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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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Specificity of the IgG response of adoptively transferred memory B cells. (A) C57BL/6 mice were immunized with HCMV-DBs and TBEV particles, and CD19+ cells were isolated and transferred to RAG-1−/− mice according to the legend for Fig. 1 A. On day 4 posttransfer, recipients were challenged with 10 μg HCMV-DBs (▪) or 2 μg TBEV particles (•). Blood was taken at the days indicated, and sera were analyzed by ELISA for TBEV- (left) and HCMV-specific (right) IgG reactivity, respectively. On day 90 posttransfer, recipients were immunized again (arrows), this time using the reciprocal antigen, and IgG titers were determined. (B) Sera taken at day 40 poststimulation (dilution, 1:250) and sera of donor mice (dilution, 1:40,00) were analyzed by Western blot for IgG specificities against lysates of TBEV particles. (C) C57BL/6 mice were immunized with either a recombinant monomeric form of the HCMV viral envelope protein gB or HCMV-DBs, and CD19+ cells were isolated and transferred to RAG-1−/− mice according to Fig. 1 A. On day 7 posttransfer, recipients were challenged with either 2 μg HCMV-DBs or 10 μg recombinant gB. The bars represent the mean of RI values; dots are individual values. The experiment was repeated twice with similar results.
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fig6: Specificity of the IgG response of adoptively transferred memory B cells. (A) C57BL/6 mice were immunized with HCMV-DBs and TBEV particles, and CD19+ cells were isolated and transferred to RAG-1−/− mice according to the legend for Fig. 1 A. On day 4 posttransfer, recipients were challenged with 10 μg HCMV-DBs (▪) or 2 μg TBEV particles (•). Blood was taken at the days indicated, and sera were analyzed by ELISA for TBEV- (left) and HCMV-specific (right) IgG reactivity, respectively. On day 90 posttransfer, recipients were immunized again (arrows), this time using the reciprocal antigen, and IgG titers were determined. (B) Sera taken at day 40 poststimulation (dilution, 1:250) and sera of donor mice (dilution, 1:40,00) were analyzed by Western blot for IgG specificities against lysates of TBEV particles. (C) C57BL/6 mice were immunized with either a recombinant monomeric form of the HCMV viral envelope protein gB or HCMV-DBs, and CD19+ cells were isolated and transferred to RAG-1−/− mice according to Fig. 1 A. On day 7 posttransfer, recipients were challenged with either 2 μg HCMV-DBs or 10 μg recombinant gB. The bars represent the mean of RI values; dots are individual values. The experiment was repeated twice with similar results.

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)

Specificity of the IgG response of adoptively transferred memory B cells. (A) C57BL/6 mice were immunized with HCMV-DBs and TBEV particles, and CD19+ cells were isolated and transferred to RAG-1−/− mice according to the legend for Fig. 1 A. On day 4 posttransfer, recipients were challenged with 10 μg HCMV-DBs (▪) or 2 μg TBEV particles (•). Blood was taken at the days indicated, and sera were analyzed by ELISA for TBEV- (left) and HCMV-specific (right) IgG reactivity, respectively. On day 90 posttransfer, recipients were immunized again (arrows), this time using the reciprocal antigen, and IgG titers were determined. (B) Sera taken at day 40 poststimulation (dilution, 1:250) and sera of donor mice (dilution, 1:40,00) were analyzed by Western blot for IgG specificities against lysates of TBEV particles. (C) C57BL/6 mice were immunized with either a recombinant monomeric form of the HCMV viral envelope protein gB or HCMV-DBs, and CD19+ cells were isolated and transferred to RAG-1−/− mice according to Fig. 1 A. On day 7 posttransfer, recipients were challenged with either 2 μg HCMV-DBs or 10 μg recombinant gB. The bars represent the mean of RI values; dots are individual values. The experiment was repeated twice with similar results.
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Related In: Results  -  Collection

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fig6: Specificity of the IgG response of adoptively transferred memory B cells. (A) C57BL/6 mice were immunized with HCMV-DBs and TBEV particles, and CD19+ cells were isolated and transferred to RAG-1−/− mice according to the legend for Fig. 1 A. On day 4 posttransfer, recipients were challenged with 10 μg HCMV-DBs (▪) or 2 μg TBEV particles (•). Blood was taken at the days indicated, and sera were analyzed by ELISA for TBEV- (left) and HCMV-specific (right) IgG reactivity, respectively. On day 90 posttransfer, recipients were immunized again (arrows), this time using the reciprocal antigen, and IgG titers were determined. (B) Sera taken at day 40 poststimulation (dilution, 1:250) and sera of donor mice (dilution, 1:40,00) were analyzed by Western blot for IgG specificities against lysates of TBEV particles. (C) C57BL/6 mice were immunized with either a recombinant monomeric form of the HCMV viral envelope protein gB or HCMV-DBs, and CD19+ cells were isolated and transferred to RAG-1−/− mice according to Fig. 1 A. On day 7 posttransfer, recipients were challenged with either 2 μg HCMV-DBs or 10 μg recombinant gB. The bars represent the mean of RI values; dots are individual values. The experiment was repeated twice with similar results.
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