Gating strategy for the identification of antigen-specific mouse memory B cells from IL-25 immunised mice.

<p>Following cell enrichment using CD45R microbeads (Miltenyi Biotech), cells were analysed in a BD FACS ARIA III. A gate was drawn around the lymphocyte population (gated population represented 47.6% of events) (A). FSC-W and FSC-A were then used to eliminate doublets (gated population represented 99% of events) (B). T cells, macrophages, neutrophils and 7AAD⁺ dead cells were eliminated in the “dump channel” (gated population represented 95.8% of events) (C). CD19⁺ B cells were identified (gated population represented 99% of events) (D). IgG⁺/IgM^- B cells were then gated on (gated population represented 1.49% of events) (E). To further eliminate naïve B cells, IgD staining allowed gating for IgG⁺/IgD^- cells, (gated population represented 91% of events) (F). Finally, dual-colour antigen staining allowed a gate (P1) to be drawn around the double-positive population (gated population P1 represented 0.283% of events) (G). Single cells from gate P1 were sorted into a 96-well plate for single-cell RT-PCR.</p

Schematic representation of the single-B cell sorting protocol used for antibody discovery from immunised mice.

<p>The following steps were undertaken: 1. Mice were immunised and a splenocyte suspension prepared. 2. B cells from the mouse splenocyte preparation were enriched using anti-mouse CD45R microbeads (Miltenyi Biotech) and LS MACS columns (Miltenyi Biotech) according to manufacturers’ instructions. 3. Following enrichment, cells were stained with the following antibodies: rat anti-mouse IgG brilliant violet 421 (BD biosciences), rat anti-mouse IgM PE-Cy7 (Bio legend), rat anti-mouse IgD APC-Cy7 (Bio legend), rat anti-mouse CD19 AF700 (BD biosciences) (2 μg per 108 Cells), and rat anti-mouse CD4, CD8, GR1 and F4/80 FITC (BD biosciences)(dump channel). Human TNFR2 extracellular domain was labelled with PE and APC using Lightning Link PE and APC labelling kits (Innova Bioscience) and added to the cell suspension. 4. FACS was performed on a BD FACS ARIA III with single human TNFR2-specific IgG⁺ B cells being deposited into the well of a 96-well PCR plate. 5. cDNA from single B cells was prepared using Superscript III reverse transcriptase (Invitrogen) primed with oligo (dT). Antibody variable-region genes were then recovered via two rounds of PCR followed by a third round to generate transcriptionally-active PCR (TAP) products in a manner similar to that described in Clargo <i>et al</i>.⁹ employing an Aviso Onyx liquid handling robot to facilitate set-up. 6. Heavy and light chain TAP fragments were transiently co-transfected into Expi293 cells using ExpiFectamine (Life Technologies). After 7 days expression, supernatants were harvested for further characterisation.</p

Gating strategy for identification of antigen-specific mouse memory B cells from TNFR2 immunised mice.

<p>Following cell enrichment using CD45R microbeads (Miltenyi Biotech), cells were analysed in a BD FACS ARIA III. A gate was drawn around the lymphocyte population (gated population represented 34.9% of events) (A). FSC-W and FSC-A were then used to eliminate doublets (gated population represented 95.1% of events) (B). T cells, macrophages, neutrophils and 7AAD⁺ dead cells were eliminated in the “dump channel” (gated population represented 95.4% of events) (C). CD19⁺ B cells were identified (gated population represented 97.2% of events) (D). IgG⁺/IgM^- B cells were then gated on (gated population represented 0.899% of events) (E). To further eliminate naïve B cells, IgD staining allowed gating for IgG⁺/IgD^- cells (gated population (gate P1) represented 96.3% of events) (F). In order to demonstrate the importance of the antigen staining step, we sorted single cells from the total IgG⁺/IgD^- cell population (gate P1). Finally, dual-colour antigen staining allowed a gate (P2) to be drawn around the double-positive antigen-specific population (gated population P2 represented 0.95%of events) (G). Single cells from gate P2 were sorted into a 96-well plate for single-cell RT-PCR.</p

Summary of recombinant IgG expression and antigen binding activity of antibodies derived from the IL-25-specific IgG⁺ B cell population.

<p>Summary of recombinant IgG expression and antigen binding activity of antibodies derived from the IL-25-specific IgG⁺ B cell population.</p

Diversity assessment of mouse anti-human IL-25 antibodies.

<p>For each antibody, VH CDR3 sequences were aligned in a pairwise manner to generate a sequence distance value. We performed a Principal Components Analysis (PCA)³⁴ as a means to reduce down the dimensionality of this data and generate an easy to interpret 2-dimensional data plot that illustrated the extent of diversity in our recombinant antibody panel. Data for principle component (PC) 1 and 2 are shown on the X and Y axis respectively. Clustering analysis was performed and families of closely related sequences were assigned on the basis of sequence identity in the VH CDR3 regions of 80% or higher. Each separate antibody family has been represented by a unique colour.</p

Summary of recombinant IgG expression and binding recoveries.

<p>Summary of recombinant IgG expression and binding recoveries.</p

Summary of antibody reagents used for identification of IgG⁺ antigen-specific mouse B cells.

<p>Summary of antibody reagents used for identification of IgG⁺ antigen-specific mouse B cells.</p

Summary of recombinant IgG expression and antigen binding activity of antibodies derived from the total IgG⁺ population and the TNFR2-specific subset of cells.

<p>Summary of recombinant IgG expression and antigen binding activity of antibodies derived from the total IgG⁺ population and the TNFR2-specific subset of cells.</p

Recombinant IgG concentration range produced from IL-25 B cell sorting experiment.

<p>Heavy and light chain TAP fragments produced from single-sorted B cells were transiently co-transfected into Expi293 using ExpiFectamine (Life Technologies). After 7 days expression, supernatants were harvested and the concentration of mouse IgG measured using an sandwich ELISA with a purified mouse IgG standard. Data is shown for all wells that produced measurable antibody production. Green bars indicate those that were determined to bind IL-25 and red and black-check bars represent those which did not show binding to IL-25.</p

Gating strategy for identification of antigen-specific rabbit memory B cells.

<p>Cells were analysed in a BD FACS ARIA III. A gate was drawn around the lymphocyte population (gated population represented 59.8% of events) (A). FSC-W and FSC-A were then used to eliminate doublets (gated population represented 95.7% of events) (B). 7AAD⁺ dead cells and T cells were eliminated in the “dump channel” (gated population represented 97.1% of events) (C). IgG⁺/ IgM^- B cells were identified and gated on (gated population represented 2.67% of events) (D). Finally, a gate (P1) was drawn around the double-positive mWISP-1 antigen-specific population (gated population represented 0.262% of events) (E). Cells from gate P1 were sorted into a 96-well PCR plate at either one or three cells per well for subsequent RT-PCR.</p