Investigator & Head, Center for Oncology & Cell Biology, The Feinstein Institute for Medical Research
Professor of Medicine & Professor of Molecular Medicine, Hofstra Northwell School of Medicine
Phone: (516) 562-3436
Thomas L. Rothstein MD, PhD, graduated from Duke University with MD and PhD degrees under the auspices of the Medical Scientist Training Program. He pursued internship and residency training in internal medicine at George Washington University Hospital and at Beth Israel Hospital/Harvard Medical School, respectively, in addition to a research fellowship at the National Cancer Institute with Dr. Michael G. Mage.
Dr. Rothstein then completed a fellowship in hematology/oncology at Beth Israel Hospital/Harvard Medical School, the research portion of which was carried out at the Massachusetts Institute of Technology with Dr. Malcolm L. Gefter. He subsequently joined the hematology section in the Department of Medicine at Boston University School of Medicine as an assistant professor.
Over the next 24 years, Dr. Rothstein would rise to the rank of professor of medicine and fill a number of roles, including service as associate chief for research in the combined section of hematology/oncology, director of the NIH-supported T32 Training Program in Blood Diseases and Resources and director of the Immunobiology Unit. He also served as a member and then chair of the Cellular Immunology Study Section of the Arthritis Foundation, as a member of the Novel Research Task Force of the Lupus Research Institute and as an ad hoc member of many NIH regular and special-emphasis study sections. His journal experience includes work as an associate editor of The Journal of Immunology and as an editorial board member of the Open Autoimmunity Journal, He currently is editor-in-chief of Frontiers in B Cell Biology.
In 2006, Dr. Rothstein was recruited to The Feinstein Institute for Medical Research as investigator and head of the Center for Oncology and Cell Biology. He is also a professor in the Elmezzi Graduate School of Molecular Medicine at The Feinstein Institute and professor of medicine and molecular medicine at Hofstra Northwell School of Medicine.
Dr. Rothstein is an elected member of the American Society for Clinical Investigation, the Association of American Physicians and the Henry Kunkel Society and has mentored 19 graduate students and 29 postdoctoral fellows.
Dr. Rothstein leads the immunobiology laboratory at The Feinstein Institute for Medical Research. His laboratory team aims to elucidate the role and function of B lymphocytes in health and disease. Recent work has focused on human B1 cells, a unique B lymphocyte subset that produces “natural” antibody and regulates T-cell activity. Although B1 cells were well described in mice, the markers defining the corresponding population in the human system eluded investigators for more than a quarter century, until recently when the phenotype of human B1 cells was defined here.
Human B1 cells may be a key to development of effective therapeutic antibodies and may be important in understanding and treating autoimmune diseases. Recent work in the immunobiology laboratory has also encompassed exploration of alternate intracellular signaling pathways activated by antigen receptor triggering; the regulation and role of the multifaceted cytokine, osteopontin; and the mechanisms and outcomes of signaling enhancement and anti-apoptosis produced by the novel gene, FAIM.
B1 cells represent a phenotypically distinct subset of B cells that manifests a number of functional features that are different in comparison to the characteristics of the more numerous conventional (B2) 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. Natural antibody has also been reported to serve a housekeeping function by binding to and disposing of cellular debris and toxic molecules.
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 the team’s laboratory and elsewhere, including enhanced allogeneic T cell stimulation, induction of Th17 cell differentiation and constitutive activation of STAT3 and ERK. Study of activated ERK led to the team’s finding that B1 cells are continually activated as a result of tonic intracellular signaling, presumably triggered by BCR specificity for self-antigens, that leads to increased expression of CD86 and thus increased T cell stimulation. They have elucidated other differences between B1 and B2 cells in a number of ways, examining transcriptomic differences by DNA microarray and proteomic differences by mass spectrometry. In the course of this work, they found that murine splenic and peritoneal B1 cells differ from each other, as well as from B2 cells, by numerous criteria including the magnitude of spontaneous IgM secretion.
Some of Dr. Rothstein’s team’s recent work has focused on the origin of B1 cells. Although initial studies suggested that B1 cells develop early in ontogeny and not in adult life, they and others have shown that adult bone marrow can give rise to B1 cells. Important to note — the antibody produced in adulthood by bone marrow-derived B1 cells differs from that produced by B1 cells generated early in ontogeny both in structure (N-region addition) and repertoire. This has led the team to hypothesize that B1 cells turn over slowly as individuals age, and that this process is accompanied by erosion of the initial B1 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. Because natural antibody has been shown to be protective against atherosclerosis in animal models, changes with age in B1 cells and the natural antibodies they produce could play a role as well in susceptibility to non-infectious degenerative diseases of the elderly.
In their continuing studies, they have found that a subset of B1 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 B1 cells into PD-L2+ and PD-L2- subsets. Remarkably, PD-L2+ B1 cells manifest — to a much greater extent than PD-L2- B1 cells — several well-known B1 cell features, including enhanced allogeneic T cell stimulation and repertoire skewing toward autoreactive antibody.
PD-L2 expression is regulated uniquely in B1 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- B1 cells may differ in origin or function. The lab’s future studies of B1 cells will focus on the role of PD-L2 in dictating B1 cell development, selection, and activity.
Although the characteristics of B1 cells have been well established in mice, an equivalent population of B cells in humans eluded investigators for over two decades. As a result, it had been thought by some that human B1 cells do not exist. Dr. Rothstein’s team recently approached this issue in a new way – they established functional criteria for what human B1 cells should do, based on work in the murine system, and then sort-purified distinct populations of B cells to identify those that did.
They 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. They 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 to the original qualifying features, these CD20+CD27+CD43+ B cells recapitulate murine B1 cell characteristics in expressing phosphorylcholine (PC) and DNA binding specificities, and in secreting IL-10. These B1 cells are increased in number and in activity in autoimmune diseases such as lupus and rheumatoid arthritis. They are decreased in number with advancing age; inasmuch as B1 cells produce natural antibody against PC, which is a major antigenic determinant of pneumococci, the loss of B1 cells in elderly individuals may be responsible for enhanced susceptibility to pneumococcal pneumonia in this population.
Recently, clinical treatment designed to deplete B cells with anti-CD20 antibody has shown clear efficacy in patients afflicted with rheumatoid arthritis and some efficacy in patients with lupus. However, depletion of all B cells runs the risk of crippling the immune system and its normal protective function against infectious disease. The increased number and activity of B1 cells in autoimmune diseases suggests a pathogenic link in which case successful therapy might be based on depletion of the B1 cell subset of B cells rather than depletion of all B cells, leaving the bulk of the serological immune system intact.
The phenotype of human B1 cells reflects on the general understanding of memory B cells, which are normally identified by CD27 expression. Because B1 cells also express CD27, it now appears that much previous work on memory B cells has been confused by analysis of a heterogeneous mixture of B cells consisting of B1 cells and true memory B cells. In fact, B1 cells express several characteristics previously attributed to memory B cells, which are lost from memory B cells after removal of B1 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+.
Dr. Rothstein’s team’s future studies will focus on elucidating the progenitor for human B1 cells, on understanding the role of B1 cells in instigating or perpetuating autoimmune disease, and on finding ways to regulate B1 cell number and activity. In addition, they will rescue from B1 cells antibodies directed against microbial pathogens that can be used therapeutically to prevent or treat infection. They will also work to rescue antibodies that may be beneficial in treating coronary artery disease and neurodegenerative diseases.
The activity and fate of B cells is determined by antigen binding to the B cell receptor. Dr. Rothstein and others have studied early events in BCR signaling, including examination of transcription factor activation and pharmacologic mimicry of signal propagation. Over time, a consensus has evolved regarding the absolute requirement for certain signaling mediators grouped together as the signalosome (eg, Btk, PI-3K, BLNK, PLCg2, PKCb) to mediate BCR-triggered downstream events.
Recently, they 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 imprinting. Thus, when B cells are treated first with CD40L, or IL-4, or LPS, or CpG, and then subsequently stimulated with anti-Ig, BCR triggered ERK phosphorylation is resistant to signalosome inhibitors such as LY294002 (PI-3K), U73122 (PLC) and Go6976 (PKCb), 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.
Dr. Rothstein’s team has studied the alternate pathway induced by IL-4 most extensively, and there they 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, they found that anti-Ig fails to induce ERK phosphorylation in naïve B cells deficient in PKCb (a signalosome mediator), but does so in the same PKCb-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-kB activation (which the classical pathway does).
Remarkably, Dr. Rothstein’s team found that the combined action of the alternate and classical BCR signaling pathways results in B cell secretion of osteopontin, a pleomorphic 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. eam’s future work will focus on identifying the precise components of the alternate pathway, determining the role of alternate pathway signaling in B cell responses, and elucidating the regulation and role of B cell-produced osteopontin.
B cells, like other cell types, are susceptible to apoptosis by engagement of the Fas (CD95) death receptor. Dr. Rothstein’s team demonstrated some time ago that engagement of various other receptors regulates the susceptibility of B cells to Fas-mediated apoptosis. In that work, they 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, they found that IL-4 also induces Fas-resistance and, incidentally, the team 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, Dr. Rothstein’s team 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). Others have shown that expression of FAIM-Long in the brain produces neuronal resistance to apoptosis, and that loss of FAIM produces apoptosis susceptibility in T cells and liver cells.
In more recent work, they 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 over express FAIM. Thus, FAIM has another activity beyond anti-apoptosis, namely, enhancement of B cell signaling for differentiation and immunoglobulin secretion.
Dr. Rothstein’s lab is currently in the process of analyzing a FAIM KO mouse it constructed for abnormalities, both in central nervous system and B lymphocyte function. Their 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. They will also carry out structure/function analysis of the FAIM protein to identify a presumably novel, but evolutionarily conserved, effector motif.
Sehba Dsilva, MD
Education: MD, 2001, Karnatak University, India
Research: Regulation and role of osteopontin
Elisa Keefe, MD
Fellow in Pediatric Hematology/Oncology
Education: MD, 2007, University of Oklahoma
Research: Early human B cell development
Tã¢m Quach, PhD
Education: PhD, 2011, University of Rochester, Rochester, NY
Research: Identification of the human B-1 cell progenitor
Duke University, Durham, NC
Field of Study: Physiology
Duke University School of Medicine, Durham, NC
Field of Study: Medicine
1968-1974 American Cancer Society Predoctoral Scholarship
1979-1980 American Cancer Society Regular Clinical Fellowship
1979-present Diplomate, American Board of Internal Medicine
1982-1984 Research Fellow, The Medical Foundation
1988-present Election to American Society for Clinical Investigation
2006-present Election to American Association of Physicians
2006-present Appointment to Board of Advisors, American Cancer Society, Nassau Region, New York
2010-present Election to the Henry Kunkel Society