RESUMEN
Immune cells rely on transient physical interactions with other immune and non-immune populations to regulate their function1. To study these 'kiss-and-run' interactions directly in vivo, we previously developed LIPSTIC (labelling immune partnerships by SorTagging intercellular contacts)2, an approach that uses enzymatic transfer of a labelled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ T helper cells and antigen-presenting cells, however. Here we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the steady-state cellular partners of regulatory T cells and identify germinal centre-resident T follicular helper cells on the basis of their ability to interact cognately with germinal centre B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalogue of the immune populations that physically interact with intestinal epithelial cells at the steady state and profile the evolution of the interactome of lymphocytic choriomeningitis virus-specific CD8+ T cells in multiple organs following systemic infection. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.
Asunto(s)
Linfocitos B , Linfocitos T CD8-positivos , Comunicación Celular , Células Dendríticas , Células Epiteliales , Células T Auxiliares Foliculares , Linfocitos T Reguladores , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Comunicación Celular/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Ligandos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Células T Auxiliares Foliculares/citología , Células T Auxiliares Foliculares/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Centro Germinal/citología , Análisis de Expresión Génica de una Sola Célula , Células Epiteliales/citología , Células Epiteliales/inmunología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Especificidad de ÓrganosRESUMEN
Cellular interactions are essential for tissue organization and functionality. In particular, immune cells rely on direct and usually transient interactions with other immune and non-immune populations to specify and regulate their function. To study these "kiss-and-run" interactions directly in vivo, we previously developed LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts), an approach that uses enzymatic transfer of a labeled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ helper T cells and antigen presenting cells, however. Here, we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the cellular partners of regulatory T cells in steady state, and identify germinal center (GC)-resident T follicular helper (Tfh) cells based on their ability to interact cognately with GC B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalog of the immune populations that physically interact with intestinal epithelial cells (IECs) and find evidence of stepwise acquisition of the ability to interact with IECs as CD4+ T cells adapt to residence in the intestinal tissue. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.
RESUMEN
Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures.
Asunto(s)
Linfocitos B , Centro Germinal , Infecciones por Virus ARN , Animales , Ratones , Linfocitos B/citología , Linfocitos B/inmunología , Células Clonales , COVID-19 , Centro Germinal/citología , Centro Germinal/inmunología , SARS-CoV-2 , Gripe Humana , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/patología , Infecciones por Virus ARN/virologíaRESUMEN
Germinal centers (GCs) are the site of antibody affinity maturation, a fundamental immunological process that increases the potency of antibodies and thereby their ability to protect against infection. GC biology is highly dynamic in both time and space, making it ideally suited for intravital imaging. Using multiphoton laser scanning microscopy (MPLSM), the field has gained insight into the molecular, cellular, and structural changes and movements that coordinate affinity maturation in real time in their native environment. On the other hand, several limitations of MPLSM have had to be overcome to allow full appreciation of GC events taking place across different timescales. Here, we review the technical advances afforded by intravital imaging and their contributions to our understanding of GC biology.
Asunto(s)
Linfocitos B , Centro Germinal , Anticuerpos , Afinidad de Anticuerpos , Diagnóstico por Imagen , HumanosRESUMEN
Germinal centers (GCs) are microanatomical structures in secondary lymphoid organs where B cells undergo affinity maturation for antigen during the course of an immune response. This process is driven by a subset of T cells termed T follicular helper cells (Tfh) that through a multistep process gain access to the GC niche within the B cell follicle. This protocol details how to study Tfh behavior in vivo, on a single cell level, using two-photon intravital microscopy of the murine popliteal lymph node.
Asunto(s)
Células T Auxiliares Foliculares , Animales , Linfocitos B , Centro Germinal , Ganglios Linfáticos , Ratones , Linfocitos T Colaboradores-InductoresRESUMEN
Germinal centers (GCs) are the site of immunoglobulin somatic hypermutation and affinity maturation, processes essential to an effective antibody response. The formation of GCs has been studied in detail, but less is known about what leads to their regression and eventual termination, factors that ultimately limit the extent to which antibodies mature within a single reaction. We show that contraction of immunization-induced GCs is immediately preceded by an acute surge in GC-resident Foxp3+ T cells, attributed at least partly to up-regulation of the transcription factor Foxp3 by T follicular helper (TFH) cells. Ectopic expression of Foxp3 in TFH cells is sufficient to decrease GC size, implicating the natural up-regulation of Foxp3 by TFH cells as a potential regulator of GC lifetimes.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Factores de Transcripción Forkhead/genética , Centro Germinal/inmunología , Células T Auxiliares Foliculares/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Linfocitos T CD4-Positivos/fisiología , Factores de Transcripción Forkhead/metabolismo , Genes Codificadores de los Receptores de Linfocitos T , Centro Germinal/citología , Inmunización , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de la Célula Individual , Células T Auxiliares Foliculares/inmunología , Linfocitos T Reguladores/fisiología , Regulación hacia ArribaRESUMEN
Enhancing the germinal center (GC) reaction is a prime objective in vaccine development. Targeting of antigen to MHCII on APCs has previously been shown to increase antibody responses, but the underlying mechanism has been unclear. We have here investigated the GC reaction after targeting antigen to MHCII in (i) a defined model with T and B cells of known specificity using adjuvant-free vaccine proteins, and (ii) an infectious disease model using a DNA vaccine. MHCII-targeting enhanced presentation of peptide: MHCII on APCs, and increased the numbers of GC B cells, TFH, and plasma cells. Antibodies appeared earlier and levels were increased. BCR of GC B cells and serum antibodies had increased avidity for antigen. The improved responses required cross-linking of BCR and MHCII in either cis or trans. The enhanced GC reaction induced by MHCII-targeting of antigen has clear implications for design of more efficient subunit vaccines.
RESUMEN
We developed a method for rapid generation of B cell receptor (BCR) monoclonal mice expressing prerearranged Igh and Igk chains monoallelically from the Igh locus by CRISPR-Cas9 injection into fertilized oocytes. B cells from these mice undergo somatic hypermutation (SHM), class switch recombination (CSR), and affinity-based selection in germinal centers. This method combines the practicality of BCR transgenes with the ability to study Ig SHM, CSR, and affinity maturation.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Cambio de Clase de Inmunoglobulina , Cadenas Pesadas de Inmunoglobulina , Cadenas kappa de Inmunoglobulina , Receptores de Antígenos de Linfocitos B , Animales , Linfocitos B/citología , Centro Germinal/citología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/inmunología , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
Immune-compromised mouse models allow for testing the preclinical efficacy of human cell transplantations and gene therapy strategies before moving forward to clinical trials. However, CRISPR/Cas9 gene editing of the Wsh/Wsh mouse strain to create an immune-compromised model lacking function of Rag2 and Il2rγ led to unexpected morbidity and mortality. This warranted an investigation to ascertain the cause and predisposing factors associated with the outbreak. Postmortem examination was performed on 15 moribund mice. The main lesions observed in these mice consisted of ascending urogenital tract infections, suppurative otitis media, pneumonia, myocarditis, and meningoencephalomyelitis. As Escherichia coli strains harboring polyketide synthase (pks) genomic island were recently isolated from laboratory mice, the tissue sections from the urogenital tract, heart, and middle ear were subjected to E. coli specific PNA-FISH assay that revealed discrete colonies of E. coli associated with the lesions. Microbiological examination and 16S rRNA sequencing confirmed E. coli-induced infection and septicemia in the affected mice. Further characterization by clb gene analysis and colibactin toxicity assays of the pks+ E. coli revealed colibactin-associated cytotoxicity. Rederivation of the transgenic mice using embryo transfer produced mice with an intestinal flora devoid of pks+ E. coli. Importantly, these barrier-maintained rederived mice have produced multiple litters without adverse health effects. This report is the first to describe acute morbidity and mortality associated with pks+ E. coli urosepsis and meningitis in immunocompromised mice, and highlights the importance of monitoring and exclusion of colibactin-producing pks+ E. coli.
Asunto(s)
Escherichia coli , Huésped Inmunocomprometido , Meningitis Bacterianas , Péptidos/genética , Sepsis , Infecciones Urinarias , Animales , Escherichia coli/genética , Escherichia coli/inmunología , Escherichia coli/aislamiento & purificación , Meningitis Bacterianas/genética , Meningitis Bacterianas/inmunología , Meningitis Bacterianas/microbiología , Ratones , Ratones Transgénicos , Péptidos/inmunología , Policétidos/inmunología , Sepsis/genética , Sepsis/inmunología , Sepsis/microbiología , Infecciones Urinarias/genética , Infecciones Urinarias/inmunología , Infecciones Urinarias/microbiologíaRESUMEN
During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection.
Asunto(s)
Linfocitos B/fisiología , Selección Clonal Mediada por Antígenos , Centro Germinal/inmunología , Complejos Multiproteicos/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Afinidad de Anticuerpos , Ciclo Celular , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Complejos Multiproteicos/genética , Receptores de Antígenos de Linfocitos B/genética , Sirolimus/farmacología , Hipermutación Somática de Inmunoglobulina , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Germinal centers (GCs) are inducible microanatomical structures required for the generation of high-affinity antibodies. Migration of B and T cells within and into/out of GCs plays a key role in the evolutionary process that underlies affinity maturation, and thus microanatomical location of cells is an important variable when studying GC processes. We describe a protocol in which in situ photoactivation by multiphoton microscopy can be used to add microanatomical information to flow cytometry, allowing for identification and isolation of GC cells based on their location. Cells in different microanatomical compartments can then be sorted and analyzed for surface marker and mRNA expression.
Asunto(s)
Microambiente Celular , Citometría de Flujo , Centro Germinal/citología , Centro Germinal/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biomarcadores , Citometría de Flujo/métodos , Centro Germinal/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Microscopía de Fluorescencia por Excitación MultifotónicaRESUMEN
The immune system plays a crucial role in many diseases. Activation or suppression of immunity is often related to clinical outcome. Methods to explore the dynamics of immune responses are important to elucidate their role in conditions characterized by inflammation, such as infectious disease, cancer, or autoimmunity. Immuno-PET is a noninvasive method by which disease and immune cell infiltration can be explored simultaneously. Using radiolabeled antibodies or fragments derived from them, it is possible to image disease-specific antigens and immune cell subsets. Methods: We developed a method to noninvasively image human immune responses in a relevant humanized mouse model. We generated a camelid-derived single-domain antibody specific for human class II major histocompatibility complex products and used it to noninvasively image human immune cell reconstitution in nonobese diabetic severe combined immune deficiency γ-/- mice reconstituted with human fetal thymus, liver, and liver-derived hematopoietic stem cells (BLT mice). Results: We showed imaging of infiltrating immunocytes in BLT mice that spontaneously developed a graft-versus-host-like condition, characterized by alopecia and blepharitis. In diseased animals, we showed an increased PET signal in the liver, attributable to infiltration of activated class II major histocompatibility complex+ T cells. Conclusion: Noninvasive imaging of immune infiltration and activation could thus be of importance for diagnosis and evaluation of treatment of graft-versus-host disease and holds promise for other diseases characterized by inflammation.
Asunto(s)
Enfermedad Injerto contra Huésped/diagnóstico por imagen , Enfermedad Injerto contra Huésped/inmunología , Inmunidad Innata/inmunología , Tomografía de Emisión de Positrones/métodos , Anticuerpos de Dominio Único/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Ratones , Ratones Noqueados , Radiofármacos/inmunologíaRESUMEN
Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza.
Asunto(s)
Anticuerpos/inmunología , Afinidad de Anticuerpos/inmunología , Linfocitos B/inmunología , Centro Germinal/inmunología , Imagen Molecular/métodos , Animales , Anticuerpos/genética , Afinidad de Anticuerpos/genética , VIH-1/inmunología , Humanos , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica , Orthomyxoviridae/inmunología , Análisis de Secuencia de ADN , Análisis de la Célula IndividualRESUMEN
Antibodies are currently the fastest-growing class of therapeutics. Although naked antibodies have proven valuable as pharmaceutical agents, they have some limitations, such as low tissue penetration and a long circulatory half-life. They have been conjugated to toxic payloads, PEGs, or radioisotopes to increase and optimize their therapeutic efficacy. Although nonspecific conjugation is suitable for most in vitro applications, it has become evident that site specifically modified antibodies may have advantages for in vivo applications. Herein we describe a novel approach in which the antibody fragment is tagged with two handles: one for the introduction of a fluorophore or (18)F isotope, and the second for further modification of the fragment with a PEG moiety or a second antibody fragment to tune its circulatory half-life or its avidity. Such constructs, which recognize Classâ II MHC products and CD11b, showed high avidity and specificity. They were used to image cancers and could detect small tumors.
Asunto(s)
Imagen Molecular , Anticuerpos de Dominio Único/química , Animales , Células Cultivadas , Dimerización , Semivida , Antígenos de Histocompatibilidad Clase II/inmunología , Melanoma Experimental/inmunología , Ratones , Anticuerpos de Dominio Único/sangre , Anticuerpos de Dominio Único/uso terapéuticoRESUMEN
An enormous number of B cells with different B-cell receptors (BCRs) are continuously produced in the bone marrow. BCRs are further diversified during the germinal center reaction. Due to extensive recirculation, B cells with mutually binding BCR are likely to meet in lymphoid organs. We have addressed possible outcomes of such an encounter in vitro. B lymphoma cells were transfected with complementary BCR, one transfectant expressing an Idiotype⺠(Idâº) BCR and the other an anti-Id BCR. To exclude confounding effects of secreted Ig, the transfected B lymphoma cells only expressed membrane IgD. Coincubation of paired Idâº/anti-Id lymphoma cells results in conjugate formation, signaling, activation of Caspase 3/7, and apoptosis of at least one of the two cells in the pair. Our data provide suggestive evidence for a mechanism whereby the B-cell compartment is partly purged of B cells with complementary BCRs.
Asunto(s)
Linfocitos B/inmunología , Tolerancia Inmunológica , Inmunoglobulina D/metabolismo , Región Variable de Inmunoglobulina/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/metabolismo , Apoptosis/genética , Apoptosis/inmunología , Médula Ósea/inmunología , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular Tumoral , Técnicas de Cocultivo , Inmunoglobulina D/genética , Activación de Linfocitos/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Transducción de Señal/inmunología , Transgenes/genéticaRESUMEN
The V region antigenic determinants (idiotopes (Ids)) of antibodies (Abs) have been suggested to be involved in regulating the immune system. Certain diseases such as diabetes mellitus have recently been associated with a disequilibrium between Id(+) and anti-Id Abs. However, it is unknown how Abs carrying complementary idiotypes (that is, Id(+) and anti-Id Abs) regulate each other at the level of B and T cells. In this study, we show that B lymphoma cells genetically equipped with anti-Id BCR V regions receive a signal when exposed to Id(+)Ig. Moreover, they become x 10(4) more efficient at presenting exogenous Id(+) Ab to CD4(+) T cells in vitro. Activated Id-specific T cells in turn regulated the Id-specific B lymphoma cells. Similar results were obtained in vivo in a surrogate model in which an Id-peptide was incorporated genetically into the C-region of a recombinant Ab that targeted IgD on B cells. The findings suggest that conventional T-B collaboration can explain communication between complementary Id(+) and anti-Id Ab at the cellular level. A model is suggested that integrates present and previous data on B-cell regulation by Id-specific T cells.