Identification of IFN-γ-producing innate B cells

Although B cells play important roles in the humoral immune response and the regulation of adaptive immunity, B cell subpopulations with unique phenotypes, particularly those with non-classical immune functions, should be further investigated. By challenging mice with Listeria monocytogenes, Escherichia coli, vesicular stomatitis virus and Toll-like receptor ligands, we identified an inducible CD11a^hiFcγRIII^hi B cell subpopulation that is significantly expanded and produces high levels of IFN-γ during the early stage of the immune response. This subpopulation of B cells can promote macrophage activation via generating IFN-γ, thereby facilitating the innate immune response against intracellular bacterial infection. As this new subpopulation is of B cell origin and exhibits the phenotypic characteristics of B cells, we designated these cells as IFN-γ-producing innate B cells. Dendritic cells were essential for the inducible generation of these innate B cells from the follicular B cells via CD40L-CD40 ligation. Increased Bruton''s tyrosine kinase activation was found to be responsible for the increased activation of non-canonical NF-κB pathway in these innate B cells after CD40 ligation, with the consequent induction of additional IFN-γ production. The identification of this new population of innate B cells may contribute to a better understanding of B cell functions in anti-infection immune responses and immune regulation.     

Activation of natural killer T cells inhibits the development of induced regulatory T cells via IFNγ

Recent reports have provided evidence for cross-talk between regulatory T (Treg) cells and natural killer T (NKT) cells. However, it is unclear whether NKT cells play a role in the differentiation of Treg cells. By employing NKT cell-abundant Vα14 TCR transgenic (Tg) and NKT cell-deficient CD1d knock-out (KO) mice, we examined the effects of NKT cells on the in vitro differentiation of induced Treg (iTreg) cells with IL2 and TGFβ. We found that iTreg induction from CD1d KO mice was significantly increased compared to the control. Also, the addition of isolated NKT cells from Vα14 TCR Tg mice to naïve CD4⁺ T cells from CD1d KO mice during iTreg differentiation caused a remarkable reduction of iTreg cells. Through IFNγ neutralization, we showed that this reduction was mediated by IFNγ. Furthermore, the main source of IFNγ during iTreg differentiation was NK1.1⁻CD4⁺Foxp3⁻ T cells. This finding implied that early-activated NKT cells induced Th1-type cells and subsequently underwent apoptosis. Taken together, our results suggest that NKT cells inhibit the in vitro development of iTreg cells by increasing IFNγ.     

Peritoneal cavity is dominated by IFNγ-secreting CXCR3+ Th1 cells

The chemokine receptor CXCR3, which was shown to take part in many inflammatory processes, is considered as a Th1 specific marker. Here, we show in a mouse model that CXCR3 expressing CD4(+) cells preferentially migrate to the peritoneal cavity under steady-state conditions. The peritoneal cavity milieu leads to an up-regulated expression of CXCR3. However, blocking of known ligands of this chemokine receptor did not alter the preferential migration. The peritoneal cavity environment also results in an increased percentage of memory cells producing cytokines. Up-regulation of IFNγ production occurs mostly in CXCR3(+) cells considered as Th1, whereas the up-regulation of IL-4 affects mostly in CXCR3(-) cells which are considered as Th2. We conclude that the peritoneal cavity does not change the Th-lineage of the cells, but that domination of this anatomic niche by Th1 cells rather results from preferential migration to this compartment.     

Praziquantel facilitates IFN-γ-producing CD8+ T cells (Tc1) and IL-17-producing CD8+ T cells (Tc17) responses to DNA vaccination in mice

Background

CD8⁺ cytotoxic T lymphocytes (CTLs) are crucial for eliminating hepatitis B virus (HBV) infected cells. DNA vaccination, a novel therapeutic strategy for chronic virus infection, has been shown to induce CTL responses. However, accumulated data have shown that CTLs could not be effectively induced by HBV DNA vaccination.

Methodology/Principal Findings

Here, we report that praziquantel (PZQ), an anti-schistoma drug, could act as an adjuvant to overcome the lack of potent CTL responses by HBV DNA vaccination in mice. PZQ in combination with HBV DNA vaccination augmented the induction of CD8⁺ T cell-dependent and HBV-specific delayed hypersensitivity responses (DTH) in C57BL/6 mice. Furthermore, the induced CD8⁺ T cells consisted of both Tc1 and Tc17 subtypes. By using IFN-γ knockout (KO) mice and IL-17 KO mice, both cytokines were found to be involved in the DTH. The relevance of these findings to HBV immunization was established in HBsAg transgenic mice, in which PZQ also augmented the induction of HBV-specific Tc1 and Tc17 cells and resulted in reduction of HBsAg positive hepatocytes. Adoptive transfer experiments further showed that PZQ-primed CD8⁺ T cells from wild type mice, but not the counterpart from IFN-γ KO or IL-17 KO mice, resulted in elimination of HBsAg positive hepatocytes.

Conclusions/Significance

Our results suggest that PZQ is an effective adjuvant to facilitate Tc1 and Tc17 responses to HBV DNA vaccination, inducing broad CD8⁺ T cell-based immunotherapy that breaks tolerance to HBsAg.     

in vitro generation of IFN-γ-producing Listeria-specific T cells is dependent on IFN-γ production by non-NK cells

In vitro 5-day cultures of naive spleen cells with viable Listeria monocytogenes (VLM), but not heat-killed L. monocytogenes, induced CD4⁺ T cells that produced IFN-γ upon secondary antigen stimulation. The VLM-induced Listeria-specific T cells produced IFN-γ but lacked expression of IL-2 and IL-4. To study the role of IFN-γ in the induction of the IFN-γ-producing T cells, we added anti-IFN-γ mAb to the primary culture and analyzed IFN-γ production upon secondary antigen stimulation. Addition of anti-IFN-γ mAb to the culture suppressed generation of IFN-γ-producing CD4⁺ T cells, suggesting that IFN-γ is important in the induction of IFN-γ-producing CD4⁺ T cells. Furthermore, our results showed that depletion of NK cells from spleen cells by anti-asialo GM1 antibody plus complement before culture enhanced induction of IFN-γ-producing CD4⁺ T cells. Although NK cells are known to produce IFN-γ, the results indicate that NK cell-derived IFN-γ may not be important in induction of the Listeria-specific IFN-γ-producing CD4⁺ T cells in the culture system. In addition, we demonstrated that IFN-γ expression was high in CD4⁺ T cells from cultures of spleen cells with VLM at the primary culture level. These results suggest that IFN-γ derived from T cells may enhance production of IFN-γ by CD4⁺ T cells, while NK cells rather suppress the induction of IFN-γ-producing CD4⁺ T cells.     

Autoregulatory function of interleukin-10-producing pre-na?ve B cells is defective in systemic lupus erythematosus

IntroductionPre-naïve B cells represent an intermediate stage in human B-cell development with some functions of mature cells, but their involvement in immune responses is unknown. The aim of this study was to determine the functional role of normal pre-naïve B cells during immune responses and possible abnormalities in systemic lupus erythematosus (SLE) that might contribute to disease pathogenesis.MethodsPre-naïve, naïve, and memory B cells from healthy individuals and SLE patients were stimulated through CD40 and were analyzed for interleukin-10 (IL-10) production and co-stimulatory molecule expression and their regulation of T-cell activation. Autoreactivity of antibodies produced by pre-naïve B cells was tested by measuring immunoglobulin M (IgM) autoantibodies in culture supernatants after differentiation.ResultsCD40-stimulated pre-naïve B cells produce larger amounts of IL-10 but did not suppress CD4⁺ T-cell cytokine production. Activated pre-naïve B cells demonstrated IL-10-mediated ineffective promotion of CD4⁺ T-cell proliferation and induction of CD4⁺FoxP3⁺ T cells and IL-10 independent impairment of co-stimulatory molecule expression and tumor necrosis factor-alpha (TNF-α) and IL-6 production. IgM antibodies produced by differentiated pre-naïve B cells were reactive to single-stranded deoxyribonucleic acid. SLE pre-naïve B cells were defective in producing IL-10, and co-stimulatory molecule expression was enhanced, resulting in promotion of robust CD4⁺ T-cell proliferation.ConclusionsThere is an inherent and IL-10-mediated mechanism that limits the capacity of normal pre-naïve B cells from participating in cellular immune response, but these cells can differentiate into autoantibody-secreting plasma cells. In SLE, defects in IL-10 secretion permit pre-naïve B cells to promote CD4⁺ T-cell activation and may thereby enhance the development of autoimmunity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0687-1) contains supplementary material, which is available to authorized users.     

Toll-like receptors 2 and 4 regulate the frequency of IFNγ-producing CD4+ T-cells during pulmonary infection with Chlamydia pneumoniae

TLR2 and TLR4 are crucial for recognition of Chlamydia pneumoniae in vivo, since infected TLR2/4 double-deficient mice are unable to control the infection as evidenced by severe loss of body weight and progressive lethal pneumonia. Unexpectedly, these mice display higher pulmonary levels of the protective cytokine IFNγ than wild type mice. We show here, that antigen-specific CD4(+) T-cells are responsible for the observed IFNγ-secretion in vivo and their frequency is higher in TLR2/4 double-deficient than in wild type mice. The capacity of TLR2/4 double-deficient dendritic cells to re-stimulate CD4(+) T-cells did not differ from wild type dendritic cells. However, the frequency of CD4(+)CD25(+)Foxp3(+) T-cells was considerably higher in wild type compared to TLR2/4 double-deficient mice and was inversely related to the number of IFNγ-secreting CD4(+) effector T-cells. Despite increased IFNγ-levels, at least one IFNγ-mediated response, protective NO-secretion, could not be induced in the absence of TLR2 and 4. In summary, CD4(+)CD25(+)Foxp3(+) regulatory T-cells fail to expand in the absence of TLR2 and TLR4 during pulmonary infection with C. pneumoniae, which in turn enhances the frequency of CD4(+)IFNγ(+) effector T-cells. Failure of IFNγ to induce NO in TLR2/4 double-deficient cells represents one possible mechanism why TLR2/4 double-deficient mice are unable to control pneumonia caused by C. pneumoniae and succumb to the infection.     

Differential regulation of IFNα, IFNβ and IFNε gene expression in human cervical epithelial cells

Interferonε (IFNε) is a unique type I IFN that has distinct functions from IFNα/β. IFNε is constitutively expressed at mucosal tissues, including the female genital mucosa, and is reported to be modulated by estrogen and seminal plasma. However, its regulation by cytokines, including TNFα, IL-1β, IL-6, IL-8, IL-17, IL-22 and IFNα, which are commonly present in the female genital mucosa, is not well documented in freshly isolated primary cervical cells from tissues. We determined the effect of these cytokines on gene expression of type I IFNs in an immortalized endocervical epithelial cell line (A2EN) and in primary cervical epithelial cells. Several pro-inflammatory cytokines were found to induce IFNε, and TNFα induced the strongest response in both cell types. Pretreatment of cells with the IκB inhibitor, which blocks the NF-κB pathway, suppressed TNFα-mediated IFNε gene induction and promoter activation. Expression of IFNα, IFNβ, and IFNε was differentially regulated in response to various cytokines. Taken together, our results show that regulation of these IFNs depends on cell type, cytokine concentration, and incubation time, highlighting the complexity of the cytokine network in the cervical epithelium.     

Nonplasmacytoid, high IFN-α-producing, bone marrow dendritic cells

Plasmacytoid dendritic cells (pDC) are the producers of type I IFNs in response to TLR9 ligands. However, we have found that when bone marrow is depleted of pDC, the IFN-α produced in response to TLR9 ligands is not fully removed. We assign the source of this non-pDC IFN-α as a newly described DC type. It displays the high IFN-α producing activity of pDC but to a more limited range of viruses. Unlike pDC, the novel DC display high T cell stimulation capacity. Moreover, unlike mouse pDC, they are matured with GM-CSF and are less prone to apoptosis upon activation stimuli, including viruses. We propose that these DC constitute a novel bone marrow inflammatory DC type, ideally geared to linking innate and adaptive immune responses in bone marrow via their potent IFN-α production and high T cell stimulatory capacity.     

Airway epithelial cells suppress T cell proliferation by an IFNγ/STAT1/TGFβ-dependent mechanism

Organ-specific regulation of immune responses relies on the exchange of information between nonimmune and immune cells. In a primary culture model of the lung airway, we demonstrate that T cell proliferation is potently inhibited by airway epithelial cells (ECs). This is mediated by activation of the IFNγ/STAT1 pathway in the EC and transforming growth factor-β (TGFβ)-dependent suppression of T cell proliferation. In this way, the EC can restrict the expansion of T cells. Given the constant exposure of the airway to inhaled antigen, this may be important in setting a threshold for the initiation of T cell-dependent immune responses and preventing unwanted, chronic inflammation.     

LIGHT sensitizes IFNγ-mediated apoptosis of HT-29 human carcinoma cells through both death receptor and mitochondria pathways

LIGHT [homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM/TR2)] is a new member of TNF superfamily. The HT-29 colon cancer cell line is the most sensitive to LIGHT-induced, IFNγ-mediated apoptosis among the cell lines we have examined so far. Besides downregulation of Bcl-XL, upregulation of Bak, and activation of both PARP [poly (ADP-ribose) polymerase] and DFF45 (DNA fragmentation factor), LIGHT-induced, IFNy-mediated apoptosis of HT-29 cells involves extensive caspase activation. Caspase-8 and caspase-9 activation, as shown by their cleavages appeared as early as 24 h after treatment, whereas caspase-3 and caspase-7 activation, as shown by their cleavages occurred after 72h of LIGHT treatment. Caspase-3 inhibitor Z-DEVD-FMK (benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone) and a broad range caspase inhibitor Z-VAD-FMK (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) were able to block LIGHT-induced, IFNγ-mediated apoptosis of HT-29 cells. The activity of caspase-3, which is one of the major executioner caspases, was found to be inhibited by both Z-DEVD-MFK and Z-VAD-FMK. These results suggest that LIGHT-induced, IFNy-mediated apoptosis of HT-29 cells is caspase-dependent, and LIGHT signaling is mediated through both death receptor and mitochondria pathways.     

Neutrophils and interferon-α-producing cells: who produces interferon in lupus?

Interferon-α plays a crucial role in the pathogenesis of systemic lupus erythematosus. Nevertheless, the different human cell types producing this cytokine as well as the stimuli inducing its production have not been completely characterized. So far, a subpopulation of dendritic cells activated by immune complexes has been identified as major producers of interferon-α in patients with lupus. However, those cells represent a minor population and some studies have reported the secretion of interferon-α by other cells. On the other hand, more than 50% of blood leukocytes are neutrophils and their functions are still not fully understood. Recent data suggest that neutrophils, though usually not considered interferon-α-producing cells, may represent an unexpected source of this cytokine in response to some lupus stimuli.     

Human IgM+CD27+ B cells: memory B cells or "memory" B cells?

Memory B cells are generated in germinal centers (GC) and contribute to serological immunity by rapidly differentiating into plasma cells. Human memory B cells can be identified by the expression of CD27. These cells exhibit more rapid responses than naive (CD27-) B cells following stimulation in vitro, consistent with the heightened kinetics of secondary responses in vivo. CD27+ B cells express mutated Ig V region genes; however a significant proportion continue to express IgM, suggesting the existence of IgM+ memory B cells. The observation that mutated IgM+CD27+ B cells are generated in humans who cannot form GC led to the conclusions that these cells are generated independently of GC and thus are not memory cells and that they mediate responses to T cell-independent Ag. Although some studies support the idea that IgM+CD27+ B cells participate in T cell-independent responses, many others do not. In this review we will provide alternate interpretations of the biology of IgM+CD27+ B cells and propose that they are indeed memory cells.     

IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain

It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated, and it is not known whether IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4(+) and CD8(+) T cells. Importantly, we demonstrate that IFN-γ-producing CD4(+) T cells, but not innate or CD8(+) T cells, can promote the development of ECM in normally resistant IFN-γ(-/-) mice infected with P. berghei ANKA. Adoptively transferred wild-type CD4(+) T cells accumulate within the spleen, lung, and brain of IFN-γ(-/-) mice and induce ECM through active IFN-γ secretion, which increases the accumulation of endogenous IFN-γ(-/-) CD8(+) T cells within the brain. Depletion of endogenous IFN-γ(-/-) CD8(+) T cells abrogates the ability of wild-type CD4(+) T cells to promote ECM. Finally, we show that IFN-γ production, specifically by CD4(+) T cells, is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8(+) T cell accumulation. To our knowledge, these observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4(+) T cells promote the development of ECM during P. berghei ANKA infection.     

γ-Ray irradiation induces B7.1 costimulatory molecule neoexpression in various murine tumor cells

 The use of gene-modified tumor cells as a strategy for active immunotherapy is currently undergoing intensive fundamental and clinical research. Most clinical trials use γ-ray-irradiated tumor cells as vaccine, although little is known about the effects of irradiation on the immunogenicity of tumor cells. In particular, no data have been reported so far concerning the effects of γ-ray irradiation on the expression of B7 molecules in tumor cells. In this paper, we show a neoexpression of the B7.1 molecule after γ-ray irradiation in tumor cell lines from different tissues, while the B7.2 molecule remains unexpressed in all the cell lines tested. Furthermore, the induction of B7.1 molecule membrane expression after irradiation is shown to result from the neoexpression of B7.1 mRNA, and to be reproduced with H₂O₂ oxidative stress. These data could explain the enhanced immunogenicity of many tumor cells after irradiation, and could lead to new immunotherapy protocols. Received: 27 November 1997 / Accepted: 26 February 1998     

中国鸭IFN—γ基因的克隆与表达

鸭乙型肝炎病毒（DHBV）感染鸭,是研究IFN－γ在机体自然感染过程中机体与病毒的相互作用和病毒的清除机制的良好动物模型。从PHA刺激后的鸭外周血单核细胞（PBMC）中提取RNA,通过RT－PCR获得鸭IFN－γ（DuIFN-γ）cDNA基因,构建DuIFN-γ真核表达质粒,转染COS－7细胞,经细胞病变效应（CPE）抑制分析和MTT法对重组DuIFN-γ滴度进行测定。实验表明,重组DuIFN-γ能够抑制VSV感染鸭胚成纤维细胞而产生的细胞病变效应。抗－DuIFN-γ抗体,能中和这种抗病毒活性。并且,GST－DuIFN-γ融合蛋白在大肠杆菌中得到表达和进一步纯化。