We transferred variably treated populations of hepatic iNKT and B-1 B cells into the JH−/− and CBA/N-xid mouse strains. As a positive control, we incubated naïve hepatic iNKT cells with the potent CD1d-dependent glycolipid stimulant α-GalCer, B-1 B Alisertib cell line cells with the hapten–protein complex TNP–BSA and ultimately the activated iNKT and B-1 B cells together. We found that adoptive transfer of the activated iNKT and B-1

B cells into JH−/− and CBA/N-xid mice 3 days after sensitization, and 1 day before challenge, fully reconstituted CS (Group C in Fig. 1A,B). We compared α-GalCer with hepatic lipids isolated from wild-type mice 30 min after sensitization or sham sensitization. In both JH−/− and CBA/N-xid mice, incubation of iNKT cells with lipids extracted after sensitization provided CS responses that were comparable to the positive control (Group D in Fig. 1A,B). In contrast, the use of lipids extracted after sham sensitization led to significantly impaired

CS responses (Group E in Fig. 1A,B). However, this impairment was not as marked as was seen at baseline in these strains (Group B in Fig. 1A,B). In other words, incubation of naïve hepatic iNKT cells with lipid extracts from naïve mice leads to a significant but partial reconstitution of CS, while incubation with lipid extracts from sensitized mice leads to a significant and complete reconstitution of CS. Because iNKT and B-1 B cells Ulixertinib manufacturer were co-incubated prior to adoptive transfer, almost we explored the

possibility that the ultimate differences in CS responses were secondary to direct activating effects of the lipid extracts on the B-1 B cells. We incubated LMNC derived from iNKT cell–deficient Jα18−/− mice with B-1 B cells. iNKT cells thus were absent from the cell mixture. Upon adoptive transfer, we found that CS was not even partially reconstituted in comparison with baseline levels (Group F in Fig. 1B). Evidently, hepatic lipids specifically stimulate iNKT cells, not B-1 B cells. Given that iNKT cells are stimulated by hepatic lipids, we hypothesized that CD1d is essential for iNKT cell activation in CS. We explored this via adoptive transfer of iNKT and B-1 B cells into CBA/N-xid mice that were variably treated with anti-CD1d-blocking antibody (Fig. 2). iNKT cell incubations for Groups F, G and H included anti-CD1d-blocking antibody along with α-GalCer, lipid extracts from sensitized wild-type mice and lipid extracts from naïve wild-type mice, respectively. The anti-CD1d-blocking antibody inhibited the stimulatory effects of α-GalCer and lipid extracts from sensitized mice on iNKT cells (Fig. 2, Groups F and G). Of note, the early 2-h response in the α-GalCer-positive control group was greater than in the negative controls, likely due to the known extreme potency of α-GalCer. CS responses were otherwise abrogated completely with anti-CD1d-blocking antibody.