Cells Of Adaptive Immune Response Research Articles

Matrix Protein Gene Variants of Two Distinct Serotypes of rVSV Make Effective Viral Vectors for Prime-Boost Vaccination

Recently, we developed attenuated VSV vectors by introducing temperature sensitive (ts) mutations in the M gene in both VSV Indiana and New Jersey serotypes. The newly generated M gene mutants of rVSV vectors are rVSVInd(GML) with mutations of G21E, M51R, and L111F, rVSVNJ(GMM) with mutations of G22E, M48R, and M51R, and rVSVNJ(GMML) with mutations of G22E, M48R, M51R, and L110F. Our purpose was to examine the immunogenicity of the new ts M gene mutant of rVSVInd and attenuated M gene mutants ofrVSVNJas vaccine vectors against HIV-1 proteins. We generated attenuated rVSVs carrying HIV-1 gag, pol, and env genes. We immunized mice with various prime-boost vaccination regimens. CD8+ T cell responses and humoral immune responses in the vaccinated mice were examined. Priming with rVSVInd(GML)-gag, pol, or env gene of HIV-1 and boosting with rVSVNJ(GMM)-gag, pol, or env gene or rVSVNJ(GMML)-gag, pol, or env gene induced the strongest CD8+ cytotoxic T cell responses against the HIV-1 Gag, RT, and Env proteins. The same vaccination regimen also induced strong humoral immune responses against the HIV-1 Gag and Env proteins. We conclude that rVSVInd(GML) priming followed by rVSVNJ(GMM) boosting is the best vaccination regimen for optimum B cell and T cell adaptive immune responses against inserted foreign gene products when the newly attenuated rVSVInd and rVSVNJ are used.

Permissive/Protective Interplay of Microbiota with T Cell Adaptive Immune Response in Colon Cancer

Colon microbiota, as a complex and diverse population, has been shown to be either pro- or anti-tumorigenic, depending on itscontent. The composition of microbiota critically determines the differentiation, activation, and expansion of T cells by which prooranti-tumorigenic effects of microbes are frequently reported to be mediated. In this review study, we specified an imbalance inmicrobiota and T cells in particular regulatory T cells and Th17 cells in colon cancer. We also aimed to discuss evidence, suggestingthe contribution of microbiota to carcinogenesis or anti-carcinogenesis through influencing T cells.

The Immune Response in Measles: Virus Control, Clearance and Protective Immunity.

Measles is an acute systemic viral infection with immune system interactions that play essential roles in multiple stages of infection and disease. Measles virus (MeV) infection does not induce type 1 interferons, but leads to production of cytokines and chemokines associated with nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling and activation of the NACHT, LRR and PYD domains-containing protein (NLRP3) inflammasome. This restricted response allows extensive virus replication and spread during a clinically silent latent period of 10–14 days. The first appearance of the disease is a 2–3 day prodrome of fever, runny nose, cough, and conjunctivitis that is followed by a characteristic maculopapular rash that spreads from the face and trunk to the extremities. The rash is a manifestation of the MeV-specific type 1 CD4+ and CD8+ T cell adaptive immune response with lymphocyte infiltration into tissue sites of MeV replication and coincides with clearance of infectious virus. However, clearance of viral RNA from blood and tissues occurs over weeks to months after resolution of the rash and is associated with a period of immunosuppression. However, during viral RNA clearance, MeV-specific antibody also matures in type and avidity and T cell functions evolve from type 1 to type 2 and 17 responses that promote B cell development. Recovery is associated with sustained levels of neutralizing antibody and life-long protective immunity.

NADPH Oxidase-Derived Superoxide Provides a Third Signal for CD4 T Cell Effector Responses.

Originally recognized for their direct induced toxicity as a component of the innate immune response, reactive oxygen species (ROS) can profoundly modulate T cell adaptive immune responses. Efficient T cell activation requires: signal 1, consisting of an antigenic peptide-MHC complex binding with the TCR; signal 2, the interaction of costimulatory molecules on T cells and APCs; and signal 3, the generation of innate immune-derived ROS and proinflammatory cytokines. This third signal, in particular, has proven essential in generating productive and long-lasting immune responses. Our laboratory previously demonstrated profound Ag-specific hyporesponsiveness in the absence of NADPH oxidase-derived superoxide. To further examine the consequences of ROS deficiency on Ag-specific T cell responses, our laboratory generated the OT-II.Ncf1(m1J) mouse, possessing superoxide-deficient T cells recognizing the nominal Ag OVA323-339 In this study, we demonstrate that OT-II.Ncf1(m1J) CD4 T cells displayed a severe reduction in Th1 T cell responses, in addition to blunted IL-12R expression and severely attenuated proinflammatory chemokine ligands. Conversely, IFN-γ synthesis and IL-12R synthesis were rescued by the addition of exogenous superoxide via the paramagnetic superoxide donor potassium dioxide or superoxide-sufficient dendritic cells. Ultimately, these data highlight the importance of NADPH oxidase-derived ROS in providing a third signal for adaptive immune maturation by modulating the IL-12/IL-12R pathway and the novelty of the OT-II.Ncf1(m1J) mouse model to determine the role of redox-dependent signaling on effector responses. Thus, targeting ROS represents a promising therapeutic strategy in dampening Ag-specific T cell responses and T cell-mediated autoimmune diseases, such as type 1 diabetes.

Intestinal dysbiosis and innate immune responses in axial spondyloarthritis.

Inflammatory innate and adaptive immune cell responses to commensal bacteria underlie the pathogenesis of human chronic inflammatory diseases. Intestinal dysbiosis has been described in patients with spondyloarthritis (SpA) and seems to be correlated with histologic and immunologic alterations. Purpose of this review is to discuss the relationship occurring between intestinal dysbiosis and innate immune responses in patients with axial SpA. Intestinal dysbiosis and differential activation of intestinal immune responses in patients with SpA have been demonstrated. Furthermore, innate cells that appear to be involved in the pathogenesis of SpA may control intestinal homeostasis through induction of apoptotic cell death and deletion of activated commensal bacteria-specific T cells. Although the evidence shows that dysbiosis occurs in SpA, it is not clear the role of dysbiosis in regulating innate immune responses in SpA. Relationships between cause and effect remain to be answered. http://links.lww.com/COR/A34.

Emerging roles for antigen presentation in establishing host-microbiome symbiosis.

Trillions of beneficial bacteria inhabit the intestinal tract of healthy mammals from birth. Accordingly, mammalian hosts have evolved a series of complementary and redundant pathways to limit pathologic immune responses against these bacteria, while simultaneously protecting against enteric pathogen invasion. These pathways can be generically responsive to the presence of any commensal bacteria and innate in nature, as for IL-22-related pathways. Alternatively, specific bacterial antigens can drive a distinct set of adaptive immune cell responses, including IgA affinity maturation and secretion, and a recently described pathway of intestinal selection whereby MHCII(+) ILC3 deletes commensal bacteria-reactive CD4 T cells. These pathways can either promote or inhibit colonization by specific subsets of commensal bacteria, and cooperatively maintain intestinal homeostasis. In this review, we will highlight recent developments in understanding how these diverse pathways complement each other to cooperatively shape the symbiotic relationship between commensal bacteria and mammalian hosts.

Preclinical Evaluation of Flt3 Ligand to Improve T Cell Adaptive Immune Response During Sepsis

BackgroundSepsis is a leading cause of death in the United States. Severely burned patients are highly susceptible to opportunistic infections and sepsis, leading to multi‐organ failure and death. Studies by Hotchkiss and colleagues show that T cell dysfunction significantly contributes to impaired immune response during sepsis in non‐burn subjects. However, the changes in T cell adaptive immunity after burn wound sepsis have not been investigated in detail. We used a clinically relevant mouse model of burn wound infection to assess adaptive immune system function. Furthermore, we evaluated the cytokine, Fms‐like tyrosine kinase‐3 ligand (Flt3L), to improve T cell adaptive immunity post burn wound sepsis. Flt3L is known to stimulate the expansion of hemopoietic progenitors, particularly dendritic cells.Methods8–10 weeks old male BALB/c mice were subjected to 35% total body surface area full thickness burn and the wound was infected with Pseudomonas aeruginosa on day 4 post‐burn. Flow cytometry was used to characterize the cell counts and phenotype of CD4+ and CD8+ T and dendritic cells in spleen and wound draining lymph nodes. Flt3L (10 μg, i.p.) was administered daily for 4 days following burn injury.ResultsWound infection led to severe sepsis in the mice as evidenced by detection of bacteria in blood and lungs, and multi‐organ injury (increased markers of liver and kidney injury) on day 2 after infection and mortality rate of approximately 80% by seven days. We observed a significant decline (>80%) in blood absolute lymphocyte count and CD4+ and CD8+ T cell counts in the spleen and burn‐draining lymph nodes of wound infected mice as compared to non‐infected burn mice on day 2 post infection. Flt3L significantly attenuated the infection induced decline in CD4+ and CD8+ T cell counts in the spleen. Wound infection also caused a decline in the CD11c+ dendritic cell numbers in lymph nodes. Flt3L stimulated the expansion of both the splenic and lymph node dendritic cells and protected against the dendritic cell loss in the lymph nodes. Furthermore, wound infection caused an increase in the expression of Programmed Death‐Ligand 1 (PD‐L1) expression on dendritic cells, F4/80+ macrophages and Ly6C+ inflammatory monocytes in the spleen. PD‐L1 is known to interact with its binding partner PD‐1 on T cell surface leading to T cell inhibition. Flt3L attenuated the increase in PD‐L1 expression and thus indirectly improves T cell function.ConclusionBurn wound sepsis causes significant adaptive immune system dysfunction and Flt3L represents a novel therapeutic cytokine to protect T cell function during sepsis and merits further investigation.Support or Funding InformationSupported by NIH R01 GM66885

Timing of excision after a non-severe burn has a significant impact on the subsequent immune response in a murine model

BackgroundBurn excision has emerged as the dominant clinical paradigm in treatment of deep burns. Surgical intervention is common but the timing of wound excision is a balance between wound depth assessment, avoidance of infection and unnecessary intervention. However the physiological impact of timing of excision and consequences for the immune response are not well understood. MethodsMice were subject to full-thickness burn (<8% TBSA) followed by early (day 1) or late (day 8) surgical excision. Draining lymph nodes, wound tissue and sera were collected longitudinally at day 2 and day 6 after excision and analyzed for cytokine, dendritic cell and T cell profiles using FACS and multiplex ELISA assays. ResultsDelayed excision after injury initiated acute and severe inflammatory responses, with high levels of inflammatory cytokines, increased chemokine responses, and elevated Th2 promoting cytokines compared to early excision. Cellular inflammation in the wound was exacerbated with elevated neutrophils, eosinophil and monocytes. Wound cellular innate immune response decreased after late excision with a loss of inflammatory dendritic cells (DC), decreased NKT cells, and inhibition of NK cell activation. Systemically late excision increased trafficking conventional CD8α− DC to the lymph node, but there was no apparent DC activation. This was reflected in the induction of CD4T regulatory (Treg) cells and suppression of CD8T cell proliferation after late excision. No suppression was observed with early excision. ConclusionThis data suggests early excision of the wound, during the phase of immune down-regulation initiated by the burn, maintains an innate and adaptive immune cell response. In contrast, late wound excision induced a severe inflammatory response, with subsequent down-regulation of innate and adaptive immune cell responses. Therefore timing of excision is critical in affecting the immune response to burn.

Immunomodulatory Effects and Adaptive Immune Response to Daratumumab in Multiple Myeloma

Introduction: Daratumumab (DARA) is a novel human monoclonal antibody that targets CD38, a protein that is highly expressed on multiple myeloma (MM) cells. DARA acts through multiple immune effector-mediated mechanisms, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis. In two clinical studies (NCT00574288 [GEN501] and NCT01985126 [Sirius]) of DARA monotherapy in patients with relapsed and refractory MM, overall response rates were 36% and 29%, respectively. CD38 is highly expressed in myeloma cells but also expressed in lymphocytes and other immune cell populations. Therefore, the effects of DARA on immune cell populations and adaptive immune response pathways were investigated.Methods: The patient population investigated included treated subjects with MM that were relapsed after or were refractory to ≥2 prior therapies (GEN501) or had received ≥3 prior therapies, including a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD), or were refractory to both a PI and an IMiD (Sirius). Patients assessed in this analysis were treated with 16 mg/kg DARA. When both studies were combined, median age (range) was 64 (31-84) years and median time from diagnosis was 5.12 (0.77-23.77) years. Seventy-six percent of patients had received >3 prior therapies and 91% were refractory to their last treatment. Clinical response was evaluated using IMWG consensus recommendations. Peripheral blood (PB) samples and bone marrow (BM) biopsies/aspirates were taken at prespecified time points and immunophenotyped by flow cytometry to enumerate various T-cell sub-types. T-cell clonality was measured by TCR sequencing. Antiviral T-cell response and regulatory T-cell (Treg) activity were analysed by functional in vitro assays. T-cell subpopulation counts were modelled over time with linear mixed modelling. Two group comparisons were performed using non-parametric Wilcoxon rank sum tests.Results: Data from 148 patients receiving 16 mg/kg DARA in GEN501 (n = 42) and Sirius (n = 106) were analyzed for changes in immune response. In PB, robust mean increases in CD3+ (44%), CD4+ (32%) and CD8+ (62%) T-cell counts per 100 days were seen with DARA treatment. However, responding evaluable patients (n = 45) showed significantly greater increases from baseline than nonresponders (n = 93) in CD3+ (P = 0.00012), CD4+ (P = 0.00031), and CD8+ (P = 0.00018) T cells. In BM aspirates the number of CD3+, CD4+, and CD8+ T-cells increased during treatment compared to baseline (the median percent increases were 19.95%, 5.66%, and 26.99% [n = 58]). Additionally, CD8+: CD4+ T-cell ratios significantly increased compared to baseline in both PB (P = 0.00017), and BM (P = 0.00016). T cell clonality, assessed by TCR sequencing, increased after DARA treatment compared with pretreatment (P = 0.049), with greater sums of absolute expansion in the repertoire (P = 0.037), as well as greater maximum expansion of a single clone (P = 0.048) in responders compared to nonresponders. Increased antiviral T-cell responses were observed post-DARA treatment, particularly in responders. Interestingly, a novel subpopulation of regulatory T cells was identified that expressed high levels of CD38. These cells comprised ~10% of all Tregs and were depleted by one DARA infusion. In ex vivo analyses, CD38+ Tregs appeared to be highly immune suppressive compared to CD38-Tregs.Conclusions: Robust T cell increases, increased CD8+: CD4+ ratios, increased antiviral responses, and increased T cell clonality were all observed after DARA treatment in a heavily pretreated, relapsed, and refractory patient population not expected to have strong immune responses. Improved clinical responses were associated with changes in these parameters. In addition, a sub-population of regulatory T cells expressing high CD38 levels was determined to be extremely immune suppressive and sensitive to DARA treatment. These data suggest a previously unknown immune modulatory role of DARA that may contribute to its efficacy, and a potential role for CD38 immune targeted therapies. We postulate that there are several distinct and complementary mechanisms that contribute to DARA's efficacy including increased antigen presentation through phagocytosis, targeting of immune suppressive Tregs, and increased adaptive immune responses.JK and TC contributed equally to this work. DisclosuresCasneuf:Janssen: Employment. Verbist:Janssen: Employment. Bald:Janssen: Employment. Plesner:Genmab: Membership on an entity's Board of Directors or advisory committees; Roche and Novartis: Research Funding; Janssen and Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Liu:Janssen: Employment. van de Donk:Janssen Pharmaceuticals: Research Funding; Amgen: Research Funding; Celgene: Research Funding. Weiss:Janssen and Onclave: Research Funding; Janssen and Millennium: Consultancy. Ahmadi:Janssen: Employment. Lokhorst:Genmab: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria. Mutis:Janssen: Research Funding; Genmab: Research Funding.

Abstract IA11: T cell exclusion in pancreatic ductal adenocarcinoma: The FAP+ cancer-associated fibroblast and CXCL12

Abstract Patients with pancreatic adenocarcinoma (PDA), unlike those with melanoma, non-small cell lung cancer and renal cell carcinoma, did not have objective responses to the T cell checkpoint antagonists, antibodies to CTLA-4 and PD-L1, that promote the anti-cancer function of T cells. We sought the basis for this phenomenon by examining the interaction between the immune system and PDA in a genetically engineered mouse model of this disease, the KrasLSL.G12D/+; p53R172H/+; PdxCreTg/+ (or KPC) model. PDA in KPC mice, as in human patients, is resistant to therapy with anti-CTLA-4 and anti-PD-L1, respectively. The explanation for this resistance of the PDA to immune therapy was shown not to be the absence of an anti-cancer cell adaptive immune response, but to be the exclusion of T cells from the vicinity of cancer cells within tumors. The exclusion was caused by the chemokine, CXCL12, that was produced by the carcinoma-associated fibroblasts that were identified by their expression of the membrane protein, FAP. Remarkably, CXCL12 was physically associated with the cancer cells, which were not producing the chemokine. Inhibiting the interaction of this CXCL12 with its receptor, CXCR4, on T cells by administering the drug, AMD3100, caused the rapid accumulation of T cells amongst cancer cells, uncovered the efficacy of anti-PD-L1, and greatly diminished the numbers of cancer cells within the PDA tumors after 6 days of treatment. Thus, pharmacological targeting of the CXCR4-CXCL12 interaction overcomes the dominant immune suppressive mechanism of T cell exclusion in murine PDA. This finding may be relevant to therapy of human PDA, which also exhibits T cell exclusion, coating of cancer cells with CXCL12, and, as noted above, resistance to anti-PD-L1. Citation Format: Douglas T. Fearon, Arnaud Pommier, Michael Ludwig, David Tuveson. T cell exclusion in pancreatic ductal adenocarcinoma: The FAP+ cancer-associated fibroblast and CXCL12. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA11.

ID: 14: Immune regulation at barrier surfaces

Employing models of microbial colonization, pathogen infection, chronic inflammation and tissue repair, research in the Artis lab is examining how mammalian host genetics and signals derived from the environment and commensal microbial communities influence innate and adaptive immune cell responses. We are employing gnotobiotic mice to examine the influence of defined commensal microbial communities on intestinal and peripheral immune cell development, function and influence on tissue homeostasis. Our recent findings indicate that commensal microbes have a significant regulatory influence on lymphocyte, innate lymphoid cell and granulocyte function associated with susceptibility to multiple infectious, inflammatory and metabolic disease processes. It is hoped that the results of these studies will advance understanding of the pathophysiology of multiple chronic inflammatory diseases, including asthma, allergy, inflammatory bowel disease and obesity, and provide a framework to test new therapeutic pathways to prevent and treat these diseases.

The Obese Brain--Effects of Bariatric Surgery on Energy Balance Neurocircuitry.

Obesity is a highly prevalent disease in the world and with a major impact on global health. While genetic components are also involved in its pathogenesis, in recent years, it has shown a critical role of the innate and adaptive immune cell response in many tissues triggered by excess of nutrients such as lipids and glucose. Free fatty acids and other nutrient-related signals induce damage such as insulin resistance in the peripheral tissues but also in the brain. Specifically in the hypothalamus, these metabolic signals can trigger significant changes in the control of energy balance. Recent studies have shown that saturated fat disrupts melanocortin signaling of hypothalamic neuronal subgroups pivotal to energy control. Bariatric surgery is a treatment option for obesity when other tools have failed, because it is more effective than pharmacotherapy concerning of weight loss itself and in improvement of obesity-related comorbidities. Here, we review the mechanisms by which Roux-en Y gastric bypass (RYGB) can change peripheral signals that modulate melanocortin circuits involved in the regulation of energy balance.

Abstract 278: Antibody blockade of semaphorin 4D promotes infiltration of activated tumor infiltrating leukocytes and reverses tumor growth

Abstract Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer and expression correlates with invasive disease in several human tumors. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the setting of cancer, effects on cellular movement mediated by SEMA4D/PLXNB1 interactions have been described in relation to vascular stabilization and transactivation of ERBB2. Herein, we describe a novel function of SEMA4D in regulation of immune cell infiltration and activity in the tumor microenvironment (TME). PURPOSE: Characterize immune-related and anti-tumor activity mediated by antibody neutralization of SEMA4D, as a single agent or in combination with other immunomodulatory therapies. METHODS: Blockade of SEMA4D with monoclonal murine antibody was evaluated in subcutaneous Colon26 and orthotopic ERBB2+ breast carcinoma syngeneic models. Growth kinetics were assessed following selective in vivo immune cell depletions. Anti-tumor immune response was characterized by immunohistochemistry, FACS, and functional assays. RESULTS: Strong expression of SEMA4D at the invasive margins of actively growing tumors modulates the infiltration and spatial distribution of leukocytes in the TME. Antibody neutralization of SEMA4D disrupts this gradient and enhances recruitment of activated antigen presenting cells and lymphocytes into the TME, shifting the balance of cytokines toward increased Th1 and reduced immunosuppressive cytokines. This orchestrated change in the tumor architecture was associated with durable tumor rejection and immunologic memory in Colon26 and ERBB2+ mammary carcinoma models. Efficacy was dependent on both active adaptive and innate immune cell responses. The immunomodulatory activity of anti-SEMA4D antibody can be enhanced by combination with other immunotherapies, including immune checkpoint inhibition and chemotherapy. Strikingly, the combination of anti-SEMA4D antibody with antibody to CTLA-4 acts synergistically to promote complete tumor rejection and survival, with significant 58% increase in tumor regression and &amp;gt;295% increase in survival, as compared to monotherapy. CONCLUSION: Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the tumor and inhibit tumor progression. Humanized anti-SEMA4D therapeutic antibody, VX15/2503, has completed a Phase I prospective multiple ascending dose trial in 42 adult patients with advanced refractory solid tumors, in which the highest doses were well tolerated. Patients with the longest duration of treatment, 48-55 weeks, included colorectal, breast, and a papillary thyroid patient, who had a partial response by RECIST. A phase 1b/2a trial of combination therapy is planned. Citation Format: Elizabeth E. Evans, Holm Bussler, Sebold Torno, Crystal Mallow, Laurie A, Winters, Christine Reilly, Katya Klimatcheva, Janaki Veeraraghavan, Alan S. Jonason, Maria Scrivens, Renee Kirk, Sue Giralico, Alan Howell, John E. Leonard, Mark Paris, Terrence L. Fisher, Ernest S. Smith, Maurice Zauderer. Antibody blockade of semaphorin 4D promotes infiltration of activated tumor infiltrating leukocytes and reverses tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 278. doi:10.1158/1538-7445.AM2015-278

Shigella Manipulates Host Immune Responses by Delivering Effector Proteins with Specific Roles

The intestinal epithelium deploys multiple defense systems against microbial infection to sense bacterial components and danger alarms, as well as to induce intracellular signal transduction cascades that trigger both the innate and the adaptive immune systems, which are pivotal for bacterial elimination. However, many enteric bacterial pathogens, including Shigella, deliver a subset of virulence proteins (effectors) via the type III secretion system (T3SS) that enable bacterial evasion from host immune systems; consequently, these pathogens are able to efficiently colonize the intestinal epithelium. In this review, we present and select recently discovered examples of interactions between Shigella and host immune responses, with particular emphasis on strategies that bacteria use to manipulate inflammatory outputs of host-cell responses such as cell death, membrane trafficking, and innate and adaptive immune responses.

Late stages of hematopoiesis and B cell lymphopoiesis are regulated by α-synuclein, a key player in Parkinson's disease

α-Synuclein plays a crucial role in Parkinson's disease and dementias defined as synucleinopathies. α-Synuclein is expressed in hematopoietic and immune cells, but its functions in hematopoiesis and immune responses are unknown. We utilized α-synuclein−/− (KO) mice to investigate its role in hematopoiesis and B cell lymphopoiesis. We demonstrated hematologic abnormalities including mild anemia, smaller platelets, lymphopenia but relatively normal early hematopoiesis in KO mice compared to wild-type (WT) as measured in hematopoietic stem cells and progenitors of the different cell lineages. However, the absolute number of B220+IgM+ B cells in bone marrow was reduced by 4-fold in KO mice (WT: 104±23×105 vs. KO: 27±5×105). B cells were also reduced in KO spleens associated with effacement of splenic and lymph node architecture. KO mice showed reduced total serum IgG but no abnormality in serum IgM was noted. When KO mice were challenged with a T cell-dependent antigen, production of antigen specific IgG1 and IgG2b was abolished, but antigen specific IgM was not different from WT mice. Our study shows hematologic abnormalities including anemia and smaller platelets, reduced B cell lymphopoiesis and defects in IgG production in the absence of α-synuclein. This is the first report to show an important role of α-synuclein late in hematopoiesis, B cell lymphopoiesis and adaptive immune response.

363 Human Hematopoietic Stem Cells With a Defined Immunodeficiency and Enteropathy Transfer Clinical Phenotype to a Novel Humanized Mouse Strain

Investigation into the pathogenesis of IBD has been limited by the absence of humanized murine IBD models. The development of immunodeficient mouse strains has facilitated the study of human immune reconstitution in murine hosts. However, adaptive immune responses and antibody class switching are generally weak in models that lack autologous fetal thymic grafts, possibly due to human CD4+ T cell selection in the mouse thymus occurring on murine major histocompatibility complex II (MHCII). To address this, we modified the widely utilized NOD.Prkdc.Il2rγ (NSG) immunocompromised strain to be deficient for murine MHCII (NSGAbo) and instead expressed human leukocyte antigen-DR1 under the control of the murine MHCII promoter (NSGAboDR1). We tested whether CD4+ T cell development and adaptive immune responses are improved following reconstitution using human HLA-matched CD34+ hematopoietic stem cells (HSCs) and whether immune reconstitution using HSCs from a patient with IPEX (immunodeficiency, polyendocrinopathy, enteropathy, X-linked), a well-defined immunodeficiency, that is caused by a mutation in the transcription factor FOXP3, would transfer the clinical phenotype to NSGAboDR1 mice. Results: At 20 weeks following HSC injection into radiation-conditioned neonates, we found that the development of human T cells and dendritic cells were increased in NSGAboDR1 mice and that these mice displayed delayed-type hypersensitivity response to a secondary recall antigen challenge. There was an increase in T cell clonotype diversity as determined by CDR3 sequencing of the TCRβ gene in CD4+ T cells in NSGAboDR1 mice compared to NSG mice. In addition, NSGAboDR1 mice had an increase in mature B cells and antibody class switching compared to NSG and NSGAbo mice. We then reconstituted NSGAboDR1 mice using HSCs from a patient with IPEX that is associated with functional defects in Treg and assessed if the clinical phenotype would be transferred to NSGAboDR1 mice. All mice reconstituted with IPEX CD34+ HSCs were deceased by 18 weeks while all control mice receiving healthy bone marrow CD34+ cells remained viable. IPEX mice developed lymphocytic infiltrate in lung, portal tract and bile ducts of the liver, and slight accumulation of lymphocytes in the small intestine similar to scurfy mice, which also harbor a mutation in Foxp3. CD4+ T cell in IPEX mice had an expanded effector memory T cell population and concomitant decrease in Naive CD4+ T cells. Conclusions: We demonstrate that human HSCs from a patient with a defined genetic immunodeficiency and enteropathy can transfer the disease phenoytpe in a novel humanized mouse strain. This strain does not require grafts of human fetal tissue nor is it restricted to autologous fetal HSCs allowing for reconstitution using human HSCs with defined genetics to study human immune cells and IBD pathogenesis in vivo.

TRYCAT Pathways Link Peripheral Inflammation, Nicotine, Somatization and Depression in the Etiology and Course of Parkinson’s Disease

Increased depression, somatization, gut inflammation and wider peripheral inflammation are all associated with the early stages of Parkinson's disease (PD). Classically such concurrent conditions have been viewed as "comorbidities", driven by high levels of stress in a still poorly understood and treated disorder. Here we review the data on how oxidative and nitrosative stress in association with immuno-inflammatory responses, drives alteration in tryptophan catabolites, including kynurenine, kynurenic acid and quinolinic acid that drive not only the 'comorbidities" of PD but also important processes in the etiology and course of PD per se. The induction of indoleamine 2,3-dioxygenase, leading to the driving of tryptophan into neuroregulatory tryptophan catabolite products and away from serotonin and melatonin production, has significant implications for understanding the role of nicotine, melatonin, and caffeine in regulating PD susceptibility. Tryptophan catabolite pathway activation will also regulate blood-brain barrier permeability, glia and mast cell reactivity as well as wider innate and adaptive immune cell responses, all relevant to the course of PD. As such, the "comorbidities" of PD such as depression, somatization and peripheral inflammatory disorders can all be conceptualized as being an intricate part of the biological underpinnings of both the etiology and course of PD. As a consequence, the data reviewed here has treatment implications; relevant to both the course of PD and in the management of L-DOPA induced dyskinesias.

Deletion of the Vaccinia Virus N2L Gene Encoding an Inhibitor of IRF3 Improves the Immunogenicity of Modified Vaccinia Virus Ankara Expressing HIV-1 Antigens

A modified vaccinia virus Ankara poxvirus vector expressing the HIV-1 Env, Gag, Pol, and Nef antigens from clade B (MVA-B) is currently being tested in clinical trials. To improve its immunogenicity, we have generated and characterized the immune profile of MVA-B containing a deletion of the vaccinia viral gene N2L, which codes for an inhibitor of IRF3 (MVA-B ΔN2L). Deletion of N2L had no effect on virus growth kinetics or on the expression of HIV-1 antigens; hence, the N2 protein is not essential for MVA replication. The innate immune responses triggered by MVA-B ΔN2L revealed an increase in beta interferon, proinflammatory cytokines, and chemokines. Mouse prime-boost protocols showed that MVA-B ΔN2L improves the magnitude and polyfunctionality of HIV-1-specific CD4(+) and CD8(+) T cell adaptive and memory immune responses, with most of the HIV-1 responses mediated by CD8(+) T cells. In the memory phase, HIV-1-specific CD8(+) T cells with an effector phenotype were predominant and in a higher percentage with MVA-B ΔN2L than with MVA-B. In both immunization groups, CD4(+) and CD8(+) T cell responses were directed mainly against Env. Furthermore, MVA-B ΔN2L in the memory phase enhanced levels of antibody against Env. For the vector immune responses, MVA-B ΔN2L induced a greater magnitude and polyfunctionality of VACV-specific CD8(+) T memory cells than MVA-B, with an effector phenotype. These results revealed the immunomodulatory role of N2L, whose deletion enhanced the innate immunity and improved the magnitude and quality of HIV-1-specific T cell adaptive and memory immune responses. These findings are relevant for the optimization of poxvirus vectors as vaccines. On the basis of the limited efficacy of the RV144 phase III clinical trial, new optimized poxvirus vectors as vaccines against HIV/AIDS are needed. Here we have generated and characterized a new HIV/AIDS vaccine candidate on the basis of the poxvirus MVA vector expressing HIV-1 Env, Gag, Pol, and Nef antigens (MVA-B) and containing a deletion in the vaccinia virus N2L gene. Our findings revealed the immunomodulatory role of N2L and proved that its deletion from the MVA-B vector triggered an enhanced innate immune response in human macrophages and monocyte-derived dendritic cells. Furthermore, in immunized mice, MVA-B ΔN2L induced improvements in the magnitude and quality of adaptive and memory HIV-1-specific CD4(+) and CD8(+) T cell immune responses, together with an increase in the memory phase of levels of antibody against Env. Thus, the selective deletion of the N2L viral immunomodulatory gene is important for the optimization of MVA vectors as HIV-1 vaccines.

Autophagy and Immune Response in Kidneys

Autophagy is a ubiquitous intracellular catabolic process that contributes to homeostatic maintenance and regulates the balance between health and disease. Emerging evidence from both the immunology and renal literature suggests that important relationships exist between the immune system and renal autophagy that may have significant implications for our understanding of the pathogenesis of kidney diseases. Autophagic flux in renal parenchymal cells can protect against acute and chronic kidney injury and can be stimulated via activation of innate immune receptors, cytokine secretion, and/or direct contact by immune cells. Conversely, modulation of autophagy in renal cells may influence both adaptive and innate immune cell responses. Autophagy can promote the ability of renal epithelial cells, which can act as antigen-presenting cells, to process and present self-antigen to immune cells. In addition, autophagic control of inflammasome function can modify the intrarenal inflammatory milieu, thereby preventing immune cell infiltration. Because autophagy and immune responses may promote or protect against kidney injury, further research is needed to better understand how interactions between renal parenchymal cells and the immune system are altered by autophagy. Novel agents are being developed that promote or inhibit various steps of the autophagy pathway, and it is likely that whether such agents are beneficial or harmful in the context of kidney disease will depend, at least in part, on whether and how they influence the relationship between autophagy and the immune response in the kidney.

TLR4 Signaling Shapes B Cell Dynamics via MyD88-Dependent Pathways and Rac GTPases

B cells use a plethora of TLR to recognize pathogen-derived ligands. These innate signals have an important function in the B cell adaptive immune response and modify their trafficking and tissue location. The direct role of TLR signaling on B cell dynamics nonetheless remains almost entirely unknown. In this study, we used a state-of-the-art two-dimensional model combined with real-time microscopy to study the effect of TLR4 stimulation on mouse B cell motility in response to chemokines. We show that a minimum stimulation period is necessary for TLR4 modification of B cell behavior. TLR4 stimulation increased B cell polarization, migration, and directionality; these increases were dependent on the MyD88 signaling pathway and did not require ERK or p38 MAPK activity downstream of TLR4. In addition, TLR4 stimulation enhanced Rac GTPase activity and promoted sustained Rac activation in response to chemokines. These results increase our understanding of the regulation of B cell dynamics by innate signals and the underlying molecular mechanisms.