Figure 1.
Efficient selection of CD4+ T lymphocytes in NODmini and NODβTg mice.
Lymphocytes isolated from thymii (A) and lymph nodes (B) of indicated mice were stained with monoclonal antibodies and analyzed by flow cytometry. Numbers in quadrants are representative percentages of at least six mice (6 week old) per group. The numbers of thymocytes and lymphocytes ± SD recovered from NODmini, NODβTg and NOD mice were: from thymii 78.5±16.7×106, 72.05±13.8×106 and 70.1±14.0×106, and from lymph nodes (axillary, brachial and inguinal) 17.2±5.8×106, 14.1±2.5×106, and 13.2±3.0×106, respectively. (C) Percentages (top) and total numbers (bottom) of CD4+Foxp3+ T cells in peripheral lymph nodes of 6 week old mice; each circle represents individual mouse. (D) Expression of mRNA of TCRVα genes in sorted CD4+ T cells isolated from NOD and NODβTg mice. Analysis was done by RT-PCR using primers specific to indicated Vα segments and Cα region. (E) Comparison of CD4+Foxp3+ T (Treg) cells from thymii and peripheral lymph nodes of transgenic and wild type B6 and NOD mice. Mean percentage and SD of six young mice per group are shown.
Figure 2.
NODβTg and NODmini mice do not develop T1D.
(A) Incidence of diabetes in mice shown as Kaplan–Meier survival curve. Mice with three consecutive measurements of blood glucose level above 250 mg/dL were considered diabetic. At least twelve mice per group were analyzed; p<0.05. (B) H&E staining of pancreatic tissue sections were analyzed for lymphocytic infiltrates and percentages of islets with grade of insulitis were counted. Insulitis was graded based on following criteria: no infiltrates – grade 0; peri-insulitis - grade 1; insulitis <25% - grade 2; insulitis <50% - grade 3; insulitis >50% - grade 4. At least six mice at 20 weeks of age were analyzed with the total number of 140 (NODmini), 155 (NODβTg) and 160 (NOD) islets. (C) Example of H&E staining of pancreatic tissue sections of indicated mice at 20 weeks of age.
Figure 3.
Lack of response to insulin B:9–23 peptide in transgenic NOD mice.
(A) 5×104 of CD4+ T cells sorted from lymph nodes of NOD, NODβTg and NODmini mice were cultured in the presence of 5×105 splenocytes from NOD.TCRα−/− (Ag7) or B6.TCRα−/− (Ab) mice and soluble anti-CD3 (1 µg/ml) or insulin B:9–23 peptide (50 µg/ml), as indicated. Proliferation of cells was measured after 3 days by MTT assay [38]. Experiments were done twice with 3 mice per group. (B) T-cell hybridomas specific to allo-antigens or insulin B:9–23 peptide were generated from indicated mice. For generation of B:9–23 specific hybridomas, mice were immunized with the peptide 7 days prior to isolation of lymph nodes for in vitro blasts generation [38]. Generated hybridomas were tested for their ability to respond to syngeneic (NOD.TCRα−/−) or allogeneic (B6.TCRα−/−) splenocytes with or without B:9–23 peptide or anti-CD3. Table shows numbers of identified hybridomas specific to indicated antigens and numbers of hybridomas responding to anti-CD3 stimulation. Table is representative of 3 independent experiments with two mice per group.
Figure 4.
Lymphocytic infiltrates in mandibular salivary and extraorbital lacrimal glands.
(A) H&E staining of tissue sections from indicated organs of analyzed mice, showing lymphocytic infiltrates indicated by arrows. (B) Histological score of infiltrated glands in indicated age groups. Scoring criteria: score 0, no infiltrates; score 1–1.5, 1–2 foci per section; 2–2.5, 3–5 foci per section; score 3, 6–10 foci per section; score 4, more than 10 foci per section. Infiltrate is considered as focus when number of infiltrating cells in continuous space is greater than 50. Three sections at different anatomical locations per organ were analyzed with at least 5 mice per age group. (C) FACS analysis of CD4 T cells infiltrating into salivary and lacrimal glands in 16 week old NODmini mice. Dot plots on the right show expression of transgenic TCR on CD4+ gated cells.
Figure 5.
Detection of autoantibodies and hyposalivation in NODmini and NODβTg mice.
(A) ELISA assay was performed using mouse serum (1∶100) from indicated age groups. Each graph represents mean value of OD450 and standard deviation for indicated antigens. At least six mice were used per each group. *p<0.05, **p<0.005. (B) Detection of ANAs pattern using Hep-2 cell line. Sera from mice were diluted 1∶40, incubated with HEp-2-fixed slides and evaluated under fluorescent microscope at x20 magnifications. Representative images are shown. (C) Quantitative ELISA analysis of IgG1 levels in sera (1∶50,000) from indicated mice. Each bar represents mean value and standard deviation from six mice per experimental groups. (D) Salivary flow rates after pilocarpine injection in indicated mice at 10 and 20 wks of age. Double congenic B6.NODIdd3.NODIdd5 (B6.DC) mice, that develop SS on B6 genetic background, were used as a control. Saliva volume was measured and calculated in mg per mouse body mass. Each circle represents one mouse and horizontal lines indicate mean values of the experimental groups. T-test was used to calculate differences between groups. *p<0.002, **p<0.0002.
Figure 6.
TCR repertoire of naïve and regulatory T cells in NODmini mice.
(A) Similarity between indicated populations (first 289 single cell sequences per each population) was estimated based on Morisita-Horn index (MH). TN - naïve CD4+Foxp3−CD45RB+CD62L+ T cells, TR - regulatory CD4+Foxp3+ T cells, TH - thymus, LN - lymph nodes. (B) Ratio of richness of TCRs on TN and TR cells between NODmini and B6mini mice. Abundance coverage estimator (ACE) was calculated based on 578 sequences for each population combined from thymus and peripheral lymph nodes. (C) Evenness and effective number of species (ENS) for analyzed TCR repertoires. Shannon evenness index was calculated as Shannon entropy (Hs) divided by maximum diversity Dmax, where Dmax equals natural logarithm of number of unique sequences in analyzed population. ENS (true diversity) was calculated as exponential of Shannon entropy. (D) Comparison of frequency of 20 most dominant unique protein CDR3 clonotypes found in each population indicated on the right of the heat map. Table indicates analyzed populations from lymph nodes and thymii by single cell analysis (SC) and high throughput sequencing (HT). Experimental mouse 1 and 2 are marked as m1 and m2. Numbers next to each population indicate total numbers of DNA sequences analyzed. All 86 unique CDR3α protein sequences in the heat map are shown in Table S1. (E) Accumulation curve of unique DNA clonotypes observed after accumulation of 124,730 sequences for TN cells and 124,696 for TR cells. (A–C) All indices were computed based on DNA sequences for each population using software SPADE and EstimateS8.2.