RA and TGF-β treated PEC B1 cells migrate to spleen and gut and produce serum and intestinal secretory IgA.

<p>IgA^- PEC B1a and B1b cells sorted in a fashion similar to previous experiments were cultured under various IgA CSR inducing conditions for 3 days and transferred intraperitoneally (16000 live cells/mouse) into lymphopenic Rag1^-/- recipients. (A) Unstimulated splenocytes and 2 days LPS (25 µg/ml) stimulated PECs of the recipient mice were analyzed for the presence of IgA and IgM producing cells by ELISPOT 2 weeks after adoptive B cell transfer. (B) Levels of secretory Igs in the serum and gut lavage of recipient mice were determined by ELISA. Per group, 2-3 mice were used as recipients. Cells pooled from the recipients belonging to the same group were used for ELISPOT assay and it was done in triplicates. Data show the results of one of two independent experiments. Bars represent mean ± SD.</p

RA treatment leads to up-regulation of gut homing molecules on the surface of cultured PEC B cells.

<p>Expression of surface α4β7 and CCR9 on IgA^- PEC (A) or splenic (B) B cells sorted in fashion similar to previous experiments was checked by flow cytometry after four days of culture under various combinations of stimulatory conditions. Using the same cultures (as in A and B), expression of surface IgA and IgM on PEC (C) and splenic (D) B cells was checked by flow cytometry. Data show the results of one of two independent experiments.</p

TGF-β enhances IgA switching among PEC B1b cells <i>in vitro</i>.

<p>Results of IgA and IgM ELISPOT assays performed in triplicates on sort purified PEC B cell subpopulations after 4 days of treatment with the indicated combination of stimulatory factors in culture. Surface IgA⁺ cells were excluded by sorting from B1a (IgA^-CD19^hiCD5⁺CD43⁺Mac-1⁺), B1b (IgA^-CD19^hiCD5^-CD43⁺Mac-1⁺) and B2 (IgA^-CD19^loCD5^-CD43^-Mac-1^-) B cell subpopulations. Data show the results of one of three independent experiments. Bars represent mean ± SD.</p

PEC and splenic B1 cell derived IgA VH sequences display high frequencies of nucleotide exchanges due to somatic hypermutation.

<p>Frequency of mutations observed in the IgM or IgA VH region sequences derived from PEC and splenic B1 cells cultured for 4 days with the indicated combinations of IgA CSR inducing factors. (A) Each colored sector represents a particular number of mutations found at a particular frequency. Total number of sequences is shown in the middle. (B) Number of replacement and silent mutations amongst IgM and IgA VH sequences derived from indicated B cell populations. Sequences were analyzed using SEQUENCHER™ Version 4.1 for Macintosh software (Gene Codes Corporation). Assignment of VH chain was done using VBASE2 database (<a href="http://www.vbase2.org/" target="_blank">http://www.vbase2.org/</a>). Only functional sequences were included for the mutation analysis. Bars represent mean ± SD.</p

TGF-β in combination with RA synergistically induces IgA switching by PEC B cells.

<p>(A) Results of IgA and IgM ELISPOT assays performed in triplicates on sort purified PEC B cell subpopulations after 4 days of treatment with the indicated combination of stimulatory factors in culture. Surface IgA⁺ cells were excluded by sorting from B1a (IgA^-CD19^hiCD5⁺CD43⁺Mac-1⁺), B1b (IgA^-CD19^hiCD5^-CD43⁺Mac-1⁺) and B2 (IgA^-CD19^loCD5^-CD43^-Mac-1^-) B cell subpopulations. (B) Amount of secretory Igs determined by ELISA in the supernatant of B cells cultured for 4 days under the indicated stimulatory conditions. Data show the results of one of two independent experiments. Bars represent mean ± SD.</p

Increase in cell numbers after 4 days of culture under various conditions.

<p>¹ Sorted IgA^- PEC and splenic B cells were cultured under different conditions for 4 days and the fold increase in cell number was calculated by dividing the cell count after 4 days by the number of cells at the beginning of the cell culture. The values represent mean ± SD calculated from three independent culture experiments.</p