Streptococcus pneumoniae is the most common cause of pneumonia, which claims the lives of people over the age of 65 seven times more frequently than those aged 5–49. B-1a cells provide immediate and essential protection from S. pneumoniae through production of natural immunoglobulin, which has minimal insertion of N-region additions added by the enzyme TdT. In experiments with SCID mice infected with S. pneumoniae, we found passive transfer of IgG-depleted serum from aged (18–24-month old) mice had no effect whereas IgG-depleted serum from young (3-month old) mice was protective. This suggests protective natural IgM changes with age. Using single cell PCR we found N-region addition, which is initially low in fetal-derived B-1a cell IgM developing in the absence of TdT, increased in 7–24-month old mice as compared to 3-month old mice. To determine the mechanism responsible for the age related change in B-1a cell IgM, we established a mixed chimera system in which mice were reconstituted with allotype-marked mature peritoneal B-1a cells and adult bone marrow (BM) cells. We demonstrated even in the presence of mature peritoneal B-1a cells, adult BM contributed to the mature B-1a cell pool. More importantly, using this system we found over a 10-month period peritoneal B-1a cell IgM changed, showing the number of cells lacking N-region additions at both junctions fell from 49% to 29% of sequences. These results strongly suggest selection-induced skewing alters B-1a cell derived natural antibody, which may in turn be responsible for the loss of natural IgM-mediated protection against pneumococcal infection.

Despite advances in T-cell immunotherapy against Epstein-Barr virus (EBV)-infected lymphomas that express the full EBV latency III program, a critical barrier has been that most EBV+ lymphomas express the latency I program, in which the single Epstein-Barr nuclear antigen (EBNA1) is produced. EBNA1 is poorly immunogenic, enabling tumors to evade immune responses. Using a high-throughput screen, we identified decitabine as a potent inducer of immunogenic EBV antigens, including LMP1, EBNA2, and EBNA3C. Induction occurs at low doses and persists after removal of decitabine. Decitabine treatment of latency I EBV+ Burkitt lymphoma (BL) sensitized cells to lysis by EBV-specific cytotoxic T cells (EBV-CTLs). In latency I BL xenografts, decitabine followed by EBV-CTLs results in T-cell homing to tumors and inhibition of tumor growth. Collectively, these results identify key epigenetic factors required for latency restriction and highlight a novel therapeutic approach to sensitize EBV+ lymphomas to immunotherapy.

B-1a cells constitutively secrete natural antibody that provides immediate protection against microbial pathogens and functions homeostatically to speed removal of apoptotic cell debris. Although B-1a cells are especially prominent in the peritoneal and pleural cavities, some B-1a cells reside in the spleen. A small subset of splenic B-1a cells in naïve, unimmunized mice express CD138, a recognized plasma cell antigen, whereas the bulk of splenic B-1a cells are CD138 negative. Splenic B-1a cells in toto have been shown to generate much more antibody per cell than peritoneal B-1a cells; however, specific functional information regarding CD138+ splenic B-1a cells has been lacking. Here, we find a higher proportion of CD138+ splenic B-1a cells spontaneously secrete more IgM as compared to CD138− B-1a cells. Moreover, IgM secreted by CD138+ splenic B-1a cells is skewed with respect to N-region addition, and some aspects of VH and JH utilization, as compared to CD138− splenic B-1a cells and peritoneal B-1a cells. The small population of CD138+ splenic B-1a cells is likely responsible for a substantial portion of natural IgM and differs from IgM produced by other B-1a cell subsets.

Natural antibodies produced by B-1a cells are required for immediate protection against infection. The protective capacity of natural antibodies is attributed to germ-line like structure, which includes the relative absence of N-region addition. Previous studies have shown B-1a cell immunoglobulin from aged mice contains abundant N-additions. B-1a cells have been shown to derive from a specific Lineage negative (Lin-)CD45Rlo/-CD19+ progenitor found both in fetal liver and adult bone marrow. Here, we report identification of a fetal liver population characterized phenotypically as Lin-CD45R-CD19-, which gives rise to IgM+IgDloCD45RloCD5+Mac-1+CD19hiCD43+CD23lo B-1a cells upon adoptive transfer to SCID recipients. These B-1a cells derived from the Lin-CD45R-CD19- fetal liver population produce natural antibody that binds pneumococcal antigens, but this immunoglobulin contains substantial N-addition despite initial absence of TdT. Furthermore, we show extensive N-addition is also present in B-1a cells derived from the Lin-CD45Rlo/-CD19+ B-1 progenitor found in the bone marrow. Together these results demonstrate B-1a cell N-addition depends on the type of progenitor and the location of the progenitor during its development. These findings have implications for how regulation of different progenitors from fetal liver and bone marrow may play a role in the age-related increase in N-region addition by B-1a cells in normal animals.

B1 B cells defend against infectious microorganisms by spontaneous secretion of broadly reactive “natural” immunoglobulin that appears in the absence of immunization. Among many distinguishing characteristics, B1 B cells display evidence of activation that includes phosphorylated STAT3. In order to identify the origin of pSTAT3 we examined interleukin-2 receptor (IL-2R) expression on B1 cells. We found that some (about 1/5) B1a cells express the IL-2R α chain, CD25. Although lacking CD122 and unresponsive to IL-2, B1a cells marked by CD25 express increased levels of activated signaling intermediates, interruption of which results in diminished CD25. Further, CD25+ B1a cells contain most of the pSTAT3 found in the B1a population as a whole. Moreover, CD25+ B1a cells express leukemia inhibitory factor receptor (LIFR), and respond to LIF by upregulating pSTAT3. Together, these results define a new subset of B1a cells that is marked by activation-dependent CD25 expression, expresses substantial amounts of activated STAT3, and contains a functional LIFR.