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Signaling of Human Naïve B Cells Via Toll-like Receptor 7 Induces Immunoglobulin Isotype Switch
Abstracts S101
J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 2
The Absence of HLA B8 and B44 is Associated with Decreased Absolute B Cell Numbers in Common Variable Immunodeficiency D. T. Johnston, H. W. Schroeder, Jr.; Clinical and Developmental Immunology, University of Alabama at Birmingham, Birmingham, AL. RATIONALE: Genetic susceptibility to common variable immunodeficiency (CVID) has been linked to genes within the major histocompatibility complex (MHC) and has been associated with the inheritance of part or all of two extended MHC haplotypes; HLA B8 DR3(17) and HLA B44 DR7. Our goal was to determine if the presence of these susceptible MHC haplotypes correlate with absolute B cell numbers in CVID. METHODS: A retrospective chart review of 60 consecutive CVID patients seen in our adult immunodeficiency clinic yielded 56 eligible patients. There were 4 patients excluded because of absent absolute B cell data. RESULTS: Among the 56 CVID patents, the average age was 39+2 and 79% inherited all or part of the 2 MHC susceptible haplotypes. 68% inherited MHC B8 or B44 and 61% inherited MHC DR7 or DR17(3). The mean absolute B cell count in patients who had inherited MHC B8 or B44 was 214 compared to 119 in those who had not inherited MHC B8 or B44 (p=0.01). Patients who inherited B44 had higher mean B cell numbers compared to those who did not inherited B44 or B8 (p=0.047). In patients who inherited B8, the mean absolute B cell count was higher compared to those who did not have MHC B8 or B44 (p= 0.01). The presence or absence of MHC DR antigens did not show statistically significant variation in absolute B cells. CONCLUSIONS: The absence of HLA B8 and B44 is associated with decreased absolute B cell numbers in common variable immunodeficiency. Funding: NIH
394
Impaired Maturation of Monocyte Derived Dendritic Cells from Birch Allergic Individuals R. Casas1, K. Duchén1, O. Zetterström2, O. Vaarala1; 1Molecular and Clinical Medicine, Paediatrics, Linköping University, SWEDEN, 2Molecular and Clinical Medicine, Allergy Centrum, Linköping University, SWEDEN. RATIONALE: To study whether monocytes differentiated dendritic cells (DCs) from healthy and birch allergic asthmatic individuals differs in their phenotypic receptor expression and cytokine secretion. METHODS: Monocytes were isolated from separated mononuclear cell from allergic (n= 14) and healthy (n=14) individuals. Differentiated DCs were further pulsed by adding birch and cat allergens and LPS, and nonstimulated DCs were included as controls. DCs stained with anti-CD83, -CD80, - CD1a; -CD86, -CD11c and - HLA DR antibodies were analysed by four-colour flow cytometry. Cytokines and chemokines were measure in DCs supernatant in a Bio-Plex Human 7-plex assay, including IL-6, IL-8, IL-10, IL-12p70, MCP-1/MCAF, TNF- and MIP-1. RESULTS: The percentages of DCs expressing HLA-DR, CD80, CD86, CD83, CD1a and CD11c were similar in cells from allergic and healthy individuals. However, while cells from healthy donators showed similar activation of the CD80 and double CD80/CD86 receptors after stimulation as compared to non-stimulated cells, birch allergen was unable to increase CD80 and double CD80/CD86 expression on cells from allergics. Levels of IL-12p70 and TNF- were higher in supernatant from nonstimulated cells from healthy and further increased after birch and LPS stimulation. After cat stimulation, increased levels of IL-8, IL-10, MIP-1 and MCP-1 were detected in samples from allergic while the levels after birch stimulation. CONCLUSIONS: Our results suggest an impaired specific maturation of DCs from birch allergic individuals in association to birch specific immune responses. Lower secretion of IL-12p70 indicated that the specific Th1 function seems to be affected in allergics. Funding: Swedish Allergy and Cancer Foundation, County of östergötland
395
Using the Pneumococcal Conjugate Vaccine PCV7 (Prevnar) to Evaluate the Functional Antibody Response in Adults with Recurrent Sinopulmonary Infections J. Caicedo, S. Skoda-Smith; Pediatrics, University of Florida, Gainesville, FL. RATIONALE: Measuring antibody function is a critical part of the humoral immune evaluation. Antibody response to Pneumovax has been used to determine whether polysaccharide antibody deficiency exists in children over 2 years, and in adults. In 2000, the FDA approved the use of a conjugate pneumococcal polysaccharide vaccine, Prevnar, in children under 2 years. The AAP has published recommendations related to the use of Prevnar in high-risk children, including those with primary immunodeficiency. No large scale studies have been done in immunodeficient adults. METHODS: Beginning in January 2001, we offered Prevnar vaccine challenges to selected individuals over the age of 13 with a history of recurrent sinopulmonary infections. All patients who were offered a Prevnar challenge failed to make a protective response and/or did not have a minimum two-fold rise to at least 50% of the serotypes of pneumococcal polysaccahride assessed after vaccine challenge with Pneumovax. Patients were excluded from Prevnar challenge if they had confirmed bronchiectasis, two or more bacterial pneumonias, bacterial meningitis, or CVID. RESULTS: Twenty-one patients over 18 were given Prevnar. No one developed a severe local reaction or fever. Patients who responded to vaccine challenge with Prevnar were not offered IVIG therapy. The stability of their post-vaccination titers and pattern of recurrent bacterial infections continues to be monitored. Patients who did not respond to Prevnar were offered a minimum 6 month trial of IVIG hterapy. CONCLUSIONS: Vaccine challenge using the available PCV7 vaccine may help to discriminate selected adult patients with functional antibody deficiency that might benefit from IVIG therapy.
396
SUNDAY
Signaling of Human Naïve B Cells Via Toll-like Receptor 7 Induces Immunoglobulin Isotype Switch S. Narula1, M. Glaum2, D. Song1, A. Anderson1, Y. Zheng1, A. I. Levinson1; 1Medicine, University of Pennsylvania, Philadelphia, PA, 2Medicine, University of South Florida College of Medicine, Tampa, FL. RATIONALE: Although Toll-like receptors (TLRs) are expressed on human B lymphocytes, their ability to directly induce isotype switch and immunoglobulin production remains controversial. We sought to determine if the TLR-agonist, Resiquimod, induced IgG production by naïve human B cells and if these cells demonstrated the molecular correlates of immunoglobulin class switch recombination. METHODS: Surface IgG negative/CD27 negative naïve CD19+ human B cells were isolated from healthy donors. They were cultured with Resiquimod in the presence or absence of the B cell cross-linker S. aureus Cowan I (SAC), IL-2 and IL-10. After 7 days of culture, supernatants were analyzed by ELISA for IgM and IgG. RNA, isolated from the cell pellets, was subjected to reverse transcription. The resultant cDNAs were amplified in PCR with primers specific for activation induced cytidine deaminase (AID) and I-C circle transcripts. RESULTS: Stimulation of the naive B lymphocytes with Resiquimod induced production of IgM and IgG. Also, Resiquimod additively stimulated production of IgM and IgG by naive B cells incubated with SAC and cytokines. Further evidence for Resiquimod-induced isotype switch was seen in the RT-PCRs. Amplified products of cDNAs generated from the RNA of Resiquimod-stimulated cells showed induction of both AID and I-C circle transcripts. CONCLUSIONS: These studies provide convincing evidence that direct perturbation of TLR7 on naive human B cells can lead to the induction of immunoglobulin class switch and IgG production. The additive effect of Resiquimod, SAC, and cytokines on immunoglobulin also supports a cooperative role for TLR7 ligands in the amplification of antigen-induced B cell responses.
393
J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 2
The Absence of HLA B8 and B44 is Associated with Decreased Absolute B Cell Numbers in Common Variable Immunodeficiency D. T. Johnston, H. W. Schroeder, Jr.; Clinical and Developmental Immunology, University of Alabama at Birmingham, Birmingham, AL. RATIONALE: Genetic susceptibility to common variable immunodeficiency (CVID) has been linked to genes within the major histocompatibility complex (MHC) and has been associated with the inheritance of part or all of two extended MHC haplotypes; HLA B8 DR3(17) and HLA B44 DR7. Our goal was to determine if the presence of these susceptible MHC haplotypes correlate with absolute B cell numbers in CVID. METHODS: A retrospective chart review of 60 consecutive CVID patients seen in our adult immunodeficiency clinic yielded 56 eligible patients. There were 4 patients excluded because of absent absolute B cell data. RESULTS: Among the 56 CVID patents, the average age was 39+2 and 79% inherited all or part of the 2 MHC susceptible haplotypes. 68% inherited MHC B8 or B44 and 61% inherited MHC DR7 or DR17(3). The mean absolute B cell count in patients who had inherited MHC B8 or B44 was 214 compared to 119 in those who had not inherited MHC B8 or B44 (p=0.01). Patients who inherited B44 had higher mean B cell numbers compared to those who did not inherited B44 or B8 (p=0.047). In patients who inherited B8, the mean absolute B cell count was higher compared to those who did not have MHC B8 or B44 (p= 0.01). The presence or absence of MHC DR antigens did not show statistically significant variation in absolute B cells. CONCLUSIONS: The absence of HLA B8 and B44 is associated with decreased absolute B cell numbers in common variable immunodeficiency. Funding: NIH
394
Impaired Maturation of Monocyte Derived Dendritic Cells from Birch Allergic Individuals R. Casas1, K. Duchén1, O. Zetterström2, O. Vaarala1; 1Molecular and Clinical Medicine, Paediatrics, Linköping University, SWEDEN, 2Molecular and Clinical Medicine, Allergy Centrum, Linköping University, SWEDEN. RATIONALE: To study whether monocytes differentiated dendritic cells (DCs) from healthy and birch allergic asthmatic individuals differs in their phenotypic receptor expression and cytokine secretion. METHODS: Monocytes were isolated from separated mononuclear cell from allergic (n= 14) and healthy (n=14) individuals. Differentiated DCs were further pulsed by adding birch and cat allergens and LPS, and nonstimulated DCs were included as controls. DCs stained with anti-CD83, -CD80, - CD1a; -CD86, -CD11c and - HLA DR antibodies were analysed by four-colour flow cytometry. Cytokines and chemokines were measure in DCs supernatant in a Bio-Plex Human 7-plex assay, including IL-6, IL-8, IL-10, IL-12p70, MCP-1/MCAF, TNF- and MIP-1. RESULTS: The percentages of DCs expressing HLA-DR, CD80, CD86, CD83, CD1a and CD11c were similar in cells from allergic and healthy individuals. However, while cells from healthy donators showed similar activation of the CD80 and double CD80/CD86 receptors after stimulation as compared to non-stimulated cells, birch allergen was unable to increase CD80 and double CD80/CD86 expression on cells from allergics. Levels of IL-12p70 and TNF- were higher in supernatant from nonstimulated cells from healthy and further increased after birch and LPS stimulation. After cat stimulation, increased levels of IL-8, IL-10, MIP-1 and MCP-1 were detected in samples from allergic while the levels after birch stimulation. CONCLUSIONS: Our results suggest an impaired specific maturation of DCs from birch allergic individuals in association to birch specific immune responses. Lower secretion of IL-12p70 indicated that the specific Th1 function seems to be affected in allergics. Funding: Swedish Allergy and Cancer Foundation, County of östergötland
395
Using the Pneumococcal Conjugate Vaccine PCV7 (Prevnar) to Evaluate the Functional Antibody Response in Adults with Recurrent Sinopulmonary Infections J. Caicedo, S. Skoda-Smith; Pediatrics, University of Florida, Gainesville, FL. RATIONALE: Measuring antibody function is a critical part of the humoral immune evaluation. Antibody response to Pneumovax has been used to determine whether polysaccharide antibody deficiency exists in children over 2 years, and in adults. In 2000, the FDA approved the use of a conjugate pneumococcal polysaccharide vaccine, Prevnar, in children under 2 years. The AAP has published recommendations related to the use of Prevnar in high-risk children, including those with primary immunodeficiency. No large scale studies have been done in immunodeficient adults. METHODS: Beginning in January 2001, we offered Prevnar vaccine challenges to selected individuals over the age of 13 with a history of recurrent sinopulmonary infections. All patients who were offered a Prevnar challenge failed to make a protective response and/or did not have a minimum two-fold rise to at least 50% of the serotypes of pneumococcal polysaccahride assessed after vaccine challenge with Pneumovax. Patients were excluded from Prevnar challenge if they had confirmed bronchiectasis, two or more bacterial pneumonias, bacterial meningitis, or CVID. RESULTS: Twenty-one patients over 18 were given Prevnar. No one developed a severe local reaction or fever. Patients who responded to vaccine challenge with Prevnar were not offered IVIG therapy. The stability of their post-vaccination titers and pattern of recurrent bacterial infections continues to be monitored. Patients who did not respond to Prevnar were offered a minimum 6 month trial of IVIG hterapy. CONCLUSIONS: Vaccine challenge using the available PCV7 vaccine may help to discriminate selected adult patients with functional antibody deficiency that might benefit from IVIG therapy.
396
SUNDAY
Signaling of Human Naïve B Cells Via Toll-like Receptor 7 Induces Immunoglobulin Isotype Switch S. Narula1, M. Glaum2, D. Song1, A. Anderson1, Y. Zheng1, A. I. Levinson1; 1Medicine, University of Pennsylvania, Philadelphia, PA, 2Medicine, University of South Florida College of Medicine, Tampa, FL. RATIONALE: Although Toll-like receptors (TLRs) are expressed on human B lymphocytes, their ability to directly induce isotype switch and immunoglobulin production remains controversial. We sought to determine if the TLR-agonist, Resiquimod, induced IgG production by naïve human B cells and if these cells demonstrated the molecular correlates of immunoglobulin class switch recombination. METHODS: Surface IgG negative/CD27 negative naïve CD19+ human B cells were isolated from healthy donors. They were cultured with Resiquimod in the presence or absence of the B cell cross-linker S. aureus Cowan I (SAC), IL-2 and IL-10. After 7 days of culture, supernatants were analyzed by ELISA for IgM and IgG. RNA, isolated from the cell pellets, was subjected to reverse transcription. The resultant cDNAs were amplified in PCR with primers specific for activation induced cytidine deaminase (AID) and I-C circle transcripts. RESULTS: Stimulation of the naive B lymphocytes with Resiquimod induced production of IgM and IgG. Also, Resiquimod additively stimulated production of IgM and IgG by naive B cells incubated with SAC and cytokines. Further evidence for Resiquimod-induced isotype switch was seen in the RT-PCRs. Amplified products of cDNAs generated from the RNA of Resiquimod-stimulated cells showed induction of both AID and I-C circle transcripts. CONCLUSIONS: These studies provide convincing evidence that direct perturbation of TLR7 on naive human B cells can lead to the induction of immunoglobulin class switch and IgG production. The additive effect of Resiquimod, SAC, and cytokines on immunoglobulin also supports a cooperative role for TLR7 ligands in the amplification of antigen-induced B cell responses.
393