Journal: Molecular Medicine Reports

Article Title: NF-κB signaling drives Th17-mediated lacrimal gland injury and tear dysfunction in a murine model of primary Sjögren's syndrome

doi: 10.3892/mmr.2026.13904

Figure Lengend Snippet: Characterization of lacrimal gland dysfunction and systemic inflammation in NOD/Ltj mice. (A) Tear secretion measured by the phenol red thread test. (B) Serum levels of IFN-γ, IL-17A, TNF-α and IL-6 determined by enzyme-linked immunosorbent assay. (C) Representative hematoxylin and eosin-stained lacrimal gland sections from ICR and NOD/Ltj mice presenting glandular architecture and lymphocytic infiltration (magnification, ×200 and ×400). Data are presented as mean ± SD (n=12). *P<0.05, **P<0.01 and ***P<0.001; ns vs. ICR control. ns, not significant; ICR, Institute of Cancer Research; IL-17A, interleukin-17A; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; SD, standard deviation; NOD, non-obese diabetic.

Article Snippet: To investigate the role of NF-κB signaling in lacrimal gland injury, 20 male NOD/Ltj mice (12 weeks old, weighing 24–28 g) were obtained from Beijing Huafukang Biotechnology Co., Ltd. [Experimental Animal Production License No. SCXK(Jing)2019-0008] and 16 age-matched male Institute of Cancer Research (ICR) mice (weighing 35–40 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. [Experimental Animal Production License No. SCKK(HU)2022-0004].

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Control, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: NF-κB signaling drives Th17-mediated lacrimal gland injury and tear dysfunction in a murine model of primary Sjögren's syndrome

doi: 10.3892/mmr.2026.13904

Figure Lengend Snippet: Transcriptomic analysis of lacrimal glands in NOD/Ltj mice. (A) Heatmap presenting DEGs between ICR and NOD/Ltj mice. (B) GO enrichment analysis of DEGs. (C) Volcano plot illustrates the distribution of upregulated and downregulated genes. (D) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. The six genes selected for subsequent reverse transcription-quantitative PCR validation ( CARD14, TNFSF14, TNFSF13, CCL19, CCL20 and CCL12 ) are labeled directly in (A) and (C). DEGs were defined as |log 2 FC|>1 and adjusted P-value <0.05. *P<0.05, **P<0.01 and ns. ns, not significant; ICR, Institute of Cancer Research; DEGs, differentially expressed genes; GO, Gene Ontology; FC, fold change; NOD, non-obese diabetic.

Article Snippet: To investigate the role of NF-κB signaling in lacrimal gland injury, 20 male NOD/Ltj mice (12 weeks old, weighing 24–28 g) were obtained from Beijing Huafukang Biotechnology Co., Ltd. [Experimental Animal Production License No. SCXK(Jing)2019-0008] and 16 age-matched male Institute of Cancer Research (ICR) mice (weighing 35–40 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. [Experimental Animal Production License No. SCKK(HU)2022-0004].

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Biomarker Discovery, Labeling

Journal: Molecular Medicine Reports

Article Title: NF-κB signaling drives Th17-mediated lacrimal gland injury and tear dysfunction in a murine model of primary Sjögren's syndrome

doi: 10.3892/mmr.2026.13904

Figure Lengend Snippet: JSH-23 suppresses NF-κB pathway activation in lacrimal glands of NOD/Ltj mice. (A) Representative western blotting images presenting expression levels of NF-κB pathway-related proteins and downstream inflammatory mediators in ICR, NOD/Ltj and NOD/Ltj + JSH-23 groups. (B) Densitometric quantification of protein expression normalized to β-actin. Data are presented as mean ± SD (n=4). **P<0.01 and ***P<0.001 vs. NOD/Ltj group; ns vs. ICR control. NF-κB, nuclear factor κB; ns, not significant; SD, standard deviation; ICR, Institute of Cancer Research; IKK, inhibitor of κB kinase; IκBα, inhibitor of κB α; TNF-α, tumor necrosis factor-α; p, phosphorylated; NOD, non-obese diabetic.

Article Snippet: To investigate the role of NF-κB signaling in lacrimal gland injury, 20 male NOD/Ltj mice (12 weeks old, weighing 24–28 g) were obtained from Beijing Huafukang Biotechnology Co., Ltd. [Experimental Animal Production License No. SCXK(Jing)2019-0008] and 16 age-matched male Institute of Cancer Research (ICR) mice (weighing 35–40 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. [Experimental Animal Production License No. SCKK(HU)2022-0004].

Techniques: Activation Assay, Western Blot, Expressing, Control, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: NF-κB signaling drives Th17-mediated lacrimal gland injury and tear dysfunction in a murine model of primary Sjögren's syndrome

doi: 10.3892/mmr.2026.13904

Figure Lengend Snippet: JSH-23 modulates CARD14 and CCL19 expression in lacrimal glands of NOD/Ltj mice. (A) Representative immunohistochemical staining of CARD14 and CCL19 in lacrimal gland sections from ICR, NOD/Ltj and NOD/Ltj + JSH-23 groups (magnification, ×200 and ×400). (B) Quantitative analysis of CARD14 and CCL19 staining intensity. The y-axis represents MOD. Data are presented as mean ± SD (n=4). *P<0.05 vs. NOD/Ltj group; ns vs. ICR control. ns, not significant; MOD, mean optical density; SD, standard deviation; ICR, Institute of Cancer Research; NOD, non-obese diabetic.

Article Snippet: To investigate the role of NF-κB signaling in lacrimal gland injury, 20 male NOD/Ltj mice (12 weeks old, weighing 24–28 g) were obtained from Beijing Huafukang Biotechnology Co., Ltd. [Experimental Animal Production License No. SCXK(Jing)2019-0008] and 16 age-matched male Institute of Cancer Research (ICR) mice (weighing 35–40 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. [Experimental Animal Production License No. SCKK(HU)2022-0004].

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: NF-κB signaling drives Th17-mediated lacrimal gland injury and tear dysfunction in a murine model of primary Sjögren's syndrome

doi: 10.3892/mmr.2026.13904

Figure Lengend Snippet: JSH-23 reduces systemic Th17 cell frequency and inflammatory cytokine production in NOD/Ltj mice. (A) Representative flow cytometry plots presenting CD4+ and CD4+IL-17A+ cells in peripheral blood mononuclear cells from ICR, NOD/Ltj and NOD/Ltj + JSH-23 groups. (B) Quantitative analysis of the proportion of CD4+IL-17A+ cells within the CD4+ T cell population. (C) Serum levels of IFN-γ, IL-17A and TNF-α measured by enzyme-linked immunosorbent assay. Data are presented as mean ± SD (n=6). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. NOD/Ltj group; ns, not significant vs. ICR control. SD, standard deviation; Th17, T helper 17; NOD, non-obese diabetic; ICR, Institute of Cancer Research; IL-17A, interleukin-17A; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α.

Article Snippet: To investigate the role of NF-κB signaling in lacrimal gland injury, 20 male NOD/Ltj mice (12 weeks old, weighing 24–28 g) were obtained from Beijing Huafukang Biotechnology Co., Ltd. [Experimental Animal Production License No. SCXK(Jing)2019-0008] and 16 age-matched male Institute of Cancer Research (ICR) mice (weighing 35–40 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. [Experimental Animal Production License No. SCKK(HU)2022-0004].

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: NF-κB signaling drives Th17-mediated lacrimal gland injury and tear dysfunction in a murine model of primary Sjögren's syndrome

doi: 10.3892/mmr.2026.13904

Figure Lengend Snippet: JSH-23 reduces local Th17 cell infiltration in lacrimal glands of NOD/Ltj mice. (A) Representative immunohistochemical staining of CD4+ and IL-17A+ cells in lacrimal glands (magnification, ×200 and ×400). (B) Quantitative analysis of CD4+ and IL-17A+ cell infiltration based on mean optical density (MOD). (C) Representative immunofluorescence staining of CD4+ T cells and IL-17A in lacrimal gland sections from ICR, NOD/Ltj and NOD/Ltj + JSH-23 groups (scale bars, 20 µm). (D) Quantitative analysis of CD4 + and IL-17A + fluorescence intensity. Data are presented as mean ± SD (n=4 per group). *P<0.05 and **P<0.01 vs. NOD/Ltj group; ns, not significant vs. ICR control. MOD, mean optical density; SD, standard deviation; NOD, non-obese diabetic; ICR, Institute of Cancer Research; Th17, T helper 17; IL-17A, interleukin-17A.

Article Snippet: To investigate the role of NF-κB signaling in lacrimal gland injury, 20 male NOD/Ltj mice (12 weeks old, weighing 24–28 g) were obtained from Beijing Huafukang Biotechnology Co., Ltd. [Experimental Animal Production License No. SCXK(Jing)2019-0008] and 16 age-matched male Institute of Cancer Research (ICR) mice (weighing 35–40 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. [Experimental Animal Production License No. SCKK(HU)2022-0004].

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Fluorescence, Control, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: NF-κB signaling drives Th17-mediated lacrimal gland injury and tear dysfunction in a murine model of primary Sjögren's syndrome

doi: 10.3892/mmr.2026.13904

Figure Lengend Snippet: JSH-23 reduces lacrimal gland apoptosis and improves tear secretion in NOD/Ltj mice. (A) Representative TUNEL staining of lacrimal gland sections from ICR, NOD/Ltj and NOD/Ltj + JSH-23 groups (scale bars, 50 and 20 µm). (B) Quantitative analysis of apoptotic cells expressed as percentage (%) of TUNEL + cells compared with total DAPI-stained nuclei. (C) Tear secretion measured by the phenol red thread assay. Data are presented as mean ± SD (n=4 per group). *P<0.05 and **P<0.01 vs. NOD/Ltj group; ns, not significant vs. ICR control. SD, standard deviation; NOD, non-obese diabetic; ICR, Institute of Cancer Research; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: To investigate the role of NF-κB signaling in lacrimal gland injury, 20 male NOD/Ltj mice (12 weeks old, weighing 24–28 g) were obtained from Beijing Huafukang Biotechnology Co., Ltd. [Experimental Animal Production License No. SCXK(Jing)2019-0008] and 16 age-matched male Institute of Cancer Research (ICR) mice (weighing 35–40 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. [Experimental Animal Production License No. SCKK(HU)2022-0004].

Techniques: TUNEL Assay, Staining, Control, 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 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: VH125 SD .NOD Bcl6fl/fl CD4-Cre ± , Backcrossed from transgenic strains gifted by the James W. Thomas and Alexander Dent Laboratories , See also: The Jackson Laboratory, Strains 029288 and 013234.

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: T cell expression of BCL6 supports enhanced insulitis severity, but is dispensable for tertiary lymphoid structure organization in VH125 SD .NOD mice Pancreata were harvested from female, pre-diabetic VH125 SD .NOD and VH125 SD Bcl6 ΔCD4 .NOD mice at 8–12 and 13–16 weeks of age and were formalin fixed, and paraffin embedded. (A–H) About 10 μm pancreas sections were stained with hematoxylin and eosin (H&E) and blind scored, with individual mice plotted. (A and E) Representative H&E-stained sections are shown, with arrows pointing to islets. All islets were blind scored, with average insulitis scores shown for control VH125 SD .NOD mice (black) and VH125 SD Bcl6 ΔCD4 .NOD mice (purple) at (B) 8–12 weeks and (F) 13–16 weeks. The percentage of islets, which had no lymphocytic infiltrate present were calculated for (C) 8–12 weeks and (G) 13–16 weeks. (D and H) The percentage of islets with each score for all pancreata for 8–12 and 13–16 weeks is shown. (I–L) Pancreas sections from the 8–16-week-old cohort that had the highest insulitis infiltrate ( n = 6 mice per group) were obtained and stained with antibodies reactive against CD3 (T cells) or B220 (B cells). (I) Representative images show “disorganized” T cell-B cell infiltration (top), and “organized” TLS (bottom). Islets were scored separately for T cell (CD3) and B cell (B220) infiltrate as follows: 0 (no T/B infiltrate), 1 (>25% infiltrate), 2 (25%–50% infiltrate), 3 (50%–75% infiltrate), and 4 (>75% infiltrate). Average infiltration score for (J) CD3 + and (K) B220+ cells in islets is shown. (L) Infiltrated islets that scored 2 or above were blind scored as organized (blue) or disorganized (red) in both VH125 SD .NOD mice and VH125 SD Bcl6 ΔCD4 .NOD, n = number of islets scored. (A–L) n = 6–8 mice per group, Bars represent mean ± standard deviation, ns, not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, (B, C, F, G, J, and K) Mann-Whitney U test or (L) chi-square test.

Article Snippet: VH125 SD .NOD Bcl6fl/fl CD4-Cre ± , Backcrossed from transgenic strains gifted by the James W. Thomas and Alexander Dent Laboratories , See also: The Jackson Laboratory, Strains 029288 and 013234.

Techniques: Expressing, Staining, Control, Standard Deviation, MANN-WHITNEY

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: VH125 SD .NOD Bcl6fl/fl CD4-Cre ± , Backcrossed from transgenic strains gifted by the James W. Thomas and Alexander Dent Laboratories , See also: The Jackson Laboratory, Strains 029288 and 013234.

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: VH125 SD .NOD Bcl6fl/fl CD4-Cre ± , Backcrossed from transgenic strains gifted by the James W. Thomas and Alexander Dent Laboratories , See also: The Jackson Laboratory, Strains 029288 and 013234.

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: VH125 SD .NOD Bcl6fl/fl CD4-Cre ± , Backcrossed from transgenic strains gifted by the James W. Thomas and Alexander Dent Laboratories , See also: The Jackson Laboratory, Strains 029288 and 013234.

Techniques: Binding Assay, Immunofluorescence, Staining, MANN-WHITNEY, Comparison, 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 T cells promotes formation of a phenotypically defined atypical memory B cell population in the pancreas of VH125 SD .NOD mice Pancreata were harvested from pre-diabetic, 8–12-week-old Bcl6 -sufficient (VH125 SD ) and Bcl6 -deficient (VH125 SD Bcl6 ΔCD4 .NOD) mice ( n = 12–14 mice) and flow cytometry staining was performed using the panel of markers in and (D). B220+ CD19 + CD45 + live singlets were concatenated and normalized via CyCombine and concatenated, with genotype metadata encoded. (A) t-SNE was used to perform dimensionality reduction based on phenotypic marker expression profiles. Clusters were manually defined as indicated. (B) The cluster frequency is shown for each genotype, with individual mice plotted and means indicated. ∗ p < 0.05, Kruskal-Wallis test with post hoc multiple comparison test. Bars represent mean ± standard deviation. (C) A rainbow intensity scale indicates expression levels of the phenotypic markers indicated at the top of each t-SNE plot. Insulin-binding, CD45, B220, CD19, and viability dye markers were omitted from the t-SNE analysis given they were used in parent population gating upstream of t-SNE analysis. (D) Heatmap shows relative expression of the indicated markers for each cluster defined as in (A) using a log-like arcsinh scale and appropriate arcsinh factors.

Article Snippet: VH125 SD .NOD Bcl6fl/fl CD4-Cre ± , Backcrossed from transgenic strains gifted by the James W. Thomas and Alexander Dent Laboratories , See also: The Jackson Laboratory, Strains 029288 and 013234.

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

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: NOD Bcl6 fl/fl , The Jackson Laboratory and Alexander Dent Laboratory , See also: The Jackson Laboratory, Strain 001976 and https://pubmed.ncbi.nlm.nih.gov/39556799/.

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: T cell expression of BCL6 supports enhanced insulitis severity, but is dispensable for tertiary lymphoid structure organization in VH125 SD .NOD mice Pancreata were harvested from female, pre-diabetic VH125 SD .NOD and VH125 SD Bcl6 ΔCD4 .NOD mice at 8–12 and 13–16 weeks of age and were formalin fixed, and paraffin embedded. (A–H) About 10 μm pancreas sections were stained with hematoxylin and eosin (H&E) and blind scored, with individual mice plotted. (A and E) Representative H&E-stained sections are shown, with arrows pointing to islets. All islets were blind scored, with average insulitis scores shown for control VH125 SD .NOD mice (black) and VH125 SD Bcl6 ΔCD4 .NOD mice (purple) at (B) 8–12 weeks and (F) 13–16 weeks. The percentage of islets, which had no lymphocytic infiltrate present were calculated for (C) 8–12 weeks and (G) 13–16 weeks. (D and H) The percentage of islets with each score for all pancreata for 8–12 and 13–16 weeks is shown. (I–L) Pancreas sections from the 8–16-week-old cohort that had the highest insulitis infiltrate ( n = 6 mice per group) were obtained and stained with antibodies reactive against CD3 (T cells) or B220 (B cells). (I) Representative images show “disorganized” T cell-B cell infiltration (top), and “organized” TLS (bottom). Islets were scored separately for T cell (CD3) and B cell (B220) infiltrate as follows: 0 (no T/B infiltrate), 1 (>25% infiltrate), 2 (25%–50% infiltrate), 3 (50%–75% infiltrate), and 4 (>75% infiltrate). Average infiltration score for (J) CD3 + and (K) B220+ cells in islets is shown. (L) Infiltrated islets that scored 2 or above were blind scored as organized (blue) or disorganized (red) in both VH125 SD .NOD mice and VH125 SD Bcl6 ΔCD4 .NOD, n = number of islets scored. (A–L) n = 6–8 mice per group, Bars represent mean ± standard deviation, ns, not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, (B, C, F, G, J, and K) Mann-Whitney U test or (L) chi-square test.

Article Snippet: NOD Bcl6 fl/fl , The Jackson Laboratory and Alexander Dent Laboratory , See also: The Jackson Laboratory, Strain 001976 and https://pubmed.ncbi.nlm.nih.gov/39556799/.

Techniques: Expressing, Staining, Control, Standard Deviation, MANN-WHITNEY

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: NOD Bcl6 fl/fl , The Jackson Laboratory and Alexander Dent Laboratory , See also: The Jackson Laboratory, Strain 001976 and https://pubmed.ncbi.nlm.nih.gov/39556799/.

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: NOD Bcl6 fl/fl , The Jackson Laboratory and Alexander Dent Laboratory , See also: The Jackson Laboratory, Strain 001976 and https://pubmed.ncbi.nlm.nih.gov/39556799/.

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: NOD Bcl6 fl/fl , The Jackson Laboratory and Alexander Dent Laboratory , See also: The Jackson Laboratory, Strain 001976 and https://pubmed.ncbi.nlm.nih.gov/39556799/.

Techniques: Binding Assay, Immunofluorescence, Staining, MANN-WHITNEY, Comparison, 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 T cells promotes formation of a phenotypically defined atypical memory B cell population in the pancreas of VH125 SD .NOD mice Pancreata were harvested from pre-diabetic, 8–12-week-old Bcl6 -sufficient (VH125 SD ) and Bcl6 -deficient (VH125 SD Bcl6 ΔCD4 .NOD) mice ( n = 12–14 mice) and flow cytometry staining was performed using the panel of markers in and (D). B220+ CD19 + CD45 + live singlets were concatenated and normalized via CyCombine and concatenated, with genotype metadata encoded. (A) t-SNE was used to perform dimensionality reduction based on phenotypic marker expression profiles. Clusters were manually defined as indicated. (B) The cluster frequency is shown for each genotype, with individual mice plotted and means indicated. ∗ p < 0.05, Kruskal-Wallis test with post hoc multiple comparison test. Bars represent mean ± standard deviation. (C) A rainbow intensity scale indicates expression levels of the phenotypic markers indicated at the top of each t-SNE plot. Insulin-binding, CD45, B220, CD19, and viability dye markers were omitted from the t-SNE analysis given they were used in parent population gating upstream of t-SNE analysis. (D) Heatmap shows relative expression of the indicated markers for each cluster defined as in (A) using a log-like arcsinh scale and appropriate arcsinh factors.

Article Snippet: NOD Bcl6 fl/fl , The Jackson Laboratory and Alexander Dent Laboratory , See also: The Jackson Laboratory, Strain 001976 and https://pubmed.ncbi.nlm.nih.gov/39556799/.

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

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: Bcl6 fl/fl .C57BL/6/129Sv mice (kindly provided by Dr. Alexander Dent, Indiana University) were backcrossed at least 14 generations to the NOD background and intercrossed with Cd4 -Cre.NOD mice (strain number: 013234; The Jackson Laboratory, Bar Harbor, ME ) to generate Bcl6 fl/fl .Cd4 -Cre +/− .NOD mice, as described in.

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: T cell expression of BCL6 supports enhanced insulitis severity, but is dispensable for tertiary lymphoid structure organization in VH125 SD .NOD mice Pancreata were harvested from female, pre-diabetic VH125 SD .NOD and VH125 SD Bcl6 ΔCD4 .NOD mice at 8–12 and 13–16 weeks of age and were formalin fixed, and paraffin embedded. (A–H) About 10 μm pancreas sections were stained with hematoxylin and eosin (H&E) and blind scored, with individual mice plotted. (A and E) Representative H&E-stained sections are shown, with arrows pointing to islets. All islets were blind scored, with average insulitis scores shown for control VH125 SD .NOD mice (black) and VH125 SD Bcl6 ΔCD4 .NOD mice (purple) at (B) 8–12 weeks and (F) 13–16 weeks. The percentage of islets, which had no lymphocytic infiltrate present were calculated for (C) 8–12 weeks and (G) 13–16 weeks. (D and H) The percentage of islets with each score for all pancreata for 8–12 and 13–16 weeks is shown. (I–L) Pancreas sections from the 8–16-week-old cohort that had the highest insulitis infiltrate ( n = 6 mice per group) were obtained and stained with antibodies reactive against CD3 (T cells) or B220 (B cells). (I) Representative images show “disorganized” T cell-B cell infiltration (top), and “organized” TLS (bottom). Islets were scored separately for T cell (CD3) and B cell (B220) infiltrate as follows: 0 (no T/B infiltrate), 1 (>25% infiltrate), 2 (25%–50% infiltrate), 3 (50%–75% infiltrate), and 4 (>75% infiltrate). Average infiltration score for (J) CD3 + and (K) B220+ cells in islets is shown. (L) Infiltrated islets that scored 2 or above were blind scored as organized (blue) or disorganized (red) in both VH125 SD .NOD mice and VH125 SD Bcl6 ΔCD4 .NOD, n = number of islets scored. (A–L) n = 6–8 mice per group, Bars represent mean ± standard deviation, ns, not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, (B, C, F, G, J, and K) Mann-Whitney U test or (L) chi-square test.

Article Snippet: Bcl6 fl/fl .C57BL/6/129Sv mice (kindly provided by Dr. Alexander Dent, Indiana University) were backcrossed at least 14 generations to the NOD background and intercrossed with Cd4 -Cre.NOD mice (strain number: 013234; The Jackson Laboratory, Bar Harbor, ME ) to generate Bcl6 fl/fl .Cd4 -Cre +/− .NOD mice, as described in.

Techniques: Expressing, Staining, Control, Standard Deviation, MANN-WHITNEY

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: Bcl6 fl/fl .C57BL/6/129Sv mice (kindly provided by Dr. Alexander Dent, Indiana University) were backcrossed at least 14 generations to the NOD background and intercrossed with Cd4 -Cre.NOD mice (strain number: 013234; The Jackson Laboratory, Bar Harbor, ME ) to generate Bcl6 fl/fl .Cd4 -Cre +/− .NOD mice, as described in.

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: Bcl6 fl/fl .C57BL/6/129Sv mice (kindly provided by Dr. Alexander Dent, Indiana University) were backcrossed at least 14 generations to the NOD background and intercrossed with Cd4 -Cre.NOD mice (strain number: 013234; The Jackson Laboratory, Bar Harbor, ME ) to generate Bcl6 fl/fl .Cd4 -Cre +/− .NOD mice, as described in.

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: Bcl6 fl/fl .C57BL/6/129Sv mice (kindly provided by Dr. Alexander Dent, Indiana University) were backcrossed at least 14 generations to the NOD background and intercrossed with Cd4 -Cre.NOD mice (strain number: 013234; The Jackson Laboratory, Bar Harbor, ME ) to generate Bcl6 fl/fl .Cd4 -Cre +/− .NOD mice, as described in.

Techniques: Binding Assay, Immunofluorescence, Staining, MANN-WHITNEY, Comparison, 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 T cells promotes formation of a phenotypically defined atypical memory B cell population in the pancreas of VH125 SD .NOD mice Pancreata were harvested from pre-diabetic, 8–12-week-old Bcl6 -sufficient (VH125 SD ) and Bcl6 -deficient (VH125 SD Bcl6 ΔCD4 .NOD) mice ( n = 12–14 mice) and flow cytometry staining was performed using the panel of markers in and (D). B220+ CD19 + CD45 + live singlets were concatenated and normalized via CyCombine and concatenated, with genotype metadata encoded. (A) t-SNE was used to perform dimensionality reduction based on phenotypic marker expression profiles. Clusters were manually defined as indicated. (B) The cluster frequency is shown for each genotype, with individual mice plotted and means indicated. ∗ p < 0.05, Kruskal-Wallis test with post hoc multiple comparison test. Bars represent mean ± standard deviation. (C) A rainbow intensity scale indicates expression levels of the phenotypic markers indicated at the top of each t-SNE plot. Insulin-binding, CD45, B220, CD19, and viability dye markers were omitted from the t-SNE analysis given they were used in parent population gating upstream of t-SNE analysis. (D) Heatmap shows relative expression of the indicated markers for each cluster defined as in (A) using a log-like arcsinh scale and appropriate arcsinh factors.

Article Snippet: Bcl6 fl/fl .C57BL/6/129Sv mice (kindly provided by Dr. Alexander Dent, Indiana University) were backcrossed at least 14 generations to the NOD background and intercrossed with Cd4 -Cre.NOD mice (strain number: 013234; The Jackson Laboratory, Bar Harbor, ME ) to generate Bcl6 fl/fl .Cd4 -Cre +/− .NOD mice, as described in.

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