SUMMARY Immunoglobulin M (IgM) memory cells undergo differentiation in germinal centers following antigen challenge, but the full effector cell potential of these cells is unknown. We monitored the differentiation of enhanced yellow fluorescent protein (eYFP)- labeled CD11c+ and CD11cneg T-bet+ IgM memory cells after their transfer into naive recipient mice. Following challenge infection, many memory cells differentiated into IgM-producing plasmablasts. Other donor B cells entered germinal centers, down- regulated CD11c, underwent class switch recombination, and became switched memory cells. Yet other donor cells were maintained as IgM memory cells, and these IgM memory cells retained their multi-lineage potential following serial transfer. These findings were corroborated at the molecular level using immune repertoire analyses. Thus, IgM memory cells can differentiate into all effector B cell lineages and undergo self-renewal, properties that are characteristic of stem cells. We propose that these memory cells exist to provide long-term multi-functional immunity and act primarily to maintain the production of protective antibodies.

CD11c+ T-bet+ B cells have now been detected and characterized in different experimental and clinical settings, in both mice and humans. Whether such cells are monolithic, or define subsets of B cells with different functions is not yet known. Our studies have identified CD11c+ IgM+ CD19hi splenic IgM memory B cells that appear at approximately three weeks post-ehrlichial infection, and persist indefinitely, during low-level chronic ehrlichial infection. Although the CD11c+T-bet+ B cells we have described are distinct, they appear to share many features with similar cells detected under diverse conditions, including viral infections, aging, and autoimmunity. We propose that CD11c+ T-bet+ B cells as a group share characteristics of memory B cells that are maintained under conditions of inflammation and/or low-level chronic antigen stimulation. In some cases, these cells may be advantageous, by providing immunity to re-infection, but in others may be deleterious, by contributing to aged-associated autoimmune responses.

IgM memory cells are recognized as an important component of B cell memory in mice and humans. Our studies of B cells elicited in response to ehrlichial infection identified a population of CD11c-positive IgM memory cells, and an IgM bone marrow antibody-secreting cell population. The origin of these cells was unknown, although an early T-independent spleen CD11c- and T-bet-positive IgM plasmablast population precedes both, suggesting a linear relationship. A majority of the IgM memory cells detected after day 30 post-infection, also T-bet-positive, had undergone somatic hypermutation, indicating they expressed activation-induced cytidine deaminase (AID). Therefore, to identify early AID-expressing precursor B cells, we infected an AID-regulated tamoxifen-inducible Cre-recombinase-EYFP reporter strain. Tamoxifen administration led to the labeling of both IgM memory cells and bone marrow ASCs on day 30 and later post-infection. High frequencies of labeled cells were identified on day 30 post-infection, following tamoxifen administration on day 10 post-infection, although IgM memory cells were marked when tamoxifen was administered as early as day 4 post-infection. Transcription of Aicda in the early plasmablasts was not detected in the absence of CD4 T cells, but occurred independently of TLR signaling. Unlike the IgM memory cells, the bone marrow IgM ASCs were elicited independent of T cell help. Moreover, Aicda was constitutively expressed in IgM memory cells, but not in bone marrow ASCs. These studies demonstrate that two distinct long-term IgM-positive B cell populations are generated early in response to infection, but are maintained via separate mechanisms.

The processes and factors that regulate B cell fate decisions during IgM memory cell development and differentiation are unknown, and may differ from those required by canonical memory cells. In our experimental model of Ehrlichia muris infection, we have characterized two long-lived B cell populations: IgM memory cells, and IgM bone marrow (BM) antibody secreting cells (ASCs). The IgM+ memory B cells are required for recall IgG responses, while the BM ASCs produce antigen-specific IgM responsible for maintaining long-term immunity. Both populations are derived from an early AID-expressing CD4 T cell-independent splenic CD11c+ plasmablast population. Because abundant T follicular helper cells are generated in the spleen during early infection, we investigated whether T cell help regulates B cell fate. Infection of MHC class II-deficient mice revealed that the IgM+ memory B cells required CD4+ T cell help, while the IgM+ BM ASCs did not. Moreover, IgM memory cell transfer studies indicated that T cell help was required for the generation of IgM+ memory cells, but not for the generation of BM ASCs. On the basis of these observations, we propose that T cells drive B cell fate: developing spleen plasmablasts that receive T cell signals mature to become IgM memory cells, while cells that do not receive these signals follow a default pathway to become long-lived IgM BM ASCs.

Our laboratory has been studying the differentiation of CD11c+ T-bet+ IgM memory B cells during secondary ehrlichial infection. These cells are closely related to B cells that have been described in a range of other contexts, including hepatitis, AIDs, malaria, SLE, and age-related autoimmunity. Following secondary infection, IgM memory cells, as a population, undergo self-renewal, and differentiate into effector cells, including splenic and bone marrow antibody secreting cells (ASC). Moreover, IgM memory cells enter germinal centers, where they undergo class-switch recombination and give rise to class-switched memory and effector cells. Although these data suggest that a single memory cell has multi-lineage potential, we sought to formally address this question by searching for shared clones among IgM memory cell-derived effector cells. Among the IgM memory cell-derived subsets, we identified several clones common to all effector cell populations. The number of common clones varied for each pairwise comparison of effector cells and suggested lineal relationships. IgM memory cells accumulated mutations following rechallenge; although all memory cell-derived subsets displayed similar numbers of V region mutations. Lineage analysis demonstrated that the effector cell subsets underwent varying degrees of clonal diversification, although this was clone-dependent. These studies reveal that a single IgM memory cell clone can give rise to different memory and effector cell subsets, that is, they exhibit stem cell properties. This property distinguishes these T-bet+ IgM memory cells as a unique memory cell subset.