RESUMEN
Clostridioides difficile is a leading cause of nosocomial infection responsible for significant morbidity and mortality with limited options for therapy. Secreted C. difficile toxin B (TcdB) is a major contributor to disease pathology, and select TcdB-specific Abs may protect against disease recurrence. However, the high frequency of recurrence suggests that the memory B cell response, essential for new Ab production following C. difficile reexposure, is insufficient. We therefore isolated TcdB-specific memory B cells from individuals with a history of C. difficile infection and performed single-cell deep sequencing of their Ab genes. Herein, we report that TcdB-specific memory B cell-encoded antibodies showed somatic hypermutation but displayed limited isotype class switch. Memory B cell-encoded mAb generated from the gene sequences revealed low to moderate affinity for TcdB and a limited ability to neutralize TcdB. These findings indicate that memory B cells are an important factor in C. difficile disease recurrence.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Infecciones por Clostridium/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Linfocitos B/microbiología , Células CHO , Estudios de Casos y Controles , Clostridioides difficile/inmunología , Cricetulus , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Memoria Inmunológica , Persona de Mediana Edad , Hipermutación Somática de InmunoglobulinaRESUMEN
Memory B cells that are generated during an infection or following vaccination act as sentinels to guard against future infections. Upon repeat antigen exposure memory B cells differentiate into new antibody-secreting plasma cells to provide rapid and sustained protection. Some pathogens evade or suppress the humoral immune system, or induce memory B cells with a diminished ability to differentiate into new plasma cells. This leaves the host vulnerable to chronic or recurrent infections. Single cell approaches coupled with next generation antibody gene sequencing facilitate a detailed analysis of the pathogen-specific memory B cell repertoire. Monoclonal antibodies that are generated from antibody gene sequences allow a functional analysis of the repertoire. This review discusses what has been learned thus far from analysis of diverse pathogen-specific memory B cell compartments and describes major differences in their repertoires. Such information may illuminate ways to advance the goal of improving vaccine and therapeutic antibody design.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Memoria Inmunológica , Células Plasmáticas/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Antígenos/inmunología , Subgrupos de Linfocitos B/metabolismo , Linfocitos B/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Humoral/inmunología , Células Plasmáticas/metabolismoRESUMEN
The B-cell ELISPOT assay is a sensitive tool that can be utilized to measure total immunoglobulin (Ig) and antigen-specific antibody-secreting cells. Typically, membrane-bound antigen enables binding of antibody secreted by B-cells. Bound antibody is then detected by using an anti-Ig antibody and a colorimetric substrate, resulting in colored spots on the membrane that can be easily enumerated. Here we have described a method to quantitate antigen-specific antibody-secreting cells from the spleen or bone marrow of a vaccinated mouse.
Asunto(s)
Antígenos/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Ensayo de Immunospot Ligado a Enzimas/métodos , Animales , Médula Ósea/inmunología , Separación Celular , Ratones , VacunaciónRESUMEN
OBJECTIVE: In recent years, vitamin D has been shown to possess a wide range of immunomodulatory effects. Although there is extensive amount of research on vitamin D, we lack a comprehensive understanding of the prevalence of vitamin D deficiency or the mechanism by which vitamin D regulates the human immune system. This study examined the prevalence and correlates of vitamin D deficiency and the relationship between vitamin D and the immune system in healthy individuals. METHODS: Healthy individuals (n = 774) comprised of European-Americans (EA, n = 470), African-Americans (AA, n = 125), and Native Americans (NA, n = 179) were screened for 25-hydroxyvitamin D [25(OH)D] levels by ELISA. To identify the most noticeable effects of vitamin D on the immune system, 20 EA individuals with severely deficient (<11.3 ng/mL) and sufficient (>24.8 ng/mL) vitamin D levels were matched and selected for further analysis. Serum cytokine level measurement, immune cell phenotyping, and phosphoflow cytometry were performed. RESULTS: Vitamin D sufficiency was observed in 37.5% of the study cohort. By multivariate analysis, AA, NA, and females with a high body mass index (BMI, >30) demonstrate higher rates of vitamin D deficiency (p<0.05). Individuals with vitamin D deficiency had significantly higher levels of serum GM-CSF (p = 0.04), decreased circulating activated CD4+ (p = 0.04) and CD8+ T (p = 0.04) cell frequencies than individuals with sufficient vitamin D levels. CONCLUSION: A large portion of healthy individuals have vitamin D deficiency. These individuals have altered T and B cell responses, indicating that the absence of sufficient vitamin D levels could result in undesirable cellular and molecular alterations ultimately contributing to immune dysregulation.
Asunto(s)
Etnicidad , Inmunidad , Deficiencia de Vitamina D/sangre , Población Blanca , Adolescente , Adulto , Negro o Afroamericano , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Índice de Masa Corporal , Estudios de Casos y Controles , Estrógenos/farmacología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Humanos , Inmunidad/efectos de los fármacos , Indígenas Norteamericanos , Modelos Logísticos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Análisis Multivariante , Factor de Transcripción STAT1/metabolismo , Rayos Ultravioleta , Estados Unidos , Vitamina D/análogos & derivados , Vitamina D/sangre , Adulto JovenRESUMEN
CD1d-restricted invariant NKT (iNKT) cells boost humoral immunity to T-dependent Ags that are coadministered with the CD1d-binding glycolipid Ag α-galactosylceramide (α-GC). Observations that mice lacking iNKT cells have decaying Ab responses following vaccination have led to the hypothesis that iNKT cells express plasma cell (PC) survival factors that sustain specific Ab titers. Bone marrow chimeric mice in which the entire hematopoietic compartment or iNKT cells selectively lacked BAFF, a proliferation-inducing ligand (APRIL), or both BAFF and APRIL were created and immunized with nitrophenol hapten-conjugated keyhole limpet hemocyanin adsorbed to Imject aluminum hydroxide-containing adjuvant or mixed with α-GC. In comparison with BAFF- or APRIL-sufficient bone marrow chimeras, absence of hematopoietic compartment- and iNKT-derived BAFF and APRIL was associated with rapidly decaying Ab titers and reduced PC numbers. The iNKT cell-derived BAFF or APRIL assumed a greater role in PC survival when α-GC was used as the adjuvant for immunization. These results show that iNKT cell-derived BAFF and APRIL each contribute to survival of PCs induced by immunization. This study sheds new light on the mechanisms through which iNKT cells impact humoral immunity and may inform design of vaccines that incorporate glycolipid adjuvants.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos/sangre , Factor Activador de Células B/metabolismo , Células T Asesinas Naturales/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Hidróxido de Aluminio/administración & dosificación , Hidróxido de Aluminio/inmunología , Animales , Antígenos CD1d/inmunología , Factor Activador de Células B/deficiencia , Factor Activador de Células B/genética , Células de la Médula Ósea , Femenino , Galactosilceramidas/administración & dosificación , Galactosilceramidas/inmunología , Hemocianinas/administración & dosificación , Hemocianinas/inmunología , Inmunidad Humoral , Inmunización , Ratones , Ratones Noqueados , Células Plasmáticas/metabolismo , Quimera por Trasplante , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/deficiencia , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , VacunaciónRESUMEN
Alum-based adjuvants facilitate vaccine-driven humoral immunity, but their mechanism of action remains poorly understood. Herein, we report that lack of type II NKT cells is associated with intact, mature B cells but dampened humoral immunity following immunization with Alum-adsorbed T-dependent antigen. Type II NKT cells facilitated production of IL-4, IL-5, IL-10, IL-13, and antibody by LN and splenocyte cultures following Alum/antigen administration in vivo and antigen restimulation in vitro. Addition of IL-4 and IL-5 to type II NKT-deficient cultures restored in vitro antibody production. Intracellular staining revealed that Alum-primed type II NKT cells coordinated IL-4 secretion by T cells. Alum did not significantly affect CD1d expression in vivo, but addition of CD1d-blocking mAb diminished cytokine production and in vitro antibody production. Type II NKT cells therefore function as part of the Alum-sensing apparatus and in a CD1d-dependent manner, facilitate T(H)2-driven humoral immunity. This may have important consequences for understanding the mechanism of action of Alum-containing vaccines.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Compuestos de Alumbre/farmacología , Inmunidad Humoral/efectos de los fármacos , Células T Asesinas Naturales/inmunología , Animales , Antígenos CD1d/análisis , Linfocitos B/inmunología , Células Cultivadas , Citocinas/biosíntesis , Femenino , Inmunización , Inmunoglobulina G/biosíntesis , Ratones , Ratones Endogámicos C57BL , Células Th2/inmunologíaRESUMEN
CD1d-binding glycolipids exert potent adjuvant effects on T-dependent Ab responses. The mechanisms include cognate interaction between CD1d-expressing B cells and TCR-expressing Type I CD1d-restricted natural killer T cells (NKTs). However, the critical NKT-derived factors that stimulate B cells are poorly understood. We tested the hypothesis that CD1d-driven CD40L expression by NKT cells influences humoral immunity. Bone marrow chimeras with CD40L(+/+) or CD40L(-/-) NKT cells were immunized with Ag plus CD1d ligand before measuring Ab responses. CD40L(-/-) NKT cells stimulated higher endpoint Ab titers than controls expressing CD40L. In contrast, immunization of CD40L(-/-) mice revealed that CD40L(-/-) NKT cells could not provide B cell help when Th cells lacked CD40L. We report that CD40L(-/-) NKT cells can provide help for Ab production and do so cooperatively with CD40L(+/+) Th cells. We suggest that the manner in which NKT cells provide B cell help is distinct from that of Th cells.
Asunto(s)
Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Ligando de CD40/inmunología , Linfocitos Nulos/inmunología , Células T Asesinas Naturales/inmunología , Animales , Anticuerpos/sangre , Anticuerpos/inmunología , Antígenos CD1d , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Exogenous CD1d-binding glycolipid (alpha-Galactosylceramide, alpha-GC) stimulates TCR signaling and activation of type-1 natural killer-like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis-derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.
Asunto(s)
Antígenos Bacterianos/farmacología , Antígenos CD1d/metabolismo , Toxinas Bacterianas/farmacología , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Antígenos CD1d/inmunología , Bacillus anthracis/inmunología , Citocinas/metabolismo , Femenino , Citometría de Flujo , Galactosilceramidas/farmacología , Ratones , Ratones Endogámicos C57BL , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Células T Asesinas Naturales/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Péptidos/biosíntesis , Receptores de Péptidos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
NKT cell activation with CD1d-binding glycolipid alpha-galactosylceramide (alpha-GC) enhances antibody responses to co-administered T-dependent antigen. The efficacy of alpha-GC relative to other CD1d-binding glycolipids and adjuvants is not known. There is little information on how NKT cells affect antibody production beyond initial booster-stimulated recall responses. We therefore tested the hypothesis that alpha-GC stimulates induction of plasma cells and antibody responses as effectively as Th1- and Th2-skewing variants of alpha-GC and several other adjuvants. C57BL/6 and CD1d-/- mice were immunized with nitrophenol-conjugated keyhole limpet hemocyanin (NP-KLH) plus alpha-GC or NP-KLH plus adjuvants before administration of an NP-KLH booster and assessing antibody responses and plasma cell frequency. alpha-GC boosted long-term antibody responses as efficiently as all other agents tested and induced plasma cells that were detected in bone marrow 13 weeks after immunization. We then determined whether NKT cells were required in the presence of other adjuvants. CD1d-/- mice had a reduced induction of plasma cells in response to NP-KLH/Alum as compared to C57BL/6 mice. However, NKT cells were not required for the continued presence of those cells that were induced. Although NKT cells are capable of inducing persistent plasma cell responses, they may not play a major role in supporting longevity post-induction.