B lymphocytes induce the formation of follicular dendritic cell clusters in a lymphotoxin alpha-dependent fashion

Abstract

Lymphotoxin (LT)alpha is expressed by activated T cells, especially CD4(+) T helper type 1 cells, and by activated B and natural killer cells, but the functions of this molecule in vivo are incompletely defined. We have previously shown that follicular dendritic cell (FDC) clusters and germinal centers (GCs) are absent from the peripheral lymphoid tissues of LTalpha-deficient (LTalpha-/-) mice. LTalpha-/- mice produce high levels of antigen-specific immunoglobulin (Ig)M, but very low levels of IgG after immunization with sheep red blood cells. We show here that LTalpha-expressing B cells are essential for the recovery of primary, secondary, and memory humoral immune responses in LTalpha-/- mice. It is not necessary for T cells to express LTalpha to support these immune functions. Importantly, LTalpha-expressing B cells alone are essential and sufficient for the formation of FDC clusters. Once these clusters are formed by LTalpha-expressing B cells, then LTalpha-deficient T cells can interact with B cells to generate GCs and productive class-switched antibody responses. Thus, B cells themselves provide an essential signal that induces and maintains the lymphoid microenvironment essential for GC formation and class-switched Ig responses.

Figure 1

LTα-expressing B cells can restore primary, secondary, and memory anti-SRBC IgG responses in LTα^−/− mice. Groups of three to five 8-wk-old mice were lethally irradiated and reconstituted with 1:1 mixtures of BM from LTα^−/− mice and either BCR^−/−, TCR^−/−, or RAG-1^−/− mice as indicated. Lethally irradiated wild-type mice reconstituted with BM from wild-type donors and LTα^−/− mice reconstituted with BM from LTα^−/− donors served as controls. 6 wk after BM reconstitution, all mice were immunized intravenously with 10⁸ SRBCs in PBS. Primary anti-SRBC IgG responses (A) were measured 10 d later by ELISA (16). For measurement of secondary responses (B), mice received a booster immunization of 10⁸ SRBCs in PBS and sera were collected 7 d later. For measurement of memory responses (C), mice that had been reconstituted with the indicated mixtures of BM were primed with a single dose of 10⁸ SRBCs and 7 d later, 5 × 10⁷ splenocytes were transferred to sublethally irradiated (750 rads) normal C57BL/6 mice and the recipients were immediately challenged with the same dose of SRBCs. 7 d later, serum anti-SRBC IgG responses were determined. Data shown represent the means ± SEM of triplicate determinations from three to five mice. RU, relative units. Each experiment was repeated at least twice with similar results.

Figure 1

LTα-expressing B cells can restore primary, secondary, and memory anti-SRBC IgG responses in LTα^−/− mice. Groups of three to five 8-wk-old mice were lethally irradiated and reconstituted with 1:1 mixtures of BM from LTα^−/− mice and either BCR^−/−, TCR^−/−, or RAG-1^−/− mice as indicated. Lethally irradiated wild-type mice reconstituted with BM from wild-type donors and LTα^−/− mice reconstituted with BM from LTα^−/− donors served as controls. 6 wk after BM reconstitution, all mice were immunized intravenously with 10⁸ SRBCs in PBS. Primary anti-SRBC IgG responses (A) were measured 10 d later by ELISA (16). For measurement of secondary responses (B), mice received a booster immunization of 10⁸ SRBCs in PBS and sera were collected 7 d later. For measurement of memory responses (C), mice that had been reconstituted with the indicated mixtures of BM were primed with a single dose of 10⁸ SRBCs and 7 d later, 5 × 10⁷ splenocytes were transferred to sublethally irradiated (750 rads) normal C57BL/6 mice and the recipients were immediately challenged with the same dose of SRBCs. 7 d later, serum anti-SRBC IgG responses were determined. Data shown represent the means ± SEM of triplicate determinations from three to five mice. RU, relative units. Each experiment was repeated at least twice with similar results.

Figure 1

LTα-expressing B cells can restore primary, secondary, and memory anti-SRBC IgG responses in LTα^−/− mice. Groups of three to five 8-wk-old mice were lethally irradiated and reconstituted with 1:1 mixtures of BM from LTα^−/− mice and either BCR^−/−, TCR^−/−, or RAG-1^−/− mice as indicated. Lethally irradiated wild-type mice reconstituted with BM from wild-type donors and LTα^−/− mice reconstituted with BM from LTα^−/− donors served as controls. 6 wk after BM reconstitution, all mice were immunized intravenously with 10⁸ SRBCs in PBS. Primary anti-SRBC IgG responses (A) were measured 10 d later by ELISA (16). For measurement of secondary responses (B), mice received a booster immunization of 10⁸ SRBCs in PBS and sera were collected 7 d later. For measurement of memory responses (C), mice that had been reconstituted with the indicated mixtures of BM were primed with a single dose of 10⁸ SRBCs and 7 d later, 5 × 10⁷ splenocytes were transferred to sublethally irradiated (750 rads) normal C57BL/6 mice and the recipients were immediately challenged with the same dose of SRBCs. 7 d later, serum anti-SRBC IgG responses were determined. Data shown represent the means ± SEM of triplicate determinations from three to five mice. RU, relative units. Each experiment was repeated at least twice with similar results.

Figure 2

Structure of spleen follicles in compound BM chimeric mice. After sera were collected from the mice shown in Fig. 1 A, the spleens were harvested and frozen sections were stained with anti-Thy1.2 (blue) and anti-B220 (brown) to visualize the T and B cell zones (A and B), anti-CR1 monoclonal antibody 8C12 (blue) and anti-B220 (brown) to visualize clusters of FDC (C and D), or PNA (blue) and anti-IgD (brown) to visualize the GC reaction (E and F). A, C, and E show spleen sections from LTα^−/− mice reconstituted with mixtures of bone marrow from LTα^−/− and BCR^−/− mice; B, D, and F show spleen sections from LTα^−/− mice reconstituted with mixtures of BM from LTα^−/− and TCR^−/− mice. Proper segregation of B and T cell zones was not restored in either compound BM chimera; however, both formation of FDC clusters and GC were restored in mice reconstituted with LTα-expressing B cells.

Figure 3

Presence of FDC clusters in TCR^−/− but not BCR^−/− mice. BCR^−/− (A and B), TCR^−/− (C and D), and wild-type (E and F) mice were immunized intraperitoneally with 10⁸ SRBCs, and spleens were harvested 10 d later. Frozen sections were stained with anti-Thy-1.2 (blue) and anti-B220 (brown) to visualize the T and B cell zones (A, C, and E). FDC clusters were observed by staining with the anti-CR1/2 monoclonal antibody 8C12 (blue; B, D, and E).

Figure 4

Purified LTα-expressing and LTα-deficient T cells both restore GC formation in TCR^−/− mice. Unirradiated TCR^−/− mice were treated with an infusion of 10⁷ purified T cells from the spleens of LTα^−/− (B, D, and F) or wild-type mice (A, C, and E). At the same time, the mice were immunized with 10⁸ SRBCs administered intravenously. The spleens of the recipients were harvested 10 d later, and frozen sections were stained as described for Fig. 2. A and B are stained for B (brown) and T (blue) cells; C and D are stained for B cells (brown) and FDC (blue); E and F are stained with B220 (brown) and PNA (blue).

Figure 5

Similar reconstitution of FDC clusters in RAG-1^−/− mice by transfer of BM or purified B cells. Unirradiated RAG-1^−/− mice were treated with an infusion of 2.5 × 10⁶ BM cells (A and C) or 10⁷ purified spleen B cells (B and D) from TCR^−/− donors. 3 wk later, the spleens were harvested and frozen sections were stained for B/T cell zones and FDC clusters as in Fig. 2, A–D.