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. 2000 Nov 21;97(24):13263-8.
doi: 10.1073/pnas.230417497.

Protective long-term antibody memory by antigen-driven and T help-dependent differentiation of long-lived memory B cells to short-lived plasma cells independent of secondary lymphoid organs

A F Ochsenbein  1 , D D Pinschewer, S Sierro, E Horvath, H Hengartner, R M Zinkernagel
Affiliations

Protective long-term antibody memory by antigen-driven and T help-dependent differentiation of long-lived memory B cells to short-lived plasma cells independent of secondary lymphoid organs

A F Ochsenbein et al. Proc Natl Acad Sci U S A. .

Abstract

Memory is a hallmark of immunity. Memory carried by antibodies is largely responsible for protection against reinfection with most known acutely lethal infectious agents and is the basis for most clinically successful vaccines. However, the nature of long-term B cell and antibody memory is still unclear. B cell memory was studied here after infection of mice with the rabies-like cytopathic vesicular stomatitis virus, the noncytopathic lymphocytic choriomeningitis virus (Armstrong and WE), and after immunization with various inert viral antigens inducing naive B cells to differentiate either to plasma cells or memory B cells in germinal centers of secondary lymphoid organs. The results show that in contrast to very low background levels against internal viral antigens, no significant neutralizing antibody memory was observed in the absence of antigen and suggest that memory B cells (i) are long-lived in the absence of antigen, nondividing, and relatively resistant to irradiation, and (ii) must be stimulated by antigen to differentiate to short-lived antibody-secreting plasma cells, a process that is also efficient in the bone marrow and always depends on radiosensitive, specific T help. Therefore, for vaccines to induce long-term protective antibody titers, they need to repeatedly provide, or continuously maintain, antigen in minimal quantities over a prolonged time period in secondary lymphoid organs or the bone marrow for sufficient numbers of long-lived memory B cells to mature to short-lived plasma cells.

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Figures

Figure 1
Figure 1
B cell and antibody memory after immunization with live virus vs. viral proteins. C57BL/6 mice were infected with 200 pfu of LCMV-WE (A) or 2 × 106 pfu of VSV-IND (●) or –NJ (○) (C and E) i.v. or immunized with 10 or 1 μg of Baculo-derived LCMV-NP protein (B) or UV-inactivated VSV-IND (2 × 108 pfu; D and F) and LCMV-NP binding (A and B), VSV-NP-binding (E and F) and VSV-IND-neutralizing (C and D) antibody titers were assessed. Results are shown as means ± SD of 3–4 mice per group. The experiment was repeated twice with similar results.
Figure 2
Figure 2
Life span of memory B cells and plasma cells. (A) C57BL/6 mice were immunized with UV-inactivated VSV-IND (2 × 108 pfu). At day 300, these mice (●) and a group of three naive C57BL/6 mice (○) were boosted with UV-inactivated VSV-IND (2 × 108 pfu) and the presence of neutralizing IgG antibodies was analyzed until day 6. (B and C) C57BL/6 mice were immunized with 2 × 106 pfu of VSV-IND. Thirty days later, 107 splenocytes + 107 bone marrow cells were injected into nonirradiated C57BL/6 recipient mice. Half of these mice received UV-inactivated VSV-IND (open bars; 2 × 106 pfu, yielding no IgG in naive mice) 20 min after the transfer of the cell suspension; 3 and 10 days later, spleen (B) and bone marrow (C) cells were prepared from recipient mice and IgG antibody-forming cells (AFC) were quantitatively assessed by ELISPOT assays against purified VSV-IND virions assessing largely neutralizing antibody-secreting cells. Bars indicate the means ± SD of four recipient mice and one of two similar experiments is shown.
Figure 3
Figure 3
Adoptive transfer experiments of memory bone marrow or spleen cells. Single-cell suspensions of spleen and bone marrow cells of C57BL/6 mice were prepared 60–80 days after i.v. infection with 2 × 106 pfu of VSV-IND (A and B), 200 pfu of LCMV-Armstrong (C), or 200 pfu of LCMV-WE (D) and 107 spleen cells (rows 1–4) or bone marrow cells (rows 5–8) were adoptively transferred into mice irradiated with 650 rad (irrad.) or nonirradiated recipient mice. Where indicated, antigen was additionally injected 20 min after the adoptive transfer [2 × 106 pfu of UV-inactivated VSV-IND (A and B); and 0.01 μg of Baculo-derived LCMV-NP (C and D)]. VSV-IND-neutralizing, VSV-NP-binding, and LCMV-NP-binding antibodies were assessed up to 120 days after the adoptive transfer. Symbols indicate the means ± SD of four mice per group. The experiment was repeated twice with comparable results.
Figure 4
Figure 4
Role of CD4+ T cells in maintaining long-term antibody titers. Single-cell suspensions of spleen or bone marrow cells were prepared 60–80 days after infection of C57BL/6 mice with 2 × 106 pfu of VSV-IND i.v. and were adoptively transferred (107 per recipient) into nonirradiated naive recipient mice. Donor mice and recipients were CD4+ T cell-depleted where indicated by i.p. injection of 1 mg of monoclonal anti-CD4 antibody YTS191.1 on days −3 and −1 before sacrifice or adoptive transfer, respectively. Bone marrow cells were either transferred into naive C57BL/6 recipient mice or VSV-NJ immune recipient mice (immunized with 2 × 106 pfu of VSV-NJ 12 days earlier). Where indicated, antigen (2 × 106 pfu of UV-inactivated VSV-IND) was additionally injected 20 min after the adoptive transfer. VSV-IND-neutralizing antibodies were measured up to 40 days after the transfer. Symbols indicate the means ± SD of four mice per group. The experiment was repeated twice with comparable results.
Figure 5
Figure 5
Effect of irradiation on B cell and antibody memory. C57BL/6 mice were infected with (A) 200 pfu of LCMV-WE i.v.; (B) 2 × 106 pfu of LCMV-Armstrong i.p.; or immunized with (C) 10 μg of Baculo-derived LCMV-NP protein. Thirty days after the infection/immunization, mice were irradiated with 650 or 850 rad or left nonirradiated as indicated by the arrow. Mice irradiated with 850 rad were substituted with 2 × 107 naive bone marrow and 2 × 107 naive spleen cells. LCMV-NP-binding antibodies were assessed up to day 125 after infection. (D) As in C but mice were immunized with 2 × 106 pfu of VSV-IND i.v. and irradiated 60 days later. Ten days later, 2 × 107 splenocytes isolated from the above irradiated and nonirradiated donor mice were adoptively transferred into nonirradiated recipient mice. Half of the recipient mice were also infected with 2 × 106 pfu of VSV-NJ 12 days earlier (closed symbols); 2 × 106 pfu of UV-inactivated VSV-IND was injected into all mice (+20 min) and neutralizing IgG antibody titers were analyzed (▪ are negative controls, and ●, ○ are positive controls). Results are shown as means ± SD of 3–4 mice per group. Each experiment was repeated 2–3 times with comparable results.
Figure 6
Figure 6
The role of secondary lymphoid organs in the maintenance of B cell and antibody memory. (A) Splenocytes (107) plus 107 bone marrow cells from VSV-IND-primed (2 × 106 pfu 60 days earlier) C57BL/6 mice were adoptively transferred into ALY × ALY mice. Twenty minutes later, 2 × 108 pfu of UV-inactivated VSV-IND were injected into recipient mice. Day 20 after the adoptive transfer, half of the recipient ALY × ALY mice were splenectomized. Neutralizing antibody titers were followed up to 360 days after the adoptive transfer. Antibody titers in splenectomized and nonsplenectomized ALY × ALY mice were also followed after transfer of 500 μl of VSV-IND immune serum (pooled of VSV-IND memory mice infected 60 days previously with 2 × 106 pfu of VSV-IND). (B) C57BL/6 mice were infected with 2 × 106 pfu of VSV-IND i.v. Sixty days later, 2 × 107 splenocytes from these mice or naive C57BL/6 mice were transferred into ALY × ALY recipient mice. At the same time, 2 × 107 splenocytes of VSV-NJ-primed (2 × 106 pfu i.v., 14 days earlier) mice were transferred into the same recipient mice as a source of primed T help. Four days later, half of the ALY × ALY recipient mice were splenectomized. All mice were boosted with 2 × 108 pfu of UV-inactivated VSV-IND 7 days after splenectomy and VSV-IND-neutralizing antibody titers were determined 22 days later. Results are shown as means ± SD of 3–4 mice per group. The experiments were repeated twice with comparable results.
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