Invariant natural killer T cells direct B cell responses to cognate lipid antigen in an IL-21-dependent manner

Abstract

Mouse invariant natural killer T cells (iNKT cells) provide cognate and noncognate help for lipid and protein-specific B cells, respectively. However, the long-term outcome for B cells after cognate help is provided by iNKT cells is unknown at present. Here we found that cognate iNKT cell help resulted in a B cell differentiation program characterized by extrafollicular plasmablasts, germinal-center formation, affinity maturation and a robust primary immunoglobulin G (IgG) antibody response that was uniquely dependent on iNKT cell-derived interleukin 21 (IL-21). However, cognate help from iNKT cells did not generate an enhanced humoral memory response. Thus, cognate iNKT cell help for lipid-specific B cells induces a unique signature that is a hybrid of classic T cell-dependent and T cell-independent type 2 B cell responses.

Figure 1. Cognate (lipid) and non-cognate (lipid plus protein) antigen stimulation of B cells induces splenic extrafollicular foci.

(a-d) 7 μm sections of OCT-embedded spleens from B1-8 Tg mice were labeled with anti-CD19 (green), anti-CD138 (red), NP-APC (blue) to identify CD138⁺ NP-specific plasmablasts. High resolution, knitted images of these spleen sections were captured by confocal microscope 5 days after immunization with 5 μg NP−α−GalCer (a) 100 μg NP-KLH + 5 μg α−GalCer (b), 100 μg NP-KLH + alum (c), or PBS-DMSO (d). Data are representative from 3-4mice per group in 2 independent experiments; scale bar = 100μm. (e, f) Flow cytometry analysis of splenic NP-specific IgD⁻, B220^loCD138⁺ plasmablast B cells from identically immunized WT C57BL/6 mice in representative plots (e) and as a summary (f). (g) ELISPOT enumeration of NP-specific IgG secreting splenic B cells. (h) the number of TCR_β⁺CD1dtet⁺ iNKT cells from the same mice as determined by flow cytometry. Each symbol represents an individual mouse; representative (g) or pool (f, h) of 5 mice per group from 2 independent experiments; bar = mean. Unpaired two-tailed t-tests were performed for large data sets [f, i], Mann-Whitney tests were performed for small data set [h] *P≤0.05,**P≤0.001

Figure 2. Cognate (lipid) and non-cognate (lipid plus protein) antigen stimulation of B cells leads to development of splenic germinal centers.

(a-d) 7 μm sections of OCT-embedded spleens from B1-8 transgenic mice were labeled with GL-7 (green), anti-CD3 (blue), and anti-CD19 (red). High resolution, knitted images of these spleen sections were captured by confocal microscope and represent duplicate sections of 4 mice/group collected 12 days after immunization with 5 μg NP−α−GalCer (a), 100 μg NP-KLH + 5 μg α−GalCer (b), 100 μg NP-KLH (c), and PBS-DMSO (d). (4mice/group, representative of 2-3 independent experiments) scale bar=500μm. Images enabled determination of total number of GL7⁺ GCs per spleen section (e). (f, g) Flow cytometry analysis of day 12 spleen cells from identically immunized C57BL/6 WT mice was used to quantitate CD19⁺CD95⁺GL7⁺ gerinal cell numbers per spleen (f), and IgD⁻ CD38⁺NP-specific B cells (g). (h)ELISPOT analysis of NP-specific IgG secreting splenic B cells from WT C57BL/6 mice. (i) TCR_β⁺CD1dtet⁺ iNKT cells were also enumerated by flow cytometry. Each symbol represents an individual mouse; bar = mean. 4-5 mice per group from representative (f, h, i) or pool (e,g) of 2-3 independent experiments. Mann-Whitney test * P≤0.05,**P≤0.001.

Figure 3. Cognate and non-cognate iNKT cell help both induce antigen-specific antibody affinity maturation

Sera was collected 7 days after primary challenge and 7 days after secondary boost (day 61) from C57BL/6 WT mice immunized with 0.5 μg NP−α−GalCer, 100 μg NP-KLH/ 0.5 μg α−GalCer, 100 μg NP-KLH + alum, or 30 μg NP⁶⁸-Ficoll and tested by ELISA on plates coated with NIP⁵BSA vs NIP¹⁵BSA. Affinity maturation is expressed as ratio of NP⁴/NP²⁵ binding as assessed by O.D. Each symbol represents an individual mouse; bar = mean. 8-10 mice per group, representative of 3 independent experiments. * P≤0.0001.

Figure 4. IL-21 receptor signaling is required for cognate iNKT cell-mediated anti-NP antibody responses.

(a, b) C57BL/6 WT and Il21r deficient mice were immunized with 0.5 μg NP−α−GalCer, 100 μg NP-KLH + 0.5 μg α−GalCer, 100 μg NP-KLH + alum, or 30 μg NP(68)-Ficoll. Mice were bled on days 0, 7, 14 and 21 and assessed for IgG (a) and IgM (b) anti-NIP titers. (Summary of 2-5 independent experiments with 3-5 mice per group in each experiment; mean and s.e.m.) * p≤0.05 **P≤0.001 for comparisons between WT and IL-21R-KO pairs. (c) TCRβ⁺CD19⁻CD1d tetramer–positive iNKT cells were sorted by flow cytometry from V_α14 transgenic mice at 1 day, 1 week (days 5-7), and 2 weeks (days 11-13), following IP immunization with 0.5 μg NP−α−GalCer per mouse or DMSO. Real-time RT-PCR was performed to assess IL-21 mRNA expression in iNKT cells. (data shown are pooled from 2-3 experiments and include 5-11 mice per group; mean and s.e.m.) **P≤0.001

Figure 5. iNKT-produced IL-21 is required for cognate lipid antigen help.

(a,b,c) Mixed bone marrow chimeras with an iNKT cell compartment capable (WT) or incapable of producing IL-21 (Il21-KO) were immunized with 0.5 μg NP−α−GalCer, 100 μg NP-KLH + 0.5 μg α−GalCer, or 100 μg NP-KLH + alum. Mice were bled on day 8 (a), day 14 (b), and day 21 (c) and assessed for anti-NIP IgG titers. (Summary of 2 independent experiments, with 3-5 mice per group in each experiment; mean and s.e.m.) * P≤0.05 for comparisons between WT and Il21 deficient pairs.

Figure 6. Only non-cognate iNKT cell help induces antibody memory response after day 177 rechallenge.

(a,b) C57BL/6 WT mice were immunized IP on day 0 with PBS + alum, PBS, or 2.2 μg NP-KLH + alum (a) or 0.5 μg NP−α−GalCer (b). Mice were bled periodically up to day 166. Mice then received a secondary boost IV on day 177 with 2.2 μg NP-KLH + PBS (or IP with alum as noted), 0.5 μg NP−α−GalCer antigen, or PBS and were bled again on days 3, 7, and 14 post-boost. Legend indicates primary challenge; secondary boost. Concentration of NP-specific IgG was determined by anti-NIP⁵ ELISA. (One experiment with 10 mice per group; mean ± s.e.m.) * P≤0.05, **P≤0.001 as compared to PBS; NP-KLH + alum group (□). For full time-course see Supplementary Fig. 3.

Figure 7. Only non-cognate iNKT cell help induces antibody memory response after day 46 rechallenge.

(a,b) C57BL/6 WT mice were immunized IP on day 0 with 2.2 μg NP-KLH in alum, 2.2 μg NP-KLH plus 0.5 μg α−GalCer (a), 0.5 μg NP−α−GalCer, or 30 μg NP(68)-Ficoll (b) and were bled periodically up to day 45. Mice then received a secondary boost IP on day 46 with the same doses of NP-KLH (±alum), NP-KLH + α−GalCer (a), NP−α−GalCer, NP⁶⁸-Ficoll or PBS (b) and were bled again on days 3, 7, 14, and 29 post-boost. Key indicates primary challenge; secondary boost. Concentration of NP-specific IgG was determined by anti-NIP⁵ ELISA. (One experiment with 10mice per group; mean ± s.e.m.) * P≤0.05, **P≤0.001 as compared to relevant primary immunization group (□ or ○). For full time-course see Supplementary Fig. 4.