Journal: Mucosal immunology

Article Title: Metabolic fitness of IgA + plasma cells in the gut requires DOCK8

doi: 10.1016/j.mucimm.2023.12.001

Figure Lengend Snippet: ELISA quantification of (A) total stool IgA, IgG, and IgM in WT ( Dock8 +/+ ; closed circles), Dock8 heterozygous (Dock8 +/− ; half-filled circles), and Dock8 knockout ( Dock8 −/− ; closed squares) cohoused littermates at steady state. (B) Principal coordinate analysis (PCoA) and (C) cladogram of weighted UniFrac distances of total (presort), sorted IgA + and IgA − fecal bacteria from Dock8 +/+ , Dock8 +/− , or Dock8 −/− mice. Scale range: 0.02. (D) Frequency of IgA binding of segmented-filamentous bacteria (SFB) isolated from fecal pellets of SFB mono-colonized mice and then incubated with fecal supernatant from Dock8 +/− or Dock8 −/− cohoused littermates. Dotted line indicates frequency of IgA binding of SFB incubated with fecal supernatant from WT mice raised in SFB-free facility. ELISA quantification of (E) stool peanut-specific IgA and (F) stool cholera toxin (CT)-specific IgA in Dock8 +/+ , Dock8 +/− , and Dock8 −/− cohoused littermates one week after the 6 th weekly intragastric (i.g.) immunization with peanut and CT. Dotted lines indicate detection limit of 1.56 arbitrary units. (A, E-F) Data are representative of 3 independent experiments with 4–10 Dock8 +/+ , 4–10 Dock8 +/− , and 4–20 Dock8 −/− mice. (B-C) Data are representative of two independent experiments with 5–10 mice per group. (D) Pooled data from 2 independent experiments with 5–8 mice per group. Statistical tests: (A) Kruskal-Wallis analysis and Dunn’s post-hoc (D-F) Mann-Whitney U test, whereby * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant, and nd = not detectable, respectively. Error bars indicate SD; each symbol represents an individual mouse.

Article Snippet: After blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 1 hour at room temperature, the membrane was incubated with anti-mouse DOCK8 antibody (Takara) at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Knock-Out, Bacteria, Binding Assay, Isolation, Incubation, MANN-WHITNEY

Journal: Mucosal immunology

Article Title: Metabolic fitness of IgA + plasma cells in the gut requires DOCK8

doi: 10.1016/j.mucimm.2023.12.001

Figure Lengend Snippet: (A) Quantification of intestinal fluid accumulation (B) representative images of fluid accumulation in cecum and quantification of (C) small intestine weight or (D) cecum weight of mice 12 hours after oral CT challenge in naïve Dock8 +/− mice (CT 1° imm -) or CT-immunized (CT 1° imm +) Dock8 +/− and Dock8 −/− cohoused littermates. CT-immunized mice received 2 weekly CT i.g. and were challenged 10–14 days later. Statistical tests: (A, C-D) Kruskal-Wallis analysis with Dunn’s posthoc test, whereby * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant. Error bars indicate SD. (A-D) Pooled data from 2 independent experiments with 3–4 mice per group.

Article Snippet: After blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 1 hour at room temperature, the membrane was incubated with anti-mouse DOCK8 antibody (Takara) at 4 °C overnight.

Techniques:

Journal: Mucosal immunology

Article Title: Metabolic fitness of IgA + plasma cells in the gut requires DOCK8

doi: 10.1016/j.mucimm.2023.12.001

Figure Lengend Snippet: ELISA quantification of stool peanut-specific IgA in mice immunized orally with peanut and CT: (A) cohoused Ctrl ( Dock8 flox/flox ) and T-Dock8 −/− ( Cd4 cre Dock8 flox/flox ) littermates (B) cohoused irradiated mice reconstituted with 1:1 mixture of B cell-deficient muMt ( Ighm −/− ) and wild-type (WT) or Dock8 -deficient ( Dock8 −/− ) bone marrow cells, or (C) cohoused Ctrl ( Dock8 flox/flox ) and B-Dock8 −/− ( Cd23 cre Dock8 flox/flox ) littermates. (D) ELISA quantification of stool CT-specific IgA in peanut/CT immunized cohoused Ctrl ( Dock8 flox/flox ) and B-Dock8 −/− ( Cd23 cre Dock8 flox/flox ) littermates. Peanut and CT-specific IgA are measured one week after the 6 th weekly i.g. immunizations with peanut and CT. (E) ELISA quantification of total stool IgA from immunized cohoused Ctrl and B-Dock8 −/− littermates. (F) Representative images and enumeration of IgA-secreting cells (ASCs) by ELISpot in the small intestine LP (SiLP) from Ctrl and B-Dock8 −/− cohoused littermates. (G) ELISA quantification of stool NP15-specific, NP7-specific, and ratio of NP7/NP15 IgA in cohoused Ctrl ( Dock8 flox/flox ) and B-Dock8 −/− littermates one week after the 6 th weekly i.g. immunizations with NP19-OVA and CT. Dotted lines indicate the detection limit of the CT IgA, peanut IgA, and NP7 and NP15 IgA assay at 1.56 arbitrary units relative to standard. Statistical tests: (A-G) Mann-Whitney U test, whereby * P < 0.05, ** P < 0.01, *** P < 0.001, **** < 0.000, ns = not significant. Error bars indicate SD. (A-G) Pooled data from 2–3 independent experiments with 3–5 mice per group. Lamina propria preparation with low number of viable cells is excluded from analyses in (F).

Article Snippet: After blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 1 hour at room temperature, the membrane was incubated with anti-mouse DOCK8 antibody (Takara) at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation, Enzyme-linked Immunospot, IgA Assay, MANN-WHITNEY

Journal: Mucosal immunology

Article Title: Metabolic fitness of IgA + plasma cells in the gut requires DOCK8

doi: 10.1016/j.mucimm.2023.12.001

Figure Lengend Snippet: Representative immunofluorescent images depicting localization of TCRβ + (white), IgD + (green), IgA + (red), or CD11c + (blue) cells in (A) PP or (B) MLN at steady state in Ctrl and B-Dock8 −/− cohoused littermates. GC = germinal centers, SED = subepithelial dome, TCZ = T cell zone, and dotted lines demarcates BCZ = B cell zone. Scale bars, 100 μm for 10x images and 4x images. (C-D) Representative flow cytometry plots and frequency of surface CCR9 + and α β + cells among live IgA + B cells in (C) PP and (D) MLN in cohoused Ctrl ( Dock8 flox/flox ) and B-Dock8 −/− littermates. (E) Transwell migration assay of Ctrl and Dock8 -deficient IgA + CCR9 + B cells towards CCL25. (F) Quantification of migration distance of Ctrl and Dock8 -deficient in vitro differentiated IgA + B cells within 3D collagen gel towards CCL25. Statistical tests: (C-D) Mann-Whitney U test or (E-F) Paired t test; ns = not significant. Error bars indicate SD. (A) Representative images from 3 independent experiments with 3 mice per group. (C-D) Representative data from 2 independent experiments with 3–5 mice per group. (E-F) Combined data from 2 independent experiments.

Article Snippet: After blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 1 hour at room temperature, the membrane was incubated with anti-mouse DOCK8 antibody (Takara) at 4 °C overnight.

Techniques: Flow Cytometry, Transwell Migration Assay, Migration, In Vitro, MANN-WHITNEY

Journal: Mucosal immunology

Article Title: Metabolic fitness of IgA + plasma cells in the gut requires DOCK8

doi: 10.1016/j.mucimm.2023.12.001

Figure Lengend Snippet: (A) Representative images with quantitation of CT-specific and peanut-specific IgA antibody-secreting cells (ASCs) by ELISpot in the small intestine lamina propria (LP). (B-D) Numbers of peanut-specific IgA ASCs in the (B) MLN and (C) PP (D) BM from Ctrl and B-Dock8 −/− cohoused littermates. (E) Frequency of NP-specific IgA + CD138 + PCs in LP pre-gated on CD19 − Lin − (NK1.1 − CD3 − TCRβ − F4/80 − IgD − ) cells. (F-G) Frequency of NP-specific IgA + CD138 + PCs pre-gated on live cells in (F) MLN and (G) PP of Ctrl and B-Dock8 −/− cohoused littermates. All tissues were analyzed 8 days after 6 th weekly i.g. immunizations with (A-D) peanut+CT or (E-G) NP19-OVA+CT. Statistical tests: Mann-Whitney U test, whereby * P < 0.05, ** P < 0.01, and ns = not significant. Error bars indicate SD. (A-B) Pooled data from 3 independent experiments with 3–4 mice per group. (C-D) Pooled data from 2 independent experiments with 2–3 mice per group. (E-G) Pooled data from 3 independent experiments with 3–5 mice per group. Lamina propria preparation with low number of viable cells is excluded from analyses in (E). Dotted lines represent baseline signal of CT-specific or peanut-specific IgA ASC in naïve mice.

Article Snippet: After blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 1 hour at room temperature, the membrane was incubated with anti-mouse DOCK8 antibody (Takara) at 4 °C overnight.

Techniques: Quantitation Assay, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: Mucosal immunology

Article Title: Metabolic fitness of IgA + plasma cells in the gut requires DOCK8

doi: 10.1016/j.mucimm.2023.12.001

Figure Lengend Snippet: (A) IgA + B cells were generated from PPs of DOCK8-TurboID mice as in . Heatmap of enriched proteins proximal to DOCK8 from proteomics analysis of biotinylated targets. Selected proteins were grouped into functional classes based on gene ontology and IPA analysis. (B) Proximity ligation assay (PLA) showing protein-protein interactions between Pyruvate kinase M1/2 (PKM) and DOCK8 in B cells from IgA + B cell cultures. Representative images from PLA experiments of wild-type (WT DOCK8) and DOCK8-TurboID (DOCK8-HA) cells (left) and quantification of the number of PLA spots per cell (right). Data were pooled data from 2 independent experiments and numbers in parentheses indicate the number of individual nuclei scored for each genotype. Error bars indicate SEM. (C) Representative plots of oxygen consumption rate (OCR) and glycolysis proton efflux rate (glycoPER) of in vitro generated IgA + B cells from Ctrl and Dock8 -deficient mice. Data are representative of 2 independent experiments.

Article Snippet: After blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 1 hour at room temperature, the membrane was incubated with anti-mouse DOCK8 antibody (Takara) at 4 °C overnight.

Techniques: Generated, Functional Assay, Proximity Ligation Assay, Protein-Protein interactions, In Vitro