Natural killer T (NKT)-B-cell interactions promote prolonged antibody responses and long-term memory to pneumococcal capsular polysaccharides

Abstract

Innate-like natural killer T (NKT) cells critically enhance cell and humoral immunity against infections through recognition of conserved microbial lipid antigens presented by CD1d-expressing antigen-presenting cells, and provision of CD40L and cytokine signals. Whereas NKT cells efficiently licensed dendritic cells to prime potent effector and memory T cells, studies based on model antigens such as alphagalactosylceramide-nitrophenyl conjugates concluded that help to B cells was associated with NKT follicular helper differentiation, but limited to short-term responses without induction of memory. We revisited this surprising conclusion in the context of the extracellular encapsulated pathogen Streptococcus pneumoniae, where recognition of lipid and capsular polysaccharide antigens by NKT cells and B cells, respectively, provide critical host protection. Using liposomal nanoparticles displaying synthetic lipid and polysaccharide antigens to elicit pure and direct NKT-B-cell interactions in vivo, we observed intense and prolonged antibody responses with isotype switch, affinity maturation, and long-lasting B-cell memory, despite modest or absent NKT follicular helper differentiation. Furthermore, conditional ablation of Cd1d demonstrated a requirement for a two-step process involving first cognate interactions with dendritic cells, for NKT cell activation, and then with B cells, for induction of isotype switch and memory. Thus, NKT help to B cells represents both a major arm of antimicrobial defense and a promising target for B-cell vaccines.

Fig. 1.

DAG-anchored pneumococcal capsular polysaccharide. The tetrasaccharide repeat of S. pneumoniae capsular polysaccharide serotype 14 (Upper) was linked to a diacylglycerol as shown (Lower, compound PBS150) for insertion into liposomal membranes.

Fig. 2.

Anti-PS antibody response elicited by pneumococcal liposomes. (A) Kinetics of serum anti-PS14 antibody isotypes after immunization of B6 mice at indicated time points (arrows) with pneumococcal liposomes carrying 4 μg PS14 and 1 μg NKT lipid. Titers are shown as arbitrary units per milliliter by reference to a standard hyperimmune serum. Results represent two independent experiments with 10 mice each. (B, Left) FACS analysis of serum IgG1 antibodies binding to glass beads coated with PS14-carrying liposomes compared with empty liposomes and liposomes carrying the NKT ligand PBS57. (Right) ELISA analysis of serum antibodies against purified pneumococcal polysaccharide in individual naïve or immunized mice. (C) Serum IgM and IgG1 antibodies against PS14 measured 20 wk after immunization with liposomes carrying both PBS57 and PBS150, or PBS150 alone.

Fig. 3.

Cognate NKT cell interactions with both DC and B cells are required for isotype-switched anti-PS antibody response. (A) Serum anti-PS antibodies after immunization of Cd1d^fl/flCd19-cre (CD19ΔΔ), Cd1d^fl/flCd11c-cre (CD11cΔΔ), and littermate controls (LM) with pneumococcal liposomes carrying 4 μg PS14 and 1 μg NKT lipid at indicated times (arrows). (B) Early serum IL-4 (2 h) and IFN-γ (24 h) after pneumococcal liposome injection in Cd1d conditional mutants and their littermate controls. Results are representative of two independent experiments with four to seven mice per group. (C) FACS analysis of surface expression of CD1d by CD19-gated splenocytes of wild type (LM), CD1d KO (KO), and CD19ΔΔ mice.

Fig. 4.

The NKT follicular helper response requires CD1d expression by DCs but not B cells. (A and B) FACS staining of splenic NKT cells after pneumococcal liposome immunization of Cd1d conditional mutants and their littermate (LM) controls, as indicated. Results are shown as percentages for representative mice (Left) and absolute numbers for each individual mouse (Right) of ICOS^hiPD-1^hi cells among gated CD1d-αGC tetramer⁺ NKT cells at day 5 (Upper) and day 8 (Lower). Results are representative of three independent experiments with five to seven mice per group. (C) Same analysis as in A for CXCR5 and PD-1. Staining controls used to set the PD-1^hiCXCR5^hi GC-Tfh gate included WT immunized NKT cells stained with IgG isotype instead of anti-CXCR5, as indicated, and Peyer’s patch CD4 T cells (rightmost dot plot), which are naturally enriched in GC-Tfh cells.

Fig. 5.

Affinity maturation of the NKT cell-helped anti-PS antibody response. (Left) FACS analysis of serum IgG3 and IgG1 antibody (1/200 dilution) binding to liposomes carrying low (0.5%) or high (20%) density PS in the early and late (after boost) response of a representative mouse showing affinity maturation. (Right) Ratios of reciprocal antibody titers to PS-20% over PS-0.5% for individual mice in the early and late response, as indicated. Results are representative of two independent experiments with five to seven mice each.

Fig. 6.

Cognate NKT cell help promotes long-term B-cell memory. Mice immunized twice with pneumococcal liposomes (as in Fig. 1) and unimmunized, naïve littermates were reinjected 8 mo later with pneumococcal liposomes. The plots show individual titers of PS-specific IgM and IgG1 after challenge. Note that preimmunized mice had detectable residual PS-specific IgM and IgG1 antibodies before challenge. Results are representative of two independent experiments with a total of 20 mice.