulatory molecule expression of human monocyte-derived dendritic cells.

Dendritic cells (DC) excel at presenting antigen to T cells and thus make a key contribution to the induction of primary and secondary immune responses. Sinomenine has been used for centuries in the treatment of patients with autoimmune diseases as it possesses immunosuppressive and anti-inflammatory activities. However, the effect of sinomenine on the differentiation, maturation, and functionality of DC derived from monocytes has not been studied. We show here that DC differentiation is promoted when monocytes are treated with GM-CSF and IL-4 (IL-4) in the presence of sinomenine (200 microg/ml), as evidenced by the upregulation of CD1a while CD14 was decreased. In addition, incubation of immature DC with sinomenine significantly blunted lipopolysaccharide (LPS)-induced DC maturation, as shown by the reduction of expression of the maturation marker CD83 and co-stimulatory molecules, including CD86, B7-H1, and CD40. Moreover, sinomenine also prevented decreases in antigen (FITC-Dextran or Lucifer Yellow) uptake by LPS-treated DC. Mixed lymphocyte reactions (MLRs) revealed that sinomenine-treated DC impede the secretion of the cytokines IL-2 and IFN-gamma by co-cultured CD4(+) T cells. Therefore, modulation of DC differentiation, maturation, and functionality by sinomenine is of potential relevance to its immunomodulatory effects in controlling specific immune responses in autoimmune diseases, transplantation, and other immune-mediated conditions.

Intrahepatic expression of programmed death-1 and its ligands in patients with HBV-related acute-on-chronic liver failure.

Hepatitis B virus (HBV) infection is a major public health problem, and HBV-related acute-on-chronic liver failure (HBV-ACLF) has an extremely poor prognosis due to a lack of understanding of pathogenesis as well as a lack of effective treatments. Signals from the inhibitory receptor programmed death-1 (PD-1) have been demonstrated to be involved in regulating the pathogenesis of infectious diseases. However, the expression of PD-1 and its ligands in HBV-ACLF patients has yet to be evaluated. In this study, the expression of PD-1 and its ligands, PD-L1 and PD-L2, in liver biopsies from HBV-ACLF as well as chronic hepatitis B (CHB) patients were analyzed by immunohistochemistry. The results showed that all three molecules were observed in the HBV-ACLF samples and their levels were significantly higher than they were in CHB. Immunofluorescence double-staining showed that PD-1 was found on CD3(+), CD8(+) T cells, CD56(+) NK cells, CD68(+) macrophages, CK-18(+) epithelial cells, and CD16(+) monocytes. The PD-L1 expression was observed on all cell types detected and the PD-L2 was chiefly on CK-18(+) epithelial cells and CD31(+) endothelial cells. Interestingly, high levels of virus-induced procoagulant molecule fibrinogen-like protein 2 (FGL2) were observed in liver sections from HBV-ACLF, and PD-L1 and PD-L2 expression was also observed on FGL2(+) cells in these patients. Our combined results suggest that the expression of PD-L1 and PD-L2 may be biomarkers to identify and diagnose ACLF, and a clear understanding of their functional roles should further elucidate the pathogenesis of this disease.

[Expression of B7-H1 on peripheral blood mononuclear cells from chronic hepatitis B patients].

OBJECTIVE: To investigate the expression of B7-H1 on peripheral blood mononuclear cells from patients with chronic HBV infection. METHODS: Immunofluorescent staining and flow cytometry assay were used to measure the expression of B7-H1 on peripheral blood CD3high T cells, CD19high B cells and CD14high monocytes from chronic HBV infected patients. RESULTS: No significant difference was observed in B7-H1 expression on T cells and B cells between chronic HBV infected patients (CHB) and health controls (HC). B7-H1-expressing CD14high cells were significantly increased in chronic HBV-infected patients (19.17-/+11.64)% as compared with healthy controls [(7.30-/+5.49)%, P<0.01]. A significant positive correlation was found between B7-H1 expression on CD14high monocytes and serum ALT levels. CONCLUSION: There is no significant difference in B7-H1 expression on T cells and B cells between CHB patients and healthy subjects. B7-H1, which is up-regulated on monocytes from chronic HBV-infected patients, in positively correlated to serum ALT levels, and may play a role in the persistence of HBV infection.

Terminal complement complex C5b-9-treated human monocyte-derived dendritic cells undergo maturation and induce Th1 polarization.

Sublytic C5b-9 has been described as a pro-inflammatory mediator that triggers cell activation rather than inducing cell death. Dendritic cells (DC) play a critical role in controlling antigen-specific immune responses. Although DC maturation induced by various stimuli has been well characterized, the role of C5b-9 in DC function has not been described. In this report, we use in vitro assembled functional C5b-9 based on purified distal complement protein to show that DC maturation is promoted by sublytic C5b-9. This was demonstrated by up-regulation of CD83, HLA-antigens and costimulatory molecules, including CD80, D86, B7-H1, B7-H3, B7-H4 and BTLA. In addition, secretion of cytokines such as interleukin (IL)-12 and tumor necrosis factor-alpha was increased while the capacity for antigen uptake (FITC-Dextran and Lucifer Yellow) was reduced in C5b-9-treated DC. Mixed lymphocyte reactions indicated that C5b-9-activated DC acted as stimulators that significantly promoted CD4+ T cell activation and elicited production of cytokines, including interferon-gamma and IL-2. Interestingly, C5b-9-treated DC also orient CD4+ CD45RA+ naïve T cells toward Th1 polarization. Our results are the first to report that DC are potential immunoregulatory targets of C5b-9, suggesting that C5b-9 bridges innate and acquired immunity by inducing DC maturation.