RESUMEN
Germinal centers (GCs) are the primary sites of clonal B cell expansion and affinity maturation, directing the production of high-affinity antibodies. This response is a central driver of pathogenesis in autoimmune diseases, such as systemic lupus erythematosus (SLE), but the natural history of autoreactive GCs remains unclear. Here, we present a novel mouse model where the presence of a single autoreactive B cell clone drives the TLR7-dependent activation, expansion, and differentiation of other autoreactive B cells in spontaneous GCs. Once tolerance was broken for one self-antigen, autoreactive GCs generated B cells targeting other self-antigens. GCs became independent of the initial clone and evolved toward dominance of individual clonal lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease.
Asunto(s)
Linfocitos B/inmunología , Evolución Clonal , Centro Germinal/citología , Centro Germinal/inmunología , Tolerancia Inmunológica , Animales , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Linfocitos B/citología , Quimera/inmunología , Epítopos/inmunología , Riñón/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Systemic lupus erythematosus (SLE) is an incurable autoimmune disease characterized by autoantibody deposition in tissues such as kidney, skin and lungs. Notably, up to 75% of patients with SLE experience neuropsychiatric symptoms that range from anxiety, depression and cognitive impairment to seizures and, in rare cases, psychosis-collectively this is referred to as central nervous system (CNS) lupus. In some cases, certain autoantibodies, such as anti-NMDAR or anti-phospholipid antibodies, promote CNS lupus. However, in most patients, the mechanisms that underlie these symptoms are unknown. CNS lupus typically presents at lupus diagnosis or within the first year, suggesting that early factors contributing to peripheral autoimmunity may promote CNS lupus symptoms. Here we report behavioural phenotypes and synapse loss in lupus-prone mice that are prevented by blocking type I interferon (IFN) signalling. Furthermore, we show that type I IFN stimulates microglia to become reactive and engulf neuronal and synaptic material in lupus-prone mice. These findings and our observation of increased type I IFN signalling in post-mortem hippocampal brain sections from patients with SLE may instruct the evaluation of ongoing clinical trials of anifrolumab, a type I IFN-receptor antagonist. Moreover, identification of IFN-driven microglia-dependent synapse loss, along with microglia transcriptome data, connects CNS lupus with other CNS diseases and provides an explanation for the neurological symptoms observed in some patients with SLE.
Asunto(s)
Interferón Tipo I/inmunología , Vasculitis por Lupus del Sistema Nervioso Central/inmunología , Vasculitis por Lupus del Sistema Nervioso Central/patología , Microglía/inmunología , Microglía/patología , Sinapsis/patología , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Conducta Animal , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Interferón Tipo I/antagonistas & inhibidores , Vasculitis por Lupus del Sistema Nervioso Central/psicología , Masculino , Ratones , Microglía/metabolismo , Fenotipo , Transducción de Señal , Sinapsis/inmunología , TranscriptomaRESUMEN
Ig heavy chain (IgH) isotypes (e.g., IgM, IgG, and IgE) are generated as secreted/soluble antibodies (sIg) or as membrane-bound (mIg) B cell receptors (BCRs) through alternative RNA splicing. IgH isotype dictates soluble antibody function, but how mIg isotype influences B cell behavior is not well defined. We examined IgH isotype-specific BCR function by analyzing naturally switched B cells from wild-type mice, as well as by engineering polyclonal Ighγ1/γ1 and Ighε/ε mice, which initially produce IgG1 or IgE from their respective native genomic configurations. We found that B cells from wild-type mice, as well as Ighγ1/γ1 and Ighε/ε mice, produce transcripts that generate IgM, IgG1, and IgE in an alternative splice form bias hierarchy, regardless of cell stage. In this regard, we found that mIgµ > mIgγ1 > mIgε, and that these BCR expression differences influence respective developmental fitness. Restrained B cell development from Ighγ1/γ1 and Ighε/ε mice was proportional to sIg/mIg ratios and was rescued by enforced expression of the respective mIgs. In addition, artificially enhancing BCR signal strength permitted IgE+ memory B cells-which essentially do not exist under normal conditions-to provide long-lived memory function, suggesting that quantitative BCR signal weakness contributes to restraint of IgE B cell responses. Our results indicate that IgH isotype-specific mIg/BCR dosage may play a larger role in B cell fate than previously anticipated.
Asunto(s)
Linfocitos B/fisiología , Cambio de Clase de Inmunoglobulina , Inmunoglobulina E/metabolismo , Inmunoglobulina G/metabolismo , Cadenas Pesadas de Inmunoglobulina/metabolismo , Inmunoglobulina M/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Linfocitos B/citología , Femenino , Perfilación de la Expresión Génica , Inmunoglobulina E/genética , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulina M/genética , Masculino , RatonesRESUMEN
Unlike T cells that recognize digested peptides, B cells recognize their cognate antigen in its native form. The B cell receptor used in recognition can also be secreted to bind to antigens and initiate multiple effector functions such as phagocytosis, complement activation, or neutralization of receptors. While B cells can interact with soluble antigens, it is now clear that the presentation of membrane-bound antigen plays a crucial role in B cell activation, and in particular during affinity-maturation, the process during which high-affinity B cells are selected. In this review we discuss how native antigen is presented to B cells and its impact at several stages of B cell responses.
Asunto(s)
Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Linfocitos B/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Afinidad de Anticuerpos , Antígenos/inmunología , Selección Clonal Mediada por Antígenos , Activación de Complemento , Humanos , FagocitosisRESUMEN
The uptake of Ag-Ab immune complexes (IC) after the ligation of activating FcγR on dendritic cells (DC) leads to 100 times more efficient Ag presentation than the uptake of free Ags. FcγRs were reported to facilitate IC uptake and simultaneously induce cellular activation that drives DC maturation and mediates efficient T cell activation. Activating FcγRs elicit intracellular signaling via the ITAM domain of the associated FcRγ-chain. Studies with FcRγ-chain knockout (FcRγ(-/-)) mice reported FcRγ-chain ITAM signaling to be responsible for enhancing both IC uptake and DC maturation. However, FcRγ-chain is also required for surface expression of activating FcγRs, hampering the dissection of ITAM-dependent and independent FcγR functions in FcRγ(-/-) DCs. In this work, we studied the role of FcRγ-chain ITAM signaling using DCs from NOTAM mice that express normal surface levels of activating FcγR, but lack functional ITAM signaling. IC uptake by bone marrow-derived NOTAM DCs was reduced compared with wild-type DCs, but was not completely absent as in FcRγ(-/-) DCs. In NOTAM DCs, despite the uptake of ICs, both MHC class I and MHC class II Ag presentation was completely abrogated similar to FcRγ(-/-) DCs. Secretion of cytokines, upregulation of costimulatory molecules, and Ag degradation were abrogated in NOTAM DCs in response to FcγR ligation. Cross-presentation using splenic NOTAM DCs and prolonged incubation with OVA-IC was also abrogated. Interestingly, in this setup, proliferation of CD4(+) OT-II cells was induced by NOTAM DCs. We conclude that FcRγ-chain ITAM signaling facilitates IC uptake and is essentially required for cross-presentation, but not for MHC class II Ag presentation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Receptores de IgG/metabolismo , Animales , Complejo Antígeno-Anticuerpo/inmunología , Antígenos CD/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Reactividad Cruzada/genética , Endocitosis/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Estructura Terciaria de Proteína/genética , Receptores de IgG/genética , Transducción de SeñalRESUMEN
In autoreactive germinal centers (GC) initiated by a single rogue B cell clone, wild-type B cells expand and give rise to clones that target other autoantigens, known as epitope spreading. The chronic, progressive nature of epitope spreading calls for early interventions to limit autoimmune pathologies, but the kinetics and molecular requirements for wild-type B cell invasion and participation in GC remain largely unknown. With parabiosis and adoptive transfer approaches in a murine model of systemic lupus erythematosus, we demonstrate that wild-type B cells join existing GCs rapidly, clonally expand, persist, and contribute to autoantibody production and diversification. The invasion of autoreactive GCs by wild-type B cells required TLR7, B cell receptor specificity, antigen presentation, and type I interferon signaling. The adoptive transfer model provides a tool for identifying early events in the breaking of B cell tolerance in autoimmunity.
Asunto(s)
Linfocitos B , Lupus Eritematoso Sistémico , Ratones , Animales , Centro Germinal , Autoinmunidad , EpítoposRESUMEN
IgG FcRs are important mediators of immunity and play a key role during Ab-based immunotherapy. Within the leukocyte IgG receptor family, only FcγRI is capable of IgG binding with high affinity. FcγRI exists as a complex of a ligand binding α-chain and an FcR γ-chain. The receptors' α-chain can, furthermore, elicit several functions independent of the ITAM-bearing FcR γ-chain. Functional implications of high-affinity IgG binding and mechanisms underlying FcR γ-chain-independent signaling remain unclear to this day. In this paper, we provide an overview of past literature on FcγRI and address the implications of recently described interactions between cytosolic proteins and the FcγRI α-chain, as well as cytokine-enhanced FcγRI immune complex binding. Furthermore, an analysis of potential polymorphisms within the FCGR1A gene is provided.
Asunto(s)
Sitios de Unión de Anticuerpos , Receptores de IgG/fisiología , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/genética , Complejo Antígeno-Anticuerpo/fisiología , Sitios de Unión de Anticuerpos/genética , Modelos Animales de Enfermedad , Humanos , Datos de Secuencia Molecular , Unión Proteica/genética , Unión Proteica/inmunología , Estructura Terciaria de Proteína/genética , Receptores de IgG/química , Receptores de IgG/genéticaRESUMEN
Circumstantial evidence suggests that B cells may instruct T cells to break tolerance. Here, to test this hypothesis, we used a murine model in which a single B cell clone precipitates an autoreactive response resembling systemic lupus erythematosus (SLE). The initiating clone did not need to enter germinal centers to precipitate epitope spreading. Rather, it localized to extrafollicular splenic bridging channels early in the response. Autoantibody produced by the initiating clone was not sufficient to drive the autoreactive response. Subsequent epitope spreading depended on antigen presentation and was compartmentalized by major histocompatibility complex (MHC). B cells carrying two MHC haplotypes could bridge the MHC barrier between B cells that did not share MHC. Thus, B cells directly relay autoreactivity between two separate compartments of MHC-restricted T cells, leading to inclusion of distinct B cell populations in germinal centers. Our findings demonstrate that B cells initiate and propagate the autoimmune response.
Asunto(s)
Presentación de Antígeno , Lupus Eritematoso Sistémico , Ratones , Animales , Epítopos , Antígenos de Histocompatibilidad Clase II/genética , Linfocitos B , Complejo Mayor de Histocompatibilidad , Antígenos de HistocompatibilidadRESUMEN
FcγRI is the sole high-affinity immunoglobulin G (IgG) receptor on leukocytes. Its role in immunity and the clearance of opsonized particles has been challenged, as the receptor function may well be hindered by serum IgG. Here, we document immune complex binding by FcγRI to be readily enhanced by cytokine stimulation, whereas binding of monomeric IgG only modestly increased. Enhanced immune complex binding was independent of FcγRI surface expression levels. FcγRI, saturated with prebound IgG, was found capable of effective immune complex binding upon cytokine stimulation. Cytokine-enhanced binding was observed across a variety of immune complexes, including huIgG3- or mIgG2a-opsonized red blood cells, rituximab- or ofatumumab-opsonized B-cell lymphoma, and cetuximab-opsonized glioblastoma cells. This study contributes to our understanding of how FcγRI can participate in the clearance of opsonized particles despite saturation by monomeric IgG.
Asunto(s)
Complejo Antígeno-Anticuerpo/metabolismo , Citocinas/farmacología , Inmunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Animales , Línea Celular , Eritrocitos/inmunología , Glioblastoma/inmunología , Humanos , Linfoma de Células B/inmunología , Ratones , Proteínas Opsoninas/metabolismo , Unión ProteicaRESUMEN
BACKGROUND: CD20 monoclonal antibodies are widely used in clinical practice. Antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and direct cell death have been suggested to be important effector functions for CD20 antibodies. However, their specific contributions to the in vivo mechanism of action of CD20 immunotherapy have not been well defined. DESIGN AND METHODS: Here we studied the in vivo mechanism of action of type I (rituximab and ofatumumab) and type II (HuMab-11B8) CD20 antibodies in a peritoneal, syngeneic, mouse model with EL4-CD20 cells using low and high tumor burden. RESULTS: Interestingly, we observed striking differences in the in vivo mechanism of action of CD20 antibodies dependent on tumor load. In conditions of low tumor burden, complement was sufficient for tumor killing both for type I and type II CD20 antibodies. In contrast, in conditions of high tumor burden, activating FcγR (specifically FcγRIII), active complement and complement receptor 3 were all essential for tumor killing. Our data suggest that complement-enhanced antibody-dependent cellular cytotoxicity may critically affect tumor killing by CD20 antibodies in vivo. The type II CD20 antibody 11B8, which is a poor inducer of complement activation, was ineffective against high tumor burden. CONCLUSIONS: Tumor burden affects the in vivo mechanism of action of CD20 antibodies. Low tumor load can be eliminated by complement alone, whereas elimination of high tumor load requires multiple effector mechanisms.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos CD20 , Antineoplásicos/farmacología , Linfoma/tratamiento farmacológico , Carga Tumoral/efectos de los fármacos , Animales , Anticuerpos Monoclonales Humanizados , Humanos , Linfoma/patología , Ratones , Ratones Noqueados , Trasplante de Neoplasias , RituximabRESUMEN
Pathogenic autoantibodies contribute to tissue damage and clinical decline in autoimmune disease. Follicular T cells are central regulators of germinal centers, although their contribution to autoantibody-mediated disease remains unclear. Here we perform single cell RNA and T cell receptor (TCR) sequencing of follicular T cells in a mouse model of autoantibody-mediated disease, allowing for analyses of paired transcriptomes and unbiased TCRαß repertoires at single cell resolution. A minority of clonotypes are preferentially shared amongst autoimmune follicular T cells and clonotypic expansion is associated with differential gene signatures in autoimmune disease. Antigen prediction using algorithmic and machine learning approaches indicates convergence towards shared specificities between non-autoimmune and autoimmune follicular T cells. However, differential autoimmune transcriptional signatures are preserved even amongst follicular T cells with shared predicted specificities. These results demonstrate that follicular T cells are phenotypically distinct in B cell-driven autoimmune disease, providing potential therapeutic targets to modulate autoantibody development.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos B/inmunología , Perfilación de la Expresión Génica/métodos , Centro Germinal/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/metabolismo , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Células Clonales/inmunología , Células Clonales/metabolismo , Centro Germinal/citología , Centro Germinal/metabolismo , Ratones Endogámicos C57BL , Microscopía Confocal , RNA-Seq/métodos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de la Célula Individual/métodos , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Systemic lupus erythematosus (SLE) is a severe autoimmune disease mediated by pathogenic autoantibodies. While complement protein C4 is associated with SLE, its isoforms (C4A and C4B) are not equal in their impact. Despite being 99% homologous, genetic studies identified C4A as more protective than C4B. By generating gene-edited mouse strains expressing either human C4A or C4B and crossing these with the 564lgi lupus strain, we show that, overall, C4A-like 564Igi mice develop less humoral autoimmunity than C4B-like 564Igi mice. This includes a decrease in the number of GCs, autoreactive B cells, autoantibodies, and memory B cells. The higher efficiency of C4A in inducing self-antigen clearance is associated with the follicular exclusion of autoreactive B cells. These results explain how the C4A isoform is protective in lupus and suggest C4A as a possible replacement therapy in lupus.
Asunto(s)
Linfocitos B/inmunología , Complemento C4a/metabolismo , Lupus Eritematoso Sistémico/inmunología , Secuencia de Aminoácidos , Animales , Apoptosis , Autoanticuerpos/metabolismo , Autoantígenos/metabolismo , Secuencia de Bases , Complemento C4a/química , Complemento C4b/química , Complemento C4b/metabolismo , Modelos Animales de Enfermedad , Edición Génica , Humanos , Tolerancia Inmunológica , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
The C-terminal domain of protein 4.1G was identified to interact with the cytosolic tail of the high affinity IgG receptor, Fc gamma RI, in yeast two-hybrid screens. Proteins of the 4.1 family have previously been found to mediate receptor/cytoskeleton interactions. In the study presented here, we show an alternatively spliced 4.1G product to be associated with increased Fc gamma RI binding in yeast two-hybrid assays, and to be selectively enriched in most immune cells at the transcript level. In addition, a detailed analysis of the 4.1G 'docking site' within Fc gamma RI is provided by examining Fc gamma RI-CY-truncated and alanine-substituted mutants. These pointed to an Fc gamma RI membrane-proximal core motif of HxxBxxxBB (H represents hydrophobic residues, B basic residues and x represents any residue), followed by hydrophobic and (potentially) negatively charged residues to be central for interaction with protein 4.1G.
Asunto(s)
Secuencias de Aminoácidos , Citoplasma/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de IgG/química , Receptores de IgG/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Humanos , Inmunoprecipitación , Ratones , Datos de Secuencia Molecular , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
Epitope-focused approaches for selective clonal induction of broadly neutralizing antibodies (bnAbs) inform most current vaccine strategies for influenza virus and other rapidly evolving pathogens. The two conserved epitopes on the influenza hemagglutinin (HA) - the "stem" and the receptor-binding site (RBS) on the "head" - are the focus of the current "universal" influenza vaccine development efforts. Because stem-directed serum bnAbs are much less abundant than head-directed ones, we hypothesized that the HA stem bnAbs may be autoreactive and thus eliminated through the mechanisms of self-tolerance. We compared autoreactivity profiles of a set of stem and head-directed bnAbs. Most of the stem bnAbs we examined bound autoantigens; several showed staining of HEp-2 cells. A smaller proportion of the head-directed bnAbs were polyreactive. Gene usage did not correlate with autoreactivity. We suggest that complex foreign antigens may often have surface patches resembling some host epitope; our results indicate that HA stem epitopes resemble a host epitope more frequently than does the RBS.
Asunto(s)
Anticuerpos Antivirales/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Reacciones Antígeno-Anticuerpo , Autoantígenos/inmunología , Línea Celular , Epítopos/inmunología , Humanos , Virus de la Influenza A/metabolismo , Vacunas contra la Influenza/inmunología , Análisis por Matrices de ProteínasRESUMEN
Follicular dendritic cells (FDCs), a rare and enigmatic stromal cell type in the B cell follicles of secondary lymphoid organs, store and present antigen to B cells. While essential for germinal center (GC) responses, their exact role during GC B cell selection remains unknown. FDCs upregulate the inhibitory IgG Fc receptor FcγRIIB during GC formation. We show that the stromal deficiency of FcγRIIB does not affect GC B cell frequencies compared to wild-type mice. However, in the absence of FcγRIIB on FDCs, GCs show aberrant B cell selection during autoreactive and selective foreign antigen responses. These GCs are more diverse as measured by the AidCreERT2 -confetti system and show the persistence of IgM+ clones with decreased numbers of IgH mutations. Our results show that FDCs can modulate GC B cell diversity by the upregulation of FcγRIIB. Permissive clonal selection and subsequent increased GC diversity may affect epitope spreading during autoimmunity and foreign responses.
Asunto(s)
Células Dendríticas Foliculares/inmunología , Centro Germinal/inmunología , Receptores de IgG/genética , Animales , Diferenciación Celular , Humanos , RatonesRESUMEN
Absence of the mouse cell surface receptor SLAMF3 in SLAMF3-/- mice suggested that this receptor negatively regulates B cell homeostasis by modulating activation thresholds of B cell subsets. Here, we examine whether anti-SLAMF3 affects both B and T cell subsets during immune responses to haptenated ovalbumin [NP-OVA] and in the setting of chronic graft vs. host disease (cGVHD) induced by transferring B6.C-H2bm12/KhEg (bm12) CD4+ T cells into B6 WT mice. We find that administering αSLAMF3 to NP-OVA immunized B6 mice primarily impairs antibody responses and Germinal center B cell [GC B] numbers, whilst CXCR5+, PD-1+, and ICOS+ T follicular helper (TFH) cells are not significantly affected. By contrast, administering αSLAMF3 markedly enhanced autoantibody production upon induction of cGVHD by the transfer of bm12 CD4+ T cells into B6 recipients. Surprisingly, αSLAMF3 accelerated both the differentiation of GC B and donor-derived TFH cells initiated by cGVHD. The latter appeared to be induced by decreased numbers of donor-derived Treg and T follicular regulatory (TFR) cells. Collectively, these data show that control of anti-SLAMF3-induced signaling is requisite to prevent autoantibody responses during cGVHD, but reduces responses to foreign antigens.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Proliferación Celular , Enfermedad Injerto contra Huésped/inmunología , Transducción de Señal/inmunología , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/inmunología , Animales , Subgrupos de Linfocitos B/patología , Femenino , Centro Germinal/inmunología , Centro Germinal/patología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Ratones , Ratones Noqueados , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Follicular dendritic cells (FDCs) are stromal cells that are centrally located within B cell follicles of lymph nodes and other lymphoid organs such as the spleen. Due to their relative low abundance and difficulty to isolate, FDCs are still largely an enigma. Here we describe how to isolate FDCs for ex vivo cell culture, sorting by flow cytometry and how to load them in vivo or in vitro with immune complexes.