We have previously reported that B cells that are activated by multivalent but not bivalent membrane Ig cross-linking ligands synergize with various B cell activators culminating in enhanced B cell proliferation. In this study we asked whether B cells that are activated by a multivalent mIg cross-linking agonist could respond to oligodeoxynucleotides (ODN) containing non-stimulatory motifs. Earlier reports have shown that ODN containing a CpG motif in which the cytosine is unmethylated and is flanked by two 5' purines and two 3' pyrimidines induce high levels of B cell activation, while ODN whose CpG are methylated or flanked by sequences other than the optimal two 5' purines and two 3' pyrimidines were non-stimulatory. In this manuscript we show that when B cells are stimulated in vitro with dextran-conjugated anti-IgD antibodies (anti-IgD-dex), as the multivalent mIg ligand, their proliferation is enhanced and they can be induced to secrete Ig in response to ODN containing various non-optimal motifs, both methylated and non-methylated. Furthermore we could induce synergistic levels of proliferation with concentrations of anti-IgD-dex that were in the picomolar concentration range and with concentrations of ODN that were 10- to 100-fold less than previously reported to be necessary for mitogenic activity. These data provided a model to explain how low concentrations of a multi-epitope-expressing microorganism in the context of mammalian (methylated) or microorganism (non-methylated) DNA can lead to dysregulated B cell proliferation and Ig secretion.

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NK cells not only function as cytotoxic effector cells, but also have immunoregulatory roles including the enhancement of Ig secretion. To have a stable and uniform population of NK cells to study their role in Ig secretion, we generated murine NK clones. Thus, culture of splenocytes from mice that were homozygous for a mutation in the p53 tumor suppressor gene (p53-KO) with IL-2 and poly(IC) resulted in a long-term NK line, from which four stable clones were derived. This approach also yielded a long-term NK line from splenocytes of normal C57BL/6 mice. Identification of the clones as members of the NK lineage was based on large granular morphology, expression of NK-TR and absence of TCR gene rearrangement. Flow cytometry revealed that all clones expressed IL-2R alpha and beta, chains and B220, but no CD3, NK1.1, DX5 or Ly-49. RT-PCR analysis showed heterogeneity in NK1.1 gene expression, and demonstrated expression of perforin and several granzymes in all clones. Three out of four clones lysed YAC-1, but not P815 target cells, corresponding to a pattern of NK specificity. All NK clones enhanced Ig secretion in an in vitro model for T cell-independent type 2 antigens, albeit to varying degrees. We found no correlation between the degree of helper activity of the NK clones and the level of their cytotoxic activity on YAC-1 targets. Thus, we established murine NK clones, and show that they mediate both cytotoxicity and enhancement of Ig secretion.

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