Journal: iScience

Article Title: BCL6 in T cells promotes type 1 diabetes by redirecting fates of insulin-autoreactive B lymphocytes

doi: 10.1016/j.isci.2026.115990

Figure Lengend Snippet: BCL6 in T cells promotes spontaneous GC formation, anti-insulin B cell infiltration of islets, and spontaneous diabetes development in VH125 SD .NOD mice Cells from spleen, pLNs, mLNs, and pancreas were isolated from 8 to 12-week-old VH125 SD .NOD and VH125 SD .Bcl6 ΔCD4 .NOD mice (genotypes fully defined in ). (A) A representative flow cytometry plot from spleen shows the frequency of insulin-binding B cells (insulin+) identified using biotinylated human insulin/streptavidin fluorochrome as in among total B cells (live singlet CD45 + CD19 + lymphocytes). (B) Diabetes was monitored in cohorts of female VH125 SD .NOD mice ( n = 14, black line) and VH125 SD . Bcl6 ΔCD4 .NOD littermates ( n = 14, purple line) from 10 to 35 weeks of age. Mice were considered diabetic after two consecutive blood glucose readings >250 mg/dL, p < 0.0001, log-rank test. (C–G) Representative flow cytometry plots of pancreatic draining lymph nodes and pancreata gating on anti-insulin B cells as in (A) and as described in for pancreas are shown (C). The frequency of anti-insulin B cells (among total B cells) in (D) spleen, (E) mesenteric lymph nodes (mLNs), (F) pancreatic draining lymph nodes, and (G) pancreata are plotted for individual mice of the indicated genotypes. (H–L) Representative flow cytometry plots from the pancreatic lymph nodes (pLNs) of (H) Tfh cells (live singlet CD45 + CD4 + PD-1 hi CXCR5 hi Foxp3 − lymphocytes) are shown with (I and J) frequencies among total CD4 + Foxp3- CD45+ cells and (K and L) numbers of Tfh cells in pLNs and pancreata plotted for individual mice. (M–Q) Representative flow plots of (M) GC B cells from the pancreatic lymph nodes (live singlet CD45 + CD19 + Fas + GL7 + lymphocytes) with (N and O) frequencies of GC B cells among total B cells and (P and Q) numbers shown for pLNs and pancreata. (C–Q) n = 6–8 mice per group, 5 independent experiments, Mann-Whitney U test, bars representative of mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant.

Article Snippet: Regions of B cell zone, T cell zone, germinal center, and extrafollicular sites were drawn by blinded experimenter, and the proportion of insulin+ B cells was quantified utilizing Imaris once borders were drawn for VH125 SD -BCL6 sufficient mice.

Techniques: Isolation, Flow Cytometry, Binding Assay, MANN-WHITNEY, Standard Deviation

Journal: iScience

Article Title: BCL6 in T cells promotes type 1 diabetes by redirecting fates of insulin-autoreactive B lymphocytes

doi: 10.1016/j.isci.2026.115990

Figure Lengend Snippet: BCL6 in T cells increases activation and proliferation markers of insulin-binding B cells relative to non-insulin-binding B cells Cells were isolated from 8 to 12-week-old, female, pre-diabetic VH125 SD .NOD with and without Cd4 -Cre Bcl6 deletion from (A–G) spleen, pancreatic lymph nodes (pLNs) and pancreata. (A–C) Representative flow cytometry plots show Ki67 staining overlays of insulin-binding (left) or non-insulin-binding (right) B cells (identified as in ) from each genotype within live singlet GL7- CD95- CD45 + CD19 + lymphocytes in spleen (A), pancreatic lymph nodes (B), and pancreata (C). (D) Non-GC (Fas − GL7 − ) B cells were further gated on insulin-binding (ins+) and non-insulin-binding (ins−) and the frequency of cells that were Ki67+ (a marker of proliferation). (E–G) Insulin+ or insulin− B cell expression of (E) CD86 (T cell co-stimulatory molecule), (F) CD44 (activation marker), and (G) CD69 (activation marker) is shown for spleen (left), pancreatic lymph nodes (middle), and pancreata (right), with n = 6–8 individual mice plotted per group. (D–G) Bars represent mean ± standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns = not significant, Kruskal-Wallis test with post hoc test of multiple comparisons. All other comparisons not shown are not significant.

Article Snippet: Regions of B cell zone, T cell zone, germinal center, and extrafollicular sites were drawn by blinded experimenter, and the proportion of insulin+ B cells was quantified utilizing Imaris once borders were drawn for VH125 SD -BCL6 sufficient mice.

Techniques: Activation Assay, Binding Assay, Isolation, Flow Cytometry, Staining, Marker, Expressing, Standard Deviation

Journal: iScience

Article Title: BCL6 in T cells promotes type 1 diabetes by redirecting fates of insulin-autoreactive B lymphocytes

doi: 10.1016/j.isci.2026.115990

Figure Lengend Snippet: BCL6 loss in CD4 + T cells impairs CD4 + T cell activation and promotes Treg formation in some sites but does not affect anti-insulin B cell proliferation in the pancreas CD4 + T cells were isolated from 12 to 14 week-old female donors and ∼5 × 10 6 BCL6+ or ΔBCL6 CD4 + T cells were CellTrace Violet (CTV)-labeled and adoptively transferred into 8–12 week-old VH125 SD .Bcl6 ΔCD4 .NOD recipients. Seven days after transfer, spleen, pancreatic lymph nodes, and pancreata were assayed as follows. (A) Experimental schematic. (B) (Left) Representative flow plots of CD4 + CTV+ labeled cells in FMO control, spleen, pancreatic lymph nodes, and pancreas. (Right) The proportions of CTV+ labeled cells in spleen, pancreatic lymph nodes, and pancreata are shown for individual recipients following BCL6+ or ΔBCL6 CD4 + transfers. (C–F) CTV+ (transferred) or CTV- (endogenous, non-transferred) CD4 + T cells were assessed regarding: (C) CD44 MFI levels, (D) % CD69 + , (E) % Tfh (CXCR5+ PD-1+), and (F) % Treg (FoxP3+). (G–H) % GC B cells (CD95 + GL7+) is shown among total B cells (CD19 + B220+ CD45 + live cells) and (H) % Ki67+ among non-GC (CD95 − GL7 − CD19 + B220+ CD45 + live) B cells in either insulin-binding (INS+) or non-insulin binding (INS−) B cells. (B–H) n = 5–6 mice per group, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, not significant, Kruskal-Wallis test with post hoc test of multiple comparisons (B–F and H) or Mann-Whitney U test (G) was used for analyses. All other comparisons not shown are not significant. Bars represent mean ± standard deviation.

Article Snippet: Regions of B cell zone, T cell zone, germinal center, and extrafollicular sites were drawn by blinded experimenter, and the proportion of insulin+ B cells was quantified utilizing Imaris once borders were drawn for VH125 SD -BCL6 sufficient mice.

Techniques: Activation Assay, Isolation, Labeling, Control, Binding Assay, MANN-WHITNEY, Standard Deviation

Journal: iScience

Article Title: BCL6 in T cells promotes type 1 diabetes by redirecting fates of insulin-autoreactive B lymphocytes

doi: 10.1016/j.isci.2026.115990

Figure Lengend Snippet: Insulin-binding B cells exist in extrafollicular niches and skew toward CD11c + T-bet + and CD11b + CD11c + atypical B cell subsets, some of which are reduced by loss of Bcl6 Spleen, pancreatic draining lymph nodes, and pancreata were harvested from pre-diabetic, 8–12-week-old VH125 SD .NOD mice. (A) Around 10 μm sections of spleen form VH125 SD .NOD underwent immunofluorescence staining with IgD, Ki67, insulin, and CD3. Representative merge image shown on left with identified borders of B cell zone (BCZ), T cell zone (TCZ), germinal center (GC) and extrafollicular area (EF). Circled cells are representative insulin-autoreactive B cells, which express both IgD and insulin. Quantification of % insulin+ B cells in predefined niches on the left graph. Quantification of insulin+ IgD + colocalization, or insulin+ IgD-. n = 4–6 individual splenic sections per group. (B) Representative flow plots show CD11c+/CD11b+ cells among B220+ CD19 + live singlet lymphocytes for both non-insulin-reactive (insulin−, left) and insulin-autoreactive (insulin+, middle, right) B cell populations in the pancreas. (C) Quantification of the proportion of CD11c+ CD11b+ B cells in pancreata for both insulin− and insulin+ B cells for VH125 SD (black) and VH125 SD Bcl6 ΔCD4 (purple) mice. (D) Representative flow plots of CD11c+/Tbet+ cells among B220+ CD19 + live singlet lymphocytes for both insulin− and insulin+ B cell populations in the pancreas. (E) Quantification of the proportion of CD11c+/Tbet+ cells among both insulin− and insulin+ B cells in the pancreas of VH125 SD (black) and VH125 SD Bcl6 ΔCD4 (purple) mice. (F and G) Quantification of the proportion of CD11c+/Tbet+ B cells and CD11b+/CD11c+ B cells in pancreatic draining lymph nodes (F) and spleen (G). (A–G) One-way ANOVA (A, left), Mann-Whitney U test (A, right) (A) or Kruskal-Wallis test (C–G) with post hoc multiple comparison test were used, n = 4–8 mice per group. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns, not significant. All other comparisons not shown are not significant. Bars represent mean ± standard deviation.

Article Snippet: Regions of B cell zone, T cell zone, germinal center, and extrafollicular sites were drawn by blinded experimenter, and the proportion of insulin+ B cells was quantified utilizing Imaris once borders were drawn for VH125 SD -BCL6 sufficient mice.

Techniques: Binding Assay, Immunofluorescence, Staining, MANN-WHITNEY, Comparison, Standard Deviation

Journal: iScience

Article Title: Type 2 and type 1 diabetes have opposing effects on the systemic murine complement alternative pathway

doi: 10.1016/j.isci.2026.116359

Figure Lengend Snippet: HFD leads to downregulation of the expression of adipose Cfd and hepatic C3 and Cfb (A and B) C3-tdTomato reporter mice were placed on LFD or HFD, and expression of inflammatory markers and complement genes were assessed by qPCR in (A) subcutaneous inguinal adipose tissue and (B) visceral epididymal adipose tissue. (C) Western blot for FD in serum from female LFD and HFD mice after 25 weeks of diet. (D) Western blot for FD in serum from male mice after 15 weeks of diet. (E) Left: representative western blot from sequential serum samples from 2 male mice on HFD. Right: densitometry for serum FD signal in western blots of 6 male mice over time. (F–H) Serum FD levels, measured by ELISA, plotted against individual mouse body weight (F), fasting insulin (G), and fasting blood glucose levels (H). (I) Expression of main alternative pathway components in livers of LFD and HFD mice. (J) Protein levels of C3 in liver homogenates from lean control or ob/ob mice on the C57Bl/6 background. WAT, white adipose tissue. In (A)–(C), there were 4 mice per group, except for HFD visceral fat pad, which had 3 samples. In (F)–(H), measurements are from a total of 42 individual mouse samples. For (I) and (J), there were 4–5 mice per group. Data represent mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as tested by two-way ANOVA. See also .

Article Snippet: Serum insulin was measured using an ultrasensitive mouse insulin ELISA (Mercodia #10-1249) and mouse C3b was measured in serum samples using an ELISA kit from Hycult Biotech (HK216).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: iScience

Article Title: Type 2 and type 1 diabetes have opposing effects on the systemic murine complement alternative pathway

doi: 10.1016/j.isci.2026.116359

Figure Lengend Snippet: Expression changes in STZ-induced diabetes leads to increased complement in serum and increased alternative pathway activation (A) Serum levels of FD in untreated and STZ-treated male mice. (B) Serum C3 levels in untreated controls and STZ-treated male mice. (C) Western blot for FD in homogenates of inguinal adipose tissue from control mice and STZ-treated WT and C3-KO mice. Densitometry quantification shown on the right. (D) Blood glucose levels in STZ-treated WT or C3-KO male mice over time. (E) Serum FB levels in STZ-treated male mice before and after treatment. (F) Western blot for FB in serum samples of untreated and STZ-treated male mice (top), and quantification by densitometry (bottom). (G) Example flow cytometry histograms of C3 staining of zymosan beads after incubation with serum from untreated or STZ-treated cage-mate mice in EGTA buffer, allowing only AP activation. EDTA completely inhibits complement and acts as a negative control. (H) Results of C3 deposition onto zymosan beads from serum taken at different time points from untreated or STZ-treated male mice. (I) Serum C3b levels in STZ-treated or mock-treated male mice and in STZ-treated C3-KO controls, as measured by ELISA. For all images, n = 4 untreated mice and 5 STZ-treated mice, as in , except for (C), with groups of 4; (D), with groups as stated in the figure; and (I), with n = 8, 9, and 4 for untreated, STZ-treated, and STZ-treated KO groups, respectively. Data represent mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as tested by two-way ANOVA, t test (E), or one-way ANOVA (C and I).

Article Snippet: Serum insulin was measured using an ultrasensitive mouse insulin ELISA (Mercodia #10-1249) and mouse C3b was measured in serum samples using an ELISA kit from Hycult Biotech (HK216).

Techniques: Expressing, Activation Assay, Western Blot, Control, Flow Cytometry, Staining, Incubation, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Type 2 and type 1 diabetes have opposing effects on the systemic murine complement alternative pathway

doi: 10.1016/j.isci.2026.116359

Figure Lengend Snippet: T1D Akita mice have increased serum FB levels and alternative pathway activation (A) Serum FD levels in Akita and WT littermates, as measured by ELISA. (B) Serum C3 levels in the same mice. (C) Serum C3 levels in “young” versus “older” mice. (D) Serum FB levels in all Akita and WT littermate mice. (E) Serum FB levels in the same mice, stratified over time, plotting line of best fit and 95% confidence intervals. (F) C3 alternative pathway deposition onto zymosan beads from age-matched Akita or WT littermate mice. Left: example histograms from cage-mate mice; right: quantification of C3 deposition results from serum taken from age-matched pairs of mice aged from 18 to 24 weeks ( n = 5 per genotype). Each data point represents mean value from an individual mouse. Data represent mean ± SD, with ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as tested by one-way ANOVA (F) or t test (C and D). In (E), lines show best fit and 95% confidence intervals.

Article Snippet: Serum insulin was measured using an ultrasensitive mouse insulin ELISA (Mercodia #10-1249) and mouse C3b was measured in serum samples using an ELISA kit from Hycult Biotech (HK216).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay