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Summary Background Among the most consequential unknowns of the devastating COVID-19 pandemic are the durability of immunity and time to likely reinfection. There are limited direct data on SARS-CoV-2 long-term immune responses and reinfection. The aim of this study is to use data on the durability of immunity among evolutionarily close coronavirus relatives of SARS-CoV-2 to estimate times to reinfection by a comparative evolutionary analysis of related viruses SARS-CoV, MERS-CoV, human coronavirus (HCoV)-229E, HCoV-OC43, and HCoV-NL63. Methods We conducted phylogenetic analyses of the S, M, and ORF1b genes to reconstruct a maximum-likelihood molecular phylogeny of human-infecting coronaviruses. This phylogeny enabled comparative analyses of peak-normalised nucleocapsid protein, spike protein, and whole-virus lysate IgG antibody optical density levels, in conjunction with reinfection data on endemic human-infecting coronaviruses. We performed ancestral and descendent states analyses to estimate the expected declines in antibody levels over time, the probabilities of reinfection based on antibody level, and the anticipated times to reinfection after recovery under conditions of endemic transmission for SARS-CoV-2, as well as the other human-infecting coronaviruses. Findings We obtained antibody optical density data for six human-infecting coronaviruses, extending from 128 days to 28 years after infection between 1984 and 2020. These data provided a means to estimate profiles of the typical antibody decline and probabilities of reinfection over time under endemic conditions. Reinfection by SARS-CoV-2 under endemic conditions would likely occur between 3 months and 5·1 years after peak antibody response, with a median of 16 months. This protection is less than half the duration revealed for the endemic coronaviruses circulating among humans (5–95% quantiles 15 months to 10 years for HCoV-OC43, 31 months to 12 years for HCoV-NL63, and 16 months to 12 years for HCoV-229E). For SARS-CoV, the 5–95% quantiles were 4 months to 6 years, whereas the 95% quantiles for MERS-CoV were inconsistent by dataset. Interpretation The timeframe for reinfection is fundamental to numerous aspects of public health decision making. As the COVID-19 pandemic continues, reinfection is likely to become increasingly common. Maintaining public health measures that curb transmission—including among individuals who were previously infected with SARS-CoV-2—coupled with persistent efforts to accelerate vaccination worldwide is critical to the prevention of COVID-19 morbidity and mortality. Funding US National Science Foundation.

Tertiary lymphoid organs (TLOs), also known as inducible lymphoid organs, tertiary lymphoid structures, tertiary lymphoid tissues, or ectopic lymphoid organs are accumulations of cells in chronic inflammation that have been observed in most tissues in autoimmunity, infection, and cancer in mouse and man. They share many properties with secondary lymphoid organs (SLOs), particularly lymph nodes, with regard to cellular composition, function, and regulation. TLOs include T and B cells, dendritic cells, follicular dendritic cells, and many other stromal cells, and high endothelial venules (HEVs) and lymphatic vessels. They serve as sites of antigen presentation and tolerance induction; they are harmful in autoimmunity and can be both harmful and beneficial in cancer. SLO induction in ontogeny is mediated by interactions of several cell types, including CD4+ CD3- lymphoid tissue inducer (LTi) RORγt+ cells that express LTαβ and interact with mesenchymal lymphoid tissue organizer (LTo) FAP+ cells in the presence of lymphatic and blood vessels. A variety of inducer cells initiate TLOs, including bona fide LTi cells, T cells, B cells, and NK cells. The mesenchymal organizer cells are less well characterized but can include FAP+ cells. Current challenges include identification of methods to inhibit TLOs in autoimmunity without affecting SLOs, and enhancement of TLOs for defense against tumors.

Tertiary lymphoid organs (TLOs), also known as inducible lymphoid organs, tertiary lymphoid structures, tertiary lymphoid tissues, or ectopic lymphoid organs are accumulations of cells in chronic inflammation that have been observed in most tissues in autoimmunity, infection, and cancer in mouse and man. They share many properties with secondary lymphoid organs (SLOs), particularly lymph nodes, with regard to cellular composition, function, and regulation. TLOs include T and B cells, dendritic cells, follicular dendritic cells, and many other stromal cells, and high endothelial venules (HEVs) and lymphatic vessels. They serve as sites of antigen presentation and tolerance induction; they are harmful in autoimmunity and can be both harmful and beneficial in cancer. SLO induction in ontogeny is mediated by interactions of several cell types, including CD4+ CD3- lymphoid tissue inducer (LTi) RORγt+ cells that express LTαβ and interact with mesenchymal lymphoid tissue organizer (LTo) FAP+ cells in the presence of lymphatic and blood vessels. A variety of inducer cells initiate TLOs, including bona fide LTi cells, T cells, B cells, and NK cells. The mesenchymal organizer cells are less well characterized but can include FAP+ cells. Current challenges include identification of methods to inhibit TLOs in autoimmunity without affecting SLOs, and enhancement of TLOs for defense against tumors.

Treating macaques with an anti-α4β7 antibody under the umbrella of combination antiretroviral therapy (cART) during early SIV infection can lead to viral remission, with viral loads maintained at < 50 SIV RNA copies/ml after removal of all treatment in a subset of animals. Depletion of CD8+ lymphocytes in controllers resulted in transient recrudescence of viremia, suggesting that the combination of cART and anti-α4β7 antibody treatment led to a state where ongoing immune responses kept the virus undetectable in the absence of treatment. A previous mathematical model of HIV infection and cART incorporates immune effector cell responses and exhibits the property of two different viral load set-points. While the lower set-point could correspond to the attainment of long-term viral remission, attaining the higher set-point may be the result of viral rebound. Here we expand that model to include possible mechanisms of action of an anti-α4β7 antibody operating in these treated animals. We show that the model can fit the longitudinal viral load data from both IgG control and anti-α4β7 antibody treated macaques, suggesting explanations for the viral control associated with cART and an anti-α4β7 antibody treatment. This effective perturbation to the virus-host interaction can also explain observations in other nonhuman primate experiments in which cART and immunotherapy have led to post-treatment control or resetting of the viral load set-point. Interestingly, because the viral kinetics in the various treated animals differed—some animals exhibited large fluctuations in viral load after cART cessation—the model suggests that anti-α4β7 treatment could act by different primary mechanisms in different animals and still lead to post-treatment viral control. This outcome is nonetheless in accordance with a model with two stable viral load set-points, in which therapy can perturb the system from one set-point to a lower one through different biological mechanisms., Author summary Some macaques treated with an anti-α4β7 monoclonal antibody along with antiretroviral therapy during the early stages of simian immunodeficiency virus infection had their viral load become undetectable (below 50 SIV RNA copies/ml) after all treatment was stopped, whereas animals not given the antibody all had their viral loads rebound to high levels. Using a mathematical model, we examined four potential ways in which the antibody could have altered the balance between viral growth and immune control to maintain an undetectable viral load. We show that a shift to controlled infection can occur through multiple biologically reasonable mechanisms of action of the anti-α4β7 antibody.

To investigate the role of lymphotoxin (LT) in Sjogren's syndrome (SS) and in mucosal associated lymphoid tissue (MALT)-lymphoma, we made transgenic mice (Amy1-LTαβ) that targeted LTα and LTβ to the salivary and lacrimal glands. Amy1-LTαβ mice developed atrophic salivary and lacrimal glands that contained tertiary lymphoid organs (TLOs) and had reduced tear production. Amy1-LTαβ mice developed cervical lymphadenopathy but not MALT-lymphoma. TLO formation in the salivary and lacrimal glands of Amy1-LTαβ was not sufficient to induce autoimmunity as measured by autoantibody titres.

SummaryVisceral adiposity in elderly is associated with alterations in adipose tissue immune cells leading to inflammation and metabolic dysfunction. The Nlrp3 inflammasome is a critical regulator of macrophage activation, inflammation, and immunometabolism in visceral adipose tissue during aging; however, the potential contribution of adipose tissue B cells is unexplored. Here, we show that aging expands adipose-resident B cells and fat-associated lymphoid clusters (FALCs) in visceral white adipose tissue. Adipose tissue B cells exhibit a memory-like B cell profile similar to the phenotype of aged B cells that are increased in spleen of old mice. Mechanistically, the age-induced FALC formation and adipose B cell expansion, but not B cell transcriptional program, is dependent on the Nlrp3 inflammasome. Furthermore, B cell depletion in aged mice restores lipolysis and defense against loss of core body temperature during cold stress. These data reveal that inhibiting Nlrp3-dependent B cell accumulation can be targeted to reverse metabolic impairment in aging adipose tissue.Highlights- Adipose-resident aged B cells are increased in fat-associated lymphoid clusters (FALC)- FALC formation and adipose-resident B cell expansion during aging are regulated by the Nlrp3 inflammasome- Nlrp3 and B cell depletion in aging restores lipolysis and improves cold tolerancea

The ability to recognize the presence of a pathogen and respond appropriately is a fundamental aspect of innate immunity. This chapter describes the types of receptors and their ligands that are used to detect microbes that have entered the body as well as responses. Some receptors recognize conserved structures unique to microbes and not present in mammalian cells, called pathogen-associated molecular patterns (PAMPs). Other receptors recognize nucleic acids that are unique to the microbe or are in the cytoplasm where nucleic acids are not normally found. Interestingly, certain disruptions to the cell, such as microbial invasion or cell stress, can be detected and are called danger-associated molecular patterns (DAMPs). The host makes multiple types of responses appropriate for eliminating the type of pathogen. The inflammatory response consists of fluid and cells entering tissues to fight the infection and secretion of cytokines and chemokines for cell–cell communication. The complement system is an ancient system of proteins for fighting infections. Once activated, complement proteins promote inflammation and directly kill pathogens. Other effector mechanisms such as the secretion of antimicrobial peptides are discussed. The breadth of receptors and responses reflects how important it is to recognize an invading microbe, make an appropriate response for the type of pathogen, and destroy it before being harmed. However, in some cases, it is not possible to eliminate the microbe or inducer of inflammation. A state of chronic inflammation is an adaption by the host to this situation.

In this chapter, we discuss the definitions, history, and major concepts of immunology, epidemiology, and the newly emerging field of immunoepidemiology. Definitions of the central topics in this chapter are as follows: Immunology is the study of that combination of cells and compounds that protect individual multicellular organisms against foreign entities. Epidemiology is the study of the occurrence and distribution of health-related events, states, and processes in specified populations, including the study of the determinants influencing such processes, as well as the application of this knowledge to control relevant health problems. Immunoepidemiology is the study of the immune response of populations. Immunoepidemiology uses both immunological and epidemiologic tools to better understand how select populations are protected from microbial infection, malignancy, inflammatory diseases, and autoimmunity. Immunoepidemiologists study genetic polymorphisms and environmental factors to explain the diversity of the immune response.

The immune system is designed to ward off infections. In some situations, the system is dysfunctional. The causes of these dysfunctions are many and varied but can be generally classified as deficient (immunodeficiency) or disproportionate (allergy and autoimmunity). Primary immune deficiencies are genetic defects that result in inappropriate responses to infections. They may affect T cell selection and function or antibody production or involve lymphoproliferative syndromes. Treatment strategies include intravenous immunoglobulin therapy, bone marrow transplantation, or gene therapy. Allergies are classified as immediate hypersensitivity diseases, most often mediated by IgE antibody. Food allergy is one of the most common. Pathogenic mechanisms include IgE antibody, non-IgE antibody, or both, or cellular processes. Food allergies are distinguished from food intolerance and can be permanent or transient. Risk factors for food allergies include genes (a family history of atopy), gender (more common in males), and the environment (less common in individuals exposed to pets). Autoimmune diseases can be directed against a particular cell such as the insulin-producing β cell in Type I diabetes; an organ, such as the joints in rheumatoid arthritis; or the contents of all cells, as in systemic lupus erythematosus. The pathogenic mechanisms can be cell-mediated or antibody-mediated, or a combination of both. The autoimmune diseases are complex and multifactorial with both genetic and acquired etiologies––summarized as “genes, gender, and geography,” with an influence of lifestyle. HLA type is a risk factor, but sophisticated genome-wide association studies have revealed the enormous complexity of genetic involvement. A higher prevalence in females is seen in some autoimmune diseases. The role of geography includes the country of origin and the microbiome.

Innate antigen-presenting cells (APC) inform the adaptive immune that a pathogen is present and indicate the nature of the pathogen. Naive T cells interacting with APCs in secondary lymphoid organs then develop into effector cells depending on the type of pathogen and the cytokine milieu. In some cases the effector T cells function independently, such as the CD8 cytotoxic T cells that kill pathogen-infected host cells. In other cases, they function with innate cells to boost immune responses. One example is the CD4 T helper (Th1) cell that enhances macrophage response against bacteria. The cytokines secreted by the effector cells play critical roles in their functions. Other types of CD4 subsets interact with B cells to help in their activation. Thus, the immune response resembles a relay race: innate cells inform T cells that a pathogen is present, and then T cells interact with B cells to relay the information. The B cells generate antibodies of a class that is most effective against the type of infecting pathogen. Effector T and B cells are short lived; however, long-lived T and B memory cells and long-lived plasma cells are generated. Reinfection with the same pathogen leads to a more rapid and stronger response by activation of memory cells. This is the basis for vaccination. A balanced immune response requires not just activation but also mechanisms for inhibiting and terminating responses in order to clear pathogens and minimize damage to the host.

The human immune system is designed for protection against invading pathogens. The areas of the body exposed to microbes are the skin and mucosal surfaces of the intestinal, respiratory, reproductive, and urinary tracts. The immune system associated with these surfaces is called barrier immunity. Once a pathogen has entered the body, cells of the innate and adaptive immune system can be activated to fight the infection. Macrophages, dendritic cells, granulocytes, innate lymphoid cells, and invariant and conventional T and B cells function to protect the host. Characteristics of different immune cell types and some of their functions such as phagocytosis (eating) and NETosis (trapping) are presented in this chapter. In order to mount a coordinated and appropriate response, there is cross-talk between the different immune cells. Soluble mediators called cytokines and chemokines are important mechanisms for cell–cell communication. Cytokines and chemokines also provide communication between immune cells and other organs of the body. The adult lymphoid system is composed of primary (bone marrow and thymus) and secondary (lymph node, spleen, Peyer’s patches, tonsils, adenoids, lymphoid tissue associated with mucosal surfaces) lymphoid organs that are connected through blood and lymphatic vessels. The lymphatic vessels transport immune cells, fluid, antigen, and fat throughout the body. Tertiary lymphoid organs can form in the context of chronic inflammation. This chapter provides a broad overview of the components of the immune system with greater detail provided in later chapters.

The remarkable evolutionary advantage of T and B lymphocytes as a defense mechanism is their ability to specifically recognize millions of different microbes, including some that may not yet even exist. The genes that encode T and B cell receptors and as antibodies that are the soluble form of the B cell receptors, are arranged unlike any other genes in the body. The process of generating the receptors includes somatic recombination of different segments of DNA, the addition or subtraction of nucleotides during the process (junctional diversity), and pairing of different chains of the receptors (combinatorial diversity). The B cell receptor and the corresponding antibody can further diversify by changing their constant domains in a process called isotype switching so that there are five possible isotypes (IgM, IgD, IgG, IgA, and IgE). While the B cell receptor binds a variety of antigens as ligands, the T cell receptor binds to peptides presented on the cell surface by proteins of the major histocompatibility complex (MHC), named HLA proteins in humans. One advantage of the MHC system is that CD8 killer T cells can recognize and destroy host cells infected by an intracellular pathogen because peptides from pathogen proteins are brought to the cell surface by HLA proteins as a type of “flag.” In order for a population of diverse individuals to have the potential to survive an epidemic, the HLA proteins are highly polymorphic so that at least some individuals will be able to present peptides from pathogen proteins and mount an effective immune response. Because the process of generating T and B cell receptors is random, it is important to eliminate cells with receptors recognizing self-antigens. This process is called central tolerance.

Immunotherapy uses advances from the field of immunology to exploit the immune system in clinical practice. The goal is the use of directed immunotherapy with minimal side effects to combat infection, cancer, and autoimmunity. Although immunotherapy was first used as passive transfer of serum containing polyclonal antibodies, the development of monoclonal antibodies has provided reagents that are targeted, specific, and completely uniform. Monoclonal antibodies are used against several infectious organisms, including respiratory syncytial virus. Monoclonal antibodies in cancer immunotherapy were first directed against tumor cells. Later they targeted immune molecules that modulate immune response against cancer cells. For instance, starting in 2011, checkpoint inhibitors were developed that block inhibitory receptors on immune cells involved in immune regulation. This had a dramatic impact on survival in a subset of patients with cancers such as melanoma and lung cancer. Checkpoint inhibition is associated with risks in a subset of patients that develop neurological, respiratory, musculoskeletal, cardiac, ocular, and/or autoimmune side effects. Management of autoimmune diseases with cytokines and cytokine inhibitors has revolutionized the field, although treatment failure is not uncommon. This may be due to an initial unresponsiveness to the agent, a later tolerance to that agent, or adverse events such as activation of latent infections.

Human variations in immune response can result from genetic polymorphisms. Polymorphisms are the result of differences in gene sequences that can give rise to changes in gene regulation (generally in enhancers or promoters), gene function (in exon sequences), or simple intronic sequence changes with no apparent effect on function. Polymorphisms can be single-nucleotide polymorphisms (SNPs), microsatellite repeats and minisatellites, or variable number tandem insertions or deletions. The immune system is replete with polymorphisms, in part due to the necessity for response to multiple microorganisms in diverse environments. The presence of polymorphisms in some cases leads to a selective survival in the face of devastating pathogen infection. This chapter analyzes a few systems that embody these polymorphisms. Polymorphisms in pattern recognition receptors (PRR), complement, cytokines, chemokines, the major histocompatibility complex (MHC), killer cell immunoglobulin-like receptors (KIRs), T cell receptors (TCRs), and immunoglobulins (IgG) are evaluated with regard to their effects on disease and pregnancy.

Scleroderma is a group of skin-fibrosing diseases for which there are no effective treatments. A feature of the skin fibrosis typical of scleroderma is atrophy of the dermal white adipose tissue (DWAT). Adipose tissue contains adipose-derived mesenchymal stromal cells (ADSCs) that have regenerative and reparative functions; however, whether DWAT atrophy in fibrosis is accompanied by ADSC loss is poorly understood, as are the mechanisms that might maintain ADSC survival in fibrotic skin. Here, we have shown that DWAT ADSC numbers were reduced, likely because of cell death, in 2 murine models of scleroderma skin fibrosis. The remaining ADSCs showed a partial dependence on dendritic cells (DCs) for survival. Lymphotoxin β (LTβ) expression in DCs maintained ADSC survival in fibrotic skin by activating an LTβ receptor/β1 integrin (LTβR/β1 integrin) pathway on ADSCs. Stimulation of LTβR augmented the engraftment of therapeutically injected ADSCs, which was associated with reductions in skin fibrosis and improved skin function. These findings provide insight into the effects of skin fibrosis on DWAT ADSCs, identify a DC-ADSC survival axis in fibrotic skin, and suggest an approach for improving mesenchymal stromal cell therapy in scleroderma and other diseases.

During aging, visceral adiposity is often associated with alterations in adipose tissue (AT) leukocytes, inflammation, and metabolic dysfunction. However, the contribution of AT B cells in immunometabolism during aging is unexplored. Here, we show that aging is associated with an expansion of a unique population of resident non-senescent aged adipose B cells (AABs) found in fat-associated lymphoid clusters (FALCs). AABs are transcriptionally distinct from splenic age-associated B cells (ABCs) and show greater expansion in female mice. Functionally, whole-body B cell depletion restores proper lipolysis and core body temperature maintenance during cold stress. Mechanistically, the age-induced FALC formation, AAB, and splenic ABC expansion is dependent on the Nlrp3 inflammasome. Furthermore, AABs express IL-1R, and inhibition of IL-1 signaling reduces their proliferation and increases lipolysis in aging. These data reveal that inhibiting Nlrp3-dependent B cell accumulation can be targeted to reverse metabolic impairment in aging AT.

The journey from the discoveries of lymphotoxin (LT) and tumor necrosis factor (TNF) to the present day age of cytokine inhibitors as therapeutics has been an exciting one with many participants and highs and lows; the saga is compared to that in "The Wizard of Oz". This communication summarizes the contributions of key players in the discovery of the cytokines and their receptors, the changes in nomenclature, and the discovery of the LT family's crucial role in secondary and tertiary lymphoid organs. The remarkable advances in therapeutics are detailed as are remaining problems. Finally, special tribute is paid to two pioneers in the field who have recently passed away: Byron H. Waksman and Lloyd Old.

Tertiary lymphoid organs (TLOs) are accumulations of lymphoid cells in chronic inflammation that resemble LNs in their cellular content and organization, high endothelial venules, and lymphatic vessels (LVs). Although acute inflammation can result in defective LVs, TLO LVs appear to function normally in that they drain fluid and transport cells that respond to chemokines and sphingosine-1-phosphate (S1P) gradients. Molecular regulation of TLO LVs differs from lymphangiogenesis in ontogeny with a dependence on cytokines and hematopoietic cells. Ongoing work to elucidate the function and molecular regulation of LVs in TLOs is providing insight into therapies for conditions as diverse as lymphedema, autoimmunity, and cancer.

Serious infections of the head and neck cause lymphedema that can lead to airway compromise and oropharyngeal obstruction. Lymphangiogenesis occurs in the head and neck during infection and after immunization. The goal of this project was to develop tools to image lymphatic vessels in living animals and to be able to isolate individual lymphatic endothelial cells in order to quantify changes in single cells caused by inflammation.The ProxTom transgenic red-fluorescent reporter mouse was developed specifically for the purpose of imaging lymphatic vessels in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in development and for the maintenance of lymphatics in adulthood. Mice were immunized and their lymphatic vessels in lymph nodes were imaged in vivo. Individual lymphatic endothelial cells were isolated by means of their fluorescence.The ProxTom transgene has the red-fluorescent reporter td-Tomato under the control of Prox1 regulatory elements. tdTomato was faithfully expressed in lymphatic vessels coincident with endogenous Prox1 expression. We show lymphangiogenesis in vivo after immunization and demonstrate a method for the isolation of lymphatic endothelial cells by their tdTomato red-fluorescence.The faithful expression of the red-fluorescent reporter in the lymphatic vessels of ProxTom means that these mice have proven utility for in vivo study of lymphatic vessels in the immune response. ProxTom has been made available for distribution from the Jackson Laboratory: http://jaxmice.jax.org/strain/018128.html .

Tertiary lymphoid organs (TLOs), or more properly, tertiary lymphoid tissues, are accumulations of lymphoid tissues that arise in situations of chronic inflammation in chronic graft rejection, autoimmunity, chronic microbial infection, tumors, and atherosclerosis. They resemble secondary lymphoid organs (SLOs), such as lymph nodes, in their lymphoid and stromal cellular composition and arrangement, and vasculature including high endothelial venules and lymphatic vessels. They differ from conventional lymphoid organs in that they are in ectopic sites, that is, in nonlymphoid organs, and generally do not have a capsule. Antigen priming and maturation of the immune response can occur in TLOs. TLOs can be considered to be detrimental in autoimmunity where they can contribute to determinant spreading and exacerbation of clinical disease; they can be beneficial as they provide a local environment for defense against tumors; and they can provide a local site to respond to infectious organisms, preventing dispersion through the body.

While the role of T cells has been studied extensively in multiple sclerosis (MS), the pathogenic contribution of B cells has only recently attracted major attention, when it was shown that B cell aggregates can develop in the meninges of a subset of MS patients and were suggested to be correlates of late-stage and more aggressive disease in this patient population. However, whether these aggregates actually exist has subsequently been questioned and their functional significance has remained unclear. Here, we studied myelin basic protein (MBP)-proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), which is one of the few animal models for MS that is dependent on B cells. We provide evidence that B cell aggregation is reflective of lymphoid neogenesis in the central nervous system (CNS) in MBP-PLP-elicited EAE. B cell aggregation was present already few days after disease onset. With disease progression CNS B cell aggregates increasingly displayed the phenotype of tertiary lymphoid organs (TLOs). Our results further imply that these TLOs were not merely epiphenomena of the disease, but functionally active, supporting intrathecal determinant spreading of the myelin-specific T cell response. Our data suggest that the CNS is not a passive "immune-privileged" target organ, but rather a compartment, in which highly active immune responses can perpetuate and amplify the autoimmune pathology and thereby autonomously contribute to disease progression.

IFN-γ–inducible lysosomal thiol reductase (GILT) is an enzyme located in the Lamp-2–positive compartments of APC. GILT−/− mice are phenotypically normal, but their T cells exhibit reduced proliferation to several exogenously administered Ags that include cysteine residues and disulfide bonds. We undertook the present studies to determine if GILT−/− mice would process exogenously administered myelin oligodendrocyte glycoprotein (MOG), which contains disulfide bonds, to generate experimental autoimmune encephalomyelitis (EAE) to the endogenous protein. One possibility was that MOG35–55 peptide would induce EAE, but that MOG protein would not. GILT−/− mice were relatively resistant to MOG35–55-induced EAE but slightly more susceptible to rat MOG protein-induced EAE than wild-type (WT) mice. Even though MOG35–55 was immunogenic in GILT−/− mice, GILT APCs could not generate MOG35–55 from MOG protein in vitro, suggesting that the endogenous MOG protein was not processed to the MOG35–55 peptide in vivo. Immunization of GILT−/− mice with rat MOG protein resulted in a switch in pathogenic mechanism from that seen in WT mice; the CNS infiltrate included large numbers of plasma cells; and GILT−/− T cells proliferated to peptides other than MOG35–55. In contrast to WT rat MOG-immunized mice, rat MOG-immunized GILT−/− mice generated Abs that transferred EAE to MOG35–55-primed GILT−/− mice, and these Abs bound to oligodendrocytes. These studies, demonstrating the key role of a processing enzyme in autoimmunity, indicate that subtle phenotypic changes have profound influences on pathogenic mechanisms and are directly applicable to the outbred human population.

Lymphatic vessels (LVs) are important structures for antigen presentation, for lipid metabolism, and as conduits for tumor metastases, but they have been difficult to visualize in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in ontogeny and the maintenance of LVs. To visualize LVs in the lymph node of a living mouse in real time, we made the ProxTom transgenic mouse in a C57BL/6 background using red fluorescent LVs that are suitable for in vivo imaging. The ProxTom transgene contained all Prox1 regulatory sequences and was faithfully expressed in LVs coincident with endogenous Prox1 expression. The progenies of a ProxTom × Hec6stGFP cross were imaged using two-photon laser scanning microscopy, allowing the simultaneous visualization of LVs and high endothelial venules in a lymph node of a living mouse for the first time. We confirmed the expression of Prox1 in the adult liver, lens, and dentate gyrus. These intensely fluorescent mice revealed the expression of Prox1 in three novel sites: the neuroendocrine cells of the adrenal medulla, megakaryocytes, and platelets. The novel sites identified herein suggest previously unknown roles for Prox1. The faithful expression of the fluorescent reporter in ProxTom LVs indicates that these mice have potential utility in the study of diseases as diverse as lymphedema, filariasis, transplant rejection, obesity, and tumor metastasis.

To trigger an effective immune response, antigen and antigen-presenting cells travel to the lymph nodes via collecting lymphatic vessels. However, our understanding of the regulation of collecting lymphatic vessel function and lymph transport is limited. To dissect the molecular control of lymphatic function, we developed a unique mouse model that allows intravital imaging of autonomous lymphatic vessel contraction. Using this method, we demonstrated that endothelial nitric oxide synthase (eNOS) in lymphatic endothelial cells is required for robust lymphatic contractions under physiological conditions. By contrast, under inflammatory conditions, inducible NOS (iNOS)-expressing CD11b + Gr-1 + cells attenuate lymphatic contraction. This inhibition of lymphatic contraction was associated with a reduction in the response to antigen in a model of immune-induced multiple sclerosis. These results suggest the suppression of lymphatic function by the CD11b + Gr-1 + cells as a potential mechanism of self-protection from autoreactive responses during on-going inflammation. The central role for nitric oxide also suggests that other diseases such as cancer and infection may also mediate lymphatic contraction and thus immune response. Our unique method allows the study of lymphatic function and its molecular regulation during inflammation, lymphedema, and lymphatic metastasis.

Thymic B cells represent a numerically minor cell population located primarily at the cortico-medullary junction. Their biological role is unclear. B cell-deficient μMT mice exhibited reduced medullary thymic epithelial cell (mTEC) numbers and reduced MOG and insulin mRNA expression. Lymphotoxin produced by B cells was critical for normal tissue restricted antigen (TRA) expression, suggesting that B cells regulate self-antigens through their production of LT. These results reveal an unexpected role of B cells in mTEC maintenance and expression of TRAs through their production of LT.

Transgenic mouse models are valuable resources for analyzing functions of genes involved in human diseases. Mouse models provide critical insights into biological processes, including in vivo visualization of vasculature critical to our understanding of the immune system. Generating transgenic mice requires the capture and modification of large-insert DNAs representing genes of interest. We have developed a methodology using a yeast-bacterial shuttle vector, pClasper, that enables the capture and modification of bacterial artificial chromosomes (BAC)-sized DNA inserts. Numerous improvements and technical advances in the original pClasper vector have allowed greater flexibility and utility in this system. Examples of such pClasper mediated gene modifications include: Claspette-mediated capture of large-insert genomic fragments from BACs-human polycystic kidney disease-1 (PKD1); modification of pClasperA clones by the RareGap method-PKD1 mutations; Claspette-mediated modification of pClasper clones-mouse albumin-1 gene; and, of most relevance to our interest in lymph node vasculature-Claspimer-mediated modification of pClasper clones-high endothelial venule and lymphatic vessel genes. Mice that have been generated with these methods include mice with fluorescent high endothelial venules.

Lymphotoxin-α (LTα), lymphotoxin-β (LTβ), and tumor necrosis factor-α (TNFα) are inflammatory mediators that play crucial roles in lymphoid organ development. We demonstrate here that LTα also contributes to the function of lymphatic vessels and to lymphangiogenesis during inflammation. LTα−/− mice exhibited reduced lymph flow velocities and increased interstitial fluid pressure. Airways of LTβ−/− mice infected with Mycoplasma pulmonis had significantly more lymphangiogenesis than wild type (WT) or LTα−/− mice, as did the skin draining immunization sites of LTβ−/− mice. Macrophages, B cells, and T cells, known sources of LT and TNFα, were apparent in the skin surrounding the immunization sites as were LTα, LTβ, and TNFα mRNAs. Ectopic expression of LTα led to the development of LYVE-1 and Prox1-positive lymphatic vessels within tertiary lymphoid organs (TLOs). Quantification of pancreatic lymphatic vessel density in RIPLTαLTβ−/− and WT mice revealed that LTα was sufficient for inducing lymphangiogenesis and that LTβ was not required for this process. Kidneys of inducible LTα transgenic mice developed lymphatic vessels before the appearance of obvious TLOs. These data indicate that LTα plays a significant role in lymphatic vessel function and in inflammation-associated lymphangiogenesis.

Various models of experimental autoimmune encephalomyelitis (EAE) have led to insights into the pathogenesis and novel therapies for multiple sclerosis. One generalized EAE model uses immunizing the Lewis Rat with myelin oligodendrocyte glycoprotein (MOG) and complete Freund's adjuvant that induces systemic disease and inflammatory lesions at random central nervous system (CNS) locations. These lesions result from a combination of sensitized T cells and pathogenic antibodies gaining access to the CNS to cause an immune assault on myelin-expressing oligodendrocytes. We report a focal and temporal variant of the EAE model that results in immune-mediated demyelination at a predictable time and location. Lewis rats were immunized with the extracellular domain (1-125) of recombinant rat MOG in incomplete Freund's adjuvant (IFA) to induce a clinically silent humoral response. Vascular endothelial growth factor (VEGF) was then microinjected into the spinal cord to induce a transient, focal breakdown of the blood brain barrier (BBB). Clinical signs were apparent within 72 hours and began to resolve by day 21. The histopathology at the site of injection consisted of a focal region containing OX-42(+) cells, phagocytic cells with debris, extensive demyelination, and some lymphocyte infiltration. Neither intraspinal injection of PBS into immunized animals nor VEGF into animals treated with IFA alone resulted in clinical lesions. Thus, a transient, focal opening of the BBB with VEGF in animals with subclinical MOG immunization leads to a discrete inflammatory demyelinating lesion. This model may be useful for the study of transplanted myelin-forming cells in a discrete inflammatory demyelinating lesion.

OBJECTIVE The nonobese diabetic (NOD) mouse is a well-established mouse model of spontaneous type 1 diabetes, which is characterized by an autoimmune destruction of the insulin-secreting pancreatic β-cells. In this study, we address the role of tertiary lymphoid organs (TLOs) that form in the pancreas of NOD mice during disease progression. METHODS We developed a model designed to “lock” lymphocytes in the pancreatic lymph node (PLN) and pancreas by the use of FTY720, which blocks the exit of lymphocytes from lymph nodes. A combination of flow cytometry, immunofluorescence, and analysis of clinical scores was used to study the effects of long-term FTY720 treatment on TLO development and development of diabetes. RESULTS Continuous treatment of NOD mice with FTY720 prevented diabetes development even at a time of significant insulitis. Treatment withdrawal led to accelerated disease independent of the PLN. Interestingly, naive T-cells trafficked to and proliferated in the TLOs. In addition, morphological changes were observed that occurred during the development of the disease. Remarkably, although the infiltrates are not organized into T/B-cell compartments in 8-week-old mice, by 20 weeks of age, and in age-matched mice undergoing FTY720 treatment, the infiltrates showed a high degree of organization. However, in naturally and FTY720-induced diabetic mice, T/B-cell compartmentalization was lost. CONCLUSION Our data show that TLOs are established during diabetes development and suggest that islet destruction is due to a loss of TLO integrity, which may be prevented by FTY720 treatment.

A key question in the field of autoimmunity concerns the fact that experimental disease is generally induced more easily with closely related, but not completely identical, tissue-restricted antigens. Here, the possibility that naturally occurring regulatory T cells (Tregs) for self-antigens are more potent than those for related antigens was investigated. The self-antigen specificity of naturally occurring Tregs was tested in experimental autoimmune encephalomyelitis (EAE) induced with mouse (self) or closely related (rat) myelin oligodendrocyte glycoproteins (MOGs). Surprisingly, Treg depletion increased EAE severity in mice immunized with mouse, but not rat, MOG. This increase was associated with increased T-cell activation and infiltration of the central nervous system, as well as increased interleukin (IL)-17 production and a higher ratio of interferon-gamma- to IL-10-producing cells. These data suggest that Tregs are specific for self-antigen and do not "cross-protect" against autoimmunity even when disease is induced with closely related foreign antigens.

Secondary lymphoid organs (SLOs) include lymph nodes, spleen, Peyer’s patches, and mucosal tissues such as the nasal-associated lymphoid tissue, adenoids, and tonsils. Less discretely anatomically defined cellular accumulations include the bronchus-associated lymphoid tissue, cryptopatches, and isolated lymphoid follicles. All SLOs serve to generate immune responses and tolerance. SLO development depends on the precisely regulated expression of cooperating lymphoid chemokines and cytokines such as LTα, LTβ, RANKL, TNF, IL-7, and perhaps IL-17. The relative importance of these factors varies between the individual lymphoid organs. Participating in the process are lymphoid tissue initiator, lymphoid tissue inducer, and lymphoid tissue organizer cells. These cells and others that produce crucial cytokines maintain SLOs in the adult. Similar signals regulate the transition from inflammation to ectopic or tertiary lymphoid tissues.

Evaluation of lymphedema and lymph node metastasis in humans has relied primarily on invasive or radioactive modalities. While noninvasive technologies such as magnetic resonance imaging (MRI) offer the potential for true three-dimensional imaging of lymphatic structures, invasive modalities, such as optical fluorescence microscopy, provide higher resolution and clearer delineation of both lymph nodes and lymphatic vessels. Thus, contrast agents that image lymphatic vessels and lymph nodes by both fluorescence and MRI may further enhance our understanding of the structure and function of the lymphatic system. Recent applications of bimodal (fluorescence and MR) contrast agents in mice have not achieved clear visualization of lymphatic vessels and nodes. Here the authors describe the development of a nanoparticulate contrast agent that is taken up by lymphatic vessels to draining lymph nodes and detected by both modalities.A unique nanoparticulate contrast agent composed of a polyamidoamine dendrimer core conjugated to paramagnetic contrast agents and fluorescent probes was synthesized. Anesthetized mice were injected with the nanoparticulates in the hind footpads and imaged by MR and fluorescence microscopy. High resolution MR and fluorescence images were obtained and compared to traditional techniques for lymphatic visualization using Evans blue dye.Lymph nodes and lymphatic vessels were clearly observed by both MRI and fluorescence microscopy using the bimodal nanoparticulate contrast agent. Characteristic tail-lymphatics were also visualized by both modalities. Contrast imaging yielded a higher resolution than the traditional method employing Evans blue dye. MR data correlated with fluorescence and Evans blue dye imaging.A bimodal nanoparticulate contrast agent facilitates the visualization of lymphatic vessels and lymph nodes by both fluorescence microscopy and MRI with strong correlation between the two modalities. This agent may translate to applications such as the assessment of malignancy and lymphedema in humans and the evaluation of lymphatic vessel function and morphology in animal models.

Tertiary lymphoid tissues are lymph node-like cell aggregates that arise at sites of chronic inflammation. They have been observed in transplanted organs undergoing chronic rejection, but it is not known whether they contribute to the rejection process by supporting local activation of naïve lymphocytes. To answer this question, we established a murine transplantation model in which the donor skin contains tertiary lymphoid tissues due to transgenic expression of lymphotoxin-alpha(RIP-LT alpha), whereas the recipient lacks all secondary lymphoid organs and does not mount primary alloimmune responses. We demonstrate in this model that RIP-LT alpha allografts that harbor tertiary lymphoid tissues are rejected, while wild-type allografts that lack tertiary lymphoid tissues are accepted. Wild-type allografts transplanted at the same time as RIP-LT alpha skin or 60 days later were also rejected, suggesting that tertiary lymphoid tissues, similar to secondary lymphoid organs, generate both effector and memory immune responses. Consistent with this observation, naive T cells transferred to RIP-LT alpha skin allograft but not syngeneic graft recipients proliferated and differentiated into effector and memory T cells. These findings provide direct evidence that tertiary lymphoid structures perpetuate the rejection process by supporting naïve T-cell activation.

Hec-6st is a highly specific high endothelial venule (HEV) gene that is crucial for regulating lymphocyte homing to lymph nodes (LN). The enzyme is also expressed in HEV-like vessels in tertiary lymphoid organs that form in chronic inflammation in autoimmunity, graft rejection, and microbial infection. Understanding the molecular nature of Hec-6st regulation is crucial for elucidating its function in development and disease. However, studies of HEV are limited because of the difficulties in isolating and maintaining the unique characteristics of these vessels in vitro . The novel pClasper yeast homologous recombination technique was used to isolate from a BAC clone a 60-kb DNA fragment that included the Hec-6st ( Chst4 ) gene with flanking sequences. Transgenic mice were generated with the β- galactosidase ( LacZ ) reporter gene inserted in-frame in the exon II of Hec-6st within the isolated BAC DNA fragment. LacZ was expressed specifically on HEV in LN, as indicated by its colocalization with peripheral node vascular addressin. LacZ was increased in nasal-associated lymphoid tissue during development and was reduced in LN and nasal-associated lymphoid tissue by LTβR-Ig (lymphotoxin-β receptor human Ig fusion protein) treatment in a manner identical to the endogenous gene. The transgene was expressed at high levels in lymphoid accumulations with characteristics of tertiary lymphoid organs in the salivary glands of aged mice. Thus, the Hec-6s-LacZ construct faithfully reproduces Hec-6st tissue-specific expression and can be used in further studies to drive expression of reporter or effector genes, which could visualize or inhibit HEV in autoimmunity.

Accumulation of inflammatory cells in different renal compartments is a hallmark of progressive kidney diseases including glomerulonephritis (GN). Lymphotoxin β receptor (LTβR) signaling is crucial for the formation of lymphoid tissue, and inhibition of LTβR signaling has ameliorated several non-renal inflammatory models. Therefore, we tested whether LTβR signaling could also have a role in renal injury. Renal biopsies from patients with GN were found to express both LTα and LTβ ligands, as well as LTβR. The LTβR protein and mRNA were localized to tubular epithelial cells, parietal epithelial cells, crescents, and cells of the glomerular tuft, whereas LTβ was found on lymphocytes and tubular epithelial cells. Human tubular epithelial cells, mesangial cells, and mouse parietal epithelial cells expressed both LTα and LTβ mRNA upon stimulation with TNF in vitro. Several chemokine mRNAs and proteins were expressed in response to LTβR signaling. Importantly, in a murine lupus model, LTβR blockade improved renal function without the reduction of serum autoantibody titers or glomerular immune complex deposition. Thus, a preclinical mouse model and human studies strongly suggest that LTβR signaling is involved in renal injury and may be a suitable therapeutic target in renal diseases.

Ectopic expression of CC chemokine ligand 21 (CCL21) in the thyroid leads to development of lymphoid structures that resemble those observed in Hashimoto thyroiditis. Deletion of the inhibitor of differentiation 2 (Id2) gene, essential for generation of CD3-CD4+ lymphoid tissue-inducer (LTi) cells and development of secondary lymphoid organs, did not affect formation of tertiary lymphoid structures. Rather, mature CD3+CD4+ T cells were critical for the development of tertiary lymphoid structures. The initial stages of this process involved interaction of CD3+CD4+ T cells with DCs, the appearance of peripheral-node addressin-positive (PNAd+) vessels, and production of chemokines that recruit lymphocytes and DCs. These findings indicate that the formation of tertiary lymphoid structures does not require Id2-dependent conventional LTis but depends on a program initiated by mature CD3+CD4+ T cells.

Antibodies to myelin components are routinely detected in multiple sclerosis patients. However, their presence in some control subjects has made it difficult to determine their contribution to disease pathogenesis. Immunization of C57BL/6 mice with either rat or human myelin oligodendrocyte glycoprotein (MOG) leads to experimental autoimmune encephalomyelitis (EAE) and comparable titers of anti-MOG antibodies as detected by ELISA. However, only immunization with human (but not rat) MOG results in a B cell-dependent EAE. In this study, we demonstrate that these pathogenic and nonpathogenic anti-MOG antibodies have a consistent array of differences in their recognition of antigenic determinants and biological effects. Specifically, substituting proline at position 42 with serine in human MOG (as in rat MOG) eliminates the B cell requirement for EAE. All MOG proteins analyzed induced high titers of anti-MOG (tested by ELISA), but only antisera from mice immunized with unmodified human MOG were encephalitogenic in primed B cell-deficient mice. Nonpathogenic IgGs bound recombinant mouse MOG and deglycosylated MOG in myelin (tested by Western blot), but only pathogenic IgGs bound glycosylated MOG. Only purified IgG to human MOG bound to live rodent oligodendrocytes in culture and, after cross-linking, induced repartitioning of MOG into lipid rafts, followed by dramatic changes in cell morphology. The data provide a strong link betweenin vivoandin vitroobservations regarding demyelinating disease, further indicate a biochemical mechanism for anti-MOG-induced demyelination, and suggestin vitrotools for determining autoimmune antibody pathogenicity in multiple sclerosis patients.