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

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.

Figure 1.

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.

Figure 1.

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.

Figure 1.

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.

Figure 2.

IgG memory response in different host mice lacking functional CD4⁺ cells. 5 × 10⁶ sorted B cells from hyperimmunized C57BL/6 mice were transferred as described in Fig. 1, and mice were challenged with antigen 1 d after adoptive transfer of cells. The IgG antibody response in different recipient mice was measured 7 d after challenge with 2 μg HCMV-DBs i.v. Controls received no memory B cell preparations. The bars represent the mean of RI values; dots are individual values.

Figure 3.

T cell depletion and T cell numbers in recipient mice. (A) RAG-1^−/− mice adoptively transferred with immune B cells were injected with 500 μg GK1.5 antibody 2 d before and on the day of stimulation with 10 μg HCMV-DBs. Blood was taken on the days indicated, and sera were analyzed for HCMV-specific IgG (♦) compared with sera of untreated recipients (▪). (B) Spleens and blood were taken from RAG-1^−/− mice, RAG-1^−/− mice 90 d post adoptive transfer and from donor mice. Single cell suspensions were stained with FITC- or PE-conjugated antibodies and analyzed by FACScan. The specificities of the staining antibodies are indicated at the axes.

Figure 4.

Antibody responses after adoptive transfer of memory B and memory T cells. C57BL/6 mice received 5 × 10⁶ B cells from C57BL/6 mice immunized with HCMV-DBs (○), 5 × 10⁶ immune B cells together with 5 × 10⁶ CD4⁺ T cells from C57BL/6 mice previously immunized with HCMV-DBs (•) or no cells (▪). On day 6 posttransfer, the mice were challenged with 2 μg HCMV-DBs i.v. Blood was taken at the days indicated, and sera were analyzed by ELISA for HCMV-specific IgG. The values represent the mean values (± SD) of four recipient mice, and one of two similar experiments is shown.

Figure 5.

Localization of the IgG response in the presence of T cell help. Memory B cell preparations (5 × 10⁶ CD19⁺ cells) together with primed CD4⁺ cells (2.5 × 10⁶ cells), both from C57BL/6-Ly5.2 mice, were transferred into naive C57BL/6-Ly5.1 mice and challenged with HCMV-DBs 1 d after adoptive transfer. (A) 4 d after challenge, clusters of donor-derived (CD45.2 in blue), IgG^high (red) cells were observed outside the follicles (thick arrows). B220 is stained in green. (B) 10 d after challenge, clusters of donor-derived (CD45.2, blue), IgG^high (red) cells are still persistent (thick arrows); in addition IgG⁺ GCs could be observed inside the follicles (thin arrows). The cells participating in the GC reaction are CD45.2 negative. (C) A GC from day 10 after challenge is stained with peanut agglutinin (green). Donor CD45.2 cells are blue; IgG-positive cells are red. Magnification: (A and B) 50×; (C) 200×.

Figure 6.

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.

Figure 7.

IgG memory response in recipient mice with altered lymphoid architecture. 5 × 10⁶ sorted B cells from a pool of hyperimmunized C57BL/6 mice were transferred as described in the legend for Fig. 1, and mice were challenged with antigen 1 d after adoptive transfer of cells. Control C57BL/6 mice were treated with GK1.5 antibody but received no memory B cell preparations. The IgG antibody response in different recipient mice treated with GK1.5 antibody was measured 7 d after challenge with 2 μg HCMV-DBs i.v. The bars represent the mean of RI values; dots are individual values. The experiment was repeated, and similar results were obtained.