Cervical Draining Lymph Nodes Research Articles

Antibody-Mediated Depletion of Autoreactive T Lymphocytes through PD-1 Improves Disease Outcomes and Visualizes T Cell Activation in Experimental Autoimmune Encephalomyelitis.

Long-term therapeutic outcomes of multiple sclerosis (MS) remain hindered by the chronic nature of immune cell stimulation toward self-antigens. Development of novel methods to target and deplete autoreactive T lymphocytes remains an attractive target for therapeutics for MS. We developed a programmed cell death 1 (PD-1)-targeted radiolabeled mAb and assessed its ability to deplete activated PD-1+ T lymphocytes invitro and its ability to reduce disease burden of the myelin oligodendrocyte glycoprotein 35-55 experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice. We also investigated the upregulation of PD-1 on infiltrating lymphocytes in an animal model of MS. Finally, we demonstrate the (to our knowledge) first reported positron-emission tomography/computed tomography imaging of activated PD-1+ cells in the EAE animal model of MS. We found that the 177Lu radioisotope-labeled anti-PD-1 mAb demonstrated significant invitro cytotoxicity toward activated CD4+PD-1+ T lymphocytes and led to significant reduction in overall disease progression in the EAE animal model. Our results show high expression of PD-1 on infiltrating lymphocytes in the spinal cords of EAE diseased animals. Positron-emission tomography/computed tomography imaging of the anti-PD-1 mAb demonstrated significant uptake in the cervical draining lymph nodes highlighting accumulation of activated lymphocytes. Targeted depletion of T lymphocytes using T cell activation markers such as PD-1 may present a novel method to reduce autoimmune attack and inflammation in autoimmune diseases such as MS. Development of multimodal nuclear theranostic agents may present the opportunity to monitor T cell activation via imaging radioisotopes and simultaneously treat MS using therapeutic radioisotopes.

Single-cell sequencing of the retina shows that LDHA regulates pathogenesis of autoimmune uveitis

Autoimmune uveitis (AU) is a severe disorder causing poor vision and blindness. However, the cellular dynamics and pathogenic mechanisms underlying retinal injury in uveitis remain unclear. In this study, single-cell RNA sequencing of the retina and cervical draining lymph nodes in experimental autoimmune uveitis mice was conducted to identify the cellular spatiotemporal dynamics and upregulation of the glycolysis-related gene LDHA. Suppression of LDHA can rescue the imbalance of T effector (Teff) cells/T regulator (Treg) cells under inflammation via downregulation of the glycolysis-PI3K signaling circuit and inhibition of the migration of CXCR4+ Teff cells towards retinal tissue. Furthermore, LDHA and CXCR4 are upregulated in the peripheral blood mononuclear cells of Vogt-Koyanagi-Harada patients. The LDHA inhibitor suppresses CD4+ T cell proliferation in humans. Therefore, our data indicate that the autoimmune environment of uveitis regulates Teff cell accumulation in the retina via glycolysis-associated LDHA. Modulation of this target may provide a novel therapeutic strategy for treating AU.

Study the involvement of Langerin in mediating epicutaneous sensitization of atopic dermatitis-like mouse model

Objective: To explore the role of Langerin in mediating epicutaneous sensitization of atopic dermatitis (AD) in mouse model. Methods: Mice were topically treated with calcipotriol (MC903) plus ovalbumin (OVA) on the ears to establish AD mouse models, and mice were divided into wild-type control group, wild-type AD group, Langerin knockout control group, and Langerin knockout AD group. Changes of lesion were daily observed. Infiltration of inflammatory cells, mRNA expression of Tslp, Il4, Il13, Il17a, and Il22, levels of serum total IgE, OVA-specific IgE (sIgE), OVA sIgG1 and OVA sIgG2a, proportion of regulatory T (Treg) cells in cervical draining lymph nodes were evaluated at the end of model preparation. Results: Skin tumidness and thickness, dermal inflammatory cells infiltration, the mRNA expression levels of Tslp, Il4, Il13, Il17a and Il22 in wild-type AD groups were higher than those in wild-type control groups, with (1.80±0.66, 1.64±0.25, 1.71±0.54, 2.41±0.23, 2.49±0.32) and (0.53±0.45, 0.85±0.29, 0.73±0.50, 0.72±0.25, 0.56±0.29), respectively (all P<0.05). In addition, the levels of serum total IgE, OVA sIgE and OVA sIgG1 in wild-type AD groups were higher than those in wild-type control groups, with [(1 216.00±572.70) ng/ml, (597.00±538.30) ng/ml, 1.59±0.09] and [(24.22±35.04) ng/ml, (20.01±41.71) ng/ml, 1.16±0.03], respectively (all P<0.05). In Langerin knockout mice, compared to wild-type mice, skin erythema, skin tumidness, epidermal thickening, inflammatory cell infiltration were more obvious; the mRNA expression levels of Tslp, Il4, Il13, Il17a and Il22 were upregulated with (8.19±6.44, 2.53±0.69, 2.82±0.73, 3.94±1.32, 3.80±1.43) (all P<0.05); the levels of serum total IgE, OVA sIgE and OVA sIgG1 were significantly increased with (2 508.00±657.10) ng/ml, (1 808.00±470.70) ng/ml, (1.73±0.09) (all P<0.05); the number of CD4+CD25+CD127-Treg cells were decreased significantly with (13.25±0.96)% and (15.31±1.47)%, respectively (P<0.05). Conclusion: Langerin is involved in mediating epicutaneous sensitization of the AD mouse model and plays a negative immunoregulatory role.

Sleep loss potentiates Th17-cell pathogenicity and promotes autoimmune uveitis.

Sleep loss (SL) is a health issue associated with the higher risk of autoimmune and inflammatory disorders. However, the connection between SL, the immune system, and autoimmune diseases remains unknown. We conducted mass cytometry, single-cell RNA sequencing, and flow cytometry to analyze how SL influences immune system and autoimmune disease development. Peripheral blood mononuclear cells from six healthy subjects before and after SL were collected and analyzed by mass cytometry experiments and subsequent bioinformatic analysis to identify the effects of SL on human immune system. Sleep deprivation and experimental autoimmune uveitis (EAU) mice model were constructed, and scRNA-seq data from mice cervical draining lymph nodes were generated to explore how SL influences EAU development and related autoimmune responses. We found compositional and functional changes in human and mouse immune cells after SL, especially in effector CD4+ T and myeloid cells. SL upregulated serum GM-CSF levels in healthy individuals and in patients with SL-induced recurrent uveitis. Experiments in mice undergoing SL or EAU demonstrated that SL could aggravate autoimmune disorders by inducing pathological immune cell activation, upregulating inflammatory pathways, and promoting intercellular communication. Furthermore, we found that SL promoted Th17 differentiation, pathogenicity, and myeloid cells activation through the IL-23Th17GM-CSF feedback mechanism, thus promoting EAU development. Lastly, an anti-GM-CSF treatment rescued SL-induced EAU aggravation and pathological immune response. SL promoted Th17 cells pathogenicity and autoimmune uveitis development, especially through the interaction between Th17 and myeloid cells involving GM-CSF signaling, providing possible therapeutic targets for the SL-related pathological disorders.

Interaction of immune cells and lymphatic vessels in corneal transplantation

AbstractImmune‐mediated graft rejections remain the most common cause for graft failure after organ and tissue transplantation. There is a great unmet medical need for pharmacologic strategies to promote graft survival without unduly compromising the health of the recipient.Corneal allograft rejection is histologically characterized by massive infiltration of CD4+ T‐cells, macrophages and the invasion of blood and lymphatic vessels.We could show that lymphatic vessels determine the high‐risk status of a corneal graft recipient. The healthy cornea is devoid of blood and lymphatic vessels. In contrast, in the high risk situation graft rejection is highly correlated with the degree of corneal vascularisation and presence of numerous pro‐inflammatory immune cells.CD11c+ CD11b+ myeloid dendritic cells (DCs) are present throughout the anterior stroma – even in the healthy cornea. Inflammatory stimuli determine the maturation stage of DCs and their immunogenic properties. It is known that an increasing number of MHC class II‐bearing, antigen specific DCs in cervical draining lymph nodes arise only a few hours after transplantation. This implies that DCs at different maturation stages might be the main initiators of this devastating tissue destruction.The origin of donor antigen presenting cells can be either from the donor tissue or the host. Thereby allo‐antigens from the grafted tissues travel via lymphatic vessels towards the draining lymph nodes of the recipient. Here they are recognized by host T cells either by the direct pathway, which involves the recognition of intact donor MHC molecules on donor‐derived APCs, or by the indirect pathway, which involves the recognition of donor major or minor MHC‐derived peptides, processed and presented by recipient APCs to host T cells. Nowadays it is widely accepted that resident MHC class II APC populations are present in the cornea. These cells are capable of expressing MHC class II antigen and accessory molecules (CD40, CD80) after transplantation. We and others could demonstrate that the immune reaction is triggered by the above described “direct allo‐sensitisation” where APCs travel from the donor tissue via corneal pathological lymphatic vessels into the local draining lymph nodes of the recipient and present the foreign antigen.Recent studies could show that lymphatic vessels not just display an exit route for leukocytes but are in close interaction with these cells and regulate the entry and trafficking of antigen presenting cells into and via lymphatic vessels.This presentation will summarize the current knowledge about the interaction of immune cells and lymphatic vessels in corneal transplantation.

Therapeutic Effect of IL-38 on Experimental Autoimmune Uveitis: Reprogrammed Immune Cell Landscape and Reduced Th17 Cell Pathogenicity.

PurposeThe purpose of this study was to elucidate the effects of interleukin (IL)-38 on experimental autoimmune uveitis (EAU) and its underlying mechanisms.MethodsMice with EAU were treated with IL-38, and the retinas and cervical draining lymph nodes (CDLNs) were analyzed by flow cytometry. Single-cell RNA sequencing (scRNA-seq) was conducted to analyze the immune cell profiles of CDLNs from normal, EAU, and IL-38-treated mice.ResultsAdministration of IL-38 attenuated EAU symptoms and reduced the proportion of T helper 17 (Th17) and T helper 1 (Th1) cells in the retinas and CDLNs. In scRNA-seq analysis, IL-38 downregulated the IL-17 signaling pathway and reduced the expression of Th17 cell pathogenicity-related genes (Csf2 and Il23r), findings which were also confirmed by flow cytometry. In vitro, IL-38 reduced the granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation function of IL-23 and inhibited IL-23R expression in Th17 cells. Moreover, when co-cultured with Th17 cells, IL-38 prevented IL-23 production in antigen-presenting cells (APCs).ConclusionsOur data demonstrate the therapeutic effect of IL-38 on EAU, and suggest that the effect of IL-38 may be caused by dampening of the GM-CSF/IL-23R/IL-23 feedback loop between Th17 cells and APCs.

IMMU-46. ARTIFICIAL INTELLIGENCE-DIRECTED, GENE THERAPY-BASED TRANSDIFFERENTIATION OF GLIOBLASTOMA TO FUNCTIONAL DENDRITIC CELLS AS NOVEL CANCER IMMUNOTHERAPY

Abstract BACKGROUND Despite recent advances in tumor immunotherapy in solid tumors, success in GBM remains elusive, likely due to its poor immunogenicity and CNS barriers limiting immune cell trafficking. Here we describe a novel approach of stimulating glioma-specific immunity by transdifferentiating GBM cells in situ to induced dendritic cells (iDCs). METHODS We applied NETZEN, an integrated deep-learning and gene network-based ranking computational platform and identified cell fate determinants (CFDs) to convert GBM cells to DCs. CFDs were delivered using a viral vector. Transdifferentiation was assessed by immunophenotyping and iDCs functionally validated by their ability to prime naive T cells. RESULTS A four CFDs subnetwork anchored by PU.1 was sufficient to transdifferentiate mouse GBM cells to CD45+MHCII+ cells with high co-stimulatory CD80 expression and to induce nearly 98% of GBM cells to express 100-fold higher levels of MHCI. Consistent with a new identity of antigen-presenting cells (APC), the induced immune cells are growth arrested, exhibit 3-fold higher phagocytic activity and upregulate the canonical antigen processing and presenting machineries by 10-40 folds, resulting in 40-fold greater efficiency at processing ovalbumin and presenting SIINFEKL on MHCI compared to native GBM cells. Importantly, SIINFEKL-loaded iAPCs are capable of activating naive OTII-CD4+ and OTI-CD8+ T cells, indicating that they are DC-like. In addition, iDCs efficiently present tumor cell-intrinsic antigens and elicit &amp;gt;20-fold higher activation and cytotoxicity in tumor-specific T cells compared to native GBM cells. Lastly, intratumoral GBM-DC transdifferentiation in a syngeneic orthotopic GBM model produces a robust memory T cell response in deep cervical draining lymph nodes compared to control animals. CONCLUSIONS Our results comfirm that GBM-derived iDCs acquire functions similar to native DCs, and thus, lay the foundation for a novel therapeutic approach in which poorly immunogenic tumors like GBM may be forced to generate their own immunity from within through cell fate transdifferentiation.

Aging weakens Th17 cell pathogenicity and ameliorates experimental autoimmune uveitis in mice

Aging-induced changes in the immune system are associated with a higher incidence of infection and vaccination failure. Lymph nodes, which filter the lymph to identify and fight infections, play a central role in this process. However, careful characterization of the impact of aging on lymph nodes and associated autoimmune diseases is lacking. We combined single-cell RNA sequencing (scRNA-seq) with flow cytometry to delineate the immune cell atlas of cervical draining lymph nodes (CDLNs) of both young and old mice with or without experimental autoimmune uveitis (EAU). We found extensive and complicated changes in the cellular constituents of CDLNs during aging. When confronted with autoimmune challenges, old mice developed milder EAU compared to young mice. Within this EAU process, we highlighted that the pathogenicity of T helper 17 cells (Th17) was dampened, as shown by reduced GM-CSF secretion in old mice. The mitigated secretion of GM-CSF contributed to alleviation of IL-23 secretion by antigen-presenting cells (APCs) and may, in turn, weaken APCs’ effects on facilitating the pathogenicity of Th17 cells. Meanwhile, our study further unveiled that aging downregulated GM-CSF secretion through reducing both the transcript and protein levels of IL-23R in Th17 cells from CDLNs. Overall, aging altered immune cell responses, especially through toning down Th17 cells, counteracting EAU challenge in old mice.

Endogenous Toll-like Receptor 2 Modulates Th1/Treg-Promoting Dendritic Cells in Mice Corneal Transplantation Model

ABSTRACT Purpose Endogenous toll-like receptor (TLR) 2 is linked to allograft rejection in corneal transplantation. TLR2 also could modulate dendritic cell (DC) phenotype, resulting in T cell polarization. Thus, we investigated the role of endogenous TLR2 on DC development and T cell polarization during corneal rejection. Materials and Methods Corneas of BALB/c mice were transplanted into the eyes of C57BL/6 wild-type (WT) recipients and TLR2−/- (KO) recipients. Graft survival and TLR2 mRNA expression were assessed. At day 14 after transplantation, to study endogenous TLR2 effects on DC development and function, surface expression of MHC classⅡ (MHCⅡ), CD86, CD80 and CD40 in ipsilateral cervical draining lymph nodes (DLNs) is measured by flow cytometry, and DC phenotype in corneas is detected by immunofluorescence. The levels of IL-12, IL-10 and IL-4 in corneas were measured by real time-qPCR (RT-qPCR). The ability of DCs to stimulate T cell polarization was assessed by IFN-γ expressions via RT-qPCR and immunohistochemistry. Results TLR2 mRNA expression in corneas was peaked at day 14 post-transplantation in WT group. KO group improved corneal allograft survival compared to the WT group. In addition, the KO group decreased expression of CD86, CD80 and CD40 on DCs compared to the WT group. There was no difference in MHCⅡ expression in two groups. The CD11c+MHCⅡ+CD40high DCs could not be detected in corneas of the KO group. Moreover, the KO group decreased IL-12 (Th1-promoting cytokines) mRNA expression and increasing IL-10 (Treg-promoting cytokines) mRNA expression compared to the WT group. IL-4 (Th2-promoting cytokines) mRNA expression gained no difference between the two groups. The IFN-γ (Th1 cytokines) expression was significantly decreased in the KO group compared to the WT group. Conclusions Endogenous TLR2 may contribute to allogeneic corneal rejection via Th1 immunity by activating Th1-promoting DCs and suppressing Treg-promoting DCs.

TMIC-08. IDENTIFICATION OF TUMOR SPECIFIC LYMPHOCYTES IN A MURINE GLIOMA MODEL

Abstract INTRODUCTION One major challenge in the use of immunotherapy for glioblastoma is the identification of tumor specific lymphocytes within the tumor microenvironment. Previous studies in non-glioma cancer models have identified CD39 and PD1 as lymphocyte markers of tumor specificity. In this study, we demonstrate the existence of this specific T cell phenotype and show correlation with anti-tumor activity. METHODS We implanted KR158B murine glioma cells into right caudate nucleus into yellow fluorescent protein (YFP) interferon-gamma reporter mice for a total of 104 cells using stereotaxis. At 4 weeks, mice were sacrificed followed by organ harvest. Samples taken included tumor, cervical draining lymph nodes, spleen, and peripheral blood. These were processed into single cell suspensions and then stained for CD3, CD8, CD39, and PD1. Flow cytometry was performed on a BD FACSCanto II. Gating analysis was performed with FlowJo while data analysis was performed with GraphPad Prism. RESULTS Gating for CD3+ CD8+ on tumor lymphocytes reveals a significant population of CD39 and PD1 double positive (DP) cells (61.4%) not found in the peripheral organs (&lt; 1.0%). Evaluation of these cells for YFP expression demonstrated significantly greater median fluorescence intensity (MFI) in the DP group as opposed to either of the single positive (SP) or double negative (DN) groups (DP = 225.4, SP-CD39 = 40.54, SP-PD1 = 82.7, DN = 29.5, p &lt; 0.0001). CONCLUSION Here we demonstrate the existence of a unique phenotype of CD39+ and PD1+ expression on lymphocytes within the tumor microenvironment. These DP cells also had high levels of interferon gamma production compared SP and DN groups. This phenotype is not observed in peripheral circulating lymphocytes. Enrichment for this subpopulation will allow for delivery of purified lymphocyte-based immunotherapy in this murine glioma model.

Neurokinin-1 Receptor Antagonism Ameliorates Dry Eye Disease by Inhibiting Antigen-Presenting Cell Maturation and T Helper 17 Cell Activation

Neuroinflammation plays an important role in the pathogenesis of ocular surface disease, including dry eye disease (DED), but little is known about the contribution of substance P (SP) to DED. In this study, we investigated the expression of SP at the ocular surface and evaluated its effect on maturation of antigen-presenting cells (APCs), the key cell component involved in the induction of type 17 helper T-cell (Th17) response in DED. The effect of topical blockade of SP signaling was further investigated using neurokinin-1 receptor (NK1R) inhibitors on APC maturation, Th17 cell activation, and disease severity in a mouse model of DED. The results demonstrate that SP is constitutively expressed at the ocular surface, and trigeminal ganglion neurons are the major source of SP in DED. SP derived from trigeminal ganglion enhanced the expression of major histocompatibility complex class II maturation marker by bone marrow-derived dendritic cells, an effect that is abrogated by blockade of SP signaling using NK1R antagonist spantide. Finally, using a well-established murine model of DED, topical treatment of DED mice with NK1R antagonists CP-99,994 and L-733,060 suppressed APC acquisition of major histocompatibility complex class II, reduced Th17 cell activity, and ameliorated DED severity. These findings are of translational value, as they suggest that antagonizing NK1R-mediated SP signaling may be an effective strategy in suppressing Th17-mediated ocular surface disease.

Identification of Tumor Specific Lymphocytes Cells in a Murine Glioma Model

Abstract INTRODUCTION One major challenge in the use of immunotherapy for glioblastoma is the identification of tumor specific lymphocytes within the tumor microenvironment. Previous studies in nonglioma cancer models have identified CD39 and PD1 as lymphocyte markers of tumor specificity. In this study, we demonstrate the existence of this specific T cell phenotype and show correlation with antitumor activity. METHODS We implanted KR158B murine glioma cells into right caudate nucleus into yellow fluorescent protein (YFP) interferon-gamma reporter mice for a total of 104 cells using stereotaxis. At 4 wk, mice were sacrificed followed by organ harvest. Samples taken included tumor, cervical draining lymph nodes, spleen, and peripheral blood. These were processed into single cell suspensions and then stained for CD3, CD8, CD39, and PD1. Flow cytometry was performed on a BD Canto II. Gating analysis was performed with FlowJo while data analysis was performed with GraphPad Prism. RESULTS Gating for CD3+ CD8+ on tumor lymphocytes reveals a significant population of CD39 and PD1 double positive (DP) cells (61.4%) not found in the peripheral organs (&lt;1.0%). Evaluation of these cells for YFP expression demonstrated significantly greater median fluorescence intensity (MFI) in the DP group as opposed to either of the single positive (SP) or double negative (DN) groups (DP = 225.4, SP-CD39 = 40.54, SP-PD1 = 82.7, DN = 29.5, P &lt; .001). CONCLUSION Here we demonstrate the existence of a unique phenotype of CD39+ and PD1+ expression on lymphocytes within the tumor microenvironment. These DP cells also had high levels of interferon gamma production compared SP and DN groups. This phenotype is not observed in peripheral circulating lymphocytes. Enrichment for this subpopulation will allow for delivery of purified lymphocyte-based immunotherapy in this murine glioma model.

Enhanced Efficacy of Vaccination With Vaccinia Virus in Old vs. Young Mice.

Immunosenescence is believed to be responsible for poor vaccine efficacy in the elderly. To overcome this difficulty, research into vaccination strategies and the mechanisms of immune responses to vaccination is required. By analyzing the innate and adaptive immune responses to vaccination with vaccinia virus (VACV) in mice of different age groups, we found that immune cell recruitment, production of cytokines/chemokines and control of viral replication at the site of intradermal vaccination were preserved in aged mice and were comparable with younger groups. Analysis of cervical draining lymph nodes (dLN) collected after vaccination showed that numbers of germinal center B cells and follicular T helper cells were similar across different age groups. The number of VACV-specific CD8 T cells in the spleen and the levels of serum neutralizing antibodies 1 month after vaccination were also comparable across all age groups. However, following intranasal challenge of vaccinated mice, body weight loss was lower and virus was cleared more rapidly in aged mice than in younger animals. In conclusion, vaccination with VACV can induce an effective immune response and stronger protection in elderly animals. Thus, the development of recombinant VACV-based vaccines against different infectious diseases should be considered as a strategy for improving vaccine immunogenicity and efficacy in the elderly.

The BtaF Adhesin Is Necessary for Full Virulence During Respiratory Infection by Brucella suis and Is a Novel Immunogen for Nasal Vaccination Against Brucella Infection.

Brucella enters their hosts mostly through mucosae from where it spreads systemically. Adhesion to extracellular matrix (ECM) components or to host cells is important for the infectious process, and is mediated by several adhesins, including the BtaF trimeric autotransporter. Although Th1 responses and gamma interferon (IFN-γ) are important for protection, antibodies able to block adhesions might also contribute to prevent Brucella infection. We evaluated the importance of BtaF for respiratory Brucella infection, and characterized the immune response and protection from mucosal challenge induced by nasal vaccination with recombinant BtaF. While lung CFU numbers did not differ at day 1 p.i. between mice intratracheally inoculated with B. suis M1330 (wild type) and those receiving a ΔbtaF mutant, they were reduced in the latter group at 7 and 30 days p.i. For vaccination studies the BtaF passenger domain was engineered and expressed as a soluble trimeric protein. Mice were immunized by the nasal route with BtaF or saline (control group) plus the mucosal adjuvant c-di-AMP. Specific anti-BtaF antibodies (IgG and IgA) were increased in serum, including a mixed IgG2a/IgG1 response. In vitro, these antibodies reduced bacterial adhesion to A549 alveolar epithelial cells. Specific IgA antibodies were also increased in several mucosae. Spleen cells from BtaF immunized mice significantly increased their IL-2, IL-5, IL-17, and IFN-γ secretion upon antigen stimulation. In cervical draining lymph nodes, antigen-experienced CD4+ T cells were maintained mainly as central memory cells. A BtaF-specific delayed-type hypersensitivity response was detected in BtaF immunized mice. Lung cells from the latter produced high levels of IFN-γ upon antigen stimulation. Although nasal immunization with BtaF did not protect mice against B. suis respiratory challenge, it conferred significant protection from intragastric challenge; the splenic load of B. suis was reduced by 3.28 log CFU in immunized mice. This study shows that nasal vaccination with BtaF+c-di-AMP protects against intragastric challenge with B. suis by inducing local and systemic antibody responses, central memory CD4+ T cells and strong Th1 responses. Therefore, although BtaF vaccination did not protect from B. suis respiratory infection, this adhesin constitutes a promising immunogen against mucosal B. suis infection.

Endogenous Neoantigen-Specific CD8 T Cells Identified in Two Glioblastoma Models Using a Cancer Immunogenomics Approach.

The "cancer immunogenomics" paradigm has facilitated the search for tumor-specific antigens over the last 4 years by applying comprehensive cancer genomics to tumor antigen discovery. We applied this methodology to identify tumor-specific "neoantigens" in the C57BL/6-derived GL261 and VM/Dk-derived SMA-560 tumor models. Following DNA whole-exome and RNA sequencing, high-affinity candidate neoepitopes were predicted and screened for immunogenicity by ELISPOT and tetramer analyses. GL261 and SMA-560 harbored 4,932 and 2,171 nonsynonymous exome mutations, respectively, of which less than half were expressed. To establish the immunogenicities of H-2Kb and H-2Db candidate neoantigens, we assessed the ability of the epitopes predicted in silico to be the highest affinity binders to activate tumor-infiltrating T cells harvested from GL261 and SMA-560 tumors. Using IFNγ ELISPOT, we confirmed H-2Db-restricted Imp3D81N (GL261) and Odc1Q129L (SMA-560) along with H-2Kb-restricted E2f8K272R (SMA-560) as endogenous tumor-specific neoantigens that are functionally immunogenic. Furthermore, neoantigen-specific T cells to Imp3D81N and Odc1Q129L were detected within intracranial tumors as well as cervical draining lymph nodes by tetramer analysis. By establishing the immunogenicities of predicted high-affinity neoepitopes in these models, we extend the immunogenomics-based neoantigen discovery pipeline to glioblastoma models and provide a tractable system to further study the mechanism of action of T cell-activating immunotherapeutic approaches in preclinical models of glioblastoma. Cancer Immunol Res; 4(12); 1007-15. ©2016 AACR.

Short-Term Dynamic Observation of the Color Change and Enhancement Effect of Polyethylenimine-Entrapped Gold Nanoparticles Used for Indirect Lymphography

Aims: To dynamically observe the color change and enhancement effect of gadolinium-loaded polyethylenimine-entrapped gold nanoparticles (Gd-Au PENPs) as a dual-mode CT/MRI contrast agent for sentinel lymph node (SLN) identification. Methods: In 6 rabbits, Gd-Au PENPs were injected into the right side tongue submucosa, after which the color change of cervical draining lymph nodes was observed. Then the draining lymph nodes were examined by CT/MRI scan. Another 6 rabbits were randomly divided into a CT lymphography (CT-LG) and a MRI lymphography (MRI-LG) group and examined by CT/MRI scan 1 and 30 min after injection. Then SLNs were identified under the guidance of CT/MRI-LG. Results: The ipsilateral afferent lymphatic vessels, cervical draining lymph nodes and efferent lymphatic vessels were stained black orderly and constantly after Gd-Au PENPs injection. Thirty minutes after injection, all cervical lymph nodes were excised to be examined by CT/MRI scan, and only the black-stained draining lymph nodes were enhanced. The 6 rabbits with CT/MRI-LG showed clear enhancement of SLNs. With indirect lymphography, the black-stained SLNs were easily visualized at autopsy. In all rabbits, the identification rate of lingual SLNs was 100%. Conclusions: Rabbit lingual SLNs could be identified with indirect lymphography using Gd-Au PENPs as a dual-mode contrast agent.

Intracerebral Mycobacterium tuberculosis infection-induced immune response in the central nervous system

Abstract The World Health Organization declared a tuberculosis (TB) global emergency. Central nervous system (CNS) TB is a rare, highly devastating manifestation of extra-pulmonary TB. To date, research has focused on pulmonary TB, with no significant advancements being made in understanding the mechanisms associated with CNS TB. This study uses a small animal model of CNS TB that permits us to investigate the immune responses following CNS Mycobacterium tuberculosis (Mtb) infection. We use intracranial infection of mice with the H37Rv strain of Mtb which has been fluorescently tagged with a TdTomato plasmid, allowing us to track it in vivo, and analyzed the cellular composition of the subsequent immunological response in the brain. At 7 days post infection (PI), we show a robust infiltration of immune cells into the CNS including the infiltration of CD11c+ dendritic cells. We can also see Mtb-containing granuloma-like lesions. We adoptively transferred Mtb-specific P25 CD4+ T and found that these cells also accumulate in the brain and were found activated in the peripheral lymphoid tissue, including the spleen and cervical draining lymph nodes. The P25 T cells present in the brain display extensive proliferation. Moreover, P25 T cells present in the peripheral lymphoid organs of Mtb infected mice show increased proliferation when compared to non-infected controls suggesting a systemic sampling of CNS Mtb antigen. We also show that at 7 days PI there is a significantly higher percentage of activated, INFγ producing, CD4+ T in the brain. Both INFγ and IL-17 producing T cell populations increase in size by day 21. This study will provide knowledge into the pathogenesis of CNS TB, and provide possible treatment strategies to lessen its destructive outcome.

IL-33 Exacerbates Periodontal Disease through Induction of RANKL

Cytokines mediate the balance between protective and destructive immunity in periodontitis. We sought to investigate the role of IL-33 in periodontitis. The expression of IL-33 in gingival tissue from healthy controls (n = 10) and patients with chronic periodontitis (n = 17) was investigated. Based on a murine model of periodontal disease, the function of IL-33 was determined first by administration of exogenous IL-33 and second by inhibition of IL-33 signaling using mice deficient in the IL-33 receptor ST2. Alveolar bone level, serum antibody, and lymphocyte responses were assessed in the murine model. Expression of IL-33 and ST2 was elevated in gingival tissues from patients with chronic periodontitis as compared with healthy tissues (P < 0.05). Similarly, Il33 expression was higher in periodontal tissues of Porphyromonas gingivalis–infected mice as compared with sham-infected controls (P < 0.05). IL-33 treatment of P. gingivalis–infected mice significantly exacerbated alveolar bone loss when compared with infection or IL-33 treatment alone (P < 0.001). Conversely, P. gingivalis infection–induced alveolar bone loss was attenuated in mice lacking ST2. The percentages of T and B lymphocytes expressing nuclear factor κB ligand (RANKL) in the gingival tissues and T lymphocytes expressing RANKL in the cervical draining lymph nodes were higher in IL-33-treated P. gingivalis–infected mice versus phosphate buffered saline–treated P. gingivalis–infected controls (all P < 0.001). Targeting the RANKL pathway by osteoprotegerin administration abrogated periodontal bone destruction in P. gingivalis–infected, IL-33-treated mice. These data demonstrate a previously unrecognized role for IL-33 in exacerbating bone loss in a RANKL-dependent manner in the context of bacterial infection and suggest that this pathway may be amenable to manipulation as a novel therapeutic target in periodontitis.

Periodontitis in the absence of B cells and specific anti‐bacterial antibody

Periodontitis (PD) results from complex interactions between a dysbiotic oral microbiota and a dysregulated host immune response. The inflammatory infiltrate in the gingiva of PD patients includes an abundance of B cells, implicating these cells in the immunopathology. We sought to investigate the role of B cells in PD using a murine model. Wild-type or B-cell-deficient (μMT) mice were orally infected with Porphyromonas gingivalis. One or six weeks following infection, lymphocyte populations in the gingiva and cervical draining lymph nodes (dLN) were analysed by flow cytometry; serum anti-P. gingivalis IgG antibody titers were measured by enzyme-linked immunosorbent assay, and alveolar bone loss was determined. In wild-type mice, the percentage of gingival B cells expressing receptor activator of nuclear factor-κB ligand (RANKL) was significantly increased 1 week post-infection (5.36% control versus 11% PD, P < 0.01). The percentage of Fas(+) GL7(+) germinal centre B cells in the dLN was significantly increased at both 1 week (2.03% control versus 6.90% PD, P < 0.01) and 6 weeks (4.45% control versus 8.77% PD, P < 0.05) post-infection. B-cell-deficient mice were protected from P. gingivalis-induced alveolar bone loss, with a lack of B-cell proliferation and lack of CD4(+) CD44(+) CD62L(-) T-cell generation in the dLN, and absence of serum anti-P. gingivalis antibodies. Our data imply a pathological role for B cells in PD, and that selective targeting of this immune axis may have a role in treating severe periodontal disease.

Intranasal DNA Vaccination Induces Potent Mucosal and Systemic Immune Responses and Cross-protective Immunity Against Influenza Viruses

The induction of potent virus-specific immune responses at mucosal surfaces where virus transmission occurs is a major challenge for vaccination strategies. In the case of influenza vaccination, this has been achieved only by intranasal delivery of live-attenuated vaccines that otherwise pose safety problems. Here, we demonstrate that potent mucosal and systemic immune responses, both cellular and humoral, are induced by intranasal immunization using formulated DNA. We show that formulation with the DNA carrier polyethylenimine (PEI) improved by a 1,000-fold the efficiency of gene transfer in the respiratory track following intranasal administration of luciferase-coding DNA. Using PEI formulation, intranasal vaccination with DNA-encoding hemagglutinin (HA) from influenza A H5N1 or (H1N1)2009 viruses induced high levels of HA-specific immunoglobulin A (IgA) antibodies that were detected in bronchoalveolar lavages (BALs) and the serum. No mucosal responses could be detected after parenteral or intranasal immunization with naked-DNA. Furthermore, intranasal DNA vaccination with HA from a given H5N1 virus elicited full protection against the parental strain and partial cross-protection against a distinct highly pathogenic H5N1 strain that could be improved by adding neuraminidase (NA) DNA plasmids. Our observations warrant further investigation of intranasal DNA as an effective vaccination route.