(A) CFSE-labeled Thy1.1⁺ clone-4 T-cells (3×10⁶) were injected into either NOD-InsHA mice (controls) or Human CD20 Tg NOD-InsHA mice that had been injected with Rituxan or 2H7 mAb with or without anti-CD4 on days −10 and −3. Four days after clone-4 cell transfer PcLN were analyzed by FACS (three pooled experiments). (B) NOD-InsHA recipients were injected with anti-Asialo GM1 Ab with or without anti-CD4 on days −10 and −3 before transfer of CFSE labeled clone-4 cells and analysis of PcLN on day 4 (two pooled experiments).

Purified CSFE-labeled CD8⁺Thy1.1⁺ clone-4 NOD T-cells (3×10⁶) were injected into NOD-InsHA, Idd3/5-InsHA, Idd3-InsHA and Idd5-InsHA recipients with or without anti-CD4 treatment on days −10 and −3. Four days after clone-4 cell transfer PcLN were analyzed by FACS for divided clone-4 cells (pooled data from three to seven experiments). ***p<0.0001, **p<0.01,*p<0.05, ns: not significant.

(A) NOD-SCID or Idd3/5-SCID mice were reconstituted with spleen and lymph node cells depleted of CD11c+ DCs isolated from 3-week-old donor NOD or Idd3/5 mice. After 6-8 weeks CFSE labeled 8.3 Thy1.1+ CD8 T-cells were transferred (3×10⁶) and the PcLN analyzed by FACS on day 4 for divided 8.3 cells (three pooled experiments). **p<0.005. (B) H&E stained pancreas sections from the following mice were scored for insulitis: NOD->NOD-SCID n=6, NOD->Idd3/5-SCID n=8, Idd3/5->NOD-SCID n=14, Idd3/5->Idd3/5-SCID n=15. Some reconstituted SCID mice did not receive CFSE labeled 8.3 cells. Mice receiving 8.3 cells and those not injected with the cells had similar degrees of insulitis and pooled data are shown. Data represent mean +/− SEM.

(A) Thy1.1+ NOD clone-4 cells (1×10⁴) were transferred into NOD, Idd3/5, NOD-InsHA and Idd3/5-InsHA mice. After 5 weeks the mice were infected with Vac-K^dHA and 7 days later the frequency of Thy1.1+ clone-4 cells was determined in the spleen by FACS analysis (two pooled experiments). (B) On days −10 and −3, anti-CD4 or isotype control Abs were injected into NOD-InsHA or Idd3/5-InsHA mice. On day 0 CSFE-labeled Thy1.1⁺ NOD clone-4 cells (3×10⁶), were injected and PcLN analyzed by FACS four days later (three to four pooled experiments). ***p<0.0001, ns: not significant. (C) Adult or 4 week old NOD-InsHA mice were injected with anti-CD4 or anti-CD40L mAb or isotype control on days −10, −3 and 0 before transfer of CFSE labeled clone-4 cells and analysis of PcLN on day 4 (two pooled experiments). (D) NOD-InsHA mice were injected with anti B7.1and anti B7.2 mAbs was on days 0, 1, 2 and 3 with or without CD4 depletion before transfer of CFSE labeled clone-4 cells and analysis of PcLN on day 4 (two pooled experiments). ***p<0.0001.

(A) Idd3/5-InsHA mice were irradiated and reconstituted with BM from Idd3/5, mixed Idd3/5+NOD, mixed Idd3/5+ NOD β2M−/−, mixed NOD+ NOD β2M−/− or NOD. After 7 weeks anti-CD4 was injected on days −10 and −3 and 3×10⁶ purified CSFE-labeled CD8⁺Thy1.1⁺ clone-4 NOD T-cells were transferred on day 0. PcLN were analyzed on day 4, data pooled from 2 experiments. ***P<0.0001. (B) NOD-CD45.2 congenic mice were irradiated and reconstituted with BM from the following mice: mixed NOD-CD11c-DTR and NOD, mixed NOD-CD11c-DTR and Idd3/5 or NOD-CD11c-DTR alone. After 6 weeks anti-CD4 was injected on days −10 and −3, DT or saline was injected on days −4, −1 and +2 and 3×10⁶ purified CSFE-labeled CD8⁺Thy1.1⁺ 8.3 NOD T-cells were transferred on day 0. PcLN were analyzed on day 4 for divided 8.3 cells (left panels) and spleen cells enriched for DCs and analyzed for CD11c and GFP expression (right panels).

(A) NOD and Idd3/5 BM derived DCs and macrophages were unstimulated or stimulated with _γIFN and LPS for 48 hours and analyzed by western blot for SLC11a1 protein expression. Lane E shows the positive control, a lysate from the Slc11a1-expressing NAMALWA cell line (Santa Cruz Biotech). This is one of two experiments yielding similar results. (B) BM derived DC were analyzed by qRT-PCR for ACADL mRNA levels. Representative data from one experiment of three performed is shown. Comparisons of the delta Ct values using β2M as the normalization control of ACADL mRNA levels obtained from NOD vs Idd3/5 DCs stimulated with _γIFN and LPS (or LPS alone) demonstrated significant differences in both unstimulated and stimulated samples (P = 0.031 using the nonparametric Wilcoxon matched pairs test). Data are represented as mean +/− SEM. (C) NOD and Idd3/5 BM derived DCs were unstimulated or stimulated with zymosan for 5 hours before analysis of supernatants for IL-2 protein. Representative data from one experiments of 7 performed using various NOD strains having the B6 versus NOD allele at Idd3. DCs were stimulated with zymosan (10 μg/ml) or LPS (1 μg/ml) for 5 hours in these seven experiments. DCs from strains having the B6 allele at Idd3 consistently produced more IL-2 than DCs having the NOD allele (P = 0.0156) using the nonparametric Wilcoxon matched pairs test).