Adaptive Immune System Research Articles

T Cell Dysfunction in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune inflammatory disease that can affect many organs in the body with very diverse clinical appearances. The prevalence of SLE in each country varies. The Lupus Foundation of America estimates that around 1.5 million cases occur in America and at least 5 million cases occur worldwide. The pathophysiology of SLE is very complex. The involvement of innate and adaptive immunity in the initiation and pathophysiology of SLE disease shows that there are interactions between leukocytes, cytokines, chemokines and tissue cells. T cells are the main component of the adaptive immune system which can kill infected host cells, activate other immune cells, produce cytokines and regulate immune responses. T cell dysfunction in SLE includes triggering inflammation through the secretion of pro-inflammatory cytokines, helping B cells produce autoantibodies and the accumulation of autoreactive T cells. Aberrations in T cells could be a therapeutic target for development and a potential SLE therapy.

Photodynamic Therapy and Adaptive Immunity Induced by Reactive Oxygen Species: Recent Reports.

Cancer is one of the most significant causes of death worldwide. Despite the rapid development of modern forms of therapy, results are still unsatisfactory. The prognosis is further worsened by the ability of cancer cells to metastasize. Thus, more effective forms of therapy, such as photodynamic therapy, are constantly being developed. The photodynamic therapeutic regimen involves administering a photosensitizer that selectively accumulates in tumor cells or is present in tumor vasculature prior to irradiation with light at a wavelength corresponding to the photosensitizer absorbance, leading to the generation of reactive oxygen species. Reactive oxygen species are responsible for the direct and indirect destruction of cancer cells. Photodynamically induced local inflammation has been shown to have the ability to activate an adaptive immune system response resulting in the destruction of tumor lesions and the creation of an immune memory. This paper focuses on presenting the latest scientific reports on the specific immune response activated by photodynamic therapy. We present newly discovered mechanisms for the induction of the adaptive response by analyzing its various stages, and the possible difficulties in generating it. We also present the results of research over the past 10 years that have focused on improving the immunological efficacy of photodynamic therapy for improved cancer therapy.

BCG vaccination and multiple sclerosis risk: A Norwegian cohort study

Introduction: Bacillus Calmette–Guérin (BCG) vaccination induces long-lasting effects on the adaptive and innate immune systems and prevents development of experimental autoimmune encephalomyelitis and possibly also inflammatory disease activity in multiple sclerosis (MS). Objective: The objective is to examine if BCG given in early adulthood decreases MS risk. Methods: From 791,369 (52% females) Norwegians participating in a national tuberculosis screening program from 1963 to 1975, we collected information on BCG vaccination and tuberculosis disease status. Later, MS disease was ascertained through both the Norwegian MS Registry and Biobank and the Norwegian Death Registry. We used logistic regression models to assess the relationship between BCG vaccination and MS risk. Results: In those BCG vaccinated, mean age at vaccination was 15.6 (standard deviation (SD) = 5.5) years. A total of 2862 (65% females) MS cases were retrieved. Overall, we found no association between MS risk and BCG vaccination. Compared to non-BCG-vaccinated individuals with no signs of tuberculosis infection, odds ratio (OR) for MS was 1.00 (95% confidence interval (CI) = 0.80–1.25) in the BCG-vaccinated group. In those not BCG vaccinated because of latent tuberculosis infection, the corresponding OR was 0.86 (95% CI = 0.66–1.13). Conclusion: We found no evidence of BCG vaccination or latent tuberculosis infection in young adulthood being linked to MS risk.

ITAM-based receptors in natural killer cells.

The ability of cells of the immune system to acquire features such as increased longevity and enhanced secondary responses was long thought to be restricted to cells of the adaptive immune system. Natural killer (NK) cells have challenged this notion by demonstrating that they can also gain adaptive features. This has been observed in both humans and mice during infection with cytomegalovirus (CMV). The generation of adaptive NK cells requires antigen-specific recognition of virally infected cells through stimulatory NK receptors. These receptors lack the ability to signal on their own and rather rely on adaptor molecules that contain ITAMs for driving signals. Here, we highlight our understanding of how these receptors influence the production of adaptive NK cells and propose areas in the field that merit further investigation.

Complex changes in serum protein levels in COVID-19 convalescents

The COVID-19 pandemic, triggered by severe acute respiratory syndrome coronavirus 2, has affected millions of people worldwide. Much research has been dedicated to our understanding of COVID-19 disease heterogeneity and severity, but less is known about recovery associated changes. To address this gap in knowledge, we quantified the proteome from serum samples from 29 COVID-19 convalescents and 29 age-, race-, and sex-matched healthy controls. Samples were acquired within the first months of the pandemic. Many proteins from pathways known to change during acute COVID-19 illness, such as from the complement cascade, coagulation system, inflammation and adaptive immune system, had returned to levels seen in healthy controls. In comparison, we identified 22 and 15 proteins with significantly elevated and lowered levels, respectively, amongst COVID-19 convalescents compared to healthy controls. Some of the changes were similar to those observed for the acute phase of the disease, i.e. elevated levels of proteins from hemolysis, the adaptive immune systems, and inflammation. In contrast, some alterations opposed those in the acute phase, e.g. elevated levels of CETP and APOA1 which function in lipid/cholesterol metabolism, and decreased levels of proteins from the complement cascade (e.g. C1R, C1S, and VWF), the coagulation system (e.g. THBS1 and VWF), and the regulation of the actin cytoskeleton (e.g. PFN1 and CFL1) amongst COVID-19 convalescents. We speculate that some of these shifts might originate from a transient decrease in platelet counts upon recovery from the disease. Finally, we observed race-specific changes, e.g. with respect to immunoglobulins and proteins related to cholesterol metabolism.

Trypanosoma brucei Invariant Surface Glycoprotein 75 Is an Immunoglobulin Fc Receptor Inhibiting Complement Activation and Antibody-Mediated Cellular Phagocytosis.

Various subspecies of the unicellular parasite Trypanosoma brucei cause sleeping sickness, a neglected tropical disease affecting millions of individuals and domestic animals. Immune evasion mechanisms play a pivotal role in parasite survival within the host and enable the parasite to establish a chronic infection. In particular, the rapid switching of variant surface glycoproteins covering a large proportion of the parasite's surface enables the parasite to avoid clearance by the adaptive immune system of the host. In this article, we present the crystal structure and discover an immune-evasive function of the extracellular region of the T. brucei invariant surface gp75 (ISG75). Structural analysis determined that the ISG75 ectodomain is organized as a globular head domain and a long slender coiled-coil domain. Subsequent ligand screening and binding analysis determined that the head domain of ISG75 confers interaction with the Fc region of all subclasses of human IgG. Importantly, the ISG75-IgG interaction strongly inhibits both activation of the classical complement pathway and Ab-dependent cellular phagocytosis by competing with C1q and host cell FcγR CD32. Our data reveal a novel immune evasion mechanism of T. brucei, with ISG75 able to inactivate the activities of Abs recognizing the parasite surface proteins.

CD200 genotype is associated with clinical outcome of patients with multiple myeloma.

Immune dysfunction in patients with MM affects both the innate and adaptive immune system. Molecules involved in the immune response pathways are essential to determine the ability of cancer cells to escape from the immune system surveillance. However, few data are available concerning the role of immune checkpoint molecules in predicting the myeloma control and immunological scape as mechanism of disease progression. We retrospectively analyzed the clinical impact of the CD200 genotype (rs1131199 and rs2272022) in 291 patients with newly diagnosed MM. Patients with a CD200 rs1131199 GG genotype showed a median overall survival (OS) significantly lower than those with CC+CG genotype (67.8 months versus 94.4 months respectively; p: 0.022) maintaining significance in the multivariate analysis. This effect was specially detected in patients not receiving an autologous stem cell transplant (auto-SCT) (p < 0.001). In these patients the rs1131199 GG genotype negatively influenced in the mortality not related with the progression of MM (p: 0.02) mainly due to infections events.

Paraprobiotic derived from Bacillus velezensis GV1 improves immune response and gut microbiota composition in cyclophosphamide-treated immunosuppressed mice.

Paraprobiotics that benefit human health have the capacity to modulate innate and adaptive immune systems. In this study, we prepared the paraprobiotic from Bacillus velezensis GV1 using the heat-killing method and investigated its effects on immunity and gut microbiota in vitro and in vivo. The morphology of inactivated strain GV1 was observed using scanning electron microscopy. Treatment with GV1 promoted nitric oxide production and augmented cytokine (IL-6, IL-1β, and TNF-α) expression and secretion in RAW 264.7 macrophages. Moreover, the strain GV1 could alleviate cyclophosphamide monohydrate (CTX)-induced immunosuppression by reversing spleen damage and restoring the immune organ index, as well as by increasing the expression of immune-related cytokines (TNF-α, IL-1β, IFN-γ, and IL-2) in the spleen and thymus, respectively. Furthermore, GV1 treatment dramatically healed the CTX-damaged colon and regulated gut microbiota by increasing the relative abundance of beneficial bacterial families (Lactobacillaceae, Akkermansiaceae, and Coriobacteriaceae) and decreasing that of harmful bacterial families (Desulfovibrionaceae, Erysipelotrichaceae, and Staphylococcaceae). Thus, the heat-killed GV1 can be considered a potential immunoregulatory agent for use as a functional food or immune-enhancing medicine.

Cbp1 and Cren7 form chromatin-like structures that ensure efficient transcription of long CRISPR arrays

CRISPR arrays form the physical memory of CRISPR adaptive immune systems by incorporating foreign DNA as spacers that are often AT-rich and derived from viruses. As promoter elements such as the TATA-box are AT-rich, CRISPR arrays are prone to harbouring cryptic promoters. Sulfolobales harbour extremely long CRISPR arrays spanning several kilobases, a feature that is accompanied by the CRISPR-specific transcription factor Cbp1. Aberrant Cbp1 expression modulates CRISPR array transcription, but the molecular mechanisms underlying this regulation are unknown. Here, we characterise the genome-wide Cbp1 binding at nucleotide resolution and characterise the binding motifs on distinct CRISPR arrays, as well as on unexpected non-canonical binding sites associated with transposons. Cbp1 recruits Cren7 forming together ‘chimeric’ chromatin-like structures at CRISPR arrays. We dissect Cbp1 function in vitro and in vivo and show that the third helix-turn-helix domain is responsible for Cren7 recruitment, and that Cbp1-Cren7 chromatinization plays a dual role in the transcription of CRISPR arrays. It suppresses spurious transcription from cryptic promoters within CRISPR arrays but enhances CRISPR RNA transcription directed from their cognate promoters in their leader region. Our results show that Cbp1-Cren7 chromatinization drives the productive expression of long CRISPR arrays.

The Role of Innate and Adaptive Immune System in the Pathogenesis of Schizophrenia.

Schizophrenia is one of the most severely debilitating mental disorders that affects 1.1% of the world's population. The exact cause of the disease is not known, but genetics, environmental factors (such as infectious agents, season and region of birth, exposure to viruses, low birth weight, advanced paternal age, and tobacco), and immune system dysfunction can all contribute to the development of schizophrenia. Recently, the role of the immune system in schizophrenia has received much attention. Both acquired and innate immune systems are involved in the pathogenesis of schizophrenia and facilitate the disease's progression. Almost all cells of the immune system including microglia, B cells, and T cells play an important role in the blood-brain barrier damage, inflammation, and in the progression of this disease. In schizophrenia, the integrity of the blood-brain barrier is reduced and then the immune cells are recruited into the endothelium following an increase in the expression of cell adhesion molecules. The entry of immune cells and cytokines leads to inflammation and antibody production in the brain. Accordingly, the results of this study strengthen the hypothesis that the innate and acquired immune systems are involved in the pathogenesis of schizophrenia.

PD-L1 Expression Is Increased in LPS-Induced Acute Respiratory Distress Syndrome by PI3K-AKT-Egr-1/C/EBPδ Signaling Pathway.

The role of programmed death ligand 1 (PD-L1) has been extensively investigated in adaptive immune system. However, increasing data show that innate immune responses are also affected by the immune checkpoint molecule. It has been demonstrated that regulation of PD-L1 signaling in macrophages may be a potential therapeutic method for acute respiratory distress syndrome (ARDS). However, the PD-L1 expression pattern in local macrophages and whole lung tissues remains mysterious, hindering optimization of the potential treatment program. Therefore, we aim to determine the PD-L1 expression pattern during ARDS. Our findings show that PD-L1 levels are markedly increased in lipopolysaccharide (LPS)-stimulated lung tissues, which might be attributable to an increase in the gene expression by immune cells, including macrophages and neutrophils. In vitro experiments are performed to explore the mechanism involved in LPS-induced PD-L1 production. We find that PD-L1 generation is controlled by transcription factors early growth response 1 (Egr-1) and CCAAT/enhancer binding protein delta (C/EBPδ). Strikingly, PD-L1 production is enhanced by phosphoinositide-3 kinase (PI3K)-protein kinase B (AKT) signaling pathway via up-regulation of Egr-1 and C/EBPδ expressions. Additionally, we observe that expressions of Egr-1 and C/EBPδ mutually reinforce each other. Moreover, we observe that PD-L1 is protective for ARDS due to its regulatory role in macrophage-associated inflammatory response. In summary, during LPS-induced ARDS, PD-L1 expression, which is beneficial for the disease, is increased via the PI3K-AKT1-Egr-1/C/EBPδ signaling pathway, providing theoretical basis for application of methods controlling PD-L1 signaling in macrophages for ARDS treatment in clinic.

Endotoxin Inflammatory Action on Cells by Dysregulated-Immunological-Barrier-Linked ROS-Apoptosis Mechanisms in Gut-Liver Axis.

Our study highlighted the immune changes by pro-inflammatory biomarkers in the gut-liver-axis-linked ROS-cell death mechanisms in chronic and acute inflammations when gut cells are exposed to endotoxins in patients with hepatic cirrhosis or steatosis. In duodenal tissue samples, gut immune barrier dysfunction was analyzed by pro-inflammatory biomarker expressions, oxidative stress, and cell death by flow cytometry methods. A significant innate and adaptative immune system reaction was observed as result of persistent endotoxin action in gut cells in chronic inflammation tissue samples recovered from hepatic cirrhosis with the A-B child stage. Instead, in patients with C child stage of HC, the endotoxin tolerance was installed in cells, characterized by T lymphocyte silent activation and increased Th1 cytokines expression. Interesting mechanisms of ROS-cell death were observed in chronic and acute inflammation samples when gut cells were exposed to endotoxins and immune changes in the gut-liver axis. Late apoptosis represents the chronic response to injury induction by the gut immune barrier dysfunction, oxidative stress, and liver-dysregulated barrier. Meanwhile, necrosis represents an acute and severe reply to endotoxin action on gut cells when the immune system reacts to pro-inflammatory Th1 and Th2 cytokines releasing, offering protection against PAMPs/DAMPs by monocytes and T lymphocyte activation. Flow cytometric analysis of pro-inflammatory biomarkers linked to oxidative stress-cell death mechanisms shown in our study recommends laboratory techniques in diagnostic fields.

Advances in application of CRISPR-Cas13a system.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins serve as an adaptive immune system that safeguards prokaryotes and some of the viruses that infect prokaryotes from foreign nucleic acids (such as viruses and plasmids). The genomes of the majority of archaea and about half of all bacteria contain various CRISPR-Cas systems. CRISPR-Cas systems depend on CRISPR RNAs (crRNAs). They act as a navigation system to specifically cut and destroy foreign nucleic acids by recognizing invading foreign nucleic acids and binding Cas proteins. In this review, we provide a brief overview of the evolution and classification of the CRISPR-Cas system, focusing on the functions and applications of the CRISPR-Cas13a system. We describe the CRISPR-Cas13a system and discuss its RNA-directed ribonuclease function. Meanwhile, we briefly introduce the mechanism of action of the CRISPR-Cas13a system and summarize the applications of the CRISPR-Cas13a system in pathogen detection, eukaryotes, agriculture, biosensors, and human gene therapy. We are right understanding of CRISPR-Cas13a has been broadened, and the CRISPR-Cas13a system will be useful for developing new RNA targeting tools. Therefore, understanding the basic details of the structure, function, and biological characterization of CRISPR-Cas13a effector proteins is critical for optimizing RNA targeting tools.

The Evolving Role of Dendritic Cells in Atherosclerosis.

Atherosclerosis, a major contributor to cardiovascular morbidity and mortality, is characterized by chronic inflammation of the arterial wall. This inflammatory process is initiated and maintained by both innate and adaptive immunity. Dendritic cells (DCs), which are antigen-presenting cells, play a crucial role in the development of atherosclerosis and consist of various subtypes with distinct functional abilities. Following the recognition and binding of antigens, DCs become potent activators of cellular responses, bridging the innate and adaptive immune systems. The modulation of specific DC subpopulations can have either pro-atherogenic or atheroprotective effects, highlighting the dual pro-inflammatory or tolerogenic roles of DCs. In this work, we provide a comprehensive overview of the evolving roles of DCs and their subtypes in the promotion or limitation of atherosclerosis development. Additionally, we explore antigen pulsing and pharmacological approaches to modulate the function of DCs in the context of atherosclerosis.

Clinically approved immunomodulators ameliorate behavioral changes in a mouse model of hereditary spastic paraplegia type 11.

We have previously demonstrated that neuroinflammation by the adaptive immune system acts as a robust and targetable disease amplifier in a mouse model of Spastic Paraplegia, type 11 (SPG11), a complicated form of Hereditary Spastic Paraplegia (HSP). While we identified an impact of neuroinflammation on distinct neuropathological changes and gait performance, neuropsychological features, typical and clinically highly relevant symptoms of complicated HSPs, were not addressed. Here we show that the corresponding SPG11 mouse model shows distinct behavioral abnormalities, particularly related to social behavior thus partially reflecting the neuropsychological changes in patients. We provide evidence that some behavioral abnormalities can be mitigated by genetic inactivation of the adaptive immune system. Translating this into a clinically applicable approach, we show that treatment with the established immunomodulators fingolimod or teriflunomide significantly attenuates distinct behavioral abnormalities, with the most striking effect on social behavior. This study links neuroinflammation to behavioral abnormalities in a mouse model of SPG11 and may thus pave the way for using immunomodulators as a treatment approach for SPG11 and possibly other complicated forms of HSP with neuropsychological involvement.

Time-resolved role of P2X4 and P2X7 during CD8+ T cell activation.

CD8+ T cells are a crucial part of the adaptive immune system, responsible for combating intracellular pathogens and tumor cells. The initial activation of T cells involves the formation of highly dynamic Ca2+ microdomains. Recently, purinergic signaling was shown to be involved in the formation of the initial Ca2+ microdomains in CD4+ T cells. In this study, the role of purinergic cation channels, particularly P2X4 and P2X7, in CD8+ T cell signaling from initial events to downstream responses was investigated, focusing on various aspects of T cell activation, including Ca2+ microdomains, global Ca2+ responses, NFAT-1 translocation, cytokine expression, and proliferation. While Ca2+ microdomain formation was significantly reduced in the first milliseconds to seconds in CD8+ T cells lacking P2X4 and P2X7 channels, global Ca2+ responses over minutes were comparable between wild-type (WT) and knockout cells. However, the onset velocity was reduced in P2X4-deficient cells, and P2X4, as well as P2X7-deficient cells, exhibited a delayed response to reach a certain level of free cytosolic Ca2+ concentration ([Ca2+]i). NFAT-1 translocation, a crucial transcription factor in T cell activation, was also impaired in CD8+ T cells lacking P2X4 and P2X7. In addition, the expression of IFN-γ, a major pro-inflammatory cytokine produced by activated CD8+ T cells, and Nur77, a negative regulator of T cell activation, was significantly reduced 18h post-stimulation in the knockout cells. In line, the proliferation of T cells after 3 days was also impaired in the absence of P2X4 and P2X7 channels. In summary, the study demonstrates that purinergic signaling through P2X4 and P2X7 enhances initial Ca2+ events during CD8+ T cell activation and plays a crucial role in regulating downstream responses, including NFAT-1 translocation, cytokine expression, and proliferation on multiple timescales. These findings suggest that targeting purinergic signaling pathways may offer potential therapeutic interventions.

Autonomous transposons tune their sequences to ensure somatic suppression

Transposable elements (TEs) are a major constituent of human genes, occupying approximately half of the intronic space. During pre-messenger RNA synthesis, intronic TEs are transcribed along with their host genes but rarely contribute to the final mRNA product because they are spliced out together with the intron and rapidly degraded. Paradoxically, TEs are an abundant source of RNA-processing signals through which they can create new introns1, and also functional2 or non-functional chimeric transcripts3. The rarity of these events implies the existence of a resilient splicing code that is able to suppress TE exonization without compromising host pre-mRNA processing. Here we show that SAFB proteins protect genome integrity by preventing retrotransposition of L1 elements while maintaining splicing integrity, via prevention of the exonization of previously integrated TEs. This unique dual role is possible because of L1’s conserved adenosine-rich coding sequences that are bound by SAFB proteins. The suppressive activity of SAFB extends to tissue-specific, giant protein-coding cassette exons, nested genes and Tigger DNA transposons. Moreover, SAFB also suppresses LTR/ERV elements in species in which they are still active, such as mice and flies. A significant subset of splicing events suppressed by SAFB in somatic cells are activated in the testis, coinciding with low SAFB expression in postmeiotic spermatids. Reminiscent of the division of labour between innate and adaptive immune systems that fight external pathogens, our results uncover SAFB proteins as an RNA-based, pattern-guided, non-adaptive defence system against TEs in the soma, complementing the RNA-based, adaptive Piwi-interacting RNA pathway of the germline.

A193 DECIPHERING THE INFLUENCE OF CROHN’S DISEASE-ASSOCIATED GENETIC RISK FACTOR, NOD2 IN INTESTINAL FIBRO-STENOSIS

Abstract Background Intestinal fibrosis (IF) is a common debilitating complication associated with Crohn’s disease (CD), an idiopathic inflammatory bowel disease (IBD). IF results in severe strictures that affect the entire gastrointestinal tract, often requiring surgery due to limited or no effective treatment. In general, IF is a consequence of chronic inflammation, excessive extracellular matrix (ECM) deposition, aberrant cellular functions and tissue remodeling involving the innate and adaptive immune system. At the cellular level, fibrosis has been proposed to involve the crosstalk between epithelial cells, mesenchymal stromal cells and immune cells. However, the cascade of events that establish IF is poorly understood as it comprises a dynamic interplay between host genetics, immunity, gut microbiome and aberrant environment insults. Notably, polymorphisms in nucleotide-binding oligomerization domain-containing protein 2 (NOD2; a bonafide cytosolic innate immune receptor), strong genetic risk factors for CD, also increase the incidence of IF. In this context, how NOD2 polymorphisms govern intestinal fibrostenosis in CD remains to be investigated. Aims To investigate how CD-associated risk gene, NOD2 contributes to intestinal fibrostenotic disease Methods We employed a single-cell RNA sequencing (scRNA-seq) approach to explore the cellular landscape in our CD-mouse model, where we challenged Nod2-deficient mice with a chronic inflammatory insult regime, using dextran sulfate sodium (cDSS) for 3 cycles. Subsequently, changes in inflammatory, metabolic and fibrotic markers in the gut were analyzed by quantitative real-time PCR (qRT-PCR), immunohistochemistry (IHC) or western blot analyses. Further, RNA-In-situ hybridization (RNA-ISH) and flow cytometric analyses were performed to validate cell populations contributing to, and the molecular signatures linked with intestinal fibrostenosis. Results We found that Nod2-deficient mice show increased expression of pro-inflammatory cytokines (TNF, IL6) and excessive ECM deposition (fibrosis) in the gut compared to wild-type littermate mice post-cDSS. Curiously, scRNA-seq analysis uncovered altered stromal-immune-cell crosstalk along with the identification of a novel subset of the Dpt+ interstitial stromal population with distinct gene signatures regulated by chronic inflammation. Further, we observed increased senescence (Cdkn2a) mediated trans-differentiation of stroma into fibroblast contributing to IF in Nod2-deficient mice. Conclusions The study outlined will deepen our understanding of cellular and molecular mediators as well as inter and intra-cellular networks that drive IF. This will be transformative in devising therapeutic strategies to prevent IF in progressive CD patients. Funding Agencies CCC, CIHRThe American Association of Immunologist (AAI)

A227 EXPLORING THE ROLE OF MICROBIAL AND GENETIC FACTORS IN DEFINING THE INTESTINAL IMMUNE SYSTEM USING A HUMANIZED GNOTOBIOTIC MOUSE MODEL

Abstract Background Studies of the gut microbial ecosystem and its role in disease are often complicated by the intricate network of interactions between its microbial members and the host. Therefore, mouse models in which the microbiota diversity can be strictly controlled, and the functional potential be easily assessed, are needed. Aims Establishing a taxonomically and functionally diverse human gnotobiotic microbiota that recapitulates many microbe-host interactions of the complex human microbiome. To validate the effects of the defined bacterial gut community on the host, we will assess host physiology, intestinal immunity and the response to inflammatory stimuli. Methods Germ-free (GF) mice were inoculated with a defined bacterial community isolated from the stool of a healthy human donor. Stability of colonization across multiple generations and spatial organization of the bacterial community in the intestine were assessed using 16S rRNA gene sequencing and species-specific quantitative PCR. Immune cells were isolated from the colonic lamina propria and characterized by flow cytometry. Results Inoculated GF mice were reproducibly colonized with 8 bacterial species, collectively termed Human Defined Community 1 (HDC1). The HDC1 is vertically transmitted from mothers to offspring and the relative abundances remain stable in the colony over an extended period in a gnotobiotic isolator. We observed spatial stratification of the HDC1 microbiota between the small intestine and colon or cecum. HDC1 mice form a colonic inner mucus layer comparable in thickness to SPF mice suggesting physiological spatial organization of the bacterial community over the cross-sectional axis of the colon. Characterization of colonic lamina propria immune cells in HDC1 mice revealed myeloid and lymphoid cell abundances closely resembling those of mice harboring a complex microbiota (specific pathogen-free [SPF] mice), including normal macrophage and dendritic cell counts and normal proportions of CD4+ cells (regulatory T, Th1 and Th17 cells). Finally, we showed that HDC1 mice can be transplanted with human gut commensals and Crohn's disease (CD)-associated bacteria, demonstrating the tractability of this human defined microbiota. Conclusions In summary, HDC1 gnotobiotic mice are stably colonized with a human defined bacterial community and harbor an intestinal innate and adaptive immune system similar to SPF mice. Therefore, HDC1 mice represent a reliable and tractable tool for host-microbe interaction studies. Ultimately, we will employ the HDC1 model to decipher the role of Nod2 – the strongest genetic risk factor for CD development – in shaping both the microbiota and host immunity. Funding Agencies CIHROGS

TCRosetta: An Integrated Analysis and Annotation Platform for T-cell Receptor Sequences.

T cells and T-cell receptors (TCRs) are essential components of the adaptive immune system. Characterization of the TCR repertoire offers a promising and highly informative source for understanding the functions of T cells in the immune response and immunotherapy. Although TCR repertoire studies have attracted much attention, there are few online servers available for TCR repertoire analysis, especially for TCR sequence annotation or advanced analyses. Therefore, we developed TCRosetta, a comprehensive online server that integrates analytical methods for TCR repertoire analysis and visualization. TCRosetta combines general feature analysis, large-scale sequence clustering, network construction, peptide-TCR binding prediction, generation probability calculation, and k-mer motif analysis for TCR sequences, making TCR data analysis as simple as possible. The TCRosetta server accepts multiple input data formats and can analyze ∼ 20,000 TCR sequences in less than 3 min. TCRosetta is the most comprehensive web server available for TCR repertoire analysis and is freely available at https://guolab.wchscu.cn/TCRosetta/.