Requirement for CD1d expression by B cells to stimulate NKT cell-enhanced antibody production

Abstract

Activation of natural killer-like T (NKT) cells with the CD1d ligand alpha-galactosylceramide enhances T-dependent humoral immune responses against coadministered T-dependent Ag. At present, there is little information on the mechanisms involved other than a dependence on CD1d expression by antigen-presenting cells and/or development of the NKT subset. We therefore tested the hypothesis that direct presentation of alpha-GC by B cells was required for NKT-enhanced Ab responses against T-dependent Ag. We reconstituted B cell-deficient microMT mice with B cells from C57Bl/6 donors or CD1d(-/-) donors before immunization with NP-KLH alone or NP-KLH mixed with alpha-GC. We made the surprising observation that B-cell expression of CD1d is absolutely required for the NKT-enhanced Ab response. Our data show that the mechanism by which NKT cells enhance humoral immune responses involves interaction with CD1d-expressing B cells.

Figure 1

μMT mice as a tool for examining NKT-enhanced Ab production. (A) Thymocytes (THY) and splenocytes (SPN) were assessed for TCRβ⁺, CD1d tetramer⁺ cells (dot plots) and CD1d⁺ splenocytes (histograms). Data are representative of several (> 6) determinations. (B) Mice were immunized as described in “Immunization and sera collection,” and sera were collected at times indicated before Bio-Plex analysis. Data show serum IL-4 and IFNγ concentration (mean ± SEM, n = 2 for vehicle, n = 5 for C57Bl/6 and n = 2 for μMT). Samples were analyzed in duplicate. No response was elicited with α-GC in CD1d^−/− mice (not shown). Data are representative of 2 independent experiments. (C) Complement-mediated lysis of Thy1.2- and CD4-labeled cells depletes T and NKT cells resulting in a B cell–enriched preparation. Data are representative of several (> 6) determinations. (D) CFSE-labeled C57Bl/6 B cells were adoptively transferred to μMT mice. Cells were detected in recipient spleen after 18 hours (top panel) and 2 weeks after immunization with NP-KLH/Alum (bottom panel). (E) B cells were transferred into μMT recipients, which were then untreated (naive) or immunized 24 hours later with NP-KLH/Alum by the intraperitoneal or subcutaneous route. Sera were obtained after 2 weeks and anti-NP IgG1 detected by enzyme-linked immunosorbent assay. Data show A405 at a 1 of 1000 dilution of serum. (F) After T/NKT-depletion, CD19⁺ cells and CD19⁻ (non-B/non-T cells) were resolved by magnetic sorting. C57Bl/6 mice were adoptively transferred with 2 × 10⁷ CD19⁺ cells or 10⁶ CD19⁻ cells before immunization with NP-KLH/α-GC. The graph shows that the resulting Ab response was the result of the CD19⁺ fraction. The CD19⁻ cellular fraction was determined by flow cytometry to be approximately 50% granulocytes and 50% DCs (not shown). Data in panels D-F show the results from single experiments.

Figure 2

CD1d expression by B cells is required for NKT-enhanced Ab recall responses. (A) Splenocytes from C57Bl/6 and CD1d^−/− mice were assessed for TCRβ⁺, CD1d tetramer⁺ cells (dot plots) and CD1d⁺ (histograms). Empty histograms show anti-CD1d and filled show isotype control mAb. Data are representative of several (> 20) determinations. (B) Shows CD1d status of the T- and NKT-depleted B220⁺ cells used for adoptive transfer. Data are representative of several (> 12) determinations. (C) B cells from C57Bl/6 and CD1d^−/− donors were adoptively transferred to μMT recipients before immunization with NP-KLH or NP-KLH plus α-GC 24 hours after transfer. All groups were boosted with NP-KLH on day 28 and sera collected on day 33. Each mouse was immunized and boosted with 10 μg (experiment 1) and 20 μg (experiment 2) NP-KLH. Data show endpoint IgG1 titer (mean ± SD) for 3 mice per group from 2 independent experiments. * Significant differences in titer between mice receiving cells from C57Bl/6 and CD1d^−/− donors. (D) CFSE-labeled B cells from C57Bl/6 and CD1d^−/− donors were adoptively transferred into μMT mice. After 18 hours, splenocytes were prepared and assessed for CFSE⁺ cells. Data are representative of 3 independent experiments. (E) Experiment was performed as in panel C, except that mice were immunized with NP-KLH/Alum on day 0, bled (primary bleed), and then boosted with NP-KLH on day 21 and bled again on day 26 (secondary bleed). Results from a single experiment are shown in panel E.