Natural history of invariant iNKT cells. (A) Quantitation of Vα4Jα281 TCR transcripts in individual NOD/LtJ female, NOD/LtJ male, and NOR/LtJ female mice. cDNA prepared from pancreatic islets and spleens of mice aged 5, 10, and 15 weeks were assayed for TCR content using primer sets specific for the invariant TCR α-chain by Taqman assay and are reported as a ratio to total TCR β-chain content. (B) Quantitation of total TCR β-chain in islets isolated from NOD/LtJ female, NOD/LtJ male, and NOR/LtJ female mice that are reported relative to GAPDH internal controls. No invariant Vα14Jα281 TCR transcript was detected in RNA prepared from CD1d^(−/−)/NOD, or Ja281^(−/−)/BALB/c mice (data not shown).

Treatment with α-GalCer recruits iNKT cells to the islets and pancreatic lymph nodes. Relative frequency of invariant Vα14Jα281 TCR transcripts in pancreatic islets (A), spleen (B), pancreatic LN (C), and inguinal LN (D). Starting at 5–6 weeks of age, Female NOD/LtJ mice were treated with α-GalCer (100 μg/kg), α-ManCer (100 μg/kg), or vehicle as 2 i.p. at day 0 and day 4 (n = 10–13/treatment). On d10, islets and spleens were harvested from individual mice, and the relative frequency of invariant Vα14Jα281 TCR transcripts was determined as in Fig. 1. The frequency of invariant Vα14Jα281 TCR transcripts in islets (P < 0.05, Student's t test) and pancreatic LN (P < 0.05) was significantly increased relative to vehicle control after α-GalCer injection. (A and C) Priming of mice in vivo with α-GalCer augments the in vitro secretion of IFN-γ and IL-4 by α-GalCer. Female NOD mice were treated as above with vehicle or increasing doses, 2, 4, or 8 μg of α-GalCer. After 10 days, inguinal and pancreatic lymph nodes were then harvested and cultured in vitro with either vehicle or α-GalCer for 5 days, and levels of IL-4 (E) or IFN-γ (F) present in the culture supernatants were determined by ELISA. Similar results were seen on day 5 and day 20 (data not shown).

Transfer of myeloid DC isolated from pancreatic LN protects female NOD mice from developing diabetes. Transfer of myeloid DC from pancreatic LN (n = 6 recipients) or inguinal DC cultured in media alone (n = 15 recipients) or loaded with sonicated islet cell preparations (n = 15 recipients) significantly (P < 0.002) protected NOD female mice from diabetes. Myeloid DC from either inguinal or pancreatic lymph nodes were isolated (18) from nondiabetic female NOD mice. Pancreatic DC were cultured in media alone, and inguinal DC were preincubated with media or sonicated islet preparations for 2 h before transfer. Individual NOD naïve female mice received 50,000 DC as a one-time injection in the footpad and were then followed for diabetes development. Control experiments demonstrated these DC preparations to be essentially pure populations of myeloid DC and that no viable cells remained after sonication of isolated islets (K.B., unpublished results, and data not shown).

Diabetes progression in NOD mice is regulated by iNK T cells. Female NOD/LtJ and CD1d^(−/−)/NOD mice (age 3–4 weeks) were treated with weekly injections of α-GalCer (100 μg/kg i.p., NOD/LtJ, n = 18; CD1d^(−/−)/NOD, n = 16) or vehicle (NOD/LtJ, n = 17; CD1d^(−/−)/NOD, n = 15). Treatment with α-GalCer significantly protected wild-type NOD females (P = 0.001, Kaplan–Meier method) from diabetes, whereas the introgression of the CD1d^(−/−) phenotype exacerbated disease and rendered therapy with α-GalCer ineffective (P = 0.0002 for vehicle, P = 0.006 for α-GalCer treatment).

Treatment with α-GalCer results in the preferential accumulation of myeloid CD8α−/CD11c+ dendritic cells in the lymph nodes draining the pancreas. (A) CD1d is preferentially expressed on CD8α+/CD11c+ dendritic cells in lymph nodes draining the pancreas and is unaffected by treatment. Five to six 6-week-old female NOD mice were treated as in Fig. 3, and inguinal and pancreatic lymph nodes were isolated. Median fluorescence for CD1d was determined on cells gated to be DC by FSC and SSC and CD11c+/MHC class II bright (n = 6 mice/group; data are shown as Box-and-Whisker plots; mean, −; median, filled bars; first and third quartiles, empty bars; error bars, standard deviation). (B) In vitro LPS-induced IL-12 secretion by PLN DC is suppressed by prior in vivo α-GalCer treatment. Mice were treated with vehicle, or α-GalCer (100 μg/kg), i.p. on day 0 and day 4 (n = 6/treatment, for a total of six experiments). On day 10, inguinal and pancreatic LN were harvested. Isolated cells were challenged with LPS/anti-CD40 (32) or LPS (data not shown) and incubated for 48 h. The amount of IL-12p40 in the culture supernatants was determined by ELISA, and the results are reported as normalized IL-12 secreted per total DC (P = 0.006, student's t test comparing vehicle with α-GC-treated PLN). DC frequency was determined by cell count and FACS. The absolute values for IL-12 were vehicle-treated inguinal LN, 408 ± 108 pg/ml; PLN 180 ± 64 pg/ml; and α-GalCer-treated inguinal, 580 ± 80 pg/ml; PLN 210 ± 35 pg/ml. (C and D) The total number (×10³) of CD8α+/CD11c+ and CD8α−/CD11c+ DC in pancreatic and inguinal lymph nodes was determined 10 or 20 (not shown) days after mice were treated with vehicle, α-ManCer (100 μg/kg), or α-GalCer (100 μg/kg), i.p. on day 0 and day 4 (n = 6/treatment, for a total of six experiments). Treatment with α-GalCer resulted in significantly more CD8α−/CD11c+ DC in the pancreatic lymph node (P = 0.005, student's t test) compared with vehicle. The various treatments did not alter the frequencies of CD8α+/CD11c+ DC. No significant differences in mean fluorescent index for MHC class II, CD80, or CD86 was observed for the various treatments, and CD11c staining was not observed on T, B, or NK cells (data not shown). (E–H) Immunohistochemical staining for CD11c+ cells (brown deposits) in representative lymph nodes from mice treated with α-GalCer (E), α-ManCer (F), or vehicle (G). A representative inguinal node from an animal treated with α-GalCer is also shown (H). CD11c staining for vehicle-treated mice was identical to α-ManCer-treated mice, and isotype control staining was negative (data not shown).