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. Author manuscript; available in PMC: 2016 Oct 15.
Published in final edited form as: J Immunol. 2015 Sep 9;195(8):3838–3848. doi: 10.4049/jimmunol.1500203

Figure 3. Repetitive injection or increased dose can augment iNKT cell hypo-responsiveness.

Figure 3

(A, B) C57BL/6 (B6) mice were either left untreated or injected i.v. once with 4μg of αGalCer (1xαGC) or three times every other day with 4μg OCH (3xOCH) or C-Gly (3xC-Gly) as indicated. One month later mice were injected i.v. with 1μg αGalCer and 90 min later splenic iNKT cells were analyzed for the expression of surface makers (A) and of intracellular cytokines (B). (C, D) C57BL/6 (B6) mice were either left untreated or injected i.v. once with 4μg of αGalCer (αGC), once with 12μg of C-Gly (1xC-Gly) or three times every other day with 4μg C-Gly (3xC-Gly, i.e. 12μg in total) as indicated. One month later mice were injected i.v. with 1μg αGalCer and 90 min later splenic iNKT cells were analyzed for the expression of surface makers (C) and of intracellular cytokines (D). (E, F) C57BL/6 (B6) mice were either left untreated or injected i.v. once with 4μg of αGalCer (1xαGC) or three times every other day with 1μg αCD3ε (145.2C11) antibodies (3x αCD3εAb) as indicated. One month later mice were injected i.v. with 1μg αGalCer, and 90 min later splenic iNKT cells were analyzed for the expression of CD69 (D) and of intracellular cytokines (E). Differences in the amount of IFN□+ iNKT cells detectable in different experiments depended largely on the fluorochrome conjugated to the utilized antibody in the particular experiment (e.g. for IFNγ AF700 (3B, 3F) vs. PE-C594 (3D)). Regardless, within an experiment, consistent differences were observed between groups, and statistically significant differences are indicated. Representative data from one of at least two independent experiments are shown.