International Immunology - recent issues

IN THIS ISSUE

Wed, 18 Nov 2009 15:15:03 PST

IL-5- and eosinophil-mediated inflammation: from discovery to therapy

Kouro, T., Takatsu, K. — Wed, 18 Nov 2009 15:15:03 PST

IL-5 was originally defined as a T-cell-derived cytokine that triggers activated B cells for terminal differentiation into antibody-secreting plasma cells, at least in mice. Concurrently, IL-5 was recognized as the major maturation and differentiation factor for eosinophils in mice and humans. Over-expression of IL-5 significantly increases eosinophil numbers and antibody levels in vivo. Conversely, mice lacking a functional gene for IL-5 or the IL-5 receptor alpha chain (IL-5R) display a number of developmental and functional impairments in B-cell and eosinophil lineages. In addition to the Janus kinase–signal transducer and activator of transcription pathway, the tyrosine kinases Lyn and Btk (Bruton agammaglobulinemia tyrosine kinase) are involved, and Ras GTPase–extracellular signal-regulated kinase (Ras–ERK) signals are important for IL-5-dependent cell proliferation and survival. IL-5 critically regulates expression of genes involved in proliferation, cell survival and maturation and effector functions of B cells and eosinophils. Thus, IL-5 plays a pivotal role in innate and acquired immune responses and eosinophilia. In humans, the biologic effects of IL-5 are best characterized for eosinophils. The recent expansion in our understanding of the mechanisms of eosinophil development and activation in the context of IL-5 has led to advances in therapeutic options. A new therapy currently in clinical trials uses humanized mAbs against IL-5 or the IL-5R.

The study of allergy by Japanese researchers: a historical perspective

Takai, T., Karasuyama, H. — Wed, 18 Nov 2009 15:15:03 PST

It has been over a hundred years since Shibasaburo Kitasato and Emil Adolf von Boehring's finding of a serum component that neutralizes bacterial toxins and the subsequent development of antiserum therapy. Over that time, many Japanese researchers have greatly contributed to our understanding of the molecular mechanisms for allergic and inflammatory diseases. This article is aimed at introducing such individual work and how these areas have contributed to our understanding of the mechanisms of allergic reactions.

Expression of fully assembled TCR-CD3 complex on double positive thymocytes: synergistic role for the PRS and ER retention motifs in the intra-cytoplasmic tail of CD3{varepsilon}

Brodeur, J.-F., Li, S., Damlaj, O., Dave, V. P. — Wed, 18 Nov 2009 15:15:03 PST

TCR expression on double-positive (DP) thymocytes is a prerequisite for thymic selection that results in the generation of mature CD4⁺ and CD8⁺ single-positive T cells. TCR is expressed at very low level on preselection DP thymocytes and is dramatically up-regulated on positively selected thymocytes. However, mechanism governing TCR expression on developing thymocytes is not understood. In the present report, we demonstrate that the intra-cytoplasmic (IC) domain of CD3 plays a critical role in regulating TCR expression on DP thymocytes. We provide genetic and biochemical evidence to show that the CD3 IC domain mutations result in elevated expression of fully assembled TCR on DP thymocytes. We also demonstrate that TCR up-regulation on DP thymocytes in these transgenic mice occurs in a ligand-independent manner. Further, we show that the proline-rich sequence and endoplasmic reticulum (ER) retention motifs in the IC domain of CD3 play synergistic role in regulating TCR surface expression on DP thymocytes.

Regulatory and pro-inflammatory phenotypes of myelin basic protein-autoreactive T cells in multiple sclerosis

Wed, 18 Nov 2009 15:15:03 PST

MBP-specific autoreactive T cells are considered pro-inflammatory T cells and thought to play an important role in the pathogenesis of multiple sclerosis (MS). Here, we report that MBP_83–99-specific T cells generated from MS patients (n = 7) were comprised of pro-inflammatory and regulatory subsets of distinct phenotypes. The pro-inflammatory phenotype was characterized by high production of IFN-, IL-6, IL-21 and IL-17 and low expression of FOXP3, whereas the regulatory subset expressed high levels of FOXP3 and exhibited potent regulatory functions. The regulatory subset of MBP-specific T cells appeared to expand from the CD4⁺CD25^– T-cell pool. Their FOXP3 expression was stable, independent of the activation state and it correlated with suppressive function and inversely with the production of IFN-, IL-6, IL-21 and IL-17. In contrast, the phenotype and function of FOXP3^low MBP-specific T cells were adaptive and dependent on IL-6. The higher frequency of FOXP3^high MBP-specific T cells was observed when IL-6 was neutralized in the culture of PBMC with MBP. The study provides new evidence that MBP-specific T cells are susceptible to pro-inflammatory cytokine milieu and act as either pro-inflammatory or regulatory T cells.

Induction of TNF-alpha-converting enzyme-ectodomain shedding by pathogenic autoantibodies

Wed, 18 Nov 2009 15:15:03 PST

The release of the soluble form of tumor necrosis factor (TNF)-alpha from the plasma membrane occurs through the activation of the secretase tumor necrosis factor-alpha-converting enzyme (TACE). The current study was designed to examine whether the anti-Ro/SSA autoantibodies (Abs) are capable to regulate TACE expression in non-neoplastic human salivary gland epithelial cells (SGEC) cultures. We investigated the effect of anti-Ro/SSA Abs on the localization and abundance of cell-surface TACE and on TACE pro-domain-shedding and activation. In addition, the potential physiological consequences of TNF-alpha blockage by the biological agent Adalimumab on post-translational regulation of TACE are discussed. Anti-Ro/SSA Abs were purified from IgG fractions of patients with primary Sjögren's syndrome, using Sepharose 4B-Ro/SSA affinity columns. Flow cytometry, reverse transcription–PCR, western blot and immunohistochemistry were used to study TACE expression on SGEC and TACE regulation by Abs. Our study demonstrated a dose-dependent increase of TACE messenger RNA (mRNA) expression in anti-Ro/SSA Abs-treated SGEC, followed by internalization, pro-domain shedding and activation of TACE protein, suggesting that increased TACE activity is necessary for the release of TNF-alpha observed in anti-Ro/SSA Abs-stimulated SGEC. Adalimumab treatment brought TACE mRNA and surface TACE expression to levels than those observed in untreated SGEC. These data suggest that the effect of anti-Ro/SSA Abs on TACE expression and intracellular distribution is exerted by TNF-alpha production.

hnRNP-K is a nuclear target of TCR-activated ERK and required for T-cell late activation

Chang, J.-W., Koike, T., Iwashima, M. — Wed, 18 Nov 2009 15:15:03 PST

Sustained extracellular signal-regulated kinase (ERK)-signaling plays a critical role in T-cell-mediated IL-2 production. Although many downstream targets are known for ERK, details remain unknown about which molecules play functional roles in IL-2 production. Here, we addressed this question using proteomic analysis of nuclear proteins from TCR-activated T cells and identified hnRNP-K as one of the ERK targets essential for IL-2 production. hnRNP-K was previously shown by others to be a direct substrate of ERK and form complexes with multiple signaling proteins as well as DNA and RNA. Our data showed a clear ERK-dependent increase in one form of hnRNP-K after TCR stimulation. Small interfering RNA-mediated gene knockdown of hnRNP-K expression abrogated IL-2 production by T cells. Moreover, reduction of hnRNP-K expression caused a notable increase in proteolysis of Vav1, a binding target of hnRNP-K. Since Vav1 is an essential molecule for T-cell activation, the data suggest that ERK signaling is required for T-cell activation partly by inhibiting activation-induced proteolysis of Vav1.

IN THIS ISSUE

Mon, 26 Oct 2009 06:19:26 PDT

Molecular basis of canonical and bactericidal autophagy

Noda, T., Yoshimori, T. — Mon, 26 Oct 2009 06:19:26 PDT

Autophagy is a catabolic process by which cells degrade their own cytoplasmic constituents. Cells respond to the stress response of nutrient deficiency by degrading a portion of their cellular components to produce amino acids and energy. Recently, it became evident that the autophagic machinery is also involved in a kind of innate immune system. Some bacteria that invade mammalian cells are eventually entrapped in an autophagic membrane structure. In this review, we describe the current understanding of three of the basic components of the canonical autophagy machinery—LC3, the Atg16L complex and phosphatidylinositol 3-phosphate (PI3P)—which are dynamically associated with the autophagic structure. LC3 is proposed to function in autophagosome closure, whereas the Atg16L complex functions as an E3-like protein in ubiquitination-like reactions in the LC3 lipidation system. PI3P is a key determinant of the autophagic membrane. Further, their relation to bactericidal autophagy (i.e. xenophagy) will be introduced.

Morbus Crohn--a disease of failing macroautophagy in the immune system?

Meixlsperger, S., Munz, C. — Mon, 26 Oct 2009 06:19:26 PDT

Mutations in genes involved in macroautophagy have been found to be associated with Morbus Crohn, also called Crohn's disease (CD), an inflammatory bowel disease. Taking this disease as an example for pathogenesis due to altered macroautophagy, we discuss here how macroautophagy supports innate and adaptive immunity. This support ranges from maintenance of components of the immune system, antigen processing for presentation to the immune system, to education of the immune system in order to distinguish self from dangerous non-self. A better understanding of these mechanisms should allow us not only to develop therapeutical strategies for CD but also to utilize macroautophagy for enhanced immunity against pathogens and tumors.

CD4 T cell cooperation is required for the in vivo activation of CD4 T cells

Peters, N. C., Kroeger, D. R., Mickelwright, S., Bretscher, P. A. — Mon, 26 Oct 2009 06:19:26 PDT

We address here the role of CD4 T cell cooperation in the activation of CD4 T cells. Administration of aggregated hen egg lysozyme (HEL) without microbial adjuvant to BALB/c mice normally generates cytokine-producing CD4 T cells specific for the HEL major peptide, HEL_105–120, as well as CD4 T cells specific for HEL non-major peptides. The prior administration of HEL_105–120 ablates the generation of cytokine-secreting CD4 T cells specific for HEL_105–120, as well as the CD4 T cells specific for HEL non-major peptides, normally generated upon HEL challenge. Thus, the activation of HEL non-major peptide-specific CD4 T cells appears to depend upon the HEL_105–120-specific CD4 T cell population. In contrast, when HEL_105–120 and saline-treated mice are challenged with HEL coupled to ovalbumin (OVA), CD4 T cell responses to HEL non-major peptides and to OVA are the same, whereas treated mice still do not generate cytokine-secreting cells specific for HEL_105–120. We infer that the administration of HEL_105–120 does not generate regulatory cells capable of down-regulating CD4 T cell responses to HEL and OVA peptides. OVA-specific CD4 T cells restore the generation of HEL non-major peptide-specific T cells in the absence of HEL major peptide-specific T cells. We conclude that the generation of CD4 T cells producing IL-2, IFN- and IL-4 requires CD4 T cell cooperation and that this cooperation is not mediated simply by CD40–CD40L interactions. We also conclude from these observations that there is no requirement for a microbial or danger signal for CD4 T cell activation.

Enhanced B cell activation in the absence of CD81

Sanyal, M., Fernandez, R., Levy, S. — Mon, 26 Oct 2009 06:19:26 PDT

CD81 is a component of the CD19/CD21 co-receptor complex in B cells. However, the role of CD81 in B cell activation has not been clearly elucidated. Here, we demonstrate that Cd81^–/– B cells stimulated via their B cell receptor fluxed higher intracellular-free calcium ion along with increased phosphorylation of spleen tyrosine kinase and phospholipase gamma 2. Additionally, Cd81^–/– B cells responded to toll like receptor 4 stimulation with increased nuclear factor-kappa B activation, cell proliferation and antibody secretion compared with wild-type B cells. Cd81^–/– mice also mounted a significantly higher immune response to T-independent antigens than their wild-type counterparts. Finally, analysis of Cd81^–/– B cells that were generated by bone marrow transplantation into Rag1^–^/– mice confirmed that the hyperactive phenotype is not dependent on the CD81-deficient environment. Taken together, these results indicate that CD81 plays a negative role in B cell activation in vitro and in vivo.

Recombinant YopJ induces apoptosis in murine peritoneal macrophages in vitro: involvement of mitochondrial death pathway

Pandey, A. K., Sodhi, A. — Mon, 26 Oct 2009 06:19:26 PDT

Yersinia species during infection adhere to host immune cells primarily to macrophages and employ its secretary proteins known as Yersinia outer proteins to trigger death in infected cells. In the present study, it is shown that recombinant Yersinia outer protein J (rYopJ) could induce apoptosis in murine peritoneal macrophages in vitro as assessed by morphological features, internucleosomal DNA fragmentation, change in mitochondrial membrane potential (MMP) (m), activation of caspases and Annexin V binding. rYopJ-induced cell death was dose and time dependent. Pre-treatment with broad-spectrum caspase inhibitor Z-VAD-FMK, caspase-3 inhibitor Ac-DEVD-CHO and caspase-8 inhibitor Z-IETD-FMK prevented the change in MMP and DNA fragmentation, suggesting caspase-dependent apoptosis of rYopJ-treated macrophages. Blocking the endocytosis by pre-treatment of cells with cytochalasin B did not prevent the rYopJ-induced macrophages apoptosis. The data further suggest that rYopJ-induced apoptosis is mediated by molecules upstream of caspase-8 and relay through mitochondrial pathway involving Bax, Bcl-2, activation of caspase-8 and caspase-3, Bid and polyadenosine diphosphate-ribose polymerase cleavage, cytochrome c release and DNA fragmentation.

Apoptosis of lymphocytes and monocytes infected with influenza virus might be the mechanism of combating virus and causing secondary infection by influenza

Xie, D., Bai, H., Liu, L., Xie, X., Ayello, J., Ma, X., Zhang, J. — Mon, 26 Oct 2009 06:19:26 PDT

Influenza affects most of the world's population annually, often causing a secondary infection, but pathological mechanisms of influenza virus infection remain unclear. We have found that influenza viruses have a selective preference for infecting monocytes and mature immune effector cells. This paper provides evidence that influenza virus infection increases the expression of granzyme B (GrB) in monocytes, activated T and B cells. All GrB⁺ cells had cytolytic function. GrB⁺CD62L^high central memory (T_CM) cells were fast response population to virus infection when compared with GrB⁺CD62L^low population. The influenza virus-infected PBMC could be killed by GrB⁺ cells. We propose the following mechanism for influenza: (i) influenza virus within the respiratory tract overcomes humoral defenses; (ii) free virus is directly engulfed by the immune system effector cells and free virus also infects epithelial cells; (iii) virus-infected epithelial cells and the immune system cells are killed by cytotoxic cells. These indicated that an immune system that was combating a virus infection needs to sacrifice some of its immune system cells. Therefore, influenza viruses might temporally destroy the human immune system's line of defense, resulting in susceptibility to a secondary infection. This might be a prevalent mechanism existing in cell-mediated immune responses.

Histidine decarboxylase but not histamine receptor 1 or 2 deficiency protects from K/BxN serum-induced arthritis

Mon, 26 Oct 2009 06:19:26 PDT

Serum transfer from arthritic K/BxN mice into naive animals results in arthritis. Mast cells have been shown to be essential since mice lacking these cell type do not develop arthritis upon serum injection. Mast cell function depends on the release of granules filled with mediators such as histamine. Mice deficient in histidine decarboxylase (HDC^–/–) that do not produce histamine and mice deficient for histamine receptor 1 (H1R^–/–) or histamine receptor 2 (H2R^–/–) were injected with arthritogenic sera from the K/BxN mice, and the progression of arthritis was observed through the next 2 weeks. HDC^–/– mice that are histamine free developed a milder form of arthritis in comparison with the wild-type controls. In both receptor-deficient mice as well as in wild-type controls, the onset and severity of clinical arthritis and ankle thickening occurred during day 1 to 3. These results indicate that histamine is required but not indispensable for the development of serum-induced arthritis and histamine receptors other than those studied here may be involved.

Mitochondrial translocation of the glucocorticoid receptor in double-positive thymocytes correlates with their sensitivity to glucocorticoid-induced apoptosis

Mon, 26 Oct 2009 06:19:26 PDT

Glucocorticoid receptor (GR) signaling plays an important role in the selection and apoptosis of thymocytes. Besides nuclear translocation, mitochondrial translocation of the ligand-bound GR in lymphoid cells was also shown, which might determine glucocorticoid (GC)-induced apoptosis sensitivity. In the present work, we followed the ligand-induced GR trafficking in CD4+CD8+ double-positive (DP) thymocytes. Using confocal microscopy, we found that upon short-term in vitro GC analog [dexamethasone (DX)] treatment, the GR translocates into the mitochondria but not into the nucleus in DP cells. We also analyzed the GR redistribution in cytosolic, nuclear and mitochondrial fractions of unseparated thymocytes by western blot and confirmed that in DX-treated cells a significant fraction of the GR translocates into the mitochondria. DX reduced the mitochondrial membrane potential of DP cells within 30 min, measured by flow cytometry, which refers to a direct modulatory activity of mitochondrial GR translocation. The abundant mitochondrial GR found in DP cells well correlates with their high GC-induced apoptosis sensitivity.

Naive CD4 T cells from aged mice show enhanced death upon primary activation

Mon, 26 Oct 2009 06:19:26 PDT

Poor T cell immunity is one of the many defects seen in elderly humans and aged (Ad) mice. We report that naive CD4 T cells from aged mice (ANCD4 cells) showed greater apoptosis upon primary activation than those from young (Yg) mice, with loss of mitochondrial membrane potential, poor activation of Rel family transcription factors and increased DNA damage. Their ability to enhance glycolysis, produce lactate and induce autophagy following activation was also compromised. ANCD4 cells remained susceptible to death beyond first cell division. Activated ANCD4 cells also showed poor transition to a ‘central memory’ (CM) CD44^high, CD62L^high phenotype in vitro. This correlated with low proportions of CM cells in Ad mice in vivo. Functionally, too, IFN-gamma responses recalled from T cells of immunized Ad mice, poor to begin with, worsened with time as compared with Yg mice. Thus, ANCD4 cells handle activation-associated stress very poorly due to multiple defects, possibly contributing to poor formation of long-lasting memory.

Critical role of IFN-{gamma} in CFA-mediated protection of NOD mice from diabetes development

Mori, Y., Kodaka, T., Kato, T., Kanagawa, E. M., Kanagawa, O. — Mon, 26 Oct 2009 06:19:26 PDT

IFN- signaling-deficient non-obese diabetic (NOD) mice develop diabetes with similar kinetics to those of wild-type NOD mice. However, the immunization of IFN- signaling-deficient NOD mice with CFA failed to induce long-term protection, whereas wild-type NOD mice receiving CFA remained diabetes-free. CFA also failed to protect IFN- receptor-deficient (IFN-R^–/–) NOD mice from the autoimmune rejection of transplanted islets, as it does in diabetic NOD mice, and from disease transfer by spleen cells from diabetic NOD mice. These data clearly show that the pro-inflammatory cytokine IFN- is necessary for the CFA-mediated protection of NOD mice from diabetes. There is no difference in the T_h1/T_h17 balance between IFN-R^–/– NOD and wild-type NOD mice. There is also no difference in the total numbers and percentages of regulatory T (Treg) cells in the lymph node CD4⁺ T-cell populations between IFN-R^–/– NOD and wild-type NOD mice. However, pathogenic T cells lacking IFN-R are resistant to the suppressive effect of Treg cells, both in vivo and in vitro. Therefore, it is likely that CFA-mediated protection against diabetes development depends on a change in the balance between Treg cells and pathogenic T cells, and IFN- signaling seems to control the susceptibility of pathogenic T cells to the inhibitory activity of Treg cells.

Anti-vascular endothelial growth factor (VEGF) specific activity of intravenous immunoglobulin (IVIg)

Damianovich, M., Blank, M., Raiter, A., Hardy, B., Shoenfeld, Y. — Mon, 26 Oct 2009 06:19:26 PDT

IN THIS ISSUE

Thu, 24 Sep 2009 05:39:09 PDT

The study of regulatory T cells and NKT cells in Japan: a historical perspective

Arase, H., Seino, K.-i. — Thu, 24 Sep 2009 05:39:09 PDT

Immune regulation plays an important role in maintaining homeostasis of the immune system. A number of Japanese immunologists have made significant contributions to the elucidation of the mechanisms of immune regulation. In particular, lymphocyte populations that could regulate immune responses—for example regulatory T cells and NKT cells—have been extensively analyzed. Here, we present an overview of research on immune regulation by highlighting the work of several Japanese contributors.

Regulatory T cells: how do they suppress immune responses?

Sakaguchi, S., Wing, K., Onishi, Y., Prieto-Martin, P., Yamaguchi, T. — Thu, 24 Sep 2009 05:39:09 PDT

Regulatory T cells (Tregs), either natural or induced, suppress a variety of physiological and pathological immune responses. One of the key issues for understanding Treg function is to determine how they suppress other lymphocytes at the molecular level in vivo and in vitro. Here we propose that there may be a key suppressive mechanism that is shared by every forkhead box p3 (Foxp3)⁺ Treg in vivo and in vitro in mice and humans. When this central mechanism is abrogated, it causes a breach in self-tolerance and immune homeostasis. Other suppressive mechanisms may synergistically operate with this common mechanism depending on the environment and the type of an immune response. Further, Treg-mediated suppression is a multi-step process and impairment or augmentation of each step can alter the ultimate effectiveness of Treg-mediated suppression. These findings will help to design effective ways for controlling immune responses by targeting Treg suppressive functions.

Nitric oxide cooperates with glucocorticoids in thymic epithelial cell-mediated apoptosis of double positive thymocytes

Thu, 24 Sep 2009 05:39:09 PDT

T cell development in the thymus is controlled by thymic epithelial cells (TE). While it is accepted that TE interact with maturing T cells, the mechanisms by which they trigger ‘death by neglect’ of double-positive (DP) thymocytes are poorly understood. We and others have demonstrated a role for TE-derived glucocorticoids (GCs) in this process. We have studied TE-induced apoptosis using an in vitro system based on co-culturing a thymic epithelial cell line (TEC) with DP thymic lymphoma cells or thymocytes (DP thymic cells). Here, we demonstrate that nitric oxide (NO·) is also involved in this death process. The inducible nitric oxide synthase (iNOS) inhibitors N^G-methyl-L-arginine and 1,4-PBIT attenuated TEC-induced apoptosis of DP thymic cells. Co-cultivation of TEC with DP thymic cells increased the expression of iNOS in TEC. A concomitant increase in NO· was detected by staining with DAF-FM diacetate. Moreover, the iNOS-regulating cytokines IL-1, IL-1β and IFN were up-regulated upon interaction of TEC with DP thymic cells. Neutralizing IL-1R or IFN reduced TEC-induced apoptosis of DP thymic cells. Cardinally, NO· synergizes with GCs in eliciting apoptosis of DP thymic cells. Our data indicate that a cross-talk between DP thymic cells and TEC is required for proper induction of iNOS-up-regulating cytokines with a subsequent increase in iNOS expression and NO· production in TEC. NO·, in turn, cooperates with GCs in promoting death by neglect. We suggest that NO· together with GCs fine-tune the T cell selection process.

Protective role of mouse MBL-C on intestinal mucosa during Shigella flexneri invasion

Zuo, D.-M., Zhang, L.-Y., Lu, X., Liu, Y., Chen, Z.-L. — Thu, 24 Sep 2009 05:39:09 PDT

Mannan-binding lectin (MBL) is a C-type serum lectin, which is believed to play an important role in the innate immunity against a variety of pathogens. MBL can bind to sugar determinants of a wide variety of microorganisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Given that small intestine is a predominant site of extrahepatic expression of MBL, here we addressed the question whether MBL is involved in mucosal innate immunity. The carbohydrate recognition domain (CRD) genes of mouse MBL-C (mMBL-C) were cloned and expressed in Escherichia coli. Recombinant mMBL-C-CRD binds to Shigella flexneri 2a in a calcium-dependent manner and that interaction could be blocked by the anti-mMBL-C-CRD antibody. mMBL-C-CRD protein could inhibit the adhesion of S. flexneri 2a to intestinal mucosa, while administration of anti-mMBL-C-CRD antibody caused an increased level of bacteria adhesion in vitro. Administration of recombinant mMBL-C-CRD protein reduced the secretion of IL-6 and monocyte chemoattractant protein 1 from primary intestinal epithelial cells stimulated with S. flexneri 2a. Furthermore, neutralization of MBL activity by anti-MBL-C-CRD resulted in a significant increase in the number of S. flexneri 2a that colonized the intestines of BALB/c mice and attenuated the severity of inflammation seen in the areas of bacterial invasion. These findings suggest that mMBL-C may protect host intestinal mucosa by directly binding to the bacteria.

A novel approach to induce human DCs from monocytes by triggering 4-1BBL reverse signaling

Thu, 24 Sep 2009 05:39:09 PDT

Dendritic cells (DCs) are responsible for the initiation of immune responses. Our study demonstrates a new pathway for generating a large quantity of stimulatory monocyte-derived DCs (Mo-DCs) from human monocytes using anti-4-1BB ligand (4-1BBL) mAb to trigger reverse signaling. The anti-4-1BBL-driven Mo-DCs (DCs_-4-1BBL) not only express higher levels of CD86, CD83 and HLA-DR, when compared with the Mo-DCs matured by tumor necrosis factor , but also exhibit a unique phenotype that expresses lower levels of PD-L1. High levels of GM-CSF, M-CSF and Flt3 ligand (FL) were found in the anti-4-1BBL-differentiation culture. Neutralizing M-CSF, GM-CSF and FL inhibited Mo-DC proliferation stimulated by anti-4-1BBL mAb, suggesting that M-CSF, GM-CSF and FL are involved in cell proliferation stimulated by anti-4-1BBL. Further analysis of the DCs_-4-1BBL showed increased secretion of T_h1-type cytokines IL-12 and IFN- and decreased secretion of IL-10. DCs_-4-1BBL induced much stronger proliferative responses in the mixed lymphocyte reaction assay when compared with DCs derived by GM-CSF. Moreover, DCs_-4-1BBL preferentially induced T_h1 responses. We have further demonstrated that anti-4-1BBL antibody stimulated nuclear translocation of NF-B from the cytoplasm in monocytes, suggesting that reverse signaling by 4-1BBL is likely responsible for mediating DC differentiation. Collectively, we have found that reverse signaling of 4-1BBL promotes the differentiation of potent T_h1-inducing DCs from human monocytes.

Time-lapse observation of cellular function with fluorescent probe reveals novel CTL-target cell interactions

Tomura, M., Mori, Y. S., Watanabe, R., Tanaka, M., Miyawaki, A., Kanagawa, O. — Thu, 24 Sep 2009 05:39:09 PDT

Fluorescent protein that detects caspase-3 activation was used for the time-lapse observation of CTL–target cell interaction. In the target cells transfected with SCAT3.1 (caspase-3-sensitive fusion protein) complementary DNA, caspase-3 activation can be detected significantly earlier than the commonly used annexin-V binding that detects membrane change in apoptotic cells. Moreover, during the cytolytic interaction between OE4 CTL and W3 tumor target cells, detachment of CTL from the target cells occurred prior to caspase-3 activation and death of the target cells, indicating very early sensing of apoptotic target cells by CTL. This early detachment of OE4 CTL from W3 target cells was inhibited by the expression of CD80 co-stimulatory molecule on the target cells. Taken together, time-lapse observation of cellular interaction with functional probe, SCAT3.1 provides new kinetic information and demonstrates that co-stimulatory molecules regulate the kinetics of CTL–target cell interaction.

Ectopically expressed PIR-B on T cells constitutively binds to MHC class I and attenuates T helper type 1 responses

Thu, 24 Sep 2009 05:39:09 PDT

Activated mature T cells induce various inhibitory receptors implicated in maintaining peripheral tolerance in response to the trans-acting ligands. Interestingly, paired Ig-like receptor (PIR)-B, an inhibitory MHC class I receptor on B cells and myeloid cells, could be involved in regulating early T cell development because epitope for PIR is detected on pre-thymic T/NK progenitors but not on thymocytes or mature T cells. We hypothesized that PIR-B is not only a regulator for T cell development but is also detrimental if expressed on mature T cells. Here we demonstrated, using PIR-B-deficient fetuses, that PIR-B is indeed expressed on the T cell progenitors but failed to identify its distinctive roles in the development. Forced expression of PIR-B in thymocytes and mature T cells also resulted in no abnormalities in development. However, upon antigenic or allogeneic stimulation, peripheral T cells with the ectopic PIR-B showed reduced T_h type 1 responses due to the suppression of proximal TCR signaling by constitutive binding of PIR-B to MHC class I on the same cell surface. Our findings suggest that T cell expression of PIR-B with the cis-interacting MHC class I is strictly prohibited in periphery so as to secure prompt immune responses.

Amyloid precursor family proteins are expressed by thymic and lymph node stromal cells but are not required for lymphocyte development

Laky, K., Annaert, W., Fowlkes, B. J. — Thu, 24 Sep 2009 05:39:09 PDT

Pharmacological inhibitors that block amyloid precursor protein (APP) cleavage and the formation of senile plaques are under development for the treatment of familial Alzheimer's disease. Unfortunately, many inhibitors that block -secretase-mediated cleavage of APP also have immunosuppressive side effects. In addition to APP, numerous other proteins undergo -secretase-mediated cleavage. In order to develop safer inhibitors, it is necessary to determine which of the -secretase substrates contribute to the immunosuppressive effects. Because APP family members are widely expressed and are reported to influence calcium flux, transcription and apoptosis, they could be important for normal lymphocyte maturation. We find that APP and amyloid precursor-like protein 2 are expressed by stromal cells of thymus and lymph nodes, but not by lymphocytes. Although signals provided by thymic stromal cells are critical for normal T cell differentiation, lymphocyte development proceeds unperturbed in mice deficient for these APP family members.

Recombinant nucleocapsid-like particles from dengue-2 virus induce protective CD4+ and CD8+ cells against viral encephalitis in mice

Thu, 24 Sep 2009 05:39:09 PDT

Virus-like particles are a highly effective type of subunit vaccine that mimics the overall structure of virus particles without containing infectious genetic material. In this work, a particulate form of the recombinant capsid protein from dengue-2 was evaluated in mice to determine the level of protection against viral challenge and to measure the antigen-induced cell-mediated immunity (CMI). The nucleocapsid-like particles (NLPs) adjuvanted with alum did not induce antiviral antibodies. However, splenocytes from the immunized animals secreted high levels of IFN- upon virus stimulation, and a significant protection rate was achieved after challenge with lethal dengue-2 virus. Finally, both IFN- secretion and protection against viral encephalitis were demonstrated to be dependent on CD4⁺ and CD8⁺ cells. This study provides new evidences regarding the protective role of the CMI in the mouse model without the induction of neutralizing antibodies. Further studies in non-human primates or humanized mice should be carried out to elucidate the usefulness of the NLPs as a potential vaccine candidate against dengue disease.

Lactoferrin modulation of BCG-infected dendritic cell functions

Hwang, S.-A., Actor, J. K. — Thu, 24 Sep 2009 05:39:09 PDT

Lactoferrin, an 80-kDa iron-binding protein with immune modulating properties, is a unique adjuvant component able to enhance efficacy of the existing Mycobacterium bovis Bacillus Calmette Guerin (BCG) vaccine to protect against murine model of tuberculosis. Although identified as having effects on macrophage presentation events, lactoferrin's capability to modulate dendritic cells (DCs) function when loaded with BCG antigens has not been previously recognized. In this study, the potential of lactoferrin to modulate surface expression of MHC II, CD80, CD86 and CD40 from bone marrow-derived dendritic cells (BMDCs) was examined. Generally, lactoferrin decreased pro-inflammatory cytokines [tumor necrosis factor (TNF)-, IL-6 and IL-12p40] and chemokines [macrophage inflammatory protein (MIP)-1 and MIP-2] and increased regulatory cytokine, transforming growth factor-β1 and a T-cell chemotatic factor, monocyte chemotactic protein-1, from uninfected or BCG-infected BMDCs. Culturing BCG-infected BMDCs with lactoferrin also enhanced their ability to respond to IFN- activation through up-regulation of maturation markers: MHC I, MHC II and the ratio of CD86:CD80 surface expression. Furthermore, lactoferrin-exposed BCG-infected DCs increased stimulation of BCG-specific CD3⁺CD4⁺ splenocytes, as defined by increasing IFN- production. Finally, BCG-/lactoferrin-vaccinated mice possessed an increased pool of BCG antigen-specific IFN- producing CD3⁺CD4⁺CD62L^– splenocytes. These studies suggest a mechanism in which lactoferrin may exert adjuvant activity by enhancing DC function to promote generation of antigen-specific T cells.