Posttransplant Tertiary Lymphoid Organs.

Tertiary lymphoid organs (TLOs), also known as tertiary or ectopic lymphoid structures or tissues, are accumulations of lymphoid cells in sites other than canonical lymphoid organs, that arise through lymphoid neogenesis during chronic inflammation in autoimmunity, microbial infection, cancer, aging, and transplantation, the focus of this review. Lymph nodes and TLOs are compared regarding their cellular composition, organization, vascular components, and migratory signal regulation. These characteristics of posttransplant TLOs (PT-TLOs) are described with individual examples in a wide range of organs including heart, kidney, trachea, lung, artery, skin, leg, hand, and face, in many species including human, mouse, rat, and monkey. The requirements for induction and maintenance of TLOs include sustained exposure to autoantigens, alloantigens, tumor antigens, ischemic reperfusion, nephrotoxic agents, and aging. Several staging schemes have been put forth regarding their function in organ rejection. PT-TLOs most often are associated with organ rejection, but in some cases contribute to tolerance. The role of PT-TLOs in cancer is considered in the case of immunosuppression. Furthermore, TLOs can be associated with development of lymphomas. Challenges for PT-TLO research are considered regarding staging, imaging, and opportunities for their therapeutic manipulation to inhibit rejection and encourage tolerance.

Regulation, Maintenance, and Remodeling of High Endothelial Venules in Homeostasis, Inflammation, and Cancer.

High endothelial venules (HEVs), high walled cuboidal blood vessels, through their expression of adhesion molecules and chemokines, allow the entrance of lymphoid cells into primary, secondary, and tertiary lymphoid structures (aka tertiary lymphoid organs). HEV heterogeneity exists between various lymphoid organs in their expression of peripheral node addressin (PNAd) and mucosal vascular addressin adhesion molecule 1(MAdCAM-1). Transcriptomic analyses reveal extensive heterogeneity, plasticity, and regulation of HEV gene expression in ontogeny, acute inflammation, and chronic inflammation within and between lymphoid organs. Rules regulating HEV development are flexible in inflammation. HEVs in tumor tertiary lymphoid structures are diagnostic of favorable clinical outcome and response to Immunotherapy, including immune check point blockade. Immunotherapy induces HEVs and provides an entrance for naïve, central memory, and effector cells and a niche for stem like precursor cells. Understanding HEV regulation will permit their exploitation as routes for drug delivery to autoimmune lesions, rejecting organs, and tumors.

The durability of immunity against reinfection by SARS-CoV-2: a comparative evolutionary study.

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.

Mechanistic basis of post-treatment control of SIV after anti-α4ß7 antibody therapy.

Treating macaques with an anti-α4ß7 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-α4ß7 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-α4ß7 antibody operating in these treated animals. We show that the model can fit the longitudinal viral load data from both IgG control and anti-α4ß7 antibody treated macaques, suggesting explanations for the viral control associated with cART and an anti-α4ß7 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-α4ß7 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.

Basics of Inducible Lymphoid Organs.

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 LTαß 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.

Lymphotoxin targeted to salivary and lacrimal glands induces tertiary lymphoid organs and cervical lymphadenopathy and reduces tear production.

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

Aging Induces an Nlrp3 Inflammasome-Dependent Expansion of Adipose B Cells That Impairs Metabolic Homeostasis.

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.

High Endothelial Venules and Lymphatic Vessels in Tertiary Lymphoid Organs: Characteristics, Functions, and Regulation.

High endothelial venules (HEVs) and lymphatic vessels (LVs) are essential for the function of the immune system, by providing communication between the body and lymph nodes (LNs), specialized sites of antigen presentation and recognition. HEVs bring in naïve and central memory cells and LVs transport antigen, antigen-presenting cells, and lymphocytes in and out of LNs. Tertiary lymphoid organs (TLOs) are accumulations of lymphoid and stromal cells that arise and organize at ectopic sites in response to chronic inflammation in autoimmunity, microbial infection, graft rejection, and cancer. TLOs are distinguished from primary lymphoid organs - the thymus and bone marrow, and secondary lymphoid organs (SLOs) - the LNs, spleen, and Peyer's patches, in that they arise in response to inflammatory signals, rather than in ontogeny. TLOs usually do not have a capsule but are rather contained within the confines of another organ. Their structure, cellular composition, chemokine expression, and vascular and stromal support resemble SLOs and are the defining aspects of TLOs. T and B cells, antigen-presenting cells, fibroblast reticular cells, and other stromal cells and vascular elements including HEVs and LVs are all typical components of TLOs. A key question is whether the HEVs and LVs play comparable roles and are regulated similarly to those in LNs. Data are presented that support this concept, especially with regard to TLO HEVs. Emerging data suggest that the functions and regulation of TLO LVs are also similar to those in LNs. These observations support the concept that TLOs are not merely cellular accumulations but are functional entities that provide sites to generate effector cells, and that their HEVs and LVs are crucial elements in those activities.

Dendritic cells maintain dermal adipose-derived stromal cells in skin fibrosis.

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 ß (LTß) expression in DCs maintained ADSC survival in fibrotic skin by activating an LTß receptor/ß1 integrin (LTßR/ß1 integrin) pathway on ADSCs. Stimulation of LTßR 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.

The lymphotoxin ß receptor is a potential therapeutic target in renal inflammation.

Accumulation of inflammatory cells in different renal compartments is a hallmark of progressive kidney diseases including glomerulonephritis (GN). Lymphotoxin ß receptor (LTßR) signaling is crucial for the formation of lymphoid tissue, and inhibition of LTßR signaling has ameliorated several non-renal inflammatory models. Therefore, we tested whether LTßR signaling could also have a role in renal injury. Renal biopsies from patients with GN were found to express both LTα and LTß ligands, as well as LTßR. The LTßR protein and mRNA were localized to tubular epithelial cells, parietal epithelial cells, crescents, and cells of the glomerular tuft, whereas LTß was found on lymphocytes and tubular epithelial cells. Human tubular epithelial cells, mesangial cells, and mouse parietal epithelial cells expressed both LTα and LTß mRNA upon stimulation with TNF in vitro. Several chemokine mRNAs and proteins were expressed in response to LTßR signaling. Importantly, in a murine lupus model, LTßR 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 LTßR signaling is involved in renal injury and may be a suitable therapeutic target in renal diseases.

A dendritic-cell-stromal axis maintains immune responses in lymph nodes.

Within secondary lymphoid tissues, stromal reticular cells support lymphocyte function, and targeting reticular cells is a potential strategy for controlling pathogenic lymphocytes in disease. However, the mechanisms that regulate reticular cell function are not well understood. Here we found that during an immune response in lymph nodes, dendritic cells (DCs) maintain reticular cell survival in multiple compartments. DC-derived lymphotoxin beta receptor (LTßR) ligands were critical mediators, and LTßR signaling on reticular cells mediated cell survival by modulating podoplanin (PDPN). PDPN modulated integrin-mediated cell adhesion, which maintained cell survival. This DC-stromal axis maintained lymphocyte survival and the ongoing immune response. Our findings provide insight into the functions of DCs, LTßR, and PDPN and delineate a DC-stromal axis that can potentially be targeted in autoimmune or lymphoproliferative diseases.

Lymphatic vessels and tertiary lymphoid organs.

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.

Lymphotoxin and TNF: how it all began-a tribute to the travelers.

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.

Cell-selective knockout and 3D confocal image analysis reveals separate roles for astrocyte-and endothelial-derived CCL2 in neuroinflammation.

BACKGROUND: Expression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood-brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s). METHODS: Mice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or endothelial cells (Endo KO) were used to evaluate the respective contributions of these sources to neuroinflammation, i.e., clinical disease progression, BBB damage, and parenchymal leukocyte invasion in a myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE model. High-resolution 3-dimensional (3D) immunofluorescence confocal microscopy and colloidal gold immuno-electron microscopy were employed to confirm sites of CCL2 expression, and 3D immunofluorescence confocal microscopy utilized to assess inflammatory responses along the CNS microvasculature. RESULTS: Cell-selective loss of CCL2 immunoreactivity was demonstrated in the respective KO mice. Compared to wild-type (WT) mice, Astro KO mice showed reduced EAE severity but similar onset, while Endo KO mice displayed near normal severity but significantly delayed onset. Neither of the KO mice showed deficits in T cell proliferation, or IL-17 and IFN-γ production, following MOG35-55 exposure in vitro, or altered MOG-major histocompatibility complex class II tetramer binding. 3D confocal imaging further revealed distinct actions of the two CCL2 pools in the CNS. Astro KOs lacked the CNS leukocyte penetration and disrupted immunostaining of CLN-5 at the BBB seen during early EAE in WT mice, while Endo KOs uniquely displayed leukocytes stalled in the microvascular lumen. CONCLUSIONS: These results point to astrocyte and endothelial pools of CCL2 each regulating different stages of neuroinflammation in EAE, and carry implications for drug delivery in neuroinflammatory disease.

A humanized mouse model of autoimmune insulitis.

Many mechanisms of and treatments for type 1 diabetes studied in the NOD mouse model have not been replicated in human disease models. Thus, the field of diabetes research remains hindered by the lack of an in vivo system in which to study the development and onset of autoimmune diabetes. To this end, we characterized a system using human CD4(+) T cells pulsed with autoantigen-derived peptides. Six weeks after injection of as few as 0.5 × 10(6) antigen-pulsed cells into the NOD-Scid Il2rg(-/-) mouse expressing the human HLA-DR4 transgene, infiltration of mouse islets by human T cells was seen. Although islet infiltration occurred with both healthy and diabetic donor antigen-pulsed CD4(+) T cells, diabetic donor injections yielded significantly greater levels of insulitis. Additionally, significantly reduced insulin staining was observed in mice injected with CD4(+) T-cell lines from diabetic donors. Increased levels of demethylated ß-cell-derived DNA in the bloodstream accompanied this loss of insulin staining. Together, these data show that injection of small numbers of autoantigen-reactive CD4(+) T cells can cause a targeted, destructive infiltration of pancreatic ß-cells. This model may be valuable for understanding mechanisms of induction of human diabetes.

Identification of a new stromal cell type involved in the regulation of inflamed B cell follicles.

Lymph node (LN) stromal cells provide survival signals and adhesive substrata to lymphocytes. During an immune response, B cell follicles enlarge, questioning how LN stromal cells manage these cellular demands. Herein, we used a murine fate mapping system to describe a new stromal cell type that resides in the T cell zone of resting LNs. We demonstrated that upon inflammation, B cell follicles progressively trespassed into the adjacent T cell zone and surrounded and converted these stromal cells into CXCL13 secreting cells that in return delineated the new boundaries of the growing follicle. Acute B cell ablation in inflamed LNs abolished CXCL13 secretion in these cells, while LT-ß deficiency in B cells drastically affected this conversion. Altogether, we reveal the existence of a dormant stromal cell subset that can be functionally awakened by B cells to delineate the transient boundaries of their expanding territories upon inflammation.

Lymphatic vessel function in head and neck inflammation.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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 .

Follicular dendritic cells, conduits, lymphatic vessels, and high endothelial venules in tertiary lymphoid organs: Parallels with lymph node stroma.

In this communication, the contribution of stromal, or non-hematopoietic, cells to the structure and function of lymph nodes (LNs), as canonical secondary lymphoid organs (SLOs), is compared to that of tertiary lymphoid tissue or organs (TLOs), also known as ectopic lymphoid tissues. TLOs can arise in non-lymphoid organs during chronic inflammation, as a result of autoimmune responses, graft rejection, atherosclerosis, microbial infection, and cancer. The stromal components found in SLOs including follicular dendritic cells, fibroblast reticular cells, lymphatic vessels, and high endothelial venules and possibly conduits are present in TLOs; their molecular regulation mimics that of LNs. Advances in visualization techniques and the development of transgenic mice that permit in vivo real time imaging of these structures will facilitate elucidation of their precise functions in the context of chronic inflammation. A clearer understanding of the inflammatory signals that drive non-lymphoid stromal cells to reorganize into TLO should allow the design of therapeutic interventions to impede the progression of autoimmune activity, or alternatively, to enhance anti-tumor responses.

Tertiary lymphoid organ development coincides with determinant spreading of the myelin-specific T cell response.

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.