Journal: bioRxiv

Article Title: Chronic inflammation drives epididymal tertiary lymphoid structure formation and autoimmune fertility disorders

doi: 10.1101/2024.11.12.623224

Figure Lengend Snippet: A) Immunolabeling of CD19 (red) and CD20 (green) in the IS, caput, and cauda regions of DT-injected WT and Foxp3-DTR mice. B) tSNE and FlowSOM analysis of proximal ( i ) and distal ( ii ) epididymides from WT and Foxp3-DTR mice. C) Flow cytometry gating strategy and absolute count (#) of total B cells (CD3 - B220 + , CD3 - CD19 + , and CD3 - CD20 + cells) per gram of proximal and distal epididymis. D) Absolute count (#) of tissue-resident B cells (CD3 - B220 + , CD3 - CD19 + , and CD3 - CD20 + cells) per gram of proximal and distal epididymis. Flow cytometry gating strategy and relative abundance of total (E) and tissue-resident (F) Plasma B cells (CD3⁻CD19⁺IgD⁻CD138⁺ of live cells) in the proximal and distal epididymides. Flow cytometry gating strategy and relative abundance of total (G) and tissue-resident (H) Germinal center (GC) B cells (CD3⁻B220⁺IgD⁻GL7⁺ of live cells) in the proximal and distal epididymides. Flow cytometry gating strategy and relative abundance of total (I) and tissue-resident (J) Regulatory B cells (Bregs, CD3 - CD19 + CD5 + CD62L - CD1d hi of live cells) in the proximal and distal epididymides. K) Flow cytometry gating strategy and relative abundance of total Follicular Dendritic cells (FDC, CD4 - CD19 - CD21 + of live cells) in the proximal and distal epididymides. Flow cytometry gating strategy and absolute count (#) of total CD8 + T cells (CD4 - CD8 + ) ( L ) and activated CD8 + T cells (CD4 - CD8 + CD44 + ) ( M ) per gram of proximal and distal epididymis. Nuclei are labeled with DAPI (blue). Bars: 20 μm. Data were analyzed using Student’s t-test (C i , D i , F ii , G i , H i , K i ), or Mann-Whitney test ( Cii-vi , Dii-vi , E, F i , G ii , H ii , I, J, K ii , L, M). Data are shown as means ± SEM.

Article Snippet: Primary antibodies utilized were rat anti-mouse-F4/80 , chicken anti-V-ATPase B1 subunit , rabbit anti-AQP9 , goat anti-mouse-IgA (1mg/mL, 62-6720, Invitrogen), goat anti-mouse-IgG1 (1mg/mL, A10551, Invitrogen), goat anti-mouse-IgG2b (1mg/mL, M32407, Invitrogen), goat anti-mouse-IgG2c (0.8mg/mL, 115-035-208, Jackson ImmunoResearch), goat anti-mouse-IgG3 (M32707, Invitrogen), donkey anti-mouse-IgM (0.8mg/mL, 715-035-020, Jackson ImmunoResearch), rabbit anti-mouse-CD19 (0.2mg/mL: sc-8500-R, Santa Cruz), rat anti-mouse-CD21/CD35 (0.5mg/mL, 14-0211-81, Invitrogen), Armenian Hamster anti-mouse- CD3 (0.5mg/mL, 100303, Bioegend), rat anti-human/mouse-AID (0.5mg/mL, 14-5959-80, Invitrogen), rabbit anti-alpha smooth muscle actin (SMA) (0.2mg/mL, ab5694, Abcam), rabbit anti-mouse-CD31 (ab28364, Abcam), rabbit anti-Ki67 (0.031 mg/mL, MA5-14520, Thermofisher Scientific), rat anti-mouse-IgD (0.5mg/mL, 405702, Biolegend), goat anti-mouse-CD20 (0.5mg/mL, sc-7735, Santa Cruz), and donkey anti-human-IgG (1.5mg/mL, 709-545-149, Jackson ImmunoResearch).

Techniques: Immunolabeling, Injection, Flow Cytometry, Labeling, MANN-WHITNEY

Journal: bioRxiv

Article Title: Chronic inflammation drives epididymal tertiary lymphoid structure formation and autoimmune fertility disorders

doi: 10.1101/2024.11.12.623224

Figure Lengend Snippet: A) Immunolabeling of CD19 (red) and CD20 (green) B cells in the IS, caput, corpus, and cauda regions of DT-injected WT and Foxp3-DTR mice. Double-head arrows indicate double positive cells. B) tSNE and FlowSOM analysis of the B cell population of proximal epididymis from Foxp3-DTR mice ( i ) and its relative marker expression heatmap ( ii ). C) Absolute count (#) of local-circulating B cells (CD3 - B220 + ( i ), CD3 - CD19 + ( ii ), and CD3 - CD20 + ( iii ) cells) per gram of proximal and distal epididymis by flow cytometry. D) Relative abundance of local-circulating Plasma B cells (CD3⁻CD19⁺IgD⁻CD138⁺ of live cells) in the proximal and distal epididymides. E) Relative abundance of local-circulating GC B cells (CD3⁻B220⁺IgD⁻GL7⁺ of live cells) in the proximal and distal epididymides. F) Relative abundance of local-circulatory Bregs (CD3 - CD19 + CD5 + CD62L - CD1d hi of live cells) in the proximal and distal epididymides. G) Absolute count (#) of tissue-resident and local-circulating FDC (CD4 - CD19 - CD21 + cells) in the proximal and distal epididymides. Absolute count (#) of tissue-resident and local-circulating CD8 + (CD4 - CD8 + ) ( H ) and CD8 + CD44 + T cells ( I ) per gram of proximal and distal epididymis. Nuclei are labeled with DAPI (blue). Bars: 20 μm. L: Lumen. Data were analyzed using Student’s t-test (E i , G i , iii , iv ) or Mann-Whitney test (C i-iii, D, E ii , F, G ii , H i-iv , I i-iv ). Data are shown as means ± SEM.

Article Snippet: Primary antibodies utilized were rat anti-mouse-F4/80 , chicken anti-V-ATPase B1 subunit , rabbit anti-AQP9 , goat anti-mouse-IgA (1mg/mL, 62-6720, Invitrogen), goat anti-mouse-IgG1 (1mg/mL, A10551, Invitrogen), goat anti-mouse-IgG2b (1mg/mL, M32407, Invitrogen), goat anti-mouse-IgG2c (0.8mg/mL, 115-035-208, Jackson ImmunoResearch), goat anti-mouse-IgG3 (M32707, Invitrogen), donkey anti-mouse-IgM (0.8mg/mL, 715-035-020, Jackson ImmunoResearch), rabbit anti-mouse-CD19 (0.2mg/mL: sc-8500-R, Santa Cruz), rat anti-mouse-CD21/CD35 (0.5mg/mL, 14-0211-81, Invitrogen), Armenian Hamster anti-mouse- CD3 (0.5mg/mL, 100303, Bioegend), rat anti-human/mouse-AID (0.5mg/mL, 14-5959-80, Invitrogen), rabbit anti-alpha smooth muscle actin (SMA) (0.2mg/mL, ab5694, Abcam), rabbit anti-mouse-CD31 (ab28364, Abcam), rabbit anti-Ki67 (0.031 mg/mL, MA5-14520, Thermofisher Scientific), rat anti-mouse-IgD (0.5mg/mL, 405702, Biolegend), goat anti-mouse-CD20 (0.5mg/mL, sc-7735, Santa Cruz), and donkey anti-human-IgG (1.5mg/mL, 709-545-149, Jackson ImmunoResearch).

Techniques: Immunolabeling, Injection, Marker, Expressing, Flow Cytometry, Labeling, MANN-WHITNEY

Journal: bioRxiv

Article Title: Chronic inflammation drives epididymal tertiary lymphoid structure formation and autoimmune fertility disorders

doi: 10.1101/2024.11.12.623224

Figure Lengend Snippet: tSNE and FlowSOM pan-populations analysis of testis from WT and Foxp3-DTR mice ( A ) and of the B cell populations of testis from Foxp3- DTR mice (B i ) and its relative marker expression heatmap (B ii ). Flow cytometry gating strategy and absolute count (#) of total ( C ), resident ( D ), and local circulating ( E ) B cells (CD3 - B220 + , CD3 - CD19 + , and CD3 - CD20 + ) per gram of testis. Flow cytometry gating strategy and relative abundance of total ( F ), resident ( G ), and local circulating ( H ) Plasma B cells (CD3⁻CD19⁺IgD⁻CD138⁺ of live cells) in the testes. Flow cytometry gating strategy and relative abundance of total ( I ), resident ( J ), and local circulating ( K ) GC B cells (CD3⁻B220⁺IgD⁻GL7⁺ of live cells) in the testes. Flow cytometry gating strategy and relative abundance of total ( L ), resident ( M ), and local circulating ( N ) Bregs (CD3-CD19+CD5+CD62L-CD1d hi of live cells) in the testes. Flow cytometry gating strategy and absolute count (#) of total ( O ), resident ( P ), and local circulating ( Q ) FDC (CD4 - CD19 - CD21 + cells) in the testes. Flow cytometry gating strategy and absolute count (#) of total ( R ), resident ( S ), and local circulating ( T ) CD8 + T cells (CD4 - CD8 + ) per gram of testis. Flow cytometry gating strategy and absolute count (#) of total ( U ), resident ( V ), and local circulating ( W ) activated CD8 + T cells (CD4 - CD8 + CD44 + ) per gram of testis. Data were analyzed using Student’s t-test (C i , ii , D i-iii , E ii , F, G, I, J, N, P, Q) or the Mann-Whitney test (C iii , E i , iii , H, K-M, O, R-W). Data are shown as means ± SEM.

Article Snippet: Primary antibodies utilized were rat anti-mouse-F4/80 , chicken anti-V-ATPase B1 subunit , rabbit anti-AQP9 , goat anti-mouse-IgA (1mg/mL, 62-6720, Invitrogen), goat anti-mouse-IgG1 (1mg/mL, A10551, Invitrogen), goat anti-mouse-IgG2b (1mg/mL, M32407, Invitrogen), goat anti-mouse-IgG2c (0.8mg/mL, 115-035-208, Jackson ImmunoResearch), goat anti-mouse-IgG3 (M32707, Invitrogen), donkey anti-mouse-IgM (0.8mg/mL, 715-035-020, Jackson ImmunoResearch), rabbit anti-mouse-CD19 (0.2mg/mL: sc-8500-R, Santa Cruz), rat anti-mouse-CD21/CD35 (0.5mg/mL, 14-0211-81, Invitrogen), Armenian Hamster anti-mouse- CD3 (0.5mg/mL, 100303, Bioegend), rat anti-human/mouse-AID (0.5mg/mL, 14-5959-80, Invitrogen), rabbit anti-alpha smooth muscle actin (SMA) (0.2mg/mL, ab5694, Abcam), rabbit anti-mouse-CD31 (ab28364, Abcam), rabbit anti-Ki67 (0.031 mg/mL, MA5-14520, Thermofisher Scientific), rat anti-mouse-IgD (0.5mg/mL, 405702, Biolegend), goat anti-mouse-CD20 (0.5mg/mL, sc-7735, Santa Cruz), and donkey anti-human-IgG (1.5mg/mL, 709-545-149, Jackson ImmunoResearch).

Techniques: Marker, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Chronic inflammation drives epididymal tertiary lymphoid structure formation and autoimmune fertility disorders

doi: 10.1101/2024.11.12.623224

Figure Lengend Snippet: A) Diagram of Treg depletion protocol, in vivo staining of CD45 + circulatory cells, and subsequent analysis 8 weeks after DT treatment. B) Flow cytometry gating strategy and absolute count (#) of total CD45 + cells per gram of proximal and distal epididymis. tSNE and FlowSOM analysis of proximal ( C ) and distal ( D ) epididymides from WT and Foxp3-DTR mice. E) Flow cytometry gating strategy ( i ) and absolute count (#) of total Tregs (CD4 + Foxp3 + ) ( ii ) and T CD4 + cells (CD4 + Foxp3 - ) ( iii ) per gram of proximal and distal epididymis. F) Confocal microscopy of distal regions of Foxp3-DTR-eGFP mice. Notice the Treg Foxp3 + (green, arrows) cell accumulation in the interstitium of the DT-treated Foxp3-DTR mice. G) Flow cytometry gating strategy and relative abundance of T follicular helper cells (Tfh, CD4 + CD19 - CD8 - CXCR5 + of live cells) in the proximal and distal epididymides. H) Flow cytometry gating strategy and absolute count (#) of total follicular dendritic cells (FDC, CD4 - CD19 - CD21 + live cells) per gram of proximal and distal epididymides. I) Flow cytometry gating strategy ( i ), absolute count (#) of total CD8 + T cells (CD4 - CD8 + ) ( ii ) and activated CD8 + T cells (CD4 - CD8 + CD44 + ) ( iii ) per gram of proximal and distal epididymis. Nuclei are labeled with DAPI (blue). L: Lumen. Bars: 20 μm. Data were analyzed using the Mann-Whitney test. Data are shown as means ± SEM.

Article Snippet: Primary antibodies utilized were rat anti-mouse-F4/80 , chicken anti-V-ATPase B1 subunit , rabbit anti-AQP9 , goat anti-mouse-IgA (1mg/mL, 62-6720, Invitrogen), goat anti-mouse-IgG1 (1mg/mL, A10551, Invitrogen), goat anti-mouse-IgG2b (1mg/mL, M32407, Invitrogen), goat anti-mouse-IgG2c (0.8mg/mL, 115-035-208, Jackson ImmunoResearch), goat anti-mouse-IgG3 (M32707, Invitrogen), donkey anti-mouse-IgM (0.8mg/mL, 715-035-020, Jackson ImmunoResearch), rabbit anti-mouse-CD19 (0.2mg/mL: sc-8500-R, Santa Cruz), rat anti-mouse-CD21/CD35 (0.5mg/mL, 14-0211-81, Invitrogen), Armenian Hamster anti-mouse- CD3 (0.5mg/mL, 100303, Bioegend), rat anti-human/mouse-AID (0.5mg/mL, 14-5959-80, Invitrogen), rabbit anti-alpha smooth muscle actin (SMA) (0.2mg/mL, ab5694, Abcam), rabbit anti-mouse-CD31 (ab28364, Abcam), rabbit anti-Ki67 (0.031 mg/mL, MA5-14520, Thermofisher Scientific), rat anti-mouse-IgD (0.5mg/mL, 405702, Biolegend), goat anti-mouse-CD20 (0.5mg/mL, sc-7735, Santa Cruz), and donkey anti-human-IgG (1.5mg/mL, 709-545-149, Jackson ImmunoResearch).

Techniques: In Vivo, Staining, Flow Cytometry, Confocal Microscopy, Labeling, MANN-WHITNEY

Journal: bioRxiv

Article Title: Chronic inflammation drives epididymal tertiary lymphoid structure formation and autoimmune fertility disorders

doi: 10.1101/2024.11.12.623224

Figure Lengend Snippet: A) tSNE and FlowSOM analysis of distal epididymides from Foxp3-DTR mice. B) Flow cytometry gating strategy ( i ) and absolute count (#) of total ( ii ), tissue-resident ( iii ), and local-circulating ( iv ) B cells (CD3 - B220 + cells) per gram of distal epididymis. C) Flow cytometry gating strategy ( i ) and relative abundance of total ( ii ), tissue-resident ( iii ), and local-circulating ( iv ) plasma B cells (CD3⁻CD19⁺IgD⁻CD138⁺ of live cells) in the distal epididymides. D) Flow cytometry gating strategy ( i ) and relative abundance of total ( ii ), tissue-resident ( iii ), and local-circulating ( iv ) Germinal center (GC) B cells (CD3⁻B220⁺IgD⁻GL7⁺ of live cells) in the distal epididymides. E) Flow cytometry gating strategy ( i ) and relative abundance of total ( ii, iii ), tissue-resident ( iv, v ), and local-circulating ( vi, vii ) regulatory B cells (B regs, CD3- CD19 + CD5 + CD62L⁻CD1d hi of live cells) in the proximal and distal epididymides. F) Flow cytometry gating strategy ( i ) and relative abundance of total ( ii, iii ), tissue-resident ( iv, v ), and local-circulating ( vi, vii ) memory B cells (memB cells, CD3 - CD19 + CD27 + ) in the proximal and distal epididymides. G) Immunolabeling of CD19 (red), CD20 (green), and CD21 (magenta, arrows) in the distal epididymal region of DT-injected Foxp3-DTR mice. H) Immunolabeling of IgD (red), CD20 (green), and CD3 (magenta, arrows) in the distal epididymal region of DT-injected Foxp3-DTR mice. I) Flow cytometry gating strategy ( i ) and relative abundance of total ( ii ), tissue-resident ( iii ), and local-circulating ( iv ) IgD + CD20 + B cells (CD3⁻CD20⁺IgD⁺ of CD20 + cells) in the distal epididymides. J) Immunolabeling of CD20 (green) and CD3 (magenta) in the distal epididymal region of DT-injected Foxp3-DTR mice. K) Immunolabeling of CD19 (green) and AID (red) in the distal epididymal region of DT-injected Foxp3-DTR mice. L) Immunolabeling of CD19 (green) and Ki-67 (red) in the distal epididymal region of DT-injected Foxp3-DTR mice. M) Immunolabeling of CD20 (green) and CD31 (red) in the corpus and cauda epididymal region of DT-injected Foxp3-DTR mice. N) Immunolabeling of CD20 (green) and a-SMA (red) in the distal epididymal region of DT-injected Foxp3-DTR mice. O) Venn diagram of the soluble mediators measured in the proximal and distal epididymides at 2 and 8 weeks after DT treatment. Molecules were grouped by general functions or inflammatory abilities such as TLS formation ( & , cell recruitment, tissue remodeling, angiogenesis capacity) and pro- (*) and anti-inflammatory ( # ) mediators. See also Supp. Fig. 18. Nuclei are labeled with DAPI (blue). L: Lumen. Bars: 20 μm. Data were analyzed using Student’s t-test (E vi , F iv, I iv ), or Mann-Whitney test (B ii-iv , C ii-iv , D ii-iv , E ii-v, vii , F ii-iii,v-vii, I ii, iii ). Data are shown as means ± SEM.

Article Snippet: Primary antibodies utilized were rat anti-mouse-F4/80 , chicken anti-V-ATPase B1 subunit , rabbit anti-AQP9 , goat anti-mouse-IgA (1mg/mL, 62-6720, Invitrogen), goat anti-mouse-IgG1 (1mg/mL, A10551, Invitrogen), goat anti-mouse-IgG2b (1mg/mL, M32407, Invitrogen), goat anti-mouse-IgG2c (0.8mg/mL, 115-035-208, Jackson ImmunoResearch), goat anti-mouse-IgG3 (M32707, Invitrogen), donkey anti-mouse-IgM (0.8mg/mL, 715-035-020, Jackson ImmunoResearch), rabbit anti-mouse-CD19 (0.2mg/mL: sc-8500-R, Santa Cruz), rat anti-mouse-CD21/CD35 (0.5mg/mL, 14-0211-81, Invitrogen), Armenian Hamster anti-mouse- CD3 (0.5mg/mL, 100303, Bioegend), rat anti-human/mouse-AID (0.5mg/mL, 14-5959-80, Invitrogen), rabbit anti-alpha smooth muscle actin (SMA) (0.2mg/mL, ab5694, Abcam), rabbit anti-mouse-CD31 (ab28364, Abcam), rabbit anti-Ki67 (0.031 mg/mL, MA5-14520, Thermofisher Scientific), rat anti-mouse-IgD (0.5mg/mL, 405702, Biolegend), goat anti-mouse-CD20 (0.5mg/mL, sc-7735, Santa Cruz), and donkey anti-human-IgG (1.5mg/mL, 709-545-149, Jackson ImmunoResearch).

Techniques: Flow Cytometry, Immunolabeling, Injection, Labeling, MANN-WHITNEY

Journal: iScience

Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells

doi: 10.1016/j.isci.2024.110388

Figure Lengend Snippet: Ablation of PTEN in mature B cells led to early death in mice (A) FACS profiles of B220 versus CD5 in gated lymphocytes (left), and CD21 versus CD23 in CD5 + -gated B cells (right), from the spleens of CD23-control and CD23-cKO mice at 8–12 weeks of age. (B) Histograms showing the percentages of IM, FO, MZ, and B1a B cells in spleens from mice in (A) ( n = 3 male plus 2 female mice/group). (C) Representative images of spleens from the mice in (A). (D) Cell counts of total splenocytes (left) and splenic B cells (right) from the mice in (A). (E) Kaplan-Meier curves of female CD23-cKO ( n = 20) and female CD23-control ( n = 15) mice. (F) Representative images of spleens, mesenteric lymph nodes (MLNs), and axillary lymph nodes (ALNs) resected from the indicated mice at 34 weeks. (G) Cell counts of splenocytes (left) and splenic B cells (right) in the indicated mice ( n = 3–6 male mice/group) at 8 weeks or >16 weeks. (H) Levels of total IgM, total IgG1, and anti-nuclear and anti-dsDNA antibodies in the serum of the indicated mice ( n = 3–12 male mice/group) at 8–12 weeks or >16 weeks determined by ELISA. The samples were compared using unpaired two-tailed t test; ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005, and data are presented as mean ± SEM.

Article Snippet: PE Rat Anti-Mouse CD21/CD35 (clone 7G6) , BD Biosciences , Cat#552957; RRID: AB_394532.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells

doi: 10.1016/j.isci.2024.110388

Figure Lengend Snippet: BCR signaling and TLR9-mediated NF-κB activation were impaired in B2 B cells from mb1-cKO B cells (A) FACS profiles of CD21 versus CD23 from B220 + -gated splenocytes to show the gating of IM, FO, and MZ B cells (left) and FACS profiles of CD19 versus IgM to show IgM expression in those subsets (right) of indicated mice. Data were from 3 independent experiments. (B) Histograms to show the frequencies of IgM-expressing FO and MZ B cells as described in (A). Each group containing 3 male plus 3 female mice. (C and D) Curves to show the kinetic induction of BCR-mediated BLNK-phosphorylation (p-BLNK) on Y84 and p-Btk on Y223 in the resting state (0), or upon anti-IgM-stimulation for 1, 5, 15, and 30 min, respectively, from the indicated mice. Each group containing 3 male mice. (E) Overlaid curves showing p-p65 S536 signals by intracellular staining in splenic B cells from the indicated mice, which were left unstimulated (0) or stimulated with CpG for 1, 5, 15, and 30 min, respectively. Each group containing 3 female mice. The samples were compared using an unpaired two-tailed t test; ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005, and the data are presented mean ± SEM.

Article Snippet: PE Rat Anti-Mouse CD21/CD35 (clone 7G6) , BD Biosciences , Cat#552957; RRID: AB_394532.

Techniques: Activation Assay, Expressing, Staining, Two Tailed Test

Journal: iScience

Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells

doi: 10.1016/j.isci.2024.110388

Figure Lengend Snippet:

Article Snippet: PE Rat Anti-Mouse CD21/CD35 (clone 7G6) , BD Biosciences , Cat#552957; RRID: AB_394532.

Techniques: Recombinant, Western Blot, Staining, In Situ, Antibody Labeling, Enzyme-linked Immunosorbent Assay, Software