Elisas: Ig Serum Concentrations Were Determined by ELISA As Previously Described

Experimental Procedures

Mice. Rag2KOcKO, Hox11KO, J-chain deficient, C57Bl/6 and Balb/c animals were breed and maintained in SPF conditions at the Istituto Regina Elena (Roma, Italy).

Adoptive transfers. Rag2KOgcKO recipient mice aged 8-14 weeks were sublethally irradiated (3Gy) and injected intravenously with either sorted marginal zone B cells (B220posCD23negCD21bright) (6,3x104cells/mouse), transitional B cells (B220posCD21neg) (13,2x104cells/mouse) or follicular B cells (B220posCD23posCD21pos) (4,6x105cells/mouse). Two months after transplantation all animals were killed and bone marrow, spleen and peritoneal cavity wash single cell suspensions analyzed by FACS as described in the manuscript, section material and methods. Intestine specimens were frozen and stained as described in the manuscript.

Confocal Microscopy: Intestinal tissue samples were collected and immediately frozen in liquid nitrogen, and kept at -80°C. Multiple 5mm cryostat sections included in cryostat embedding medium (Bio-Optica, Milan, Italy) were fixed in cold acetone, washed with PBS (Sigma) and incubated for 45 min with FITC anti-mouse-IgA (clone C10-3) (BD Biosciences) or biotin anti-mouse J-chain (gift from Tomas Leanderson) followed by straptavidin-TRITC (Jackson Immunoresearch). Sections were analysed in a co-focal microscope (Olympus FV1000) and images acquired at 60x amplification.

Cell sorting. Spleen single cell suspensions from C57Bl/6 were stained for CD21, CD23 and B220 and sorted by FACS (FACSvantage SE interfaced to a PC FACSDigitalVantage 2.1.2 (Becton and Dickinson, Sunnyvale, California, U.S.A.)) for marginal zone B cells (B220posCD23negCD21bright), transitional B cells (B220posCD21neg) or follicular B cells (B220posCD23posCD21pos). Cell purity was >95%.

Peritoneal cavity washes of C57Bl/6 mice were stained for B220, CD5, IgM and IgD and B-1a cells sorted by FACS. 2x105 cells were injected i.p. in Hox11KO, n=3.

ELISAs: Ig serum concentrations were determined by ELISA as previously described. Briefly, serum antibodies were captured on F(ab’)2 Fragment Goat anti-mouse IgG+IgM (H+L) and purified anti-mouse IgA (clone C10-3) and detected with HRPO-conjugated goat anti-mouse IgM m-specific, goat anti-mouse IgG Fcg-fragment specific (Jackson Immunoresearch Laboratories) and biotin anti-mouse IgA (clone C10-1) (Pharmingen, San Diego, CA, USA) followed by peroxidase-conjugated streptavidin (Jackson Immunoresearch Laboratories), respectively. Serum dilutions used to calculate Ab titers were determined from OD450 values within the linear range of the standard curve by comparison to values obtained from dilutions of mouse IgMk anti-TNP (clone G155-228) (Pharmingen, San Diego, CA, USA), mouse IgG whole molecule (Jackson Immunoresearch Laboratories) and mouse IgA isotype (clone S107) (Southern Biotechnology Associates, Birmingham, AL).