In the most general terms, the work of the Rothstein laboratory is directed toward elucidation of the function and role of B lymphocytes in health and disease.  Over the years our efforts have focused on a number of specific, interrelated areas.  These areas currently include studies of B-1 cell population dynamics and function, B-1 cell number and activity in autoimmune diseases, BCR signaling through non-traditional alternate pathways, and FAIM function as it relates to signaling and apoptosis.

B-1 Cell Population Dynamics and Function

B-1 cells represent a phenotypically distinct subset of B cells that manifests a number of functional features that are different in comparison with the characteristics of the more numerous conventional (B-2) cell population.  Among these features is the spontaneous and constitutive secretion of IgM that is known as “natural” antibody and that serves as an initial serological line of defense against pathogens.  Many other distinctive features have been described, including repertoire skewing of expressed antibodies and IL-10–mediated immunosuppression, along with several characteristics first noted in our laboratory including, enhanced allogeneic T-cell stimulation, induction of Th17-cell differentiation, and constitutive activation of STAT3 (and ERK). Study of activated ERK led to our finding that B-1 cells are continually activated as a result of tonic intracellular signaling, presumably triggered by BCR specificity for self-antigens.  We have elucidated other differences between B-1 and B-2 cells in a number of ways, examining transcriptomic differences by DNA microarray and proteomic differences by mass spectrometry.  In the course of this work we found that murine splenic and peritoneal B-1 cells differ from each other, as well as from B-2 cells, by numerous criteria, including the magnitude of spontaneous IgM secretion.

Some of our recent work has focused on the origin of B-1 cells.  Although initial work suggested that B-1 cells develop early in ontogeny and not in adult life, we (and others) have shown that adult bone marrow can give rise to B-1 cells.  Importantly, the antibody produced by bone marrow–derived B-1 cells differs from that produced by B-1 cells generated early in ontogeny both in structure (N-region addition) and repertoire.  This has led us to hypothesize that B-1 cells turn over slowly as individuals age, and that this process is accompanied by erosion of the initial B-1 cell repertoire and consequent loss of protection against important bacterial pathogens.  This notion is currently being tested and will provide key information relevant to the susceptibility of aged individuals to bacterial pneumonia and other infections.

In our continuing studies we have found that a subset of B-1 cells normally expresses the B7 family member PD-L2, display of which had previously been attributed only to activated macrophages and dendritic cells.  PD-L2 expression divides B-1 cells into PD-L2+ and PD-L2- subsets.  Remarkably, PD-L2+ B-1 cells manifest, to a much greater extent than PD-L2- B-1 cells, several well-known B-1 cell features, including enhanced allogeneic stimulation and repertoire skewing toward autoreactive antibody.  PD-L2 expression is regulated uniquely in B-1 cells by an intronic promoter not utilized by other cell types, which appears to be controlled by the state of chromatin opening.  This, along with other information, raises the possibility that PD-L2+ and PD-L2- B-1 cells may differ in origin or function.  Our future studies of B-1 cells will focus on the role of PD-L2 in dictating B-1–cell activity.

B-1-Cell Number and Activity in Autoimmune Diseases

Although the characteristics of B-1 cells have been well established in mice, an equivalent population of B cells in humans has eluded investigators for over two decades.  As a result, it has been thought by some that human B-1 cells do not exist.  Recently we approached this issue in a new way.  We established functional criteria for what human B-1 cells should do, based on work in the murine system, and then sort-purified distinct populations of B cells to identify those that did.  We looked for human B cells that spontaneously secrete IgM, that are efficient stimulators of CD4 T cell proliferation, and that show evidence of tonic intracellular signaling.  We found B cells in umbilical cord blood and in adult peripheral blood that fulfilled these criteria and are identified by expression of CD20, CD27 and CD43.  In addition, these CD20+CD27+CD43+ B cells recapitulate murine B1-cell characteristics in expressing phosphorylcholine (PC) and DNA specificities, in secreting IL-10, and in inducing Th17-cell differentiation.  These B-1 cells are increased in both number and activity in the autoimmune diseases, lupus and rheumatoid arthritis.  They are decreased in number with advancing age; inasmuch as B-1 cells produce natural antibody against PC which is a major antigenic determinant of pneumococci, the loss of B-1 cells in elderly individuals may be responsible for enhanced susceptibility to pneumococcal pneumonia in this population.   

These results suggest that to treat autoimmune disease it may only be necessary to deplete B-1 cells, rather than to deplete all B cells (as is done with anti-CD20 antibody therapy), which would be highly beneficial in leaving the bulk of B cells intact and functioning.  These results further reflect on the general understanding of memory B cells which are normally identified by CD27 expression.  Because B-1 cells also express CD27, most previous work on memory B cells has been confused by analyzing a heterogeneous mixture of B cells consisting of B-1 cells and true memory B cells.  In fact, B-1 cells express several characteristics previously attributed to memory B cells, which are lost from memory B cells after removal of B-1 cells.  Thus, it may now be necessary to re-evaluate memory B-cell function by examining CD27+ B cells after removal of B1 cells that share CD27 expression and are CD43+.  

Our future studies will focus in understanding the role of B-1 cells in instigating or perpetuating autoimmune disease and in finding ways to regulate B-1-cell numbers and activities.  In addition, we will rescue from B-1 cells antibodies directed against microbial pathogens that can be used therapeutically to prevent or treat infection.  We will also rescue antibodies that may be beneficial in treating coronary artery disease and neurodegenerative diseases.    

BCR Signaling through Non-Traditional Alternate Pathways

The activity and fate of B cells is determined by antigen binding to the B-cell receptor.  We have studied early events in BCR signaling, including examination of transcription factor activation and pharmacologic mimicry of signal propagation. Over time a general consensus has evolved regarding the absolute requirement for certain signaling mediators grouped together as the signalosome (eg, Btk, PI-3K, BLNK, PLCγ2, PKCß) to mediate BCR-triggered downstream events. Recently we found that prior engagement of certain non-BCR receptors results in the generation of a new signalosome-independent alternate pathway for subsequent BCR signaling as a result of receptor crosstalk. Thus, when B cells are treated first with CD40L, or IL-4, or LPS, or CpG, then washed, and then stimulated with anti-Ig, BCR triggered ERK phosphorylation is resistant to signalosome inhibitors such as LY294002 (PI-3K), U73122 (PLC) and Go6976 (PKCß), in contrast to the sensitivity of anti-Ig-stimulated naïve B cells to these agents.  Exposure to CD40L/IL-4/LPS/CpG, then, establishes an alternate pathway(s) for BCR signal propagation.  

We have studied the alternate pathway induced by IL-4 most extensively, and there we found that the signalosome-dependent (classical) BCR signaling pathway, which is the only pathway present in naïve B cells, operates in parallel with the new signalosome-independent (alternate) pathway.  That is, after IL-4 treatment, two pathways co-exist, so that blockade of both is required to interrupt BCR signaling for ERK phosphorylation. Thus, in naïve B cells BCR-triggered pERK is inhibited by LY294002, but in IL-4-treated B cells, LY294002 does not block BCR-triggered pERK, nor does rottlerin (an inhibitor of the alternate pathway), but the combination of LY294002 plus rotterlin does completely block BCR-triggered pERK. Along the same lines, we found that anti-Ig fails to induce ERK phosphorylation in naïve B cells deficient in PKCß (a signalosome mediator), but does so in the same PKCß-deficient B cells when those B cells have been previously treated with IL-4, again demonstrating the ability of the IL-4-induced alternate pathway to bypass the need for signalosome elements. The IL-4-induced alternate pathway further differs from the classical pathway in requiring Lyn (which the classical pathway does not) and in failing to encompass NF-κB activation (which the classical pathway does).  

Remarkably, we found that the combined action of the alternate and classical BCR signaling pathways results in B-cell secretion of osteopontin, a pleiomorphic cytokine that polyclonally activates B cells and that is strongly associated with autoimmunity. These results all together suggest that what is known about BCR signaling may only be correct for naïve B cells and not for B cells exposed to T-cell–derived products in the midst of an ongoing immune response, and, that alternate pathway signaling may represent a B-cell adaptation designed to polyclonally strengthen BCR-triggered signaling when products produced by activated T cells are sensed in the environment, but at the risk of autoimmunity.  Our future work will focus on identifying the precise components of the alternate pathways we have identified, determining the role of alternate pathway signaling in B-cell responses, and elucidating the regulation and role of B-cell–produced osteopontin.  

FAIM Gene Expression As It Relates to Signaling and Apoptosis

B cells, like other cell types, are susceptible to apoptosis by engagement of the Fas (CD95) death receptor.  We demonstrated some time ago that engagement of various other receptors regulates the susceptibility of B cells to Fas-mediated apoptosis.  In that work, we found that treatment of B cells with CD40L upregulated Fas expression and produced marked susceptibility to FasL killing; in contrast, B-cell treatment with anti-Ig produced resistance to FasL killing, when administered coincidentally with CD40L or even 24 hours after CD40L (at which time CD40-mediated upregulation of Fas has already occurred), implying that BCR triggering is capable of reversing already-established Fas-sensitivity.  This BCR-induced Fas-resistance may come into play to protect antigen-specific B cells from activated T cells during B:T interactions that accompany immune responses.  In subsequent work we found that IL-4 also induces Fas-resistance  and, incidentally, we found that IL-4 transgenic mice express serological autoreactivity, suggesting that physiological Fas-resistance, like Fas mutation in lpr mice, results in loss of tolerance and autoantibody production.

To identify molecules that might be responsible for inducible Fas-resistance, we undertook differential display, comparing B cells stimulated with CD40L/anti-Ig with those stimulated by CD40L alone.  This led to the cloning and characterization of a novel anti-apoptotic gene termed Fas Apoptosis Inhibitory Molecule (FAIM).  FAIM is highly conserved from fly to human but contains no known effector motifs and manifests a unique protein structure.  Subsequent work showed that two alternatively spliced forms of FAIM exist, a “short” form and a “long” form, the latter being expressed only in the brain (and being longer by an additional 22 N-terminal amino acids) and producing neuronal resistance to apoptosis.  In more recent work we found that FAIM enhances B-cell signaling produced by engagement of the CD40 receptor and that this correlates with increased numbers of bone marrow plasma cells in mice that overexpress FAIM.  Thus FAIM has another activity beyond apoptosis, namely, enhancement of B-cell signaling for differentiation and immunoglobulin secretion. We are currently in the process of analyzing a FAIM KO mouse we constructed for abnormalities both in central nervous system and B-lymphocyte function.  Our future work will entail producing FAIM-L and FAIM-S transgenic mice, including FAIM-L transgenic mice in which overexpression is limited to the brain.  This will provide a model with which to examine the potential role of FAIM in neurodegenerative diseases.  We will also carry out structure/function analysis of the FAIM protein to identify a presumably novel, but evolutionarily conserved, effector motif.