Local Administration of GITR Agonistic Antibody Induces a Stronger Antitumor Immunity than Systemic Delivery.

An anti-glucocorticoid induced TNF receptor (GITR) agonistic antibody (Ab) induces an antitumor immunity with both stimulation of effector T cells and inhibition of regulatory T cell activity. To enhance GITR Ab-mediated tumor immunity, we focused on the intratumoral route, since a tumor-localized high concentration of Ab would confer activation of only tumor-infiltrating T cells. First, in a murine colon cancer model, we showed that the intratumoral delivery of Ab significantly increased the number of effector T cells infiltrated into tumors, and suppressed tumor growth more effectively than the intraperitoneal and intravenous injections did. Then, we found that the injection of Ab into the peritumoral area induced a systemic antitumor immunity at a similar level to the intratumoral injection. Therefore, we hypothesized that the transfer of locally administrated Ab into tumor-draining lymph nodes (TDLNs) plays an important role in inducing an effective immunity. In fact, intratumorally or peritumorally injected Ab was detected in TDLNs, and resection of Ab-injected TDLNs significantly reduced GITR Ab-mediated systemic tumor immunity. Intratumoral injection showed less number of auto-reactive T cells in the spleen than the intraperitoneal injection did. Intratumoral delivery of GITR Ab is a promising approach to induce an effective immunity compared to the systemic delivery.

Broad impact of extracellular DNA on biofilm formation by clinically isolated Methicillin-resistant and -sensitive strains of Staphylococcus aureus.

Staphylococcus aureus is a major causative agent for biofilm-associated infections. Inside biofilms, S. aureus cells are embedded in an extracellular matrix (ECM) composed of polysaccharide-intercellular adhesins (PIA), proteins, and/or extracellular DNA (eDNA). However, the importance of each component and the relationship among them in biofilms of diverse strains are largely unclear. Here, we characterised biofilms formed by 47 S. aureus clinical isolates. In most (42/47) of the strains, biofilm formation was augmented by glucose supplementation. Sodium chloride (NaCl)-triggered biofilm formation was more prevalent in methicillin-sensitive S. aureus (15/24) than in methicillin-resistant strain (1/23). DNase I most effectively inhibited and disrupted massive biofilms, and Proteinase K was also effective. Anti-biofilm effects of Dispersin B, which cleaves PIA, were restricted to PIA-dependent biofilms formed by specific strains and showed significant negative correlations with those of Proteinase K, suggesting independent roles of PIA and proteins in each biofilm. ECM profiling demonstrated that eDNA was present in all strains, although its level differed among strains and culture conditions. These results indicate that eDNA is the most common component in S. aureus biofilms, whereas PIA is important for a small number of isolates. Therefore, eDNA can be a primary target for developing eradication strategies against S. aureus biofilms.

Imaging of bacterial multicellular behaviour in biofilms in liquid by atmospheric scanning electron microscopy.

Biofilms are complex communities of microbes that attach to biotic or abiotic surfaces causing chronic infectious diseases. Within a biofilm, microbes are embedded in a self-produced soft extracellular matrix (ECM), which protects them from the host immune system and antibiotics. The nanoscale visualisation of delicate biofilms in liquid is challenging. Here, we develop atmospheric scanning electron microscopy (ASEM) to visualise Gram-positive and -negative bacterial biofilms immersed in aqueous solution. Biofilms cultured on electron-transparent film were directly imaged from below using the inverted SEM, allowing the formation of the region near the substrate to be studied at high resolution. We visualised intercellular nanostructures and the exocytosis of membrane vesicles, and linked the latter to the trafficking of cargos, including cytoplasmic proteins and the toxins hemolysin and coagulase. A thick dendritic nanotube network was observed between microbes, suggesting multicellular communication in biofilms. A universal immuno-labelling system was developed for biofilms and tested on various examples, including S. aureus biofilms. In the ECM, fine DNA and protein networks were visualised and the precise distribution of protein complexes was determined (e.g., straight curli, flagella, and excreted cytoplasmic molecular chaperones). Our observations provide structural insights into bacteria-substratum interactions, biofilm development and the internal microbe community.

Interaction of natural killer cells with neutrophils exerts a significant antitumor immunity in hematopoietic stem cell transplantation recipients.

Autologous hematopoietic stem cell transplantation (HSCT) can induce a strong antitumor immunity by homeostatic proliferation (HP) of T cells and suppression of regulatory T cells following preconditioning-induced lymphopenia. However, the role of innate immunity including natural killer (NK) cells is still not understood. Here, first, we examined whether NK cells exert an antitumor effect after syngeneic HSCT in a murine colon cancer model. Flow cytometry showed that NK cells as well as T cells rapidly proliferated after HSCT, and the frequency of mature NK cells was increased in tumor during HP. Furthermore, NK cells undergoing HP were highly activated, which contributed to substantial tumor suppression. Then, we found that a large number of neutrophils accumulated in tumor early after syngeneic HSCT. It was recently reported that neutrophil-derived mediators modulate NK cell effector functions, and so we examined whether the neutrophils infiltrated in tumor are associated with NK cell-mediated antitumor effect. The depletion of neutrophils significantly impaired an activation of NK cells in tumor and increased the fraction of proliferative NK cells accompanied by a decrease in NK cell survival. The results suggested that neutrophils in tumor prevent NK cells from activation-induced cell death during HP, thus leading to a significant antitumor effect by NK cells. This study revealed a novel aspect of antitumor immunity induced by HSCT and may contribute to the development of an effective therapeutic strategy for cancer using HSCT.

Proinflammatory Proteins S100A8/S100A9 Activate NK Cells via Interaction with RAGE.

S100A8/A9, a proinflammatory protein, is upregulated in inflammatory diseases, and also has a tumor-promoting activity by the recruitment of myeloid cells and tumor cell invasion. However, whether the expression of S100A8/A9 in tumors predicts a good or poor prognosis is controversial in the clinical setting. In this study, to clarify the in vivo role of S100A8/A9 in the tumor microenvironment, we s.c. inoculated Pan02 cells stably expressing S100A8 and S100A9 proteins (Pan02-S100A8/A9) in syngeneic C57BL/6 mice. Unexpectedly, after small tumor nodules were once established, they rapidly disappeared. Flow cytometry showed that the number of NK cells in the tumors was increased, and an administration of anti-asialoGM1 Ab for NK cell depletion promoted the growth of Pan02-S100A8/A9 s.c. tumors. Although the S100A8/A9 proteins alone did not change the IFN-γ expression of NK cells in vitro, a coculture with Pan02 cells, which express Rae-1, induced IFN-γ production, and Pan02-S100A8/A9 cells further increased the number of IFN-γ(+) NK cells, suggesting that S100A8/A9 enhanced the NK group 2D ligand-mediated intracellular activation pathway in NK cells. We then examined whether NK cell activation by S100A8/A9 was via their binding to receptor of advanced glycation end product (RAGE) by using the inhibitors. RAGE antagonistic peptide and anti-RAGE Ab inhibited the IFN-γ production of NK cells induced by S100A8/A9 proteins, and an administration of FPS-ZM1, a RAGE inhibitor, significantly enhanced the in vivo growth of Pan02-S100A8/A9 tumors. We thus found a novel activation mechanism of NK cells via S100A8/A9-RAGE signaling, which may open a novel perspective on the in vivo interaction between inflammation and innate immunity.

Suppression of Tregs by anti-glucocorticoid induced TNF receptor antibody enhances the antitumor immunity of interferon-α gene therapy for pancreatic cancer.

We have reported that interferon (IFN)-α can attack cancer cells by multiple antitumor mechanisms including the induction of direct cancer cell death and the enhancement of an immune response in several pancreatic cancer models. However, an immunotolerant microenvironment in the tumors is often responsible for the failure of the cancer immunotherapy. Here we examined whether the suppression of regulatory T cells (Tregs) within tumors can enhance an antitumor immunity induced by an intratumoral IFN-α gene transfer. First we showed that an intraperitoneal administration of an agonistic anti-glucocorticoid induced TNF receptor (GITR) monoclonal antibody (mAb), which is reported to suppress the function of Tregs, significantly inhibited subcutaneous tumor growth in a murine pancreatic cancer model. The anti-GITR mAb was then combined with the intratumoral injection of the IFN-α-adenovirus vector. The treatment with the antibody synergistically augmented the antitumor effect of IFN-α gene therapy not only in the vector-injected tumors but also in the vector-uninjected tumors. Immunostaining showed that the anti-GITR mAb decreased Foxp3(+) cells infiltrating in the tumors, while the intratumoral IFN-α gene transfer increased CD4(+) and CD8(+) T cells in the tumors. Therefore, the combination therapy strongly inclined the immune balance of the tumor microenvironment in an antitumor direction, leading to a marked systemic antitumor effect. The CCR5 expression on Tregs was downregulated in the antibody-treated mice, which may explain the decrease of tumor-infiltrating Tregs. The combination of Treg-suppression by GITR mAb and the tumor immunity induction by IFN-α gene therapy could be a promising therapeutic strategy for pancreatic cancer.

Vascular endothelial growth factor-D-mediated blockade of regulatory T cells within tumors is induced by hematopoietic stem cell transplantation.

Lymphopenia-induced homeostatic proliferation of T cells after autologous hematopoietic stem cell transplantation (HSCT) skews the T cell repertoire by engaging tumor-associated Ags, leading to an induction of antitumor immunity. However, how HSCT alters the immunosuppressive microenvironment in the tumors is unknown. In this study, we first analyzed the kinetics of regulatory T cells (Tregs) in the tumors after syngeneic HSCT. Unexpectedly, the frequency of CD4⁺ cells expressing Foxp3 was increased in the spleens, whereas the frequency was clearly decreased in the tumors after HSCT. The origin of reconstituted CD4⁺ and Foxp3⁺ cells in the tumors was mainly from the expansion of transferred splenic T cells. Then, to examine the mechanism of Treg suppression after HSCT, we isolated CD11c⁺ cells from tumors. A large amount of Treg-inhibitory cytokine IL-6 was secreted from the CD11c⁺ cells in the tumors, but not in the spleens in the recipient mice. Furthermore, to understand what factor affects the activity of CD11c⁺ cells in the tumors after HSCT, we analyzed the expression of various cytokines/chemokines with mouse cytokine Ab arrays, and noticed that VEGF-D concentration was increased in the tumors in the early period after HSCT. The CD11c⁺ cells produced IL-6 in response to VEGF-D stimulation, and an administration of VEGF receptor-3 neutralizing Ab significantly suppressed the production of IL-6 from CD11c⁺ cells accompanied with the increase of Tregs in the tumors of HSCT recipients. Autologous HSCT creates an environment that strongly supports the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the suppression of Tregs.

Preimmunization of donor lymphocytes enhances antitumor immunity of autologous hematopoietic stem cell transplantation.

Lymphopenia-induced homeostatic proliferation (HP) of T cells following autologous hematopoietic stem cell transplantation (HSCT) skews the T-cell repertoire by engaging tumor-associated antigens (TAAs), leading to an induction of antitumor immunity. Here, as the tumor-reactive lymphocytes preferentially proliferate during the condition of HP, we examined whether the priming of a donor lymphocytes to TAAs could enhance HP-induced antitumor immunity in autologous HSCT recipients. First, to examine whether the tumor-bearing condition of donor influences the antitumor effect of HSCT, the lymphocytes isolated from CT26 tumor-bearing mice were infused into lethally irradiated mice. The growth of tumors was substantially suppressed in the mice that received HSCT from a tumor-bearing donor compared with a naïve donor, suggesting that a fraction of donor lymphocytes from tumor-bearing mice are primed in response to TAAs and remain responsive upon transplantation. We previously reported that type I interferon (IFN) maturates the dendritic cells and promotes the priming of T cells. We then investigated whether the further priming of donor cells by IFN-α can strengthen the antitumor effect of HSCT. The intratumoral IFN-α gene transfer significantly increased the number of IFN-γ-positive lymphocytes in response to CT26 cells but not the syngeneic lymphocytes in donor mice. The infusion of primed donor lymphocytes markedly suppressed the tumor growth in recipient mice, and cured 64% of the treated mice. Autologous HSCT with the infusion of primed donor lymphocytes is a promising strategy to induce an effective antitumor immunity for solid cancers.

The growth movement in the peduncle of Eichhornia crassipes II.

The peduncle of water hyacinth (Eichhornia crassipes) showed the downward bending within 24 hours after full flowering. Previously it was suggested that the downward bending of peduncle might be induced by the differential growth of the epidermal cells of the portion because of the differential distribution of auxin in the upper side of the bending part of the peduncle. In order to investigate the effect of auxin and gravity on the peduncle bending in Water hyacinth, we examined the growth reaction of peduncle and the effects of plant hormones on the bending of peduncle under simulated microgravity, and the sedimentable amyloplast on earth and three dimensional (3D)-clinostat. As a result it was confirmed that the downward bending of peduncle in water hyacinth is the positive gravitropism, and that its phenomenon is caused by the differential distribution of auxin in the upper side of bending part of peduncle. It was found that the amyloplast sediments toward gravity direction in the bending part of the peduncle. From the present results, any direct relation between the sedimentable amyloplast and auxin transport were not cleared in the peduncle of water hyacinth. Further study should be carried out.