Figure 1. A reporter mouse marks activated B-1a cells.

(A) Schematic of targeted insertion of T2A-Cre into the Ighg3 (Iγ3) heavy chain locus to generate the Ighg3^T2A-Cre reporter mouse. (B) Representative flow cytometry plot of IgG3 and IgM expression on pregated CD19⁺Tomato⁺ splenocytes from 6 wk old Ighg3^T2A-Cre mice crossed to Rosa26^{STOP-flox-TdTomato} mice (Ighg3^{T2A-Cre:TdTomato}). (C) Percentage Tomato expression in B cell subsets from the peritoneal cavity (PerC) and spleen (Spl) of 6 wk old Ighg3^{T2A-Cre:TdTomato} mice by flow cytometry. Total B cells defined as CD19⁺; B-2 cells defined as CD19⁺CD23⁺CD43^–CD5^–; B-1a cells defined as CD19⁺CD23^–CD43⁺CD5⁺. (D) Representative flow cytometry gating of Tomato^– and Tomato⁺ splenic B-1a cells from 3 wk old Ighg3^{T2A-Cre:TdTomato} mice, pregated on Live/CD19⁺CD23^– cells. (E) Representative histogram and quantification of FSC-A and (F) IgD expression on pre-gated Tomato^– (black) and Tomato⁺ (red) splenic B-1a cells. (G) Number of IgM⁺ antibody secreting cells (ASCs)/10⁶ present in purified Tomato^– (black) or Tomato⁺ (red) splenic (closed circles) or peritoneal cavity (PerC) (open circles) B-1a cells from 6 wk old Ighg3^{T2A-Cre:TdTomato} mice, as measured by ELISpot. (H) Serum IgM titers of 7 wk old C57BL/6 (black), Ighg3^{T2A-Cre:TdTomato} (red) and Ighg3^T2A-Cre mice crossed to Rosa26^{STOP-flox-DTA} (Ighg3^T2A-Cre:DTA) (blue) mice. Error bars indicate the mean (± SEM). *p<0.05, **p<0.01, and ***p<0.001 (unpaired two-tailed Student's t-test). Each data point represents an individual mouse (C, E-H). Data are representative of at least three independent experiments (B-G) or pooled from five independent experiments (H).

Figure 1—figure supplement 1. Validation of correct targeting of knock-in mouse.

(A) Southern blot strategy to identify targeted insertion of T2A-Cre after the last transmembrane exon of Ighg3 (Iγ3) using DNA probes 5’ of Ighg3 (5‘ probe) and to the neomycin-resistance gene (Neo probe). (B) Southern blot of BglII restriction-digested ES cell DNA from clone D6, which was used to generate the Ighg3^T2A-Cre mouse using a 5’ probe to detect 10.5 Kb band corresponding to WT DNA and a 8.4 Kb band corresponding to targeted insertion of T2A-Cre. (C) Southern blot of BglII restriction-digested ES cell DNA from clone D6 using a Neo probe to detect a single 5.5 Kb band indicating a single copy, targeted insertion. Data are representative of at least two independent experiments.

Figure 1—figure supplement 2.. TdTomato expression in IgM+ cells correlates with Ighg3 germ-line transcription in Ighg3 reporter mouse.

(A) IgM expression on sorted IgM⁺Tomato⁺ splenocytes (red histogram) compared to Tomato^–CD19⁺ splenocytes (black histogram) after 48hr ex vivo culture, as measured by flow cytometry. (B) Splenocytes from 3 wk old Ighg3^{T2A-Cre:TdTomato} mice were stimulated with LPS for 72 hr, followed by single-cell sorting and subsequent RT-PCR. (C) Schematic of murine heavy chain locus depicting the generation of Ighg3 (Iγ3) germ-line transcript (GLT) prior to AID-mediated class switch recombination from IgM to IgG3. (D) RT-PCR of single-cell sorted IgG3^–IgM⁺Tomato⁺ or IgG3⁺IgM^–Tomato⁺ cells, as described in (B), for Ighm mRNA and Ighg3 mRNA, visualized by agarose gel electrophoresis. Arrows indicate primer binding sites. (E) Single-cell RT-PCR of Ighg3 germ-line transcript (GLT) and Ighm mRNA of IgG3^–IgM⁺Tomato⁺ as described in (B), visualized by agarose gel electrophoresis. Arrows indicate primer binding sites. (F) Serum IgG3 titers of 7 wk old Ighg3^{T2A-Cre:TdTomato} (black) and Ighg3^T2A-Cre mice crossed to Rosa26^{STOP-flox-DTA} (Ighg3^T2A-Cre:DTA) (blue) mice.(G) Expression of IgM versus IgG3 protein on 3-day in vitro LPS-stimulated splenocytes from Ighg3^{T2A-Cre:TdTomato} and Ighg3^-/- mice (top panel), as measured by flow cytometry. IgD and Tomato expression on pregated IgM⁺ in vitro stimulated B cells (bottom panel). FSC-A of pregated IgM⁺IgD⁺Tomato^– (gray histogram), IgM⁺IgD⁺Tomato^– (black histogram), and IgM⁺IgD^–Tomato⁺ (red histogram) LPS-stimulated Ighg3^{T2A-Cre:TdTomato} splenocytes. Error bars indicate the mean (± SEM). *p<0.05, **p<0.01, and ***p<0.001 (unpaired two-tailed Student's t-test). Data are representative of at least two independent experiments.

Figure 1—figure supplement 3. Single-cell RT-PCR Primers.

(A) Table summarizing the forward (5’) and reverse (3’) primers names and sequences for single-cell semi-nested RT-PCR of Ighm mRNA, Ighg3 mRNA, and Ighg3 germ-line transcript (GLT).

Figure 1—figure supplement 4. B cell development in bone marrow is unaltered in reporter mouse.

(A) Representative flow cyometry gating of B cell subsets in the bone marrow of 7 wk old C57BL/6 (black), Ighg3^{T2A-Cre:TdTomato} (red), and Ighg3^T2A-Cre:DTA (blue) mice. Pre-B, Late Pro-B and, Early Pro-B cells were pregated as B220⁺IgM^– and identified according to their expression of CD19 and CD43. Mature and Immature B cells were pregated as B220⁺IgM⁺ and identified according to their expression of IgM and IgD. (B) Absolute counts of B220⁺ cells in the bone marrow from one femur in 7 wk old C57BL/6 (black), Ighg3^{T2A-Cre:TdTomato} (red), and Ighg3^T2A-Cre:DTA (blue) mice. (C) Absolute counts of Early Pro-B, Late Pro-B, Pre-B, Immature, and Mature B cells in the bone marrow from one femur in 7 wk old C57BL/6 (black), Ighg3^{T2A-Cre:TdTomato} (red), and Ighg3^T2A-Cre:DTA (blue) mice. (D) Representative flow cytometry dot plot showing the expression of CD19 and CD43 on pregated B220⁺ bone marrow cells (gray) overlayed with pregated Tomato⁺B220⁺ bone marrow cells (red) from Ighg3^{T2A-Cre:TdTomato} mice (left); representative flow cytometry dot plot showing the expression of IgD and IgM on pregated B220⁺IgM⁺ bone marrow cells (gray) overlayed with pregated Tomato⁺B220⁺IgM⁺ bone marrow cells (red) from Ighg3^{T2A-Cre:TdTomato} mice (middle); representative flow cytometry dot plot showing the expression of IgD and CD43 on pregated ‘immature’ B220⁺IgM⁺IgD^– bone marrow cells (gray) overlayed with pregated ‘immature’ Tomato⁺B220⁺IgM⁺IgD^– bone marrow cells (red) from Ighg3^{T2A-Cre:TdTomato} mice (right). (E) Percentage Tomato expression in various bone marrow B cell subsets in 7 wk old Ighg3^{T2A-Cre:TdTomato} mice. Each data point represents an individual mouse. Data are representative of two independent experiments.

Figure 1—figure supplement 5. Characterization of B cell subsets in the spleen and peritoneal cavity of reporter mice.

(A) Absolute counts of CD19⁺ and CD19⁺CD23⁺ Follicular (FO) B cells from the spleens of 7 wk old C57BL/6 (black), Ighg3^{T2A-Cre:TdTomato} (red), and Ighg3^T2A-Cre:DTA (blue) mice. (B) Absolute counts of marginal zone B (MZ) (CD19⁺CD23^–CD21⁺), B-1a (CD19⁺CD23^–CD23^–CD43⁺CD5⁺), and B-1b (CD19⁺CD23^–CD23^–CD43⁺CD5^–) cells in the spleens of 7 wk old C57BL/6 (black), Ighg3^{T2A-Cre:TdTomato} (red), or Ighg3^T2A-Cre:DTA (blue) mice. (C) Percentage Tomato expression in various B cell subsets, as described in (B), in the spleens of 7 wk old Ighg3^{T2A-Cre:TdTomato} mice. (D) Representative flow cytometry plot characterizing expression of CD43 and CD19 on pregated Tomato⁺CD19⁺IgM^–IgG3^– splenocytes from a 7 wk old Ighg3^{T2A-Cre:TdTomato} mice. (E) Absolute counts of CD19⁺, B-1a (CD19⁺CD23^–CD23^–CD43⁺CD5⁺), and B-1b (CD19⁺CD23^–CD23^–CD43⁺CD5^–) cells in the peritoneal cavities (PerC) of 7 wk old C57BL/6 (black), Ighg3^{T2A-Cre:TdTomato} (red), or Ighg3^T2A-Cre:DTA (blue) mice. (F) Percentage Tomato expression in various B cell subsetsm as described in (E), in the peritoneal cavity (PerC) of 7 wk old Ighg3^{T2A-Cre:TdTomato} mice. Each data point represents an individual mouse. Data are representative of two (A, B, E) or at 5 (C, D, F) independent experiments.