TLR9 Signaling Suppresses the Canonical Plasma Cell Differentiation Program in Follicular B Cells

The relative potency and quality of mouse B cell response to Toll-like receptors (TLRs) signaling varies significantly depending on the B cell subset and on the TLR member being engaged. Although it has been shown that marginal zone cells respond faster than follicular (FO) splenic B cells to TLR4 stimulus, FO B cells retain full capacity to proliferate and generate plasmablasts and plasma cells (PBs/PCs) with 2–3 days delayed kinetics. It is not clear whether this scenario could be extended to other members of the TLR family. Here, using quantitative cell culture conditions optimized for B cell growth and differentiation, we show that TLR9 signaling by CpG, while promoting vigorous proliferation, completely fails to induce differentiation of FO B cells into PBs/PCs. Little or absent Ig secretion following TLR9 stimulus was accompanied by lack of expression of cell surface markers and canonical transcription factors involved in PB/PC differentiation. Moreover, not only TLR9 did not induce plasmocyte differentiation, but it also strongly inhibited the massive PB/PC differentiation of FO B cells triggered by LPS/TLR4. Our study reveals unexpected opposite roles for TLR4 and TLR9 in the control of plasma cell differentiation program and disagrees with previous conclusions obtained in high-density cultures conditions on the generation of plasmocytes by TRL9 signaling. The potential implications of these findings on the role of TLR9 in controlling self-tolerance, clonal sizes and regulation of humoral responses are discussed

Antibody Repertoires Identify β-Tubulin as a Host Protective Parasite Antigen in Mice Infected With Trypanosoma cruzi

Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was T. cruzi β-tubulin. The T. cruzi β-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi β-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi β-tubulin. A single immunization of mice with recombinant T. cruzi β-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease

Apoptotic lymphocytes treated with IgG from Trypanosoma cruzi infection increase TNF-alpha secretion and reduce parasite replication in macrophages.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2015-02-09T17:19:35Z No. of bitstreams: 1 Montalvao F Apoptotic...pdf: 863295 bytes, checksum: d03a35f95c14528a92900489c246a968 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-02-09T17:19:51Z (GMT) No. of bitstreams: 1 Montalvao F Apoptotic...pdf: 863295 bytes, checksum: d03a35f95c14528a92900489c246a968 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-02-09T17:40:06Z (GMT) No. of bitstreams: 1 Montalvao F Apoptotic...pdf: 863295 bytes, checksum: d03a35f95c14528a92900489c246a968 (MD5)Made available in DSpace on 2015-02-09T17:40:06Z (GMT). No. of bitstreams: 1 Montalvao F Apoptotic...pdf: 863295 bytes, checksum: d03a35f95c14528a92900489c246a968 (MD5) Previous issue date: 2010Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, BrasilUniversidade Federal Fluminense. Instituto Biomédico. Niterói, RJ, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal Fluminense. Instituto de Biologia. Niterói, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Ciências Biomédicas. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Saúde e Ambiente na Região Amazônica. Conselho Nacional de Desenvolvimento Científico e Tecnológico. Amazonas, MA, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Saúde e Ambiente na Região Amazônica. Conselho Nacional de Desenvolvimento Científico e Tecnológico. Amazonas, MA, BrasilPhagocytic removal of apoptotic lymphocytes exacerbates replication of Trypanosoma cruzi in macrophages. We investigated the presence of Ab against apoptotic lymphocytes in T. cruzi infection and the role of these Ab in parasite replication. Both control and chagasic serum contained IgG Ab that opsonized apoptotic lymphocytes. Treatment of apoptotic lymphocytes with purified IgG from chagasic, but not control serum, reduced T. cruzi replication in macrophages. The protective effect of chagasic IgG depended on Fcgamma receptors, as demonstrated by the requirement for the intact Fc portion of IgG, and the effect could be abrogated by treating macrophages with an anti-CD16/CD32 Fab fragment. Chagasic IgG displayed increased reactivity against a subset of apoptotic cell Ag, as measured by flow cytometry and immunoblot analyses. Apoptotic lymphocytes treated with chagasic IgG, but not control IgG, increased production of TNF-alpha, while decreasing production of TGF-beta1 by infected macrophages. Increased control of parasite replication required TNF-alpha production. Previous immunization with apoptotic cells or injection of apoptotic cells opsonized with chagasic IgG reduced parasitemia in infected mice. These results indicate that Ab raised against apoptotic cells could play a protective role in control of T. cruzi replication by macrophages

Image_2.tif

<p>Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was T. cruzi β-tubulin. The T. cruzi β-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi β-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi β-tubulin. A single immunization of mice with recombinant T. cruzi β-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.</p

Image_1.tif

<p>Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was T. cruzi β-tubulin. The T. cruzi β-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi β-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi β-tubulin. A single immunization of mice with recombinant T. cruzi β-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.</p