Human Lymphoid Organs Research Articles

Human implantable tertiary lymphoid organs (TLO) for solid tumor therapy: from organ chips to the clinic?

Abstract We previously created an in vitro human TLO model from patient-derived, circulating immune cells using organ-on-a-chip devices and 3D culture in extracellular matrix (ECM). B cells in these TLOs express activation induced cytidine deaminase, which is only expressed in lymphoid tissues and is required for class switching and somatic hypermutation (SHM). When challenged by vaccines, these TLO undergo SHM and produce antigen-specific antibodies and CD8 T cells. To model cancer associated TLOs, we integrated human pancreatic and lung cancer cell lines into these TLO Chips to understand how the immune context of hot and cold tumors differentially impacts TLO formation. The high PDL1/L2 expressing lung cancer cell line that displayed a “hot” phenotype in published murine studies induced high levels of cytokines and stimulated increased TLO formation in the lymphoid Organ Chip. In contrast, the cold, low PDL1/L2 expressing lung and pancreatic cell lines did not induce this cytokine signature or TLO formation. Importantly, TLO assembly correlated with increased B cell activation, anti-tumor CD8 activity, and tumor cell death. When the same mix of immune cells and ECM was injected in humanized mice bearing subcutaneous tumors from the hot lung cancer cell line, new lymphoid tissue containing dense aggregates of T and B cells was induced, showing for the first time that direct injection of synthetic TLOs at the tumor site may offer an alternative form of therapy for solid tumors.

Infection of the human lymphoid organ on-a-chip with Borrelia burgdorferi reveals novel mechanisms of immunosuppression and autoimmunity in Lyme disease

Infection of the human lymphoid organ on-a-chip with Borrelia burgdorferi reveals novel mechanisms of immunosuppression and autoimmunity in Lyme disease

Stochastic modeling of a gene regulatory network driving B cell development in germinal centers.

Germinal centers (GCs) are the key histological structures of the adaptive immune system, responsible for the development and selection of B cells producing high-affinity antibodies against antigens. Due to their level of complexity, unexpected malfunctioning may lead to a range of pathologies, including various malignant formations. One promising way to improve the understanding of malignant transformation is to study the underlying gene regulatory networks (GRNs) associated with cell development and differentiation. Evaluation and inference of the GRN structure from gene expression data is a challenging task in systems biology: recent achievements in single-cell (SC) transcriptomics allow the generation of SC gene expression data, which can be used to sharpen the knowledge on GRN structure. In order to understand whether a particular network of three key gene regulators (BCL6, IRF4, BLIMP1), influenced by two external stimuli signals (surface receptors BCR and CD40), is able to describe GC B cell differentiation, we used a stochastic model to fit SC transcriptomic data from a human lymphoid organ dataset. The model is defined mathematically as a piecewise-deterministic Markov process. We showed that after parameter tuning, the model qualitatively recapitulates mRNA distributions corresponding to GC and plasmablast stages of B cell differentiation. Thus, the model can assist in validating the GRN structure and, in the future, could lead to better understanding of the different types of dysfunction of the regulatory mechanisms.

Conserved stromal–immune cell circuits secure B cell homeostasis and function

B cell zone reticular cells (BRCs) form stable microenvironments that direct efficient humoral immunity with B cell priming and memory maintenance being orchestrated across lymphoid organs. However, a comprehensive understanding of systemic humoral immunity is hampered by the lack of knowledge of global BRC sustenance, function and major pathways controlling BRC–immune cell interactions. Here we dissected the BRC landscape and immune cell interactome in human and murine lymphoid organs. In addition to the major BRC subsets underpinning the follicle, including follicular dendritic cells, PI16+ RCs were present across organs and species. As well as BRC-produced niche factors, immune cell-driven BRC differentiation and activation programs governed the convergence of shared BRC subsets, overwriting tissue-specific gene signatures. Our data reveal that a canonical set of immune cell-provided cues enforce bidirectional signaling programs that sustain functional BRC niches across lymphoid organs and species, thereby securing efficient humoral immunity.

Functional specialization of short-lived and long-lived macrophage subsets in human tonsils.

Macrophages play a central role in tissue homeostasis and host defense. However, the properties of human macrophages in non-diseased tissues remain poorly understood. Here, we characterized human tonsil macrophages and identified three subsets with distinct phenotype, transcriptome, life cycle, and function. CD36hi macrophages were related to monocytes, while CD36lo macrophages showed features of embryonic origin and CD36int macrophages had a mixed profile. scRNA-seq on non-human primate tonsils showed that monocyte recruitment did not pre-exist an immune challenge. Functionally, CD36hi macrophages were specialized for stimulating T follicular helper cells, by producing Activin A. Combining reconstruction of ligand-receptor interactions and functional assays, we identified stromal cell-derived TNF-α as an inducer of Activin A secretion. However, only CD36hi macrophages were primed for Activin A expression, via the activity of IRF1. Our results provide insight into the heterogeneity of human lymphoid organ macrophages and show that tonsil CD36hi macrophage specialization is the result of both intrinsic features and interaction with stromal cells.

Anatomical and Histological Study of Neonatal Human Spleen

The spleen is the human body's largest lymphoid organ. The appearance of the spleen is inflated by a variety of conditions, such as cell abnormalities that lodge within the spleen or storage function disorders. As a result, the spleen's size can be used as a predictor of illness severity. Place and period of study: From July 2019 to May 2020, all of these samples were taken from autopsied bodies in the mortuary room of Kirkuk forensic medicine and the institute of forensic medicine in Baghdad, with legal authority. Study design: Cross-sectional descriptive study. Materials: The current study used 20 autopsy neonate human spleens ranging in age from 1 to 28 days that were collected from available dead undergoing post-mortem examination, were studied macroscopic and microscopic after staining with Hematoxylin and Eosin. Results: The number of notches in this study ranged from zero to five, however the top score of the specimens had one or two notches. The accessory spleen, on the other hand, was not found in the area the spleen's hilum. From the first day forward, the characteristic lymphoid follicle or white pulp could be recognized. During this time, nucleated red blood cells were observed. Purpose of study: Because no data on the standard dimensions of the normal spleen exists in the Iraqi population, the findings of this study may reveal differences in spleen morphology and provide valuable data for parameter standardization, It will be useful for correct clinical diagnosis and treatment of disease by physicians, surgeons, radiologists, and anatomists.

Tissue-resident regulatory T cells accumulate at human barrier lymphoid organs.

Regulatory T cells (Tregs) play a critical role in immune regulation and peripheral tolerance. While different types of Tregs have been identified in both mice and humans, much of our understanding about how these cells maintain immune homeostasis is derived from animal models. In this study, we examined two distinct human lymphoid organs to understand how repeated exposure to infections at the mucosal surface influences the phenotype and tissue localization of Tregs. We show that while Tregs in both tonsils and spleen express a tissue-resident phenotype, they accumulate in greater numbers in tonsils. Tonsillar-resident Tregs exhibit a highly suppressive phenotype with significantly increased expression of CD39, ICOS and CTLA-4 compared with their counterparts in circulation or in the spleen. Functionally, resident Tregs are able effectively to suppress T cell proliferation. We further demonstrate that tonsillar-resident Tregs share key features of T follicular helper cells. Spatial analysis reveals that the vast majority of resident Tregs are localized at the border of the T-zone and B cell follicle, as well as within the lymphocyte pockets enriched with resident memory T cells. Together our findings suggest that resident Tregs are strategically co-localized to maintain immune homeostasis at sites of recurrent inflammation.

Abstract 5621: Humanized rat model

Abstract The development of Novel cancer immunotherapies has relied on the use of immune humanized mice. The field of study can be enhanced using an immune humanized rat, which will provide several advantages over the currently available humanized mouse models. The SRG OncoRat®; A Rag2/Il2rg double-knockout rat supports the growth of larger tumors for serial fine needle biopsies to assess immune infiltration and serial blood draws for assessing human immune development and tumor biomarkers in real-time throughout an efficacy study. Using this rat, we developed a novel autologous human skin and immune cells-humanized rat model by co-engrafting full-thickness human-fetal skin and autologous fetal lymphoid organoids under the kidney capsule along with intravenous injection of autologous fetal-liver derived hematopoietic stem cells, thus termed, human skin-immune system humanized rat model (hSIS-humanized rat). We demonstrated the development of adult-like, full-thickness human skin and human lymphoid organoids along with human immune cells. Methicillin-resistant Staphylococcus aureus inoculation in the human skin results in infection and skin pathology, thus recapitulating clinical outcomes. This model will enable in vivo mechanistic studies for development and evaluation of novel therapeutics for skin infectious disease and may also provide a model for establishing skin grafts of patient-derived melanoma tumors to investigate melanoma metastasis and response to therapies. In addition, engrafting the rat with human lymphoid organs and human immune cells may provide a similar platform to the BLT mouse for immunotherapy studies. Furthermore, we demonstrated the presence of Human CD45+, CD3+, and CD20+ cells in peripheral blood, spleen, and bone marrow of the SRG OncoRat® engrafted with human PBMCs, hence humanizing the rat's immune system. These immune humanized rat models may be beneficial for evaluating immunotherapies in human cancer models, including assessment of immune cell infiltration through fine needle biopsies. Citation Format: Bisoye Towobola Adedeji, Fallon K. Noto, Tseten Yeshi Jamling, Yash Argawal, Cole Jamison Beatty, Sara Grace Ho, Antu Das, Rajeev Kishore Salunke, Moses Turkle Bility. Humanized rat model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5621.

Silent Infection of B and CD8+ T Lymphocytes by Influenza A Virus in Children with Tonsillar Hypertrophy.

Influenza A viruses (IAVs) cause more than 2 million annual episodes of seasonal acute respiratory infections (ARI) and approximately 500,000 deaths worldwide. Depending on virus strain and host immune status, acute infections by IAV may reach sites other than the respiratory tract. In the present study, IAV RNA and antigens were searched for in tissues of palatine tonsils and adenoids removed from patients without ARI symptoms. A real-time reverse transcriptase PCR (RT-PCR) screening revealed that 8 tissue samples from 7 patients out of 103 were positive for IAV. Positive samples were subjected to next-generation sequencing (NGS) and 3 of 8 tissues yielded complete IAV pH1N1 genomes, whereas in 5 samples, the PB1 gene was not fully assembled. Phylogenetic analysis placed tonsil-derived IAV in clusters clearly segregated from contemporaneous Brazilian viruses. Flow cytometry of dispersed tissue fragments and serial immunohistochemistry of paraffin-embedded sections of naturally infected biopsies indicated that CD20+ B lymphocytes, CD8+ T lymphocytes, and CD11c+ cells are susceptible to IAV infection. We sought to investigate whether these lymphoid tissues could be sites of viral replication and sources of viable virus particles. MDCK cells were inoculated with tissue lysates, enabling recovery of one IAV isolate confirmed by immunofluorescence, reverse transcriptase quantitative PCR (RT-qPCR), and NGS. The data indicate that lymphoid tissues not only harbor expression of IAV proteins but also contain infectious virus. Asymptomatic long-term infection raises the possibility of IAV shedding from tonsils, which may have an impact on host-to-host transmission.IMPORTANCE Influenza A virus (IAV) infections are important threats to human health worldwide. Although extensively studied, some aspects of virus pathogenesis and tissue tropism remain unclear. Here, by different strategies, we describe the asymptomatic infection of human lymphoid organs by IAV in children. Our results indicate that IAV was not only detected and isolated from human tonsils but displayed unique genetic features in comparison with those of contemporaneous IAVs circulating in Brazil and detected in swabs and nasal washes. Inside the tissue microenvironment, immune cells were shown to be carrying IAV antigens, especially B and T CD8+ lymphocytes. Taken together, these results suggest that human lymphoid tissues can be sites of silent IAV infections with possible impact on virus shedding to the population.

Abstract A007: A Rag2/Il2rg double-knockout rat supports engraftment of human immune system for immunotherapy-based cancer efficacy studies

Abstract Immune humanized mice have been valuable in the development of novel cancer immunotherapies and have demonstrated stronger efficacy when combined with standard of care chemotherapy. An immune humanized rat could provide several advantages over the currently available humanized mouse models, including supporting the growth of larger tumors for serial fine needle biopsies to assess immune infiltration and serial blood draws for assessing human immune development and tumor biomarkers in real-time throughout an efficacy study. We developed a novel autologous human skin and immune cells-humanized rat model by co-engrafting full-thickness human-fetal skin and autologous fetal lymphoid organoids under the kidney capsule along with intravenous injection of autologous fetal-liver derived hematopoietic stem cells, thus termed, human skin-immune system humanized rat model (hSIS-humanized rat). hSIS-humanized rat support development of adult-like, full-thickness human skin and human lymphoid organoids along with human immune cells. Methicillin-resistant Staphylococcus aureus inoculation in the human skin results in infection and skin pathology, thus recapitulating clinical outcomes. This model will enable in vivo mechanistic studies for development and evaluation of novel therapeutics for skin infectious disease and may also provide a model for establishing skin grafts of patient-derived melanoma tumors to investigate melanoma metastasis and response to therapies. In addition, engrafting the rat with human lymphoid organs and human immune cells may provide a similar platform to the BLT mouse for immunotherapy studies. Finally, we have demonstrated humanization of the rat immune system using human PBMCs. Human CD45+, CD3+, and CD20+ cells can be found in the peripheral blood, spleen, and bone marrow of engrafted rats. These immune humanized rat models may be beneficial for evaluating immunotherapies in human cancer models, including assessment of immune cell infiltration through fine needle biopsies. Citation Format: Fallon K Noto, Bisoye Towobola Adedeji, Yash Agarwal, Cole Beatty, Sara Ho, Antu Das, Rajeev Salunke, Moses Bility, Tseten Yeshi Jamling. A Rag2/Il2rg double-knockout rat supports engraftment of human immune system for immunotherapy-based cancer efficacy studies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A007. doi:10.1158/1535-7163.TARG-19-A007

Human lymphoid organ cDC2 and macrophages play complementary roles in T follicular helper responses.

CD4+ T follicular helper (Tfh) cells are essential for inducing efficient humoral responses. T helper polarization is classically orientated by dendritic cells (DCs), which are composed of several subpopulations with distinct functions. Whether human DC subsets display functional specialization for Tfh polarization remains unclear. Here we find that tonsil cDC2 and CD14+ macrophages are the best inducers of Tfh polarization. This ability is intrinsic to the cDC2 lineage but tissue dependent for macrophages. We further show that human Tfh cells comprise two effector states producing either IL-21 or CXCL13. Distinct mechanisms drive the production of Tfh effector molecules, involving IL-12p70 for IL-21 and activin A and TGFβ for CXCL13. Finally, using imaging mass cytometry, we find that tonsil CD14+ macrophages localize in situ in the B cell follicles, where they can interact with Tfh cells. Our results indicate that human lymphoid organ cDC2 and macrophages play complementary roles in the induction of Tfh responses.

Altered distribution and function of splenic innate lymphoid cells in adult chronic immune thrombocytopenia

Innate lymphoid cells (ILCs) have been characterized as innate immune cells capable to modulate the immune response in the mucosae. Human ILCs have been rarely described in secondary lymphoid organs except in tonsils. Moreover, their function and phenotype in human secondary lymphoid organs during autoimmune diseases have never been studied. We took advantage of splenectomy as a treatment of immune thrombocytopenia (ITP) to describe and compare splenic ILC from 18 ITP patients to 11 controls. We first confirmed that ILC3 represented the most abundant ILC subset in human non-inflamed spleens, accounting for 90% of total ILC, and that they were mostly constituted of NKp44- cells. On the contrary, proportions of ILC1 and ILC2 in spleens were lower than in blood. Splenic IL-2- and IFN-γ-producing ILC1 were increased in ITP. While the frequencies of total splenic ILC3 were similar in the two groups, splenic GM-CSF-producing ILC3 were increased in ITP.This is the first description of human ILC in a major secondary lymphoid organ during an autoimmune disease, ITP. We observed an expansion of splenic ILC1 that could participate to the Th1 skewing, while the increased production of GM-CSF by splenic ILC3 could stimulate splenic macrophages which play a key role in ITP pathophysiology.

Ultra-Sensitive HIV-1 Latency Viral Outgrowth Assays Using Humanized Mice.

In the current quest for a complete cure for HIV/AIDS, highly sensitive HIV-1 latency detection methods are critical to verify full viral eradication. Until now, the in vitro quantitative viral outgrowth assays (qVOA) have been the gold standard for assessing latent HIV-1 viral burden. However, these assays have been inadequate in detecting the presence of ultralow levels of latent virus in a number of patients who were initially thought to have been cured, but eventually showed viral rebound. In this context, new approaches utilizing in vivo mouse-based VOAs are promising. In the murine VOA (mVOA), large numbers of CD4+ T cells or PBMC from aviremic subjects are xenografted into immunodeficient NSG mice, whereas in the humanized mouse-based VOA (hmVOA) patient CD4+ T cell samples are injected into BLT or hu-hematopoetic stem cells (hu-HSC) humanized mice. While latent virus could be recovered in both of these systems, the hmVOA provides higher sensitivity than the mVOA using a fewer number of input cells. In contrast to the mVOA, the hmVOA provides a broader spectrum of highly susceptible HIV-1 target cells and enables newly engrafted cells to home into preformed human lymphoid organs where they can infect cells in situ after viral activation. Hu-mice also allow for both xenograft- and allograft-driven cell expansions with less severe GvH providing a longer time frame for potential viral outgrowth from cells with a delayed latent viral activation. Based on these advantages, the hmVOA has great potential in playing an important role in HIV-1 latency and cure research.

Infection and depletion of CD4+ group-1 innate lymphoid cells by HIV-1 via type-I interferon pathway.

Innate lymphoid cells (ILCs) are severely depleted during chronic HIV-1 infection by unclear mechanisms. We report here that human ILC1s comprising of CD4+ and CD4- subpopulations were present in various human lymphoid organs but with different transcription programs and functions. Importantly, CD4+ ILC1s expressed HIV-1 co-receptors and were productively infected by HIV-1 in vitro and in vivo. Furthermore, chronic HIV-1 infection activated and depleted both CD4+ and CD4- ILC1s, and impaired their cytokine production activity. Highly active antiretroviral (HAART) therapy in HIV-1 patients efficiently rescued the ILC1 numbers and reduced their activation, but failed to restore their functionality. We also found that blocking type-I interferon (IFN-I) signaling during HIV-1 infection in vivo in humanized mice prevented HIV-1 induced depletion or apoptosis of ILC1 cells. Therefore, we have identified the CD4+ ILC1 cells as a new target population for HIV-1 infection, and revealed that IFN-I contributes to the depletion of ILC1s during HIV-1 infection.

The expansion in lymphoid organs of IL-4+ BATF+ T follicular helper cells is linked to IgG4 class switching in vivo.

Distinct T follicular helper (TFH) subsets that influence specific class-switching events are assumed to exist, but the accumulation of isotype-specific TFH subsets in secondary lymphoid organs (SLOs) and tertiary lymphoid organs has not been hitherto demonstrated. IL-4-expressing TFH cells are surprisingly sparse in human SLOs. In contrast, in IgG4-related disease (IgG4-RD), a disorder characterized by polarized Ig class switching, most TFH cells in tertiary and SLOs make IL-4. Human IL-4+ TFH cells do not express GATA-3 but express nuclear BATF, and the transcriptomes of IL-4-secreting TFH cells differ from both PD1hi TFH cells that do not secrete IL-4 and IL-4-secreting non-TFH cells. Unlike IgG4-RD, IL-4+ TFH cells are rarely found in tertiary lymphoid organs in Sjögren's syndrome, a disorder in which IgG4 is not elevated. The proportion of CD4+IL-4+BATF+ T cells and CD4+IL-4+CXCR5+ T cells in IgG4-RD tissues correlates tightly with tissue IgG4 plasma cell numbers and plasma IgG4 levels in patients but not with the total plasma levels of other isotypes. These data describe a disease-related TFH subpopulation in human tertiary lymphoid organs and SLOs that is linked to IgG4 class switching.

Cross-Tissue Transcriptomic Analysis of Human Secondary Lymphoid Organ-Residing ILC3s Reveals a Quiescent State in the Absence of Inflammation

A substantial number of human and mouse group 3 innate lymphoid cells (ILC3s) reside in secondary lymphoid organs, yet the phenotype and function of these ILC3s is incompletely understood. Here, we employed an unbiased cross-tissue transcriptomic approach to compare human ILC3s from non-inflamed lymph nodes and spleen to their phenotypic counterparts in inflamed tonsils and from circulation. These analyses revealed that, in the absence of inflammation, lymphoid organ-residing ILC3s lack transcription of cytokines associated with classical ILC3 functions. This was independent of expression of the natural cytotoxicity receptor NKp44. However, and in contrast to ILC3s from peripheral blood, lymphoid organ-residing ILC3s express activating cytokine receptors and have acquired the ability to be recruited into immune responses by inflammatory cytokines. This comprehensive cross-tissue dataset will allow for identification of functional changes in human lymphoid organ ILC3s associated with human disease.

Fibroblastic reticular cells of the lymphoid tissues modulate T cell activation threshold during homeostasis via hyperactive cyclooxygenase-2/prostaglandin E2 axis

Fibroblastic reticular cells (FRCs) in the T cell zone of lymph nodes (LNs) are pivotal for T cell survival, mobility, and peripheral tolerance. Here, we demonstrate that during homeostasis, FRCs also suppress T cell activation via producing high level of prostaglandin E2 (PGE2) due to their thousands-fold higher cyclooxygenase-2 (COX-2) expression than immune cells. This hyperactive COX-2/PGE2-induced suppression is evident during antigen-specific and non-antigen-specific activations. It is implicated as suppressed TCR-signaling cascades, reduced alterations in activation markers, and inhibited cytokine production of freshly isolated T cells or T cells co-cultured with FRCs compared with those cultured without FRCs. Different from T cell dysfunction, this FRC-mediated suppression is surmountable by enhancing the strength of stimulation and is reversible by COX-2 inhibitors. Furthermore, T cells in the FRC environment where Cox-2 is genetic inactivated are more sensitive and rapidly activated upon stimulations than those in WT environment. Significantly, FRCs of human lymphoid organs manifest similar COX-2/PGE2 hyperactivity and T cell suppression. Together, this study identifies a previously unappreciated intrinsic mechanism of FRCs shared between mice and humans for suppressing T cell sensitivity to activation via PGE2, underscoring the importance of FRCs in shaping the suppressive milieu of lymphoid organs during homeostasis.

Hematopoietic stem cell transplantations for primary immune deficiencies associated with NFκB mutations: A review of the literature

The nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) family of transcription factors plays an instrumental role in human immunity and lymphoid organ development. Inherited defects affecting these factors or their regulation are associated with increased susceptibility to infections, as well as non-immune abnormalities. Hematopoietic stem cell transplantations (HSCT) have been shown to correct the immune abnormalities in a few patients with NFκB pathway defects. Here we review the pre-HSCT characteristics, as well as the HSCT and outcome of 35 patients who received HSCT for NFκB defects. Twenty-three patients (65.7%) were reported to have survived HSCT. Survival was higher among patients with X-linked ectodermal dysplasia and immunodeficiency (XL-EDA-ID), and those with CARD11-BCL10-MALT1 (CBM) complex defects, in comparison to patients with autosomal dominant ectodermal dysplasia and immunodeficiency (AD-EDA-ID) and IKBKB defects. Survival following myeloablative conditioning was simi...

Characterization of human fibroblastic reticular cells as potential immunotherapeutic tools

Fibroblastic reticular cells (FRCs) are essential players during adaptive immune responses not only as a structural support for the encounter of antigen-presenting cells and naive T lymphocytes but also as a source of modulatory signals. However, little is known about this cell population in humans. To address the phenotypical and functional analysis of human FRCs here we established splenic (SP) and mesenteric lymph node (LN) CD45-CD31-CD90+podoplanin+ myofibroblastic cell cultures. They shared the phenotypical characteristics distinctive of FRCs, including the expression of immunomodulatory factors and peripheral tissue antigens. Nevertheless, human FRCs also showed particular features, some differing from mouse FRCs, like the lack of nitric oxide synthase (NOS2) expression after interferon (IFN)γstimulation. Interestingly, SP-FRCs expressed higher levels of interleukin (IL)-6, BMP4, CCL2, CXCL12 and Notch molecules, and strongly adapted their functional profile to lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (Poly I:C) and IFNγ stimulation. In contrast, we found higher expression of transforming growth factor (TGF)β and Activin A in LN-FRCs that barely responded via Toll-Like Receptor (TLR)3 and constitutively expressed retinaldehyde dehydrogenase 1 enzyme, absent in SP-FRCs. This study reveals human FRCs can be valuable models to increase our knowledge about the physiology of human secondary lymphoid organs in health and disease and to explore the therapeutic options of FRCs.

Analysis of the Germinal Center Reaction in Tissue Sections.

Germinal centers are short-lived microanatomical compartments with essential roles in adaptive immunity. These lymphoid structures can be identified in secondary lymphoid organs using both flow cytometry and immunohistological analyses, but only the latter provides useful architectural and spatial information. Here we describe how to use immunofluorescence and immunohistochemistry with specific antibodies to precisely highlight the cellular and architectural features of germinal centers, both in human and mouse secondary lymphoid organs, and to study their normal development and disturbance in disease.