Journal: Scientific Reports

Article Title: Functionally Distinct Subpopulations of CpG-Activated Memory B Cells

doi: 10.1038/srep00345

Figure Lengend Snippet: We sorted normal human mBc at 60 hrs of stimulation with CpG+CK and placed isolated CD27 hi , CD27 lo , and undivided cells back into culture with fresh CpG+cytokines. Flow cytometric analysis 48 hrs later showed that CD27 hi cells remained CD27 hi , while CD27 lo cells became CD27 hi . This supports the hypothesis that CD27 lo cells are a transient population which differentiate into CD27 hi cells. N = 4 subjects in 3 separate experiments.

Article Snippet: Freshly isolated CD27+ IgG enriched human peripheral blood Bc were cultured in the presence of CpG 2006 (10 ng/ml, Oligos, etc., Wilsonville, OR), plus recombinant human cytokines IL-2 (20 IU/ml), IL-10 (50 ng/ml), IL-15 (10 ng/ml) (all from BD Biosciences, San Diego, CA), and recombinant human BAFF (75 ng/ml, Chemicon, Temecula, CA) PC-L medium (IMDM medium, lacromin (50µg/ml, Seracare, Milford, MA), insulin (5µg/ml, Sigma-Aldrich, St. Louis, MO), penicillin/streptomycin (1x, Invitrogen, Carlsbad, CA), gentamicin (15µg/ml, Invitrogen), heat-inactivated fetal bovine serum (10% v/v, Invitrogen), normocin (0.1% v/v, Invivogen, San Diego, CA) in round-bottomed 96-well plates (BD Biosciences, San Diego, CA).

Techniques: Isolation

Journal: Scientific Reports

Article Title: Functionally Distinct Subpopulations of CpG-Activated Memory B Cells

doi: 10.1038/srep00345

Figure Lengend Snippet: The same stimulation conditions can result in either CD27 lo or CD27 hi phenotypes and progression from one to the other. CpG, through TLR9, can activate ΝΦ−κΒ, a marker of receptor editing in Bc. Gene targets of NF-κB include several genes shown to be upregulated in CD27 lo cells (in gold), including AICDA. BAFF (TNFSF13) also has been shown to increase AICDA production. In a separate pathway, Jak proteins, including Jak3 can be activated through action of the common gamma chain cytokines, IL2 or IL-15, resulting in the phosphorylation of STAT proteins such as STAT3, STAT5, and STAT6. IL-10 can also activate STAT3. STAT3, by itself or with STAT6 can increase production of IRF4, a key regulator of Bc differentiation. Induction of IRF4 at low levels can stimulate the production of AICDA, a product of NF-κB and at higher levels, IRF-4 induces production of PRDM-1, a key plasmablast regulator. Thus, as IRF-4 levels increase cells may progress from CD27 lo phenotype to a CD27 hi phenotype. NF-κB can also activate transcription of SPIB which negatively regulates PRDM1 (BLIMP1). PRDM1 then represses BCL6 and AICDA. PRDM1, STAT6, and IRF4 can also enhance production of XBP1 which is necessary for immunoglobulin production. HSPA5 (BiP), a mediator of immunoglobulin folding is also a target of XBP1 and is upregulated in CD27 hi cells.

Article Snippet: Freshly isolated CD27+ IgG enriched human peripheral blood Bc were cultured in the presence of CpG 2006 (10 ng/ml, Oligos, etc., Wilsonville, OR), plus recombinant human cytokines IL-2 (20 IU/ml), IL-10 (50 ng/ml), IL-15 (10 ng/ml) (all from BD Biosciences, San Diego, CA), and recombinant human BAFF (75 ng/ml, Chemicon, Temecula, CA) PC-L medium (IMDM medium, lacromin (50µg/ml, Seracare, Milford, MA), insulin (5µg/ml, Sigma-Aldrich, St. Louis, MO), penicillin/streptomycin (1x, Invitrogen, Carlsbad, CA), gentamicin (15µg/ml, Invitrogen), heat-inactivated fetal bovine serum (10% v/v, Invitrogen), normocin (0.1% v/v, Invivogen, San Diego, CA) in round-bottomed 96-well plates (BD Biosciences, San Diego, CA).

Techniques: Marker