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Activation of virus-specific memory B cells in the absence of T cell help.

Hebeis BJ, Klenovsek K, Rohwer P, Ritter U, Schneider A, Mach M, Winkler TH - J. Exp. Med. (2004)

Bottom Line: Antigenic stimulation 4-6 d after transfer of B cells resulted in rapid IgG production.Transfer of memory B cells into immunocompetent animals indicated that presence of helper T cells did not enhance the memory B cell response.Therefore, our results indicate that activation of virus-specific memory B cells to secrete IgG is independent of cognate or bystander T cell help.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, D-91054 Erlangen, Germany.

ABSTRACT
Humoral immunity is maintained by long-lived plasma cells, constitutively secreting antibodies, and nonsecreting resting memory B cells that are rapidly reactivated upon antigen encounter. The activation requirements for resting memory B cells, particularly the role of T helper cells, are unclear. To analyze the activation of memory B cells, mice were immunized with human cytomegalovirus, a complex human herpesvirus, and tick-born encephalitis virus, and a simple flavivirus. B cell populations devoid of Ig-secreting plasma cells were adoptively transferred into T and B cell-deficient RAG-1-/- mice. Antigenic stimulation 4-6 d after transfer of B cells resulted in rapid IgG production. The response was long lasting and strictly antigen specific, excluding polyclonal B cell activation. CD4+ T cells were not involved since (a) further depletion of CD4+ T cells in the recipient mice did not alter the antibody response and (b) recipient mice contained no detectable CD4+ T cells 90 d posttransfer. Memory B cells could not be activated by a soluble viral protein without T cell help. Transfer of memory B cells into immunocompetent animals indicated that presence of helper T cells did not enhance the memory B cell response. Therefore, our results indicate that activation of virus-specific memory B cells to secrete IgG is independent of cognate or bystander T cell help.

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IgG reactivity and biological activity of recipient and donor sera. (A) Summary of the experimental protocol for immunization, adoptive transfer, and challenge with antigen. C57BL/6 mice were immunized twice with HCMV-dense bodies. 200 d later, sorted B cells from the spleen were adoptively transferred into RAG-1−/− recipients. On day 6 posttransfer, recipients were challenged with 10 μg HCMV-DBs i.v., and sera were analyzed on the days indicated. (B) Sera from recipients of B cells from immunized (♦) and naive (X) donors were analyzed by ELISA for HCMV-specific IgG. (C) Sera taken at day 40 postchallenge were analyzed in immunoblots and compared with sera of donor mice. (D) Neutralization activity of sera from RAG-1−/− mice adoptively transferred with immune (♦) or naive (X) B cells and of sera from donor mice (•) was analyzed in vitro. (E) IgG subclass composition of donor sera (•) (dilution 1:1,000) and recipient sera (○) (dilution, 1:200) analyzed by ELISA using subclass-specific antibodies. Values correspond to the dilution above detection threshold.
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fig1: IgG reactivity and biological activity of recipient and donor sera. (A) Summary of the experimental protocol for immunization, adoptive transfer, and challenge with antigen. C57BL/6 mice were immunized twice with HCMV-dense bodies. 200 d later, sorted B cells from the spleen were adoptively transferred into RAG-1−/− recipients. On day 6 posttransfer, recipients were challenged with 10 μg HCMV-DBs i.v., and sera were analyzed on the days indicated. (B) Sera from recipients of B cells from immunized (♦) and naive (X) donors were analyzed by ELISA for HCMV-specific IgG. (C) Sera taken at day 40 postchallenge were analyzed in immunoblots and compared with sera of donor mice. (D) Neutralization activity of sera from RAG-1−/− mice adoptively transferred with immune (♦) or naive (X) B cells and of sera from donor mice (•) was analyzed in vitro. (E) IgG subclass composition of donor sera (•) (dilution 1:1,000) and recipient sera (○) (dilution, 1:200) analyzed by ELISA using subclass-specific antibodies. Values correspond to the dilution above detection threshold.

Mentions: 4 wk after the last immunization, blood was taken and serum was analyzed for HCMV-specific IgG and neutralizing antibodies (a schematic outline of the experimental protocol is provided in Fig. 1 A). Sera from all immunized mice exhibited high titers of virus-specific IgG, as determined by ELISA, and considerable virus-neutralizing activity (range 1:1,000–1:3,200; a representative analysis is shown in Fig. 1 D). At different time points (42–200 d) after the last immunization, CD19-positive small resting B cells were isolated from the spleen by two rounds of cell sorting. In general, a purity of >99.8% was achieved by this procedure (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20030091/DC1). Individual RAG-1−/− mice were infused with 5 × 106 purified B cells, and 6 d later the animals were either injected with 10 μg HCMV-DBs i.v. or left untreated. Sera were analyzed for HCMV-specific IgG between days 10 and 80 after transfer of B cells. 10 d after antigen stimulation, HCMV-specific IgG was clearly detectable (Fig. 1 B). The titers remained elevated during the observation period of 80 d. In the adoptively transferred RAG-1−/− mice, HCMV-specific IgG titers were 2–3 log2 dilutions lower than in immunized animals, which was expected since only a fraction of the B cells normally present in mice were used for the transfer. In fact, transferring higher numbers of CD19+ B cells to recipient mice resulted in elevated antibody titers (not depicted). In contrast, sera from RAG-1−/− mice that had been infused with B cells from naive donors and challenged with HCMV-DBs remained negative for HCMV-specific IgG during the entire observation period (Fig. 1 B). The use of CD19, which is not present on plasma cells, as the target for B cell isolation and the absence of HCMV-specific IgG in RAG-1−/− mice on day 6 after transfer of immune B cells argued against the presence of IgG-producing plasma cells in the transplant (Fig. 1 B). The lack of plasma cells in the transplant was further corroborated by the fact that RAG-1−/− mice, which had received B cells from immune donors but were not challenged with HCMV-DBs for up to 90 d remained negative for HCMV-specific IgG (see Fig. 6 A and not depicted).


Activation of virus-specific memory B cells in the absence of T cell help.

Hebeis BJ, Klenovsek K, Rohwer P, Ritter U, Schneider A, Mach M, Winkler TH - J. Exp. Med. (2004)

IgG reactivity and biological activity of recipient and donor sera. (A) Summary of the experimental protocol for immunization, adoptive transfer, and challenge with antigen. C57BL/6 mice were immunized twice with HCMV-dense bodies. 200 d later, sorted B cells from the spleen were adoptively transferred into RAG-1−/− recipients. On day 6 posttransfer, recipients were challenged with 10 μg HCMV-DBs i.v., and sera were analyzed on the days indicated. (B) Sera from recipients of B cells from immunized (♦) and naive (X) donors were analyzed by ELISA for HCMV-specific IgG. (C) Sera taken at day 40 postchallenge were analyzed in immunoblots and compared with sera of donor mice. (D) Neutralization activity of sera from RAG-1−/− mice adoptively transferred with immune (♦) or naive (X) B cells and of sera from donor mice (•) was analyzed in vitro. (E) IgG subclass composition of donor sera (•) (dilution 1:1,000) and recipient sera (○) (dilution, 1:200) analyzed by ELISA using subclass-specific antibodies. Values correspond to the dilution above detection threshold.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211828&req=5

fig1: IgG reactivity and biological activity of recipient and donor sera. (A) Summary of the experimental protocol for immunization, adoptive transfer, and challenge with antigen. C57BL/6 mice were immunized twice with HCMV-dense bodies. 200 d later, sorted B cells from the spleen were adoptively transferred into RAG-1−/− recipients. On day 6 posttransfer, recipients were challenged with 10 μg HCMV-DBs i.v., and sera were analyzed on the days indicated. (B) Sera from recipients of B cells from immunized (♦) and naive (X) donors were analyzed by ELISA for HCMV-specific IgG. (C) Sera taken at day 40 postchallenge were analyzed in immunoblots and compared with sera of donor mice. (D) Neutralization activity of sera from RAG-1−/− mice adoptively transferred with immune (♦) or naive (X) B cells and of sera from donor mice (•) was analyzed in vitro. (E) IgG subclass composition of donor sera (•) (dilution 1:1,000) and recipient sera (○) (dilution, 1:200) analyzed by ELISA using subclass-specific antibodies. Values correspond to the dilution above detection threshold.
Mentions: 4 wk after the last immunization, blood was taken and serum was analyzed for HCMV-specific IgG and neutralizing antibodies (a schematic outline of the experimental protocol is provided in Fig. 1 A). Sera from all immunized mice exhibited high titers of virus-specific IgG, as determined by ELISA, and considerable virus-neutralizing activity (range 1:1,000–1:3,200; a representative analysis is shown in Fig. 1 D). At different time points (42–200 d) after the last immunization, CD19-positive small resting B cells were isolated from the spleen by two rounds of cell sorting. In general, a purity of >99.8% was achieved by this procedure (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20030091/DC1). Individual RAG-1−/− mice were infused with 5 × 106 purified B cells, and 6 d later the animals were either injected with 10 μg HCMV-DBs i.v. or left untreated. Sera were analyzed for HCMV-specific IgG between days 10 and 80 after transfer of B cells. 10 d after antigen stimulation, HCMV-specific IgG was clearly detectable (Fig. 1 B). The titers remained elevated during the observation period of 80 d. In the adoptively transferred RAG-1−/− mice, HCMV-specific IgG titers were 2–3 log2 dilutions lower than in immunized animals, which was expected since only a fraction of the B cells normally present in mice were used for the transfer. In fact, transferring higher numbers of CD19+ B cells to recipient mice resulted in elevated antibody titers (not depicted). In contrast, sera from RAG-1−/− mice that had been infused with B cells from naive donors and challenged with HCMV-DBs remained negative for HCMV-specific IgG during the entire observation period (Fig. 1 B). The use of CD19, which is not present on plasma cells, as the target for B cell isolation and the absence of HCMV-specific IgG in RAG-1−/− mice on day 6 after transfer of immune B cells argued against the presence of IgG-producing plasma cells in the transplant (Fig. 1 B). The lack of plasma cells in the transplant was further corroborated by the fact that RAG-1−/− mice, which had received B cells from immune donors but were not challenged with HCMV-DBs for up to 90 d remained negative for HCMV-specific IgG (see Fig. 6 A and not depicted).

Bottom Line: Antigenic stimulation 4-6 d after transfer of B cells resulted in rapid IgG production.Transfer of memory B cells into immunocompetent animals indicated that presence of helper T cells did not enhance the memory B cell response.Therefore, our results indicate that activation of virus-specific memory B cells to secrete IgG is independent of cognate or bystander T cell help.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, D-91054 Erlangen, Germany.

ABSTRACT
Humoral immunity is maintained by long-lived plasma cells, constitutively secreting antibodies, and nonsecreting resting memory B cells that are rapidly reactivated upon antigen encounter. The activation requirements for resting memory B cells, particularly the role of T helper cells, are unclear. To analyze the activation of memory B cells, mice were immunized with human cytomegalovirus, a complex human herpesvirus, and tick-born encephalitis virus, and a simple flavivirus. B cell populations devoid of Ig-secreting plasma cells were adoptively transferred into T and B cell-deficient RAG-1-/- mice. Antigenic stimulation 4-6 d after transfer of B cells resulted in rapid IgG production. The response was long lasting and strictly antigen specific, excluding polyclonal B cell activation. CD4+ T cells were not involved since (a) further depletion of CD4+ T cells in the recipient mice did not alter the antibody response and (b) recipient mice contained no detectable CD4+ T cells 90 d posttransfer. Memory B cells could not be activated by a soluble viral protein without T cell help. Transfer of memory B cells into immunocompetent animals indicated that presence of helper T cells did not enhance the memory B cell response. Therefore, our results indicate that activation of virus-specific memory B cells to secrete IgG is independent of cognate or bystander T cell help.

Show MeSH
Related in: MedlinePlus