RESUMEN
Effective vaccines induce high-affinity memory B cells and durable antibody responses through accelerated mechanisms of natural selection. Secondary changes in antibody repertoires after vaccine boosts suggest progressive rediversification of B cell receptors (BCRs), but the underlying mechanisms remain unresolved. Here, the integrated specificity and function of individual memory B cell progeny revealed ongoing evolution of polyclonal antibody specificities through germinal center (GC)-specific transcriptional activity. At the clonal and subclonal levels, single-cell expression of the genes encoding the costimulatory molecule CD83 and the DNA polymerase Polη segregated the secondary GC transcriptional program into four stages that regulated divergent mechanisms of memory BCR evolution. Our studies demonstrate that vaccine boosts reactivate a cyclic program of GC function in class-switched memory B cells to remodel existing antibody specificities and enhance durable immunological protection.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Memoria Inmunológica/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , Antígenos CD/inmunología , ADN Polimerasa Dirigida por ADN/inmunología , Cambio de Clase de Inmunoglobulina/inmunología , Inmunoglobulinas/inmunología , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Transcripción Genética/inmunología , Antígeno CD83RESUMEN
Antibodies are produced across multiple isotypes with distinct properties that coordinate initial antigen clearance and confer long-term antigen-specific immune protection. Here, we interrogate the molecular programs of isotype-specific murine plasma cells (PC) following helper T cell-dependent immunization and within established steady-state immunity. We developed a single-cell-indexed and targeted molecular strategy to dissect conserved and divergent components of the rapid effector phase of antigen-specific IgM+ versus inflammation-modulating programs dictated by type 1 IgG2a/b+ PC differentiation. During antibody affinity maturation, the germinal center (GC) cycle imparts separable programs for post-GC type 2 inhibitory IgG1+ and type 1 inflammatory IgG2a/b+ PC to direct long-term cellular function. In the steady state, two subsets of IgM+ and separate IgG2b+ PC programs clearly segregate from splenic type 3 IgA+ PC programs that emphasize mucosal barrier protection. These diverse isotype-specific molecular pathways of PC differentiation control complementary modules of antigen clearance and immune protection that could be selectively targeted for immunotherapeutic applications and vaccine design.
Asunto(s)
Diferenciación Celular , Centro Germinal , Células Plasmáticas , Animales , Antígenos , Inmunoglobulina G/genética , Inmunoglobulina M , Ratones , Células Plasmáticas/citología , Análisis de la Célula Individual , Linfocitos T Colaboradores-InductoresRESUMEN
Antibody class defines function in B cell immunity, but how class is propagated into B cell memory remains poorly understood. Here we demonstrate that memory B cell subsets unexpectedly diverged across antibody class through differences in the effects of major transcriptional regulators. Conditional genetic deletion of the gene encoding the transcription factor T-bet selectively blocked the formation and antigen-specific response of memory B cells expressing immunoglobulin G2a (IgG2a) in vivo. Cell-intrinsic expression of T-bet regulated expression of the transcription factor STAT1, steady-state cell survival and transcription of IgG2a-containing B cell antigen receptors (BCRs). In contrast, the transcription factor RORα and not T-bet was expressed in IgA(+) memory B cells, with evidence that knockdown of RORα mRNA expression and chemical inhibition of transcriptional activity also resulted in lower survival and BCR expression of IgA(+) memory B cells. Thus, divergent transcriptional regulators dynamically maintain subset integrity to promote specialized immune function in class-specific memory B cells.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Cambio de Clase de Inmunoglobulina/inmunología , Memoria Inmunológica/inmunología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/inmunología , Proteínas de Dominio T Box/inmunología , Animales , Linfocitos B/clasificación , Citometría de Flujo , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/química , ARN Mensajero/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores de Antígenos de Linfocitos B/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/inmunología , Organismos Libres de Patógenos Específicos , Proteínas de Dominio T Box/genética , Transcripción Genética/inmunologíaRESUMEN
B lymphocytes differentiate into antibody-secreting cells under the antigen-specific control of follicular helper T cells (T(FH) cells). Here we demonstrate that isotype-switched plasma cells expressed major histocompatibility complex (MHC) class II, the costimulatory molecules CD80 and CD86, and the intracellular machinery required for antigen presentation. Antigen-specific plasma cells accessed, processed and presented sufficient antigen in vivo to induce multiple helper T cell functions. Notably, antigen-primed plasma cells failed to induce interleukin 21 (IL-21) or the transcriptional repressor Bcl-6 in naive helper T cells and actively decreased these key molecules in antigen-activated T(FH) cells. Mice lacking plasma cells showed altered T(FH) cell activity, which provided evidence of this negative feedback loop. Hence, antigen presentation by plasma cells defines a previously unknown layer of cognate regulation that limits the antigen-specific T(FH) cell program that controls ongoing B cell immunity.
Asunto(s)
Presentación de Antígeno/inmunología , Activación de Linfocitos/inmunología , Células Plasmáticas/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Inmunidad Adaptativa , Animales , Separación Celular , Ensayo de Immunospot Ligado a Enzimas , Citometría de Flujo , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Helper T cell induced plasma cells (PCs) that secrete class-switched neutralizing antibody are paramount to effective immunity. Following class-switch recombination (CSR), antigen-activated B cells differentiate into extrafollicular PCs or mature in germinal centers (GCs) to produce high-affinity memory B cells and follicular PCs. Many studies focus on the core transcriptional programs that drive central PC functions of longevity and antibody secretion. However, it is becoming clear that these central programs are further subdivided across antibody isotype with separable transcriptional trajectories. Divergent functions emerge at CSR, persist through PC terminal differentiation and further assort memory PC function following antigen recall. Here, we emphasize recent work that assorts divergent isotype-specific PC function across four major modules of immune protection.
Asunto(s)
Células Plasmáticas/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , HumanosRESUMEN
How follicular helper T cells (T(FH) cells) differentiate to regulate B cell immunity is critical for effective protein vaccination. Here we define three transcription factor T-bet-expressing antigen-specific effector helper T cell subsets with distinguishable function, migratory properties and developmental programming in vivo. Expression of the transcriptional repressor Blimp-1 distinguished T zone 'lymphoid' effector helper T cells (CD62L(hi)CCR7(hi)) from CXCR5(lo) 'emigrant' effector helper T cells and CXCR5(hi) 'resident' T(FH) cells expressing the transcriptional repressor Bcl-6 (CD62L(lo)CCR7(lo)). We then show by adoptive transfer and intact polyclonal responses that helper T cells with the highest specific binding of peptide-major histocompatibility complex class II and the most restricted T cell antigen receptor junctional diversity 'preferentially' developed into the antigen-specific effector T(FH) compartment. Our studies demonstrate a central function for differences in the binding strength of the T cell antigen receptor in the antigen-specific mechanisms that 'program' specialized effector T(FH) function in vivo.
Asunto(s)
Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adyuvantes Inmunológicos/farmacología , Traslado Adoptivo , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Citocinas/biosíntesis , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Selectina L/inmunología , Ganglios Linfáticos/inmunología , Ratones , Ratones Transgénicos , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Proto-Oncogénicas c-bcl-6 , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas de Dominio T Box/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Colaboradores-Inductores/trasplante , Factores de Transcripción/biosíntesis , Factores de Transcripción/inmunologíaRESUMEN
Information is now available in real time from a multitude of sources. Twitter provides one effective means to broadcast images with short captions instantly and everywhere. Last year we began using Twitter to convey our excitement with the biological sciences, and discovered a new means to contribute, connect, and conference with a broader global scientific community and beyond. Here we share this experience, and invite you to join in the conversation.
Asunto(s)
Difusión de la Información , Ciencia/educación , Medios de Comunicación Sociales , Animales , Recursos Audiovisuales , Comunicación , Medios de Comunicación , HumanosRESUMEN
Follicular helper T (Tfh) cells are the class of effector T helper cells that regulates the step-wise development of antigen-specific B cell immunity in vivo. Deployment of CXCR5+ Tfh cells to B cell zones of lymphoid tissues and stable cognate interactions with B cells are central to the delivery of antigen-specific Tfh cell function. Here, we review recent advances that have helped to unravel distinctive elements of developmental programming for Tfh cells and unique effector Tfh cell functions focused on antigen-primed B cells. Understanding the regulatory functions of Tfh cells in the germinal center and the subsequent regulation of memory B cell responses to antigen recall represent the frontiers of this research area with the potential to alter fundamentally the design of future vaccines.
Asunto(s)
Subgrupos Linfocitarios/citología , Linfocitos T Colaboradores-Inductores/citología , Animales , Linfocitos B/inmunología , Diferenciación Celular , Linaje de la Célula , Humanos , Inmunidad , Subgrupos Linfocitarios/inmunología , Receptores CXCR5/metabolismo , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
How T cell receptor (TCR) specificity evolves in vivo after protein vaccination is central to the development of helper T (Th) cell function. Most models of clonal selection in the Th cell compartment favor TCR affinity-based thresholds. Here, we demonstrated that depot-forming vaccine adjuvants did not require Toll-like receptor (TLR) agonists to induce clonal dominance in antigen-specific Th cell responses. However, readily dispersible adjuvants using TLR-9 and TLR-4 agonists skewed TCR repertoire usage by increasing TCR selection thresholds and enhancing antigen-specific clonal expansion. In this manner, vaccine adjuvants control the local accumulation of Th cells expressing TCR with the highest peptide MHC class II binding. Clonal composition was altered by mechanisms that blocked the local propagation of clonotypes independently of antigen dose and not as a consequence of interclonal competition. This capacity of adjuvants to modify antigen-specific Th cell clonal composition has fundamental implications for the design of future protein subunit vaccines.
Asunto(s)
Adyuvantes Inmunológicos , Citocromos c/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Receptores Toll-Like/metabolismo , Vacunas/inmunología , Animales , Citocromos c/inmunología , Activación de Linfocitos , Ratones , Ratones Congénicos , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Receptores Toll-Like/agonistas , Receptores Toll-Like/inmunología , Vacunas de Subunidad/inmunologíaRESUMEN
During a T cell-dependent Ab response, B cells undergo Ab class switching and V region hypermutation, with the latter process potentially rendering previously innocuous B cells autoreactive. Class switching and hypermutation are temporally and anatomically linked with both processes dependent on the enzyme, activation-induced deaminase, and occurring principally, but not exclusively, in germinal centers. To understand tolerance regulation at this stage, we generated a new transgenic mouse model expressing a membrane-tethered gamma2a-reactive superantigen (gamma2a-macroself Ag) and assessed the fate of emerging IgG2a-expressing B cells that have, following class switch, acquired self-reactivity of the Ag receptor to the macroself-Ag. In normal mice, self-reactive IgG2a-switched B cells were deleted, leading to the selective absence of IgG2a memory responses. These findings identify a novel negative selection mechanism for deleting mature B cells that acquire reactivity to self-Ag. This process was only partly dependent on the Bcl-2 pathway, but markedly inefficient in MRL-Fas(lpr) lupus mice, suggesting that defective apoptosis of isotype-switched autoreactive B cells is central to Fas mutation-associated systemic autoimmunity.
Asunto(s)
Linfocitos B/inmunología , Inmunoglobulina G/inmunología , Receptor fas/inmunología , Traslado Adoptivo , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Línea Celular , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Ratones Endogámicos , Ratones Transgénicos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Superantígenos/genética , Superantígenos/inmunología , Superantígenos/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
Understanding how follicular helper T cells (TFH) regulate the specialization, maturation, and differentiation of adaptive B cell immunity is crucial for developing durable high-affinity immune protection. Using indexed single-cell molecular strategies, we reveal a skewed intraclonal assortment of higher-affinity T cell receptors and the distinct molecular programming of the localized TFH compartment compared with emigrant conventional effector TH cells. We find a temporal shift in B cell receptor class switch, which permits identification of inflammatory and anti-inflammatory modules of transcriptional programming that subspecialize TFH function before and during the germinal center (GC) reaction. Late collapse of this local primary GC reaction reveals a persistent post-GC TFH population that discloses a putative memory TFH program. These studies define subspecialized antigen-specific TFH transcriptional programs that progressively change with antibody class-specific evolution of high-affinity B cell immunity and a memory TFH transcriptional program that emerges upon local GC resolution.
Asunto(s)
Células T Auxiliares Foliculares , Linfocitos T Colaboradores-Inductores , Antígenos , Centro Germinal , Isotipos de Inmunoglobulinas , Receptores de Antígenos de Linfocitos BRESUMEN
Adaptive T and B lymphocytes expand, respond, and persist across a multitude of separable cell differentiation states. Small compartments of these cells present defined cell surface phenotype, but express potentially divergent immune functions. Here, we use high resolution flow cytometry to provide direct access to rare lymphocyte subpopulations for evaluation of steady-state or reactive transcriptional programs. We sort and index single cells by phenotype in 384-well format for quantification of targeted gene amplification through RNA sequencing (single cell qtSEQ). For complete details on the use and execution of this profile, please refer to Dufaud et al. (2021).
Asunto(s)
Linfocitos B , Subgrupos Linfocitarios , Citometría de Flujo , Recuento de Linfocitos , Análisis de Secuencia de ARNRESUMEN
Clonal evolution underpins all facets of adaptive immunity. In particular, antigen-specific helper T (Th) cell development is central to high-affinity B cell immunity and protective vaccination. Dendritic cell maturation and TCR affinity-based selection mechanisms control the recruitment and effective propagation of preferred antigen-specific Th cell cohorts in local lymphoid tissue. Importantly, follicular B helper T (T(FH)) cells emerge as the specialized local effector Th cells that orchestrate the stepwise development of B cell immunity in these local environments. Recent studies also introduce the role of persistent antigen in the development of effector Th cells with evidence for long-term antigen depots that might contribute to local antigen-specific Th cell memory.
Asunto(s)
Memoria Inmunológica , Activación de Linfocitos/inmunología , Modelos Inmunológicos , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linfocitos B/inmunología , Células Dendríticas/inmunología , Centro Germinal/inmunología , HumanosRESUMEN
Antigen recall can clearly induce a germinal center (GC) reaction. What has become an issue for debate are the origins of the antigen-specific B cells that form memory-response GCs (mGCs). Using antigen labeling and adoptive transfer, memory B cells expressing different antibody class can give rise to mGCs with differing efficiency. Here, we will argue that the range of class-specific memory responses reported across multiple systems represents the spectrum of memory B-cell fate and function. While the formulation of recall immunogen and location of mGCs have an important role, we propose that effective cognate regulation is the key variable influencing recall outcome. These issues remain central to contemporary efforts of rational vaccine design.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Memoria Inmunológica , Linfocitos T/inmunología , Animales , Humanos , Cambio de Clase de Inmunoglobulina , Vacunas/inmunologíaRESUMEN
Successful vaccination relies on driving the immune response towards high specificity, affinity and longevity. Germinal centers facilitate the evolution of antigen-specific B cells by iterative rounds of diversification, selection, and differentiation to memory and plasma cells. Experimental evidence points to B cell receptor affinity and amount of antigen presented to follicular helper T cells as main drivers of clonal evolution. Concurrent studies suggest that modifiers of cognate contact, temporal mechanisms, and stochastic factors can also shape diversity and influence differentiation to memory and plasma cells, but molecular pathways driving these selection decisions are unresolved. Due to rapid cycles of transcriptional change in the germinal center, single-cell resolution is imperative to dissect mechanisms dictating the mature antigen-specific repertoire. Future studies linking high-resolution analysis of this diverse evolving population with cellular outcome are needed to fully understand the complex mechanisms of selection driving antigen-specific humoral immunity.
Asunto(s)
Antígenos/inmunología , Linfocitos B/inmunología , Centro Germinal/inmunología , Inmunidad Humoral , Receptores de Antígenos de Linfocitos B/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linfocitos B/citología , Centro Germinal/citología , Humanos , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.
Asunto(s)
Subgrupos de Linfocitos B/citología , Centro Germinal/inmunología , Hemocianinas/inmunología , Memoria Inmunológica/inmunología , Células Plasmáticas/citología , Animales , Subgrupos de Linfocitos B/inmunología , Diferenciación Celular/inmunología , Centro Germinal/citología , Haptenos , Inmunización , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Células Plasmáticas/inmunología , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Bazo/citología , Bazo/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Follicular helper T (T(FH)) cells are a class of helper T cells specialized in the cognate control of antigen-specific B cell immunity. Upon first contact with antigen-primed B cells, pregerminal center effector T(FH) cells promote B cell clonal expansion, antibody isotype switch, plasma cell differentiation, and the induction of germinal centers. By contrast, within germinal centers, T(FH) cells regulate the fate of antigen-specific GC B cells expressing high-affinity variant B cell receptors to promote memory B cell and long-lived plasma cell development. Recent studies unravel multiple signals controlling T(FH) development and functional subtypes of antigen-specific T(FH) cells, including memory T(FH) cells that accelerate memory B cell responses to antigen rechallenge in vivo.
Asunto(s)
Linfocitos B/inmunología , Inmunidad Celular/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Centro Germinal/citología , Centro Germinal/inmunología , Humanos , Memoria Inmunológica/inmunología , Activación de Linfocitos/inmunología , Modelos InmunológicosRESUMEN
The unfolded protein response (UPR) is a stress response pathway that is driven by the increased load of unfolded proteins in the endoplasmic reticulum of highly secretory cells such as plasma cells (PCs). X box binding protein 1 (XBP1) is a transcription factor that mediates one branch of the UPR and is crucial for the development of antibody-secreting PCs. PCs represent only one class of terminally differentiated B cells, however, and little is known about the role for XBP1 in the other class: memory B cells. We have developed an XBP1(fl/fl) CD19(+/cre) conditional knockout (XBP1(CD19)) mouse to build upon our current understanding of the function of XBP1 in PC differentiation as well as to explore the role of XBP1 in memory cell development. Using this model, we show that XBP1(CD19) mice are protected from disease in an autoantibody-mediated mouse lupus model. We also identify a novel developmental stage at which B cells express the traditional PC marker CD138 (syndecan-1) but have yet to undergo XBP1-dependent functional and morphological differentiation into antibody-secreting cells. Finally, we show that memory B cells develop normally in XBP1(CD19) mice, demonstrating that XBP1-mediated functions occur independently of any memory cell lineage commitment.
Asunto(s)
Linfocitos B/fisiología , Proteínas de Unión al ADN/fisiología , Células Plasmáticas/citología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Memoria Inmunológica , Lupus Eritematoso Sistémico/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos MRL lpr , Ratones Endogámicos NZB , Ratones Noqueados , Factores de Transcripción del Factor Regulador X , Sindecano-1/análisis , Proteína 1 de Unión a la X-BoxRESUMEN
How vaccines control the development of antigen-specific effector and memory T helper cells is central to protective immunity but remains poorly understood. Here we found that protein vaccination selected high-affinity, CXCR5+ICOS(hi) follicular B-helper T cells (T(FH) cells) that developed in draining lymphoid tissue to regulate B cell responses. In the memory phase, reservoirs of antigen-specific CXCR5+ICOS(lo) T(FH) cells persisted with less effector activity but accelerated antigen-recall ability. This new compartment of memory T(FH) cells was retained in draining lymphoid sites with antigen-specific memory B cells, persistent complexes of peptide and major histocompatibility complex class II and continued expression of CD69. Thus, protein vaccination promotes B cell immunity by selecting high-affinity effector T(FH) cells and creating lymphoid reservoirs of antigen-specific memory T(FH) cells in vivo.