The very low level antibody-secretion by peritoneal cavity B-1 cells further indicates that they exist as “partially” activated/ differentiated cells, distinct from B-2 cells. Such partial activation might explain their rapid differentiation to antibody-producing cell following stimulation via cytokines or mitogens
34, 36–39 and is consistent with their phenotypic signs of activation, such as their larger size and constitutive expression of co-stimulatory molecules 55. The signals that induce and regulate natural IgM-secretion by spleen and BMS-907351 supplier BM B-1 cells are currently unknown. LPS-mediated differentiation of PerC B-1 cells in vitro does not seem to recapitulate the differentiation events leading to the appearance of natural IgM-producing cells in vivo, as such treatment rapidly induces BLIMP-1 expression by B-1 and B-2 cells (35 and our unpublished observations.). Spontaneous IgM-secreting B-1 cells in both spleen and BM do not appear to express high levels of BLIMP-1 (our unpublished observations). This might suggest Afatinib ic50 that B-1 cells secreting natural IgM at stimulation-independent steady-state levels differ from B-1 cells that contribute the enhanced IgM secretion following infection or mitogenic stimulation. Having identified here a distinct population of BM B-1 cells that generate steady-state natural IgM should help to answer this question
and aid the elucidation of the regulatory mechanisms underlying natural IgM secretion. Six to 12-week-old female C.B-17 (Taconic Farms, Germantown, NY, USA), BALB/c, C57BL/6, RAG-1−/− (C57BL/6) and pregnant female C.B-17 mice (The Jackson Laboratory, Bar Harbor, ME, USA) were purchased. All mice were kept under conventional housing conditions in microisolator cages for the duration of the experiments. Mice were used at 6–12 weeks of age or used to generate Ig-allotype-chimeric mice. All procedures and experiments were approved by the Animal Adenosine Use and Care Committee of the University of California, Davis. Allotype-chimeras of mice that harbor different Ig-allotype for B-1 (Igh-a) and B-2 (Igh-b) cells were generated as previously described 26. Briefly, on day 1 after
birth, 0.1 mg of anti-IgMb (AF6-78.2.5) antibody, purified by Hi-Trap Affinity Protein G Column (Amersham Biosciences, Piscataway, NJ, USA) from serum-free tissue culture supernatants was injected i.p. into newborn C.B-17 mice (Igh-b) to deplete host B (Igh-b) cells. On day 2 after birth, peritoneal cavity (PerC) washout cells from 2-month-old congenic BALB/c (Igh-a) mice were transferred i.p. into C.B-17 mice. Previous studies established that transfer of FACS-purified live CD3/4/8/ F4/80 and GR-1 negative CD19hi CD23− CD43+ cells gave the same chimera results as achieved with peritoneal cavity transfer, i.e. that only donor-derived Igh-a-expressing B-1 but not B-2 cells were found in host mice after full reconstitution of host B cells.