Journal: Frontiers in Immunology

Article Title: TLR9- and CD40-Targeting Vaccination Promotes Human B Cell Maturation and IgG Induction via pDC-Dependent Mechanisms in Humanized Mice

doi: 10.3389/fimmu.2021.672143

Figure Lengend Snippet: TLR9 agonist CpG-B promotes IgG responses in humanized mice vaccinated with CD40-targeting vaccine. Humanized mice were treated with PBS control (n=3) or vaccinated with αCD40-HIV5pep (n=3) alone or vaccinated with αCD40-HIV5pep plus CpG-B (n=4) at week0, week3 and week6. At week 7, mice were sacrificed. (A, B) The expression of IgM and IgG on B cells from PBMCs and spleens was detected by FACS. (C) The total level of IgM and IgG in the plasma was detected by ELISA. (D) Splenocyte from humanized mice were cultured ex vivo in the present of R848(1μg/ml) and IL-2(10 u/ml) for 48hours, the cells were used for total IgG detection by ELISpot. (E) Antigen specific IgG level in the plasma was detected by ELISA. (F) The expression of activation-induced cytidine deaminase (AID) in spleen cells was detected by RT-PCR. Each dot represents one individual mouse, bars indicate mean. *P < 0.05, **P < 0.01, by unpaired, two-tailed Student’s t-test comparing the two vaccinated groups.

Article Snippet: Total IgM and IgG were detected by ELISA kits, purchased from Bethyl Laboratories,int.(Cat.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Ex Vivo, Enzyme-linked Immunospot, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Normal B cell development in Igh Pax5/+ mice. (A–C) Flow-cytometric analysis of bone marrow (A), spleen (B), and lymph node (C) cells from 8–12-wk-old Igh Pax5/+ (black) and Igh +/+ (gray) littermates. The relative frequency was determined for pro-B (CD19 + Kit + CD2 – IgM – ), pre-B (CD19 + Kit – CD2 + IgM – ), immature B (CD19 + IgM + IgD – ), and recirculating B (CD19 + IgM lo IgD hi ) cells in the bone marrow (A), follicular (CD19 + CD21 lo CD23 hi ), and marginal zone (CD19 + CD21 hi CD23 lo ) B cells in the spleen (B) and mature CD4 + and CD8 + T cells in the lymph nodes (C). The flow-cytometric data were obtained in seven (A), five (B), or two (C) independent experiments. Statistical data (A–C) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; **, P < 0.01; ****, P < 0.0001. NS, not significant (P > 0.05). Each dot corresponds to one mouse. (D–F) Flow-cytometric analysis of Pax5 expression by intracellular staining of developing and recirculating B cells in the bone marrow (D), mature B cells in the spleen (E), and mature T cells in the lymph nodes of Igh Pax5/+ (black line) and Igh +/+ (gray filled) littermates. The specificity of the anti-Pax5 antibody (IgG2a) was controlled by staining of the different B cell types with a control IgG2a isotype antibody (gray dashed line).

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Two Tailed Test, Expressing, Staining

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Antibody secretion by Igh Pax5/+ plasma cells. (A and B) Antibody secretion by Igh Pax5/+ and Igh +/+ plasma cells under steady-state conditions. The numbers of IgM and IgG ASCs in the bone marrow of Igh Pax5/+ (black) and Igh +/+ (gray) mice at the age of 2–3 mo (A) and 6–8 mo (B) were determined by ELISPOT assay by incubating RBC-depleted bone marrow cells (2 × 10 5 ) on IgM- or IgG-coated plates for 6 h before determination of the number and size of the ELISPOTs. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for IgM and IgG ASCs. (C and D) Antibody secretion by Igh Pax5/+ and Igh +/+ plasma cells 14 d after immunization with NP-KLH (in alum). The numbers of anti–NP-IgM or anti–NP-IgG1 ASCs in the spleen (C) and bone marrow (D) were determined by ELISPOT assay by incubating RBC-depleted cells (2 × 10 6 ) from the spleen or bone marrow on NP 20 -BSA-coated plates before the determination of the number and size of the ELISPOTs. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for anti–NP-IgM and anti–NP-IgG1 ASCs. The data (C and D) are pooled from two independent immunization experiments. Statistical data (A–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. NS, not significant (P > 0.05). Each dot (A–D) corresponds to one mouse. (E) Electron microscope images of plasma cells isolated from the bone marrow of Igh Pax5/+ and Igh +/+ mice at the age of 6–8 mo. 1 representative image of 10 individual plasma cells analyzed per genotype is shown. The length of the indicated scale bars is 1 mm (B and D) or 0.5 µm (E).

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Enzyme-linked Immunospot, Two Tailed Test, Microscopy, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Antibody titers and morphology of plasma cells in Igh Pax5/+ mice. (A and B) Serum titers of total antibody of the IgM, IgG1, IgA, and IgE isotypes in nonimmunized Igh Pax5/+ (black) and Igh +/+ (gray) mice at the age of 2–3 mo (A) or 6–8 mo (B). The results shown are representative of three independent experiments. Statistical data (A and B) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P < 0.05. NS, not significant (P > 0.05). (C) Expression of the indicated immunoglobulin isotype mRNAs in Igh Pax5/+ (black) and Igh +/+ (gray) plasma cells. The expression data are shown as mean TPM values with SD and are based on two ( Igh +/+ ) and three ( Igh Pax5/+ ) independent RNA-seq experiments. Each dot represents one experiment performed with pooled plasma cells from 5–10 mice per genotype. The P values were calculated by the DESeq2 program; ***, P < 0.001; ****, P < 0.0001. NS, not significant (P > 0.05). (D) Higher magnification of the same two electron microscope images shown in . The two plasma cells were isolated from the bone marrow of 6–8-mo-old Igh Pax5/+ and Igh +/+ mice. (E) Comparison of ER content in B cells and plasma cells. Staining with the ER-Tracker Red dye (at a final concentration of 0.2 µM) was used to determine the ER content of B cells (CD19 + ) and plasma cells (Blimp1-GFP + CD138 + ) in the bone marrow of 6–8-mo-old Igh Pax5/+ and Igh +/+ mice at steady state (left). The quantification of the staining is shown as mean fluorescence intensity (MFI; right). Statistical data (A, B, and E) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; *, P < 0.05. NS, not significant (P > 0.05). Each dot (A, B, and E) corresponds to one mouse.

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Two Tailed Test, Expressing, RNA Sequencing Assay, Microscopy, Isolation, Staining, Concentration Assay, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Repression of the B cell identity factor Pax5 is not required for plasma cell development

doi: 10.1084/jem.20200147

Figure Lengend Snippet: Rescue of IgG1 CSR in the presence of functional T cells. (A) Experimental strategy to interrogate B-lineage–intrinsic effects of ectopic Pax5 expression in response to immunization. CD43 – B cells isolated from the spleens of Igh B1-8hi/Pax5 Prdm1 Gfp/+ or Igh B1-8hi/+ Prdm1 Gfp/+ mice (CD45.1 + CD45.2 + ) were cotransferred with splenic T cells from OT-II TCR-tg Rag2 −/− mice (CD45.2 + ) into C57BL/6 recipients (CD45.1 + ). 1 d after cell transfer, the recipients were immunized with NP-OVA (in alum) and were analyzed 6 d after immunization. (B) Flow-cytometric analysis of splenic GC B cells of Igh B1-8hi donor origin. The frequencies of IgG1 + GC B cells of the Igh B1-8hi/+ Prdm1 Gfp/+ and Igh B1-8hi/Pax5 Prdm1 Gfp/+ genotypes were determined by sequential gating on donor cells (CD45.1 + CD45.2 + ), B cells (B220 + ), and GC B cells (GL7 + Fas + ) by flow cytometry (above). The quantification of the frequencies of GC B cells and IgG1 + GC B cells is shown below. The data are pooled from three independent experiments. (C) Analysis of splenic GFP + plasma cells derived from the transferred Igh B1-8hi/+ Prdm1 Gfp/+ or Igh B1-8hi/Pax5 Prdm1 Gfp/+ B cells. The flow-cytometric analysis is shown on the left, and the frequency of the GFP + plasma cells is quantified on the right. The data are pooled from two independent experiments. (D) ELISPOT assay to determine the frequency of anti–NP-IgM and anti–NP-IgG1 ASCs derived from the transferred Igh B1-8hi/+ Prdm1 Gfp/+ or Igh B1-8hi/Pax5 Prdm1 Gfp/+ cells. Six days after immunization with NP-OVA, 500 GFP + CD138 + plasma cells of donor origin were directly sorted onto NP 20 -BSA–coated plates. Cells were incubated for 6 h, before the number and size of anti–NP-IgM and anti–NP-IgG1 ELISPOTs were determined. Images of representative ELISPOT wells (left), the mean cell number (middle), and the median ELISPOT size (right) are shown for anti–NP-IgM and anti–NP-IgG1 ASCs. Statistical data (B–D) are shown as mean values with SD and were analyzed by the two-tailed unpaired Student’s t test; **, P < 0.01; ****, P < 0.0001. NS, not significant (P > 0.05). Each dot (B–D) corresponds to one mouse. A scale bar of 1 mm length is shown.

Article Snippet: The following antibodies were used for ELISPOT and ELISA: goat anti-mouse IgM, IgG, IgG1, IgG2b, and IgA (human adsorbed; SouthernBiotech), HRP-coupled goat anti-mouse IgM, IgG, and IgG1 (human adsorbed; SouthernBiotech), and AP-coupled goat anti-mouse IgM, IgG1, IgA, and IgE (human adsorbed; SouthernBiotech).

Techniques: Functional Assay, Expressing, Isolation, Flow Cytometry, Derivative Assay, Enzyme-linked Immunospot, Incubation, Two Tailed Test

Journal: Nature Communications

Article Title: Loss of immune tolerance to IL-2 in type 1 diabetes

doi: 10.1038/ncomms13027

Figure Lengend Snippet: ( a – f ) Five-to-14-week-old male or female NOD mice were daily treated with PBS or high-doses rhIL-2 (250,000; 500,000 or 1,000,000 IU) over 30 days. ( a , b ) Kaplan-Meier survival curves of treated female ( a , top panel) or male ( b , top panel) mice; and diabetes incidence in female ( a , bottom panel) or male ( b , bottom panel) mice. ( c ) Percentage of dead, diabetic or alive and non-diabetic NOD mice after 30 days of treatment; IL-2-treated: pool of (250,000; 500,000 and 1,000,000 IU IL-2 treated mice. ( d ) Percentage of Foxp3 + among CD3 + CD4 + splenocytes of NOD mice treated for 5 to 30 days with high-doses IL-2 or PBS. ( e ) Serum anti-rhIL-2 IgG titres of untreated B6 mice and pre-diabetic NOD mice treated for 0, 7 or 30 days with high-dose IL-2. ( f ) Proliferation of CTLL-2 cells cultured for 3 days with 3 IU ml −1 rhIL-2 and serially diluted serum from B6 (closed circles) or NOD mice treated for 30 days with high-dose rhIL-2 (open circles). Proliferation is expressed as percentage of control (CTLL-2 cultured for 3 days with 3 IU ml −1 rhIL-2 without mouse serum). Data are cumulative of at least two independent experiments. ns, not significant. *** P <0.001 (non-parametric Mann-Whitney test).

Article Snippet: After extensive washing with PBS/0.1% Tween-20, biotin-labelled anti-mouse IgG, IgG1, IgG2b, IgG2c, IgG3, IgM or IgA (all 1:5000; Southern Biotech) was added to each well and the plates were kept at room temperature for 1 h. Plates were subsequently incubated with horseradish peroxidase (HRP)-conjugated streptavidin (1:2000; Invitrogen) for 30 min followed by 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (eBioscience or BD Biosciences) for 10 min.

Techniques: Cell Culture, Control, MANN-WHITNEY

Journal: Nature Communications

Article Title: Loss of immune tolerance to IL-2 in type 1 diabetes

doi: 10.1038/ncomms13027

Figure Lengend Snippet: ( a – c ) Serum samples were obtained from different mouse strains: NSG (NOD scid gamma), B6, Balb/c, pre-diabetic NOD (NOD Pre-diabetic) and diabetic NOD (NOD Diabetic). ( a , b ) Serum titres of anti-mIL-2 IgG ( a ), IgG isotypes (IgG1, 2b, 2c and 3) and IgA ( b ) in the different strains. ( c ) Proliferation of CTLL-2 cells cultured for 3 days with 1 ng ml −1 mIL-2 and different concentrations of B6 (closed circles) or NOD (open circles) sera. Proliferation is expressed as percentage of control (CTLL-2 cultured for 3 days with 1 ng ml −1 mIL-2 without mouse serum, mean c.p.m. of 84,590). ( d – f ) Sera were obtained at different ages after birth and at disease onset (Onset) in two independent cohorts of female NOD mice ( n =13 and 6, respectively) and one cohort of male NOD mice ( n =5). ( d , e ) Serum anti-mIL-2 IgG titres in NOD mice in function of the age ( d ) or of the sex ( e ). Dashed line indicates the mean value given by B6 mouse sera in the corresponding ELISA. ( f ) Correlation between anti-mIL-2 IgG titres at 6 weeks and time to onset of diabetes in female NOD mice (non-parametric Spearman correlation test). ( g , h ) Serum samples were obtained from different mouse strains (all females and age-matched): B6, wild-type NOD, NOD. Idd3 B6 , Il2 -hemizygous NOD: NOD. Idd3 NOD/NOD-IL-2null (NOD. Il2 +/− ), NOD. Idd6 C3H and their corresponding controls NOD. Idd6 NOD , as well as NOR. Serum titres of anti-mIL-2 IgG ( g ) and IL-2/anti-rhIL-2-autoantibodies complex ( h ) in the different mouse strains. Symbols and curves represent individual mice, horizontal bars are the medians and error bars represent the s.e.m. Data are cumulative of at least two independent experiments. ns, not significant. * P <0.05; ** P <0.01; *** P <0.001 (non-parametric Mann-Whitney test).

Article Snippet: After extensive washing with PBS/0.1% Tween-20, biotin-labelled anti-mouse IgG, IgG1, IgG2b, IgG2c, IgG3, IgM or IgA (all 1:5000; Southern Biotech) was added to each well and the plates were kept at room temperature for 1 h. Plates were subsequently incubated with horseradish peroxidase (HRP)-conjugated streptavidin (1:2000; Invitrogen) for 30 min followed by 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (eBioscience or BD Biosciences) for 10 min.

Techniques: Cell Culture, Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Loss of immune tolerance to IL-2 in type 1 diabetes

doi: 10.1038/ncomms13027

Figure Lengend Snippet: ( a ) Percentage of anti-rhIL-2 positive subjects among five different cohorts: serum samples were obtained from healthy donors (HD, n =249) and patients diagnosed with type 2 (T2D; n =24) or type 1 diabetes ( n =39 in cohort 1, n =15 in cohort 2 and n =21 in cohort 3). P -values were calculated using pairwise Fisher exact tests. ( b ) IgG subclass-specific anti-IL2 ELISA in T1D patients, absorbance OD measured at 450 after incubation of sera diluted 1:50. ( c ) T1D patient samples were divided into three groups based on EC 50 values for an IL-2 direct ELISA from total sera (weak, moderate and strong binders) and are plotted relative to the anti-IL-2 EC 50 value of IgG purified from the respective serum sample. Mean and s.d. overlay individual data points, and P -values were calculated using Welch's t -test. ( d ) (upper panel) rhIL-2 competition ELISA using IgG purified from the sera of T1D patients, with the control (lower panel) of an anti-influenza ELISA with competing soluble rhIL-2.

Article Snippet: After extensive washing with PBS/0.1% Tween-20, biotin-labelled anti-mouse IgG, IgG1, IgG2b, IgG2c, IgG3, IgM or IgA (all 1:5000; Southern Biotech) was added to each well and the plates were kept at room temperature for 1 h. Plates were subsequently incubated with horseradish peroxidase (HRP)-conjugated streptavidin (1:2000; Invitrogen) for 30 min followed by 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (eBioscience or BD Biosciences) for 10 min.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Direct ELISA, Purification, Control

Journal: Nature Communications

Article Title: Loss of immune tolerance to IL-2 in type 1 diabetes

doi: 10.1038/ncomms13027

Figure Lengend Snippet: ( a ) (Left) Relative frequency and (right) absolute numbers of antigen-specific IgG memory B cells across four T1D patients. Error bars represent s.d., P -values calculated using an unpaired t -test. ( b ) IL-2-specific heavy and light chain V gene segment usage, CDR3 amino acid sequences, and rhIL-2 solution equilibrium titration (SET) affinity for recombinant IgG obtained from a single T1D patient, ( c ) IFN-γ production by peripheral blood mononuclear cells (PBMCs) from healthy donors (HD) ( n =14, closed circles) or T1D patients ( n =13, open circles) quantified by ELISPOT after stimulation with rhIL-2 (Proleukin, Pro) or Pro peptides (10 μM per each), intracellular IA-2, adenovirus lysate (AdV), or PHA. The number of IFN-γ spot-forming cells (SFC)/10 6 PBMCs is depicted, the dashed line indicates the positive cut-off threshold, and the grey shaded area shows undetectable responses (that is, identical to spontaneous background responses; see material and methods for threshold determination). The percent of positive T1D (top number) and HD (bottom number) is indicated for each condition, with antigens yielding responses significantly different between HD and T1D patients in bold ( P <0.03 using the Fisher exact test).

Article Snippet: After extensive washing with PBS/0.1% Tween-20, biotin-labelled anti-mouse IgG, IgG1, IgG2b, IgG2c, IgG3, IgM or IgA (all 1:5000; Southern Biotech) was added to each well and the plates were kept at room temperature for 1 h. Plates were subsequently incubated with horseradish peroxidase (HRP)-conjugated streptavidin (1:2000; Invitrogen) for 30 min followed by 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (eBioscience or BD Biosciences) for 10 min.

Techniques: Titration, Recombinant, Enzyme-linked Immunospot

Journal: Nature Communications

Article Title: Loss of immune tolerance to IL-2 in type 1 diabetes

doi: 10.1038/ncomms13027

Figure Lengend Snippet: ( a ) Serum samples were obtained from healthy donors (HD, n =249), T1D ( n =75 in the three pooled cohorts), multiple sclerosis (MS; n =33), Sjögren syndrome (SJO; n =22), anti-JO1 positive polymyositis (JO1; n =16), rheumatoid arthritis (RA; n =33), systemic lupus erythematosus (SLE; n =20), chronic inflammatory demyelinating neuropathy (CIPD; n =51) and cancer (Cancer; n =128) patients. Cancer patients were used as controls for a non-autoimmune disease. Left panel: serum titres of anti-rhIL-2 IgG in the different cohorts. Dashed line indicates the threshold of positivity. Right panel: percentage of anti-rhIL-2 positive subjects among the different cohorts. Symbols represent individual subjects and horizontal bars are the medians. * P <0.05; *** P <0.001 (Fisher exact test). ( b ) To control the anti-rhIL-2 autoantibody specificity a competition ELISA was performed. Sera from one anti-rhIL-2-autoantibodies − healthy donor (HD) or from anti-rhIL-2-autoantibodies + samples from HD and T1D, T2D, SLE, RA, Sjögren syndrome (SJO), polymyositis (JO1) patients were pre-incubated or not for 1 h with increasing amounts of free recombinant rhIL-2 and titres of anti-rhIL-2 were then quantified by ELISA. Shown is percentage of inhibition of the ELISA signal calculated as (OD with rhIL-2 competition) × 100/(OD without competition). Curves represent individual subjects. Data are cumulative of three independent experiments. ( c ) Titres of anti-mIL-2 IgG were quantified by ELISA in the sera of B6 and 4-month-old lupus-prone mice B6/ lpr mice. Each serum was tested once or twice in duplicate. Symbols represent individual mice and horizontal bars are the medians. * P <0.05 (non-parametric Mann-Whitney test).

Article Snippet: After extensive washing with PBS/0.1% Tween-20, biotin-labelled anti-mouse IgG, IgG1, IgG2b, IgG2c, IgG3, IgM or IgA (all 1:5000; Southern Biotech) was added to each well and the plates were kept at room temperature for 1 h. Plates were subsequently incubated with horseradish peroxidase (HRP)-conjugated streptavidin (1:2000; Invitrogen) for 30 min followed by 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (eBioscience or BD Biosciences) for 10 min.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Inhibition, MANN-WHITNEY

Journal: bioRxiv

Article Title: Lung-resident memory B cells maintain allergic IgE responses in the respiratory tract

doi: 10.1101/2024.02.20.581262

Figure Lengend Snippet: (A) Schematic of local allergen sensitization. Mice were treated intranasally with ragweed pollen and ovalbumin (RWP+OVA) for five days in the first week, followed by two weeks of OVA alone, and vehicle control mice received intranasal PBS. Mice were analyzed on day 21. (B) Eosinophils (live CD45 + CD11c - CD11b + SiglecF + ) in bronchoalveolar lavage fluid (BALF) of PBS control (left) and allergen-sensitized (RWP+OVA) mice. Numbers are frequency, representative of 3 independent experiments with 3-8 mice per experiment. (C) Measurement of OVA-specific IgE and IgG1 in BALF by ELISA. (D) Intravascular labeling of lung B cells (live B220 + ) with anti-CD45 showing tissue-localized (unlabeled) and blood B cells (labeled). (E) Representative flow cytometry plots showing gating strategy for the identification of naïve (CD38 + IgD + , purple), memory (IgD - CD38 + GL7 - , green), and germinal center (GC, IgD - CD38 - GL7 + , orange) B cells (live B220 + i.v. CD45 - ) in the lungs of allergen-sensitized mice. (F) Frequencies and cell counts of naïve, memory, and GC B cells in the lungs. Representative data from four independent experiments with 3-8 mice per group in each experiment (mean ± SD).

Article Snippet: Standard curves were constructed by 2-fold dilutions of OVA-specific IgE monoclonal antibody (clone E-C1, Chondrex) diluted in 10% FBS in PBS from 200 ng/mL to 0.195 ng/mL, or with OVA-specific IgG1 antibody (clone L71, Chondrex) from 20 ng/mL to 19.5 pg/mL.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: Lung-resident memory B cells maintain allergic IgE responses in the respiratory tract

doi: 10.1101/2024.02.20.581262

Figure Lengend Snippet: (A) Schematic of Iε-tdTomato knock-in mouse reporting germline transcription at Iε locus. (B) Representative plots by flow cytometry showing Iε-tdTomato expression by B cells (live B220 + i.v. CD45 - ) in the lungs, medLN, and spleen on day 21 (left) and frequencies and cell counts of Iε-tdTomato + B cells (right). (C) Representative plots by flow cytometry showing the identification of naïve, germinal center (GC), and memory B cells (MBCs, top) and frequencies of each subset (bottom) among total Iε-tdTomato + B cells. (D) Surface expression of IgM and IgG1 on total MBCs and Iε-tdTomato + MBCs in the lungs (top) and frequencies of each population (bottom). (E) Expression of Iε-tdTomato among IgG1 + MBCs in the lungs, medLN, and spleen. Data are representative of three independent experiments with 5-9 mice in each experiment. One-way ANOVA with Tukey’s multiple comparisons test on mean ± SD; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

Article Snippet: Standard curves were constructed by 2-fold dilutions of OVA-specific IgE monoclonal antibody (clone E-C1, Chondrex) diluted in 10% FBS in PBS from 200 ng/mL to 0.195 ng/mL, or with OVA-specific IgG1 antibody (clone L71, Chondrex) from 20 ng/mL to 19.5 pg/mL.

Techniques: Knock-In, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Lung-resident memory B cells maintain allergic IgE responses in the respiratory tract

doi: 10.1101/2024.02.20.581262

Figure Lengend Snippet: (A) Immunofluorescence imaging showing localization of B cells (CD19 + ) and CD4 + T cells on day 77. Whole lung section (10x, left) and 63x magnification of indicated areas (right). (B) Number of IgM + (top) and IgG1 + (bottom) MBCs in the lungs at indicated timepoints. Mean ± SEM, representative of two independent experiments. (C) Experimental scheme for parabiosis experiments. CD45 congenic mice were surgically paired 4 weeks after allergen sensitization and tissues were analyzed for MBC chimerism 10 days later. (D) Representative flow cytometry plots and (E) aggregate frequencies of chimerism of MBCs in indicated tissues based on expression of CD45.1 and CD45.2. Mean ± SD, n=6.

Article Snippet: Standard curves were constructed by 2-fold dilutions of OVA-specific IgE monoclonal antibody (clone E-C1, Chondrex) diluted in 10% FBS in PBS from 200 ng/mL to 0.195 ng/mL, or with OVA-specific IgG1 antibody (clone L71, Chondrex) from 20 ng/mL to 19.5 pg/mL.

Techniques: Immunofluorescence, Imaging, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Lung-resident memory B cells maintain allergic IgE responses in the respiratory tract

doi: 10.1101/2024.02.20.581262

Figure Lengend Snippet: (A) Violin plots showing scaled expression of indicated genes from day 77 scRNA-seq libraries. (B) Histograms showing surface staining of indicated markers on MBCs from each tissue on day 77 by flow cytometry. Each histogram is concatenated from 8 mice. Dashed lines indicate staining from negative control. (C) Expression and gating of IgM and IgG1 on MBCs in the lungs (i.v. CD45 - ) on day 77. (D) Histograms showing surface expression of markers from day 77 libraries and (E) surface markers from A and B on IgM + and IgG1 + MBCs. Each histogram is concatenated from 8 mice. Control is only stained with CD19 and live/dead dye zombie NIR. (E) Expression of PD-L2 and CD80 and (G) CD73 and OVA-binding on IgM + and IgG1 + MBCs by flow cytometry on day 77. Representative staining from two independent experiments with 8 mice.

Article Snippet: Standard curves were constructed by 2-fold dilutions of OVA-specific IgE monoclonal antibody (clone E-C1, Chondrex) diluted in 10% FBS in PBS from 200 ng/mL to 0.195 ng/mL, or with OVA-specific IgG1 antibody (clone L71, Chondrex) from 20 ng/mL to 19.5 pg/mL.

Techniques: Expressing, Staining, Flow Cytometry, Negative Control, Control, Binding Assay

Journal: bioRxiv

Article Title: Lung-resident memory B cells maintain allergic IgE responses in the respiratory tract

doi: 10.1101/2024.02.20.581262

Figure Lengend Snippet: (A) Experimental schematic of rechallenge. Eight weeks after sensitization with RWP+OVA (or PBS control) mice were rechallenged with five consecutive days of nebulized OVA (in 1% PBS) or PBS (20 min/day) and analyzed on day 84. (B) Detection of OVA-specific IgE in bronchoalveolar lavage fluid (BALF) by ELISA. One-way ANOVA with Tukey’s multiple comparisons test (n=8). (C) Detection of OVA-specific IgE-secreting cells in single cell suspension from lungs, spleen or bone marrow (BM) by ELISpot with 1×10 6 cells plated per well. (D) Representative flow cytometry plots showing the identification of OVA-binding MBCs (i.v. CD45 - CD19 + IgD - ) in the lungs in each indicated condition. (E) Frequencies and numbers of OVA-specific MBCs in the lungs in each indicated condition. One-way ANOVA with Tukey’s multiple comparison’s test. (F) Representative (left) and summary plot (right) showing frequency of MBCs, naïve, and germinal center (GC) B cells among Iε-tdTomato + B cells in the lungs. Representative of three independent experiments, one-way ANOVA with Tukey’s multiple comparison’s test. (G) Expression of IgM and IgG1 among Iε-tdTomato + MBCs in the lungs. Representative plot (left) and aggregate frequencies by flow cytometry analyses (right). Representative of three independent experiments, one-way ANOVA with Tukey’s multiple comparison’s test. (H) Schematic of parabiosis experiments. RWP+OVA-sensitized mice were surgically paired to PBS control mice 4 or 8 weeks after sensitization. Ten days after surgery, pairs were rechallenged with five days of nebulized OVA (1% in PBS, 20 min/day). (I) Detection of OVA-specific IgE in sera and BALF of PBS or RWP+OVA-sensitized parabionts by ELISA after rechallenge with OVA. Representative data of two independent experiments. Bars are mean ± SD; *, p<0.05; ***, p<0.001; ****, p<0.0001.

Article Snippet: Standard curves were constructed by 2-fold dilutions of OVA-specific IgE monoclonal antibody (clone E-C1, Chondrex) diluted in 10% FBS in PBS from 200 ng/mL to 0.195 ng/mL, or with OVA-specific IgG1 antibody (clone L71, Chondrex) from 20 ng/mL to 19.5 pg/mL.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Suspension, Enzyme-linked Immunospot, Flow Cytometry, Binding Assay, Expressing

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Splenocytes (A-D) and tumor-infiltrating lymphocytes (TIL) (E-H) were isolated from HIS mice 16-18 days after tumor implantation and analyzed by flow cytometry. A, frequency of splenic T cells in tumor-bearing or naïve HIS mice; parent population refers to frequency (%) of CD3 + T cells within human CD45 + cells and CD4 + and CD8 + T cells within CD3 + T cells. B , CD8 + T cell differentiation defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ) in tumor-bearing or naïve HIS mice. C-D , expression of indicated markers on CD8 + T cells from spleen of tumor-bearing or naïve HIS mice. E, frequency of CD4 + and CD8 + T cells within TILs, gated on total CD3 + T cells. F, CD8 + T cell differentiation within TILs. G-H, expression of indicated markers on CD8 + T cells within TILs. I, expression of CD137 on splenic CD8 + and CD4 + T cells of tumor-bearing or naïve HIS mice. J, expression of PD-1 and CD137 on splenic CD8 + T cells of tumor-bearing mice. K , expression of PD-1 on splenic CD8 + T cells based on CD137 in tumor-bearing HIS mice or bulk CD8 + T cells from naïve HIS mice. L, CD8 + T cell differentiation in spleen of tumor-bearing HIS mice. M-O , expression of indicated markers on splenocyte-derived CD8 + T cell populations based on expression of CD137 and PD-1. Data are pooled from at least 3 independent experiments, n=10-23 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by paired t-test, one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Isolation, Tumor Implantation, Flow Cytometry, Cell Differentiation, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation, expansion and characterization of T cells from HIS mice bearing autologous LCL tumors. B , fold expansion of FACS sorted splenic CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). C , CD8 + T cell differentiation after ex vivo expansion defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ). D, IFN-ψ ELISpot of expanded T cells in 5:1 (E:T) co-culture with autologous LCL tumor cells for 24 hours. Spot count is normalized to the spots produced by expanded CD8 + bulk T cells from naïve HIS mice. E , TNF⍺ ELISA of supernatant of expanded T cells in co-culture (5:1, E:T) with autologous LCL tumor cells for 24 hours. TNF⍺ concentration is normalized to the TNF⍺ secretion from expanded CD8 + bulk T cells derived from naïve mice. Data are pooled from at least 3 independent experiments, n=6-19 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by mixed-effects analysis (paired), RM one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Cell Differentiation, Ex Vivo, Enzyme-linked Immunospot, Co-Culture Assay, Produced, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Transcriptome analysis and pathway analysis of expanded CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). A, Upset plot (intersect) showing number of differentially expressed genes between groups in bulk RNAseq. p(FDR) < 0.05, log2 FC > 1.5. B , PCA plot of RNAseq showing PC1 and PC2. C, Top 50 upregulated and D, downregulated genes of T cell subsets based on the DEG between CD137 + vs. CD137 - PD1 - CD8 + T cells. p(FDR) < 0.05, log2 FC > 1.5. E, Volcano plot showing DEG between CD137 + and CD137 - PD1 - CD8 + T cells with genes of interest highlighted in yellow. F , differential expression of genes of interest between groups. G , Overrepresentation analysis (ORA) of upregulated pathways in CD137 + CD8 + T cells. H , Gene set enrichment analysis (GSEA) of signatures described on the y-axis. Gene ratio (# genes related to GO term / total number of sig genes) is displayed on the x-axis. Signatures with an adjusted p-value <0.05 are highlighted with a red box. Shown are data from 5 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , total number of individual clonotypes found per population. Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. B, TCR (CDR3 of TRA , TRB , TRG and TRD ) sequence sample diversity estimation using Hill numbers method, with Q=1 describing the Shannon diversity. C, rare clonal proportion showing the occupied repertoire space by clonotypes with defined counts (1, 2-3, 4-10, etc.). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. D, relative abundance of clonotypes with defined frequencies (size). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. E, repertoire overlap analysis cross-comparing every population from every experiment (each with different donor). F , repertoire overlap comparing the repertoire of bulk CD8 + T cells from naïve HIS mice to the populations from tumor-bearing HIS mice from individual experiments (each with different donor; data from individual experiments are indicated by different symbols). Mixed effects analysis with Tukey’s multiple comparisons test. G , tracking of clonotypes over populations. The top 10 most abundant clonotypes of the TCR repertoire of CD137 + CD8 + T cells from one representative experiment are shown. H , proportion of the top 10 most abundant clonotypes (from repertoires of CD137 + CD8 + T cells) in the repertoire of all populations, correlated with the spot count of IFN-ψ ELISpot. Shown are data from 4 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Sequencing, Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation of tumor-reactive T cells and subsequent ACT. NSG mice were injected with 2 x 10 LCL s.c. in the flank and after three days, 10 x 10 ex vivo expanded T cells were adoptively transferred intravenously. Transferred T cells and LCL tumors were autologous to each other. B , tumor volume on the day of sacrifice in NSG recipient mice after ACT of the indicated cell populations. C, waterfall plot of tumor size in NSG recipient mice of ACT on the day of sacrifice relative to the tumor volume of control mice (no ACT). Bars depict individual mice. D , frequency of CD3 + T cells (% of human CD45 + cells) in TIL from NSG mice after ACT of the indicated cell populations, measured by flow cytometry. E , frequency of CD8 + T cells (% of total cells) in tumors of NSG mice after ACT of the indicated cell populations, measured by immunohistochemistry. F , differentiation of CD8 + T cells in TIL of NSG mice after ACT of the indicated cell populations defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ) and T EMRA (CD45RA + CD62L - ). G, correlation between tumor volume and infiltration of CD8 + T cells (measured by IHC) in tumors of NSG mice after adoptive transfer of CD137 + CD8 + T cells. H , schematic of ACT. CD137 + CD8 + T cells, CD137 - CD8 + T cells and CD137 - PD-1 - CD8 + T cells were isolated from spleen of tumor-bearing HIS mice or bulk CD8 + T cells from spleen of naïve HIS mice and expanded ex vivo . Recipient HIS mice were injected with 2 x 10 LCL s.c. in the flank and after three days, ex vivo expanded T cells were adoptively transferred intravenously. Tumor-bearing HIS recipient mice received 2 x 10 T cells without prior conditioning/lymphodepletion. Donor and recipient HIS mice as well as LCL were autologous to each other. I, tumor volume on the day of sacrifice in HIS recipient mice after ACT of the indicated cell populations. J, waterfall plot of tumor size on the day of sacrifice of HIS mice receiving ACT relative to the tumor volume of control HIS mice (no ACT). Bars depict individual mice. B-G: Data are pooled from 2-3 independent experiments, n=6-13 per group. I, data are pooled from 2-3 independent experiments (n=6-13 per group); J, data are from 1-3 independent experiments (n=3-13 per group). For each experiment, a with different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by one-way ANOVA. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Injection, Ex Vivo, Control, Flow Cytometry, Immunohistochemistry, Adoptive Transfer Assay, Isolation

Journal: Immunity

Article Title: Multivalent antigen display on nanoparticle immunogens increases B cell clonotype diversity and neutralization breadth to pneumoviruses.

doi: 10.1016/j.immuni.2023.08.011

Figure Lengend Snippet: Figure 4. Conformation- and valency-dependent activation of B cells (A) Two-dimensional classifications of negative-stain electron microscopy (nsEM) particles of DS-Cav1, postF, and DS-Cav1-I53-50A. Classes are labeled by their conformation based on the characteristic lollipop shape of postF and the oval shape of preF. Scale bars, 10 nm. (B) Proportions of DS-Cav1-I53-50A component in preF or postF conformation from nsEM analysis with corresponding models. A total of 10,657 particles were analyzed. (C) Area under the curve (AUC) quantification of sandwich ELISAs with capture of immunogens using the antibodies palivizumab (preF/postF-specific), D25 (preF- specific), or 4D7 (postF-specific). Detection was performed with biotinylated versions of the same antibodies. Representative data from two independent ex- periments. (D) Updated model of 20-mer nanoparticle co-displaying preF and postF at 15% and 85%, respectively. (E–H) Naive rhesus macaques (n = 4) were immunized by intramuscular injection with two doses of 1-mer postF immunogen in polyIC:LC adjuvant at weeks 0 and 4, and with one dose of 1-mer preF immunogen in polyIC:LC adjuvant at week 26 as depicted in (E). (F) Proportion of RSV neutralization in serum that remains after competition with excess postF or preF. (G) Proportion of preF-binding titer in plasma that is outcompeted by excess postF. (H) Multidimensional scaling projection of plasma epitope reactivity as measured by reference antibody competition. (I) Structures of antibodies D25 (PDB: 4JHW) and MPE8 (PDB: 5U68) binding to preF.

Article Snippet: ELISpot plates were washed with PBS-T, incubated with 0.25 mg/ml biotinylated goat anti-human IgG (Fcg; Jackson ImmunoResearch Laboratories) for total IgG determination, 1 mg/ml biotinylated DS-Cav1 trimer for preF-specific determination, or 1 mg/ml biotinylated ovalbumin (OVA) in PBS-T. After another round of washing, streptavidin-conjugated alkaline phosphatase (Mabtech) diluted in PBS-T was added.

Techniques: Activation Assay, Staining, Electron Microscopy, Labeling, Injection, Adjuvant, Neutralization, Binding Assay, Clinical Proteomics

Journal: PLoS ONE

Article Title: Cross Protection against Influenza A Virus by Yeast-Expressed Heterologous Tandem Repeat M2 Extracellular Proteins

doi: 10.1371/journal.pone.0137822

Figure Lengend Snippet: BALB/c mice ( n = 9) were immunized with 20 μg of M2e5x protein alone (M2e5x) or mixed with adjuvant AS04 (M2e5x.AS04). Blood samples were collected at 3 weeks after each immunization. (A) Total IgG antibody. IgG was detected by using human type M2e peptide as an ELISA-coating antigen. (B) M2e-specific IgG isotype responses of the M2e5x.AS04 group after 3rd immunization. (C) IgG antibody responses reactive to M2e peptide antigens derived from human, swine, or avian influenza A viruses. IgG antibodies specific to M2e were measured in 3rd immune sera from the M2e5x.AS04 group using human, swine, avian I, or avian II type peptide as a coating antigen. Sera were serially diluted and ELISA was performed for serum antibodies specific for M2e peptides. Error bars indicates mean ± SEM.

Article Snippet: Following washing again horseradish peroxidase (HRP)-labeled goat anti-mouse IgG, IgG1, and IgG2a (Southern Biotechnology, Birmingham, AL) were added to each well (1:2,000), and incubated for additional 2 h at 37°C.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Cross Protection against Influenza A Virus by Yeast-Expressed Heterologous Tandem Repeat M2 Extracellular Proteins

doi: 10.1371/journal.pone.0137822

Figure Lengend Snippet: Levels of IgG antibodies were determined in samples collected from mice at day 5 p.i. with A/Philippines/2/82 (H3N2) virus ( n = 5). (A) IgG antibody levels in BALF. The antibody level was determined by ELISA using M2e peptide as a coating antigen. Lung viral titers (B), IFN-γ (C), and IL-6 (D) cytokines in BALF were determined at day 5 p.i. Lung viral titers were determined by an egg infection assay. IFN-γ and IL-6 were determined by a cytokine ELISA. Data represent mean ± SEM. Statistical significances were determined by 1-way ANOVA. Asterisks indicate significant differences (* p < 0.05 and ** p < 0.01) compared with the results in the naïve group. ns, not significant.

Article Snippet: Following washing again horseradish peroxidase (HRP)-labeled goat anti-mouse IgG, IgG1, and IgG2a (Southern Biotechnology, Birmingham, AL) were added to each well (1:2,000), and incubated for additional 2 h at 37°C.

Techniques: Enzyme-linked Immunosorbent Assay, Infection

Journal: PLoS ONE

Article Title: Cross Protection against Influenza A Virus by Yeast-Expressed Heterologous Tandem Repeat M2 Extracellular Proteins

doi: 10.1371/journal.pone.0137822

Figure Lengend Snippet: (A) IFN-γ-secreting cells in lungs. (B) IFN-γ-secreting cells in spleens. Lung cells and splenocytes were isolated from mice at day 5 p.i. ( n = 5). Cytokine-producing cell spots were counted by ELISPOT reader. Spleen (C) and bone marrow cells (D) were isolated from mice at day 5 p.i. and were incubated in the presence of M2e5x protein coated on the culture plates for in vitro stimulation. Culture supernatants were harvested after 1 or 5 days of culture. M2e-specific IgG levels were determined by ELISA. Data represent mean ± SEM. Statistical significances were determined by 1-way ANOVA. Asterisks indicate significant differences (* p < 0.05 and *** p < 0.001) compared with the results in the naïve group.

Article Snippet: Following washing again horseradish peroxidase (HRP)-labeled goat anti-mouse IgG, IgG1, and IgG2a (Southern Biotechnology, Birmingham, AL) were added to each well (1:2,000), and incubated for additional 2 h at 37°C.

Techniques: Isolation, Enzyme-linked Immunospot, Incubation, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: The systemic lupus erythematosus-associated NCF1 90H allele synergizes with viral infection to cause mouse lupus but also limits virus spread

doi: 10.1038/s41467-025-56857-z

Figure Lengend Snippet: a Timeline of the MNV infection experiment with male BALB/c. Ncf1 90H mice after transferring from the SPF facility to the MNV-infected facility (day 1). b The representative appearance of arthritis in the ankle of hind paws on day 49 and mean arthritis scores from days 1 to 56 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. c The levels of proteinuria ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. d The level of anti-dsDNA, anti-Sm/RNP, anti-phospholipid (anti-PLs) specific antibodies, and β2-GP1 on day 56 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. e Correlation with anti-MNV antibodies (OD): maximum arthritis score ( p = 0.0138), proteinuria ( p = 0.0049), anti-dsDNA antibodies ( p = 0.0055) in Ncf1 90H mice on day 56 post of MNV infection ( n = 8 per group). Data were analyzed using the Pearson correlation test. f Enlarged spleen and spleen index in mice ( n = 7/group). The spleen index is defined by the spleen weight (mg) divided by the body weight ( g ) and then multiplied by 10 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. g Representative H&E stained joint and kidney sections (magnification x 10). Histological scoring of joint and kidney inflammation using a scale of 0-3 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. h Immunofluorescence images (magnification x 20) and histology scores of deposits of IgG and C3 in the glomerulus from wild-type (WT) R90 and 90H mice on day 56 ( n = 4 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. i Relative expression of Ifnα and anti-viral ISGs ( Irf1 , Irf7 , Stat1 , Mx1 , Ip10 and Isg15 ) within spleens ( n = 4 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. j Relative expression of Ifnα and anti-viral ISGs ( Irf1 , Irf7 , Stat1 , Mx1 , Ip10 and Isg15 ) within kidneys. The expression of mRNAs was normalized to the housekeeping gene β-actin ( n = 4 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. k Immunoblot analysis of p-JAK1/JAK1 and p-STAT1/STAT1 proteins in the kidneys on day 56 after MNV infection ( n = 6 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM.

Article Snippet: After washing, biotinylated goat anti-mouse IgG (1:1000 dilutions, Southern Biotech, 1030-08) or IgG2b (1:1000 dilutions, Southern Biotech, 1090-08) was added to the wells and incubated at RT for 2 h. ExtrAvidin conjugated alkaline phosphatase (Sigma-Aldrich, E2636) diluted in PBS was added and incubated for 30–45 min. 100 μL of substrate BCIP/Nitroblue Tetrazolium (Sigma-Aldrich, B3804) per well (1 tablet in 10 mL ddH 2 O and pre-filtered with 0.45 μm) was added and incubated in the dark for 10–15 min.

Techniques: Infection, Transferring, Staining, Immunofluorescence, Expressing, MANN-WHITNEY, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: The systemic lupus erythematosus-associated NCF1 90H allele synergizes with viral infection to cause mouse lupus but also limits virus spread

doi: 10.1038/s41467-025-56857-z

Figure Lengend Snippet: a Timeline of MNV infection by gavage. b The quantification of MNV in the small intestine, colon, and spleen on day 7 by RT-qPCR ( n = 9 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. c Titer of anti-MNV antibodies on days 0, 14, 28, 56, 70, and 84 in females (MNV-R90: n = 6; MNV-90H: n = 13) and males (MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. d IgG production in femals (R90: n = 6; 90H: n = 13; MNV-R90: n = 6; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 6; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. e The correlation between IgG and anti-MNV antibodies in wild-type Ncf1 R90 mice ( n = 11) and Ncf1 90H mice ( n = 12). Data were analyzed using the Pearson correlation test. f Anti-ssRNA antibodies on day 56 in females (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13). Data were analyzed using one-way ANOVA and presented as mean ± SEM. g Anti-dsDNA antibodies on day 56 in females (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. h Anti-Sm/RNP antibodies on day 56 in females (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. i Proteinuria on day 56 in females (R90: n = 6; 90H: n = 13; MNV-R90: n = 6; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 6; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. j The ratio of Tcm or Tvm cells in CD4 + Foxp3 + in the PPs (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. k, l The ratio of Tfh cells and Tfr cells in CD4 + FOXP3 + cells (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. m The ratio of Trm cells in CD4 + CD44 hi cells (MNV-R90: n = 11; MNV-90H: n = 18). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. n The ratio of GC-B cells in IgD lo B cells (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. o The ratio of LLPCs in IgD lo CD138 + Sca-1 + cells (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM.

Article Snippet: After washing, biotinylated goat anti-mouse IgG (1:1000 dilutions, Southern Biotech, 1030-08) or IgG2b (1:1000 dilutions, Southern Biotech, 1090-08) was added to the wells and incubated at RT for 2 h. ExtrAvidin conjugated alkaline phosphatase (Sigma-Aldrich, E2636) diluted in PBS was added and incubated for 30–45 min. 100 μL of substrate BCIP/Nitroblue Tetrazolium (Sigma-Aldrich, B3804) per well (1 tablet in 10 mL ddH 2 O and pre-filtered with 0.45 μm) was added and incubated in the dark for 10–15 min.

Techniques: Infection, Quantitative RT-PCR, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: The systemic lupus erythematosus-associated NCF1 90H allele synergizes with viral infection to cause mouse lupus but also limits virus spread

doi: 10.1038/s41467-025-56857-z

Figure Lengend Snippet: a Timeline of MNV infection in male mice by i.v. and i.p. injection. b Anti-MNV antibodies on days 0, 14, 28, and 56. c Anti-ssRNA antibodies on day 21 (R90: n = 6; 90H: n = 6; MNV-R90: n = 11; MNV-90H: n = 12). Data were analyzed using one-way ANOVA and presented as mean ± SEM. d IgG production on day 21 (R90: n = 8; 90H: n = 12; MNV-R90: n = 11; MNV-90H: n = 12). Data were analyzed using one-way ANOVA and presented as mean ± SEM. e Representative paws on day 21. f Mean arthritis score (MNV-R90: n = 8; MNV-90H: n = 11). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. g Arthritis incidence ( n = 9 per group). h Representative HE staining of the joints ( n = 4, magnification x 2.5 and x 10). i Anti-collagen II (COL2) IgG antibodies (R90: n = 7; 90H: n = 7; MNV-R90: n = 11; MNV-90H: n = 20). Data were analyzed using one-way ANOVA and presented as mean ± SEM. j Anti-COL2 IgG2b antibodies (R90: n = 7; 90H: n = 7; MNV-R90: n = 9; MNV-90H: n = 20). Data were analyzed using one-way ANOVA and presented as mean ± SEM. k Anti-COL2 IgM antibodies (R90: n = 8; 90H: n = 11; MNV-R90: n = 11; MNV-90H: n = 18). Data were analyzed using one-way ANOVA and presented as mean ± SEM. The level of anti-COL2 antibodies was measured in mouse sera on day 21 by ELISA. l , m Representative B cell-ELISpot and statistics of total anti-COL2 IgG and IgM ASCs in the spleen were shown (R90: n = 5; 90H: n = 5; MNV-R90: n = 8; MNV-90H: n = 12). Data were analyzed using one-way ANOVA and presented as mean ± SEM.

Article Snippet: After washing, biotinylated goat anti-mouse IgG (1:1000 dilutions, Southern Biotech, 1030-08) or IgG2b (1:1000 dilutions, Southern Biotech, 1090-08) was added to the wells and incubated at RT for 2 h. ExtrAvidin conjugated alkaline phosphatase (Sigma-Aldrich, E2636) diluted in PBS was added and incubated for 30–45 min. 100 μL of substrate BCIP/Nitroblue Tetrazolium (Sigma-Aldrich, B3804) per well (1 tablet in 10 mL ddH 2 O and pre-filtered with 0.45 μm) was added and incubated in the dark for 10–15 min.

Techniques: Infection, Injection, MANN-WHITNEY, Two Tailed Test, Staining, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal:

Article Title: Productive Infection Maintains a Dynamic Steady State of Residual Viremia in Human Immunodeficiency Virus Type 1-Infected Persons Treated with Suppressive Antiretroviral Therapy for Five Years

doi: 10.1128/JVI.77.20.11212-11219.2003

Figure Lengend Snippet: Baseline characteristics and viral kinetics of patients receiving indinavir and efavirenz ( n = 15) and HIV RNA pre- and post-therapy intensification ( n = 9) a

Article Snippet: Because these epitopes can also be recognized by HLA class II molecules ( 10 ), IFN-γ production by CD4 + was blocked by preincubating PBMC with 100 ng of neutralizing recombinant human CD4 monoclonal antibody (R&D Systems, Minneapolis, Minn.)/ml.

Techniques:

Journal:

Article Title: Productive Infection Maintains a Dynamic Steady State of Residual Viremia in Human Immunodeficiency Virus Type 1-Infected Persons Treated with Suppressive Antiretroviral Therapy for Five Years

doi: 10.1128/JVI.77.20.11212-11219.2003

Figure Lengend Snippet: HIV DNA before treatment with indinavir plus efavirenz correlates with residual viremia. Residual HIV RNA was defined as the geometric mean of all measured values after 9 months of therapy. Baseline DNA was the only significant pretreatment predictor of residual viremia in either univariate (R2 = 0.51, P = 0.003) or multivariate (P = 0.002) regression including baseline HIV RNA, CD4 counts, and patient age. These correlations remained when HIV DNA was normalized to cell counts, when residual viremia was computed using a maximum likelihood approach with censoring, and when nonparametric statistical methods were used. While the correlation does not define the mechanism connecting HIV DNA and residual RNA, the predictive value of pretreatment DNA levels suggests that long-lived cellular reservoirs of viral DNA activate to kindle HIV RNA production.

Article Snippet: Because these epitopes can also be recognized by HLA class II molecules ( 10 ), IFN-γ production by CD4 + was blocked by preincubating PBMC with 100 ng of neutralizing recombinant human CD4 monoclonal antibody (R&D Systems, Minneapolis, Minn.)/ml.

Techniques:

Journal:

Article Title: Productive Infection Maintains a Dynamic Steady State of Residual Viremia in Human Immunodeficiency Virus Type 1-Infected Persons Treated with Suppressive Antiretroviral Therapy for Five Years

doi: 10.1128/JVI.77.20.11212-11219.2003

Figure Lengend Snippet: Significant changes in cellular activation markers observed for nine patients undergoing intensification of antiretroviral therapy with abacavir and four control patients not receiving abacavir a

Article Snippet: Because these epitopes can also be recognized by HLA class II molecules ( 10 ), IFN-γ production by CD4 + was blocked by preincubating PBMC with 100 ng of neutralizing recombinant human CD4 monoclonal antibody (R&D Systems, Minneapolis, Minn.)/ml.

Techniques: Activation Assay, Control, Marker, Enzyme-linked Immunospot

Journal: Frontiers in immunology

Article Title: Evaluation of Immune and Vaccine Competence in Steroid-Sensitive Nephrotic Syndrome Pediatric Patients.

doi: 10.3389/fimmu.2021.602826

Figure Lengend Snippet: FIGURE 2 | Total and antigen-specific IgG-secreting B cells in steroid-sensitive nephrotic syndrome pediatric patients at onset. (A–E) Isolated PBMCs were stimulated for 5 days with CpG plus rhIL-21 and rhIL-4. Following stimulation, (A) total, (B,C) anti-tetanus and (D,E) anti-HBV IgG-secreting B cells were enumerated by ELISPOT in steroid-sensitive nephrotic syndrome pediatric patients at disease onset (SSNS, n=11) and in age-matched controls (CTRL, n=5). Antigen-specific memory B cells were represented as (B,D) absolute count/106 cells and as (C,E) percentage of total IgG-secreting B cells. Each plot represents a different patient. Horizontal lines indicate the means and differences between groups were compared using the unpaired t test.

Article Snippet: For the simultaneous detection of IgM and IgG we used the Human IgG/IgM Dual-Color B Cell ELISpot Kit (R&D System).

Techniques: Isolation, Enzyme-linked Immunospot