1. Induction of MHC-mismatched mixed chimerism depletes plasma and preplasma cells in NOD mice.
Induction of MHC-mismatched mixed chimerism depletes plasma and preplasma cells in NOD mice. Six-week-old WT NOD female mice were conditioned with 5 μg/g Fc receptor–nonbinding anti-CD3 (145-2C11-γ3) 12 h before injection of 5 μg/g Fc receptor–binding anti-CD3 (145-2C11) and 5 μg/g anti-CD8 ( ) on day −5 before HCT. On day 0, the mice were transplanted with CD4+ T-depleted spleen cells (12.5 × 106) and whole BM (20 × 106) from C57BL/6 donors. Control mice for these experiments received anti-CD3/8–based conditioning alone. Mice were euthanized at days 30, 45, and 60 after transplantation, and spleens were harvested and analyzed for B-cell subsets. A: Representative staining pattern of gated CD45.1+ host-type splenocytes showing CD19 vs. CD138, and gating on plasma or preplasma B-cell populations, CD138intCD19+ and CD138+CD19Lo. B: Mean ± SE of percentages and yields of each subset at each time point after HCT; n = 4–6 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < C: Representative staining pattern of gated CD45.1+ host-type BM mononuclear cells, showing CD19 vs. CD138, and gating on CD138intCD19+ and CD138+CD19Lo. D: Mean ± SE of percentages and yields of each subset at each time point after HCT; n = 4–6 mice per group. *P = ; **P < 0.01; ***P < 0.001; ****P < E: At the time point of day 60, serum was measured for anti-insulin antibodies and total IgG levels. In brief, 1 μg/mL insulin (porcine; Sigma-Aldrich) was used to coat ELISA plates overnight at 4°C. The control blank wells were coated with buffer without insulin. After two washes, plates were saturated for 2 h at room temperature with PBS-T (0.05% Tween-20). After a further three washes, 100 μL of serially diluted serum was added to each well and incubated at room temperature for 2 h. After five washes, alkaline phosphatase-conjugated goat anti-mouse IgG secondary was added for 2 h (SouthernBiotech). After an additional five washes, phosphatase substrate was added, and plates were read at 405 nm. Data are expressed as optical density (OD) after subtracting nonspecific background absorbance. Serum anti-insulin or IgG OD value is shown. n = 14 control and n = 8 chimera. *P = F: Scoring of insulitis was performed on 10 sections of hematoxylin-eosin–stained, formalin-fixed, paraffin-embedded pancreas as previously described. The percentage of no insulitis, peri-insulitis, and insulitis among total islets from each mouse (>75 islets were graded for all mice, except for one control mouse with 36 identifiable islets across 10 sections) was calculated, and the mean score of five to six combined mice is shown per group.
Jeremy J. Racine et al. Diabetes 2014;63:
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