Vaccine. 2013 Sep 23;31(41):4632-40. doi: 10.1016/j.vaccine.2013.07.030. Epub 2013 Aug 1.
Pneumococcal polysaccharide vaccination induces polysaccharide-specific B cells in adult peripheral blood expressing CD19⁺CD20⁺CD3⁻CD70⁻CD27⁺IgM⁺CD43⁺CD5⁺/⁻
Source
Department of Medicine, University of Toledo, Toledo, OH 43614, USA.
Department of Medicine, University of Toledo, Toledo, OH 43614, USA.
Abstract
Pneumococcal polysaccharide vaccines have been used to elicit a protective anti-pneumococcal polysaccharide antibody response against Streptococcus pneumoniae in healthy individuals. Identifying human B cells which respond to T-cell independent type-2 antigens, such as pneumococcal polysaccharides, has been challenging. We employed pneumococcal polysaccharides directly conjugated to fluorophores in conjunction with flow cytometry to identify the phenotype of B cells that respond to pneumococcal polysaccharide vaccination. We have previously identified that the majority of pneumococcal polysaccharide-selected cells responding to vaccination are CD27(+)IgM(+) (IgM(+) memory) cells. In this study, we further characterized pneumococcal polysaccharide-selected cells in the peripheral blood to better identify how the various B cell phenotypes responded 7 and 30 days post-immunization. We show that 7 days post-immunization the majority of pneumococcal polysaccharide-selected IgM(+) memory cells (PPS14(+) 56.5%, PPS23F(+) 63.8%) were CD19(+)CD20(+)CD27(+)IgM(+)CD43(+)CD5(+/-)CD70(-), which was significantly increased compared to pre-immunization levels. This phenotype is in alignment with recent publications describing human B-1 cells. PPS-responsive B cells receded to pre-immunization levels by day-30. These findings suggest that this B-1 like cell population plays an important role in early responses to S. pneumoniae infection and possibly other T-cell independent type-2 antigens in humans.
Pneumococcal polysaccharide vaccines have been used to elicit a protective anti-pneumococcal polysaccharide antibody response against Streptococcus pneumoniae in healthy individuals. Identifying human B cells which respond to T-cell independent type-2 antigens, such as pneumococcal polysaccharides, has been challenging. We employed pneumococcal polysaccharides directly conjugated to fluorophores in conjunction with flow cytometry to identify the phenotype of B cells that respond to pneumococcal polysaccharide vaccination. We have previously identified that the majority of pneumococcal polysaccharide-selected cells responding to vaccination are CD27(+)IgM(+) (IgM(+) memory) cells. In this study, we further characterized pneumococcal polysaccharide-selected cells in the peripheral blood to better identify how the various B cell phenotypes responded 7 and 30 days post-immunization. We show that 7 days post-immunization the majority of pneumococcal polysaccharide-selected IgM(+) memory cells (PPS14(+) 56.5%, PPS23F(+) 63.8%) were CD19(+)CD20(+)CD27(+)IgM(+)CD43(+)CD5(+/-)CD70(-), which was significantly increased compared to pre-immunization levels. This phenotype is in alignment with recent publications describing human B-1 cells. PPS-responsive B cells receded to pre-immunization levels by day-30. These findings suggest that this B-1 like cell population plays an important role in early responses to S. pneumoniae infection and possibly other T-cell independent type-2 antigens in humans.
J Infect Dis. 2013 Jul;208(1):101-8. doi: 10.1093/infdis/jit139. Epub 2013 Apr 1.
The elderly immune response to pneumococcal polysaccharides 14 and 23F consists predominantly of switched memory B cells.
Source
Department of Medicine, University of Toledo, Toledo, OH 43614, USA.
Department of Medicine, University of Toledo, Toledo, OH 43614, USA.
Abstract
The phenotype of B cells that respond to vaccination with the purified pneumococcal polysaccharide (PPS) has been a topic of debate. We have recently identified the phenotype of cells from healthy young volunteers as CD27(+)IgM(+) B cells. However, the PPS-responding B-cell population has not yet been identified in high-risk populations, such as elderly individuals. Previous studies have shown that elderly individuals have a lower percentage of immunoglobulin M memory B cells than healthy young adults. In this study, we directly characterized the phenotype of PPS-specific B cells before and after vaccination with PPS vaccine (PPV) in elderly adults, using fluorescently labeled PPS14 and PPS23F. In contrast to our observations in healthy young volunteers, the PPS-responding B-cell population consisted primarily of switched memory (CD27(+)IgM(-)) B cells. In concurrence with these findings, postvaccination immunoglobulin M concentrations were not significantly increased in this population, and the opsonophagocytic response was decreased, compared with that in young adults. These findings identify a significant shift in the phenotype of the B-cell population in response to PPV among elderly individuals.
The phenotype of B cells that respond to vaccination with the purified pneumococcal polysaccharide (PPS) has been a topic of debate. We have recently identified the phenotype of cells from healthy young volunteers as CD27(+)IgM(+) B cells. However, the PPS-responding B-cell population has not yet been identified in high-risk populations, such as elderly individuals. Previous studies have shown that elderly individuals have a lower percentage of immunoglobulin M memory B cells than healthy young adults. In this study, we directly characterized the phenotype of PPS-specific B cells before and after vaccination with PPS vaccine (PPV) in elderly adults, using fluorescently labeled PPS14 and PPS23F. In contrast to our observations in healthy young volunteers, the PPS-responding B-cell population consisted primarily of switched memory (CD27(+)IgM(-)) B cells. In concurrence with these findings, postvaccination immunoglobulin M concentrations were not significantly increased in this population, and the opsonophagocytic response was decreased, compared with that in young adults. These findings identify a significant shift in the phenotype of the B-cell population in response to PPV among elderly individuals.
Open Journal of Immunology. 2012 2;98-110. doi: 10.4236/oji.2012.23012.
Isolation and characterization of human polyreactive pneumococcal polysaccharide antibodies.
Source
Department of Medicine, University of Toledo, Toledo, OH 43614, USA.
Department of Medicine, University of Toledo, Toledo, OH 43614, USA.
Abstract
Natural antibodies serve as the body’s first line of defense against pneumococcal challenge. Polyreactive human pneumococcal polysaccharide IgG antibodies have not been extensively studied. We analyzed human polyreactive antibodies that bind multiple pneumococcal polysaccharides, including PPS14 and PPS23F. These antibodies were isolated from single pneumococcal polysaccharide specific B cells allowing for the analysis of human immunoglobulins with natively paired variable regions. Although isolated individually, these antibodies demonstrated similar characteristics. Most antibodies possessed a variable light chain with a CDR3 length made up of nine amino acids and relatively high number of flexible amino acids in combined VH/VL. While these antibodies were polyreactive and structurally alike, kinetic analysis revealed unique KD values. Variable chains are responsible for antigen recognition whereas antibody fine specificity is affected by isotype structure. To investigate the contribution of the constant region of these isotypes and their effect on antibody avidity to pneumococcal polysaccharide, the polyreactive variable regions were expressed as IgG1 or IgG2 and subjected to kinetic analysis. The IgG1 antibodies uniformly had a stronger avidity to PPS14 and PPS23F compared to IgG2. To further document the importance of the constant region in antibody avidity and fine specificity, analysis of antibody F(ab)’2 fragment binding to PPS14 and PPS23F resulted in similar KD values. These studies suggest that antigen recognition by polyreactive antibodies is determined by a conserved variable light chain CDR3 length and longer, more flexible variable heavy CDR3s when compared to pneumococcal polysaccha-ride-specific sequences while differences in specific avidities are modulated by antibody isotype.
Natural antibodies serve as the body’s first line of defense against pneumococcal challenge. Polyreactive human pneumococcal polysaccharide IgG antibodies have not been extensively studied. We analyzed human polyreactive antibodies that bind multiple pneumococcal polysaccharides, including PPS14 and PPS23F. These antibodies were isolated from single pneumococcal polysaccharide specific B cells allowing for the analysis of human immunoglobulins with natively paired variable regions. Although isolated individually, these antibodies demonstrated similar characteristics. Most antibodies possessed a variable light chain with a CDR3 length made up of nine amino acids and relatively high number of flexible amino acids in combined VH/VL. While these antibodies were polyreactive and structurally alike, kinetic analysis revealed unique KD values. Variable chains are responsible for antigen recognition whereas antibody fine specificity is affected by isotype structure. To investigate the contribution of the constant region of these isotypes and their effect on antibody avidity to pneumococcal polysaccharide, the polyreactive variable regions were expressed as IgG1 or IgG2 and subjected to kinetic analysis. The IgG1 antibodies uniformly had a stronger avidity to PPS14 and PPS23F compared to IgG2. To further document the importance of the constant region in antibody avidity and fine specificity, analysis of antibody F(ab)’2 fragment binding to PPS14 and PPS23F resulted in similar KD values. These studies suggest that antigen recognition by polyreactive antibodies is determined by a conserved variable light chain CDR3 length and longer, more flexible variable heavy CDR3s when compared to pneumococcal polysaccha-ride-specific sequences while differences in specific avidities are modulated by antibody isotype.
J Immunol. 2012 Mar 1;188(5):2455-63. doi: 10.4049/jimmunol.1102809. Epub 2012 Jan 23.
Phenotypic analysis of pneumococcal polysaccharide-specific B cells.
Source
Department of Medicine, University of Toledo, Toledo, OH 43614, USA.
Department of Medicine, University of Toledo, Toledo, OH 43614, USA.
Abstract
The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide (PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.
The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide (PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.
Dr. Rebecca Thompson 