Journal: bioRxiv

Article Title: Pathogenic IgE-fated B cell memory retains functional plasticity

doi: 10.1101/2023.11.28.567094

Figure Lengend Snippet: (A) Expression level of Ighc genes downstream of Ighg1 from scRNA-seq analysis of B cells isolated from spleens and inguinal LNs of CT-immunized mice and A4RA/IC-treated epicutaneously sensitized mice. Gene expression is plotted for cells within the MBC cluster . (B) VDJ analysis of MBC cluster. Plot depicts the assigned Ighc gene in MBCs with ≥3 nucleotide mutations compared to the germline sequence. (C) Representative contour plots of IgG1 + and IgG2c + populations from inguinal LNs of IC/A4RA-treated epicutaneously sensitized mice four weeks post-boost and antibody treatment. (D) IC/A4RA-treated mice were rested up to 15 weeks after re-exposures. IgG1 + (left) and IgG2c + (right) MBC frequency are plotted at four and 15 weeks post-boost. Pre-gated as OVA + B cells > CD38 + GL7 - > IgM - IgD - . (E) Epicutaneously sensitized mice were treated with A4RA, AIFNG, or both at the time of subcutaneous re-exposure. IgG1 + (left) and IgG2c + (right) MBC frequency are plotted at four weeks post-boost. Pre-gated as OVA + B cells > CD38 + GL7 - > IgM - IgD - . (F) OVA-specific serum immunoglobulin profiles of IC/A4RA-treated mice at zero to 15 weeks post-boost. (G) IC/A4RA-treated mice were rested for nine weeks to allow for clearance of OVA-specific IgG2c and clearance of IC/A4RA blocking antibodies. Subsequently, mice received tertiary OVA exposures and serum was collected at week 12. OVA-specific serum immunoglobulin profiles are depicted before and after tertiary exposures. (C-G) Data represents 2 experiments with at least 4 mice per group. Data are presented as mean ± SEM (F, G) or median ± min/max (D, E). * p < 0.05, ** p < 0.01, *** p < 0.001. Seq, sequences; swIg, class-switched; AIFNG, anti-IFN-ψR1; OD, optical density.

Article Snippet: The following anti-mouse fluorochrome-conjugated antibodies were used: B220-AF700 (RA3-6B2), CD138-PE/Dazzle 594 (281– ), CD3-BV711 (17A2), CD3-BV510 (17A2), F4/80-BV711 (BM8), F4/80-BV510 (BM8), CD38-PE/Cy7 (90), GL7-PerCP.Cy5.5 (GL7), GL7-BV421 (GL7; BD, Franklin Lakes, NJ), IgD-Pacific Blue (11-26c.2a), IgD-BV605 (11-26c.2a), IgM-BV786 (II/41; BD), IgM-FITC (II/41), IgG1-BV650 (RMG1-1), IgG2b-PECy7 (RMG2b-1), IgG2c-FITC (polyclonal; SouthernBiotech, Birmingham, AL), IL-4Rα-PE (REA235; Miltenyi Biotec), and CD23-BV421 (B3B4).

Techniques: Expressing, Isolation, Gene Expression, Sequencing, Blocking Assay

Journal: bioRxiv

Article Title: Pathogenic IgE-fated B cell memory retains functional plasticity

doi: 10.1101/2023.11.28.567094

Figure Lengend Snippet: (A) Adoptive transfer schematic. (B) WT mice were immunized with OVA + Alum, CT, or CpG. Representative contour plots class-switched OVA + MBCs. Pre-gated as OVA + B cells > CD38 + GL7 - > IgM - IgD - . Numbers indicate the proportion of IgG1 + , IgG2b + , and IgG2c + cells from the parent class-switched MBC population. (C) OVA-enriched B cells were adoptively transferred from WT OVA-Alum donor mice to naïve muMT mice and recipient were boosted with either OVA + CT or OVA + CpG. Summary plots show OVA + IgG1 + and OVA + IgG2c + B cell frequency in recipient mice one week post-transfer and boost. (D) OVA-specific serum immunoglobulin profiles in recipient mice one week post-transfer and boost. Data represents 2 experiments, each with 2-4 mice per group. Data are presented as median ± min/max (B) or mean ± SEM (D). * p < 0.05.

Article Snippet: The following anti-mouse fluorochrome-conjugated antibodies were used: B220-AF700 (RA3-6B2), CD138-PE/Dazzle 594 (281– ), CD3-BV711 (17A2), CD3-BV510 (17A2), F4/80-BV711 (BM8), F4/80-BV510 (BM8), CD38-PE/Cy7 (90), GL7-PerCP.Cy5.5 (GL7), GL7-BV421 (GL7; BD, Franklin Lakes, NJ), IgD-Pacific Blue (11-26c.2a), IgD-BV605 (11-26c.2a), IgM-BV786 (II/41; BD), IgM-FITC (II/41), IgG1-BV650 (RMG1-1), IgG2b-PECy7 (RMG2b-1), IgG2c-FITC (polyclonal; SouthernBiotech, Birmingham, AL), IL-4Rα-PE (REA235; Miltenyi Biotec), and CD23-BV421 (B3B4).

Techniques: Adoptive Transfer Assay

Journal: iScience

Article Title: Nanosphere pharmacodynamics improves safety of immunostimulatory cytokine therapy

doi: 10.1016/j.isci.2024.108836

Figure Lengend Snippet: List of immunophenotypic markers or gene IDs, alternative names, and their descriptions

Article Snippet: CF594 CEACAM8 antibody, polyclonal , Biorbyt , orb213728-CF594.

Techniques: Virus, Marker, Immunopeptidomics, Binding Assay

Journal: iScience

Article Title: Nanosphere pharmacodynamics improves safety of immunostimulatory cytokine therapy

doi: 10.1016/j.isci.2024.108836

Figure Lengend Snippet:

Article Snippet: CF594 CEACAM8 antibody, polyclonal , Biorbyt , orb213728-CF594.

Techniques: Recombinant, Staining, Cell Culture, Red Blood Cell Lysis, Enzyme-linked Immunosorbent Assay, Isolation, Multiplex Assay, Software, Sterility

Journal: The Journal of Experimental Medicine

Article Title: A novel M cell–specific carbohydrate-targeted mucosal vaccine effectively induces antigen-specific immune responses

doi: 10.1084/jem.20070607

Figure Lengend Snippet: Development of an M cell–targeted mucosal vaccine with NKM 16–2-4. (A) FITC-conjugated NKM 16–2-4, but not FITC-conjugated control rat IgG, was specifically attached to the apical surfaces of M cells in FAE of PPs within 10 min of inoculation in an intestinal loop assay. The NKM 16–2-4 was subsequently taken up into the cytoplasmic regions of M cells within 30 min and reached the basal membrane of M cells within 4 h. Bars, 10 μm. (B) TT conjugated with NKM 16–2-4 effectively induced high-level, TT-specific serum IgG and fecal IgA responses, unlike TT conjugated with control rat IgG or UEA-1. Furthermore, the levels were superior to those in mice immunized with 10 times the amount of noncoupled TT (500 μg). *, P < 0.01, Tukey's t test. (C) BT-conjugated NKM 16–2-4, but not BT-conjugated control rat IgG, induced brisk botulinum toxin–specific serum IgG and fecal IgA responses. (D) Mice orally immunized with BT-conjugated NKM 16–2-4 were protected from an i.p. challenge with 10,000× LD 50 type A botulinum toxin. Data are expressed as the mean ± the SD.

Article Snippet: In brief, after a blocking step with 1% BSA, 7-μm fixed frozen sections or fixed tissues containing PPs were stained with 5 μg/ml FITC-conjugated NKM 16–2-4 or FITC-conjugated isotype control (FITC-conjugated rat IgG2c; MBL International) and 1 μg/ml tetrarhodamine isothiocyanate–conjugated UEA-1 (Vector Laboratories).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: A novel M cell–specific carbohydrate-targeted mucosal vaccine effectively induces antigen-specific immune responses

doi: 10.1084/jem.20070607

Figure Lengend Snippet: Effective uptake and universality of the M cell–targeted mucosal vaccine. (A) Immunocytochemical analysis showed that an M cell–targeted OVA vaccine composed of Alexa Fluor 647–conjugated OVA, FITC-conjugated avidin, and NKM 16–2-4 specifically reacted with isolated UEA-1–positive M cells. (B) In an intestinal loop assay, the M cell-targeted OVA specifically attached to the apical surfaces of M cells (red arrows) and was immediately taken up into the cytoplasmic regions of M cells. Bars, 10 μm. (C) Orally administered OVA-conjugated NKM 16–2-4 effectively induced an OVA-specific serum IgG response, whereas an OVA-conjugated control rat IgG did not. Data are expressed as the mean ± the SD.

Article Snippet: In brief, after a blocking step with 1% BSA, 7-μm fixed frozen sections or fixed tissues containing PPs were stained with 5 μg/ml FITC-conjugated NKM 16–2-4 or FITC-conjugated isotype control (FITC-conjugated rat IgG2c; MBL International) and 1 μg/ml tetrarhodamine isothiocyanate–conjugated UEA-1 (Vector Laboratories).

Techniques: Avidin-Biotin Assay, Isolation