BAFF- and APRIL-dependent maintenance of antibody titers after immunization with T-dependent antigen and CD1d-binding ligand

Abstract

CD1d-restricted invariant NKT (iNKT) cells boost humoral immunity to T-dependent Ags that are coadministered with the CD1d-binding glycolipid Ag α-galactosylceramide (α-GC). Observations that mice lacking iNKT cells have decaying Ab responses following vaccination have led to the hypothesis that iNKT cells express plasma cell (PC) survival factors that sustain specific Ab titers. Bone marrow chimeric mice in which the entire hematopoietic compartment or iNKT cells selectively lacked BAFF, a proliferation-inducing ligand (APRIL), or both BAFF and APRIL were created and immunized with nitrophenol hapten-conjugated keyhole limpet hemocyanin adsorbed to Imject aluminum hydroxide-containing adjuvant or mixed with α-GC. In comparison with BAFF- or APRIL-sufficient bone marrow chimeras, absence of hematopoietic compartment- and iNKT-derived BAFF and APRIL was associated with rapidly decaying Ab titers and reduced PC numbers. The iNKT cell-derived BAFF or APRIL assumed a greater role in PC survival when α-GC was used as the adjuvant for immunization. These results show that iNKT cell-derived BAFF and APRIL each contribute to survival of PCs induced by immunization. This study sheds new light on the mechanisms through which iNKT cells impact humoral immunity and may inform design of vaccines that incorporate glycolipid adjuvants.

Figure 1. B cells and NKT cells in donor mice

BM cells and splenocytes were harvested from the donor B6, Jα18^−/−, BAFF^−/−, APRIL^−/− and DKO mice (n = 3–5 per group). (A) BM cells were analyzed by flow cytometry for B220⁺ versus CD93⁻ mature B cells. (B) Splenocytes were analyzed by flow cytometry for TCRβ⁺ versus CD1d tetramer⁺ cells. (C) Shows mean ± SD frequency and (D) shows absolute numbers ± SD of NKT cells. (E–F) Splenocytes from B6, Jα18^−/−, BAFF^−/−, APRIL^−/−, and DKO mice were stimulated in vitro with α-GC and incubated for 48 h before collecting supernatants and measuring IL-4 (E) and IFNγ (F) by ELISA. Data show mean ± SD for triplicate samples. Data shown in A–F are representative of two independent experiments.

Figure 2. Re-constitution of immune cell compartments in BM chimeras lacking BAFF and APRIL in the hematopoetic compartment

(A) BM cells were harvested from reconstituted chimeras and analyzed by flow cytometry. Graph depicts B220- and CD93- expressing cells as a percentage of total BM-derived cells. (B) Splenocytes were harvested from reconstituted chimeras and analyzed by flow cytometry. Graph depicts CD45.2⁺ anti-TCRβ- and CD1d tetramer-binding cells as a percentage of total splenocytes. Total BM cell and splenocyte recovery was comparable across all groups (not depicted). Data show mean ± S.D. values for analyses using 3–5 mice per group. Significantly higher iNKT frequency in the DKO than in the B6 splenocytes is indicated.

Figure 3. Immunization-induced Ab titers in chimeras lacking BAFF and APRIL in the hematopoetic compartment

Sera were collected from the (A–D) NP-KLH/Alum-immunized and (E–H) NP-KLH/α-GC-immunized chimeras on the days indicated. Samples were analyzed by ELISA and endpoint anti-NP IgG1 titers determined. Data shows mean endpoint titer ±SD. Checkered bars indicate sera collected before the booster vaccine, whereas solid bars represent sera collected after the booster. Titers in pre-bleed sera were below 1/200 (not depicted). Numbers of mice per group were: (A, D, E, H) n = 7–11, (B, C, F, G) n = 3 – 5. Statistically significant differences between experimental groups were determined by one-way ANOVA and Dunn’s post-test.

Figure 4. Reduced PC numbers in chimeras lacking BAFF and APRIL in the hematopoetic compartment

BM cells were collected on d 95 post-immunization from (A) NP-KLH/Alum-immunized chimeras and (B) NP-KLH/α-GC-immunized chimeras. Samples were then assessed by ELISPOT. Each bar shows the mean ± SD number of anti-NP IgG1 secreting cells per million total BM cells. Mean IgG1 titers at day 91 are shown for reference. For B6 and DKO chimeras, 7 and 6 mice per group were used respectively. Statistical significance was assessed using a two-tailed Mann Whitney t-test.

Figure 5. Expression of BAFF and APRIL by splenic iNKT cells

(A) NKT.3c3 hybridoma cell lysates (2.4, 1.2 and 0.6 × 10⁶ cell equivalents per lane) were resolved by SDS-PAGE and transferred to nitrocellulose membranes before immunoblotting for BAFF (upper panels) and APRIL (lower panels). Recombinant murine soluble BAFF (rmsBAFF) served as a positive control for BAFF expression. Membranes were also prepared and incubated with anti-BAFF or anti-APRIL alone or in combination with a 100-fold molar excess of blocking peptide. (B) B6 mice were immunized i.p. with PBS or PBS containing α-GC. After 16 h, splenocytes were harvested and lysates prepared before immuno-blotting as indicated. (C) B6 mice were immunized i.p. with α-GC or PBS vehicle before collecting splenocytes, culturing for 6 h and measuring BAFF by ELISA. Data shown mean ± S.D. (n=6). Data are pooled from two experiments. (D) Splenocytes were obtained from Jα18^−/−, B6, and Vα14 Tg mice and analyzed by flow cytometry (dot plots) or cultured before collecting supernatants and measuring BAFF concentrations by ELISA (bar graph). Values in bar graph represent mean ± SD for triplicate samples and are representative of two independent experiments. (E) Dot plot shows isolated iNKT cells and graph shows mean ± SD concentrations of secreted BAFF (for duplicate samples) following culture of cells with media or media containing PMA/ionomycin. Data are representative of three independent experiments.

Figure 6. Expression of BAFF by bone marrow iNKT cells

(A) Shows frequency of bone marrow iNKT cells in Jα18^−/−, B6, and Vα14 Tg mice. (B–D) Bone marrow cells were cultured in triplicate as indicated before collection of supernatants and measurement of BAFF concentrations by ELISA. (B) Cells from Jα18^−/−, B6, and Vα14 Tg mice were treated for 6 hr with vehicle or α-GC (C) Cells from Vα14 Tg mice were treated as in (B) except that PMA/ionomycin treatment was included. (B) and (C) are representative of three independent experiments that produced similar results. (D) Bone marrow cells from strains depicted were treated with vehicle or PMA/ionomycin. Data in B–D show mean ± SD BAFF concentration for triplicate samples. Data in (B) and (C) are representative of three independent experiments. (D) is from a single experiment using duplicate samples.

Figure 7. Re-constitution of immune cell compartments in BM chimeras lacking BAFF and APRIL in the iNKT compartment

(A) BM cells were harvested from reconstituted chimeras and analyzed by flow cytometry. Graph depicts B220- and CD93- expressing cells as a percentage of total BM-derived cells. (B) Splenocytes were harvested from reconstituted chimeras and analyzed by flow cytometry. Graph depicts CD45.2⁺ anti-TCRβ- and CD1d tetramer-binding cells as a percentage of total splenocytes. Total BM cell and splenocyte recovery was comparable across all groups (not depicted). Data show mean ± S.D. values for analyses using 3–5 mice per group.

Figure 8. Immunization-induced Ab titers in chimeras lacking NKT-derived BAFF and APRIL

Sera were collected from the (A–D) NP-KLH/Alum-immunized and (E–H) NP-KLH/α-GC-immunized chimeras on the ds indicated. Samples were analyzed by ELISA and endpoint anti-NP IgG1 titers determined. Data shows mean endpoint titer ±SD. Checkered bars indicate sera collected before the booster vaccine, whereas solid bars represent sera collected after the booster. Titers in pre-bleed sera were below 1/200 (not depicted). Numbers of mice per group were: (A, D, E, H) n = 7–11, (B, C, F, G) n = 4 – 6. Statistically significant differences between experimental groups were determined by one-way ANOVA and Dunn’s post-test.

Figure 9. Reduced PC numbers in chimeras lacking BAFF and APRIL in the iNKT compartment

BM cells were collected on d 95 post-immunization from (A) NP-KLH/Alum-immunized chimeras and (B) NP-KLH/α-GC-immunized chimeras. Samples were then assessed by ELISPOT. Each bar shows the mean ± SD number of anti-NP IgG1 secreting cells per million total BM cells. Mean IgG1 titers at day 91 are shown for reference. For Jα18^−/−/B6 and Jα18^−/−/DKO chimeras, 11 and 7 mice per group were used respectively. Statistical significance was assessed using a two-tailed Mann Whitney t-test.