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Dan L. Longo, M.D., Special Volunteer,
Lymphocyte Cell Biology Unit
Dan L. Longo, M.D.After completing medical school at the University of Missouri, Columbia and internal medicine training at the Peter Bent Brigham Hospital and Harvard Medical School in Boston, he obtained fellowship and laboratory training at NIH and has been here for 26 years. Before becoming Scientific Director, NIA in 1995, Dr. Longo was the Director, Biological Response Modifiers Program, and Associate Director, Division of Cancer Treatment, National Cancer Institute, Frederick, Maryland. He is the author of over 600 articles and book chapters. He is an editor of Harrison's Principles of Internal Medicine, and Cancer Chemotherapy and Biotherapy. He is an associate editor of Journal of the National Cancer Institute and Clinical Cancer Research and he sits on the editorial boards of six other peer-review journals. He has been cited by Good Housekeeping as one of the "Best Cancer Doctors in America" and listed in Best Doctors in America.

Research Interests: The Regulation of Growth Fraction in Tumor Cells: The vast majority of solid tumors have a very low growth fraction at the time they become clinically evident, usually in the range of 3-7%. When the tumor is treated, the growth fraction increases in an effort to maintain the tumor cell mass. This is reminiscent of the organization of most organ systems. Resting bone marrow stem cells are recruited into cycle under the influence of a myelotoxic stimulus. Surgical removal of a portion of the liver stimulates the recruitment of hepatocytes into the cell cycle to replace the removed tissue. Other examples could also be cited. What is of interest to us is how a tumor cell, with its many genetic abnormalities that tend to promote proliferation, is pulled out of the cell cycle in the first place. Some gene product that is working in the resting tumor cells has managed to antagonize all the oncogene mutations and missing or malfunctioning tumor suppressor gene products and stop the cell from dividing; and it does this reversibly. When the tumor perceives an attack that reduces its volume, cells can be recruited back into the cell cycle. We are separating fresh lymphoma specimens into dividing and nondividing populations, isolating cDNA, and using microarray techniques, characterizing genes that are expressed in resting cells but not in dividing cells. Such messages will be isolated, their genes identified, and then the message will be introduced into dividing cells to look for growth arrest.
Tumor-induced Immunosuppression: We initially observed, and it has been widely reproduced, that T cells from tumor-bearing hosts are defective in their signalling in response to antigen and in their function. A variety of defects are noted including defective nuclear translocation of the p65 NF-k B transcription factor, shortened half-lives for a number of cellular proteins such as TCR-e chain and signalling kinases of the src family, among others, and a deviation of the cytokine production profile toward Th2 cytokines (IL-4, IL-10) and away from Th1 cytokines (interferon-g, TNF). Evidence of suppression of immune function in mice in whom tumor is growing in hollow fibers in the peritoneal cavity without any cell-cell contact in the host suggest that a soluble tumor factor is responsible for the defect in cellular immunity. We have devised a method of reproducing these tumor-induced changes in normal T cells in vitro and are in the process of isolating the tumor-derived factor(s) responsible for the changes. In agreement with this finding, we are able to demonstrate the immunosuppressive properties of the pleural fluid isolated from cancer patients. We are in the process of isolating and characterizing the tumor-derived factor(s) from the pleural fluids of cancer patients.
Shaping the Pre-immune B Cell Repertoire: We have developed a number of transgenic mice and mice with targeted mutations as tools for understanding the mechanisms (receptor dependent and independent) that shape the B cell repertoire. These strains and their response to the antigen phosphocholine (PC) have been used to define the relationships between antigen recognition and protective, ineffective and detrimental immune responses and to understand the impact that an aging immune repertoire has on immune response. The immune response to PC is important because in mice it has been shown to confer a high degree of protection against infection by Streptococcus pneumoniae (SPn.), a pathogen that poses a significant risk to elderly, very young and immunocompromised individuals. Recently, we have shown that the mouse VH1 gene is essential for immune response to PC and PC-mediated protection against infection by SPn. Furthermore, by examining the associations between the VH1 gene and various light chains in PC-specific B cells, we have identified IgL chain structural determinants that may explain differences in the relative affinity/avidity of VH1/VLchain combinations for different PC containing antigens. Analysis of these models should provide insight into the complimentary contribution of interactions between VH and VL genes to protective versus ineffective immune responses to common determinants expressed on different pathogens.
An increase in the percentage of pneumococcal strains not represented in the current carbohydrate vaccines has been observed in individuals presenting with pneumococcal disease. In addition, the problem of emerging antibiotic resistant strains of many pathogens including S. pn. continues to worsen. We have recently developed and patented a novel, flexible and inexpensive synthesis strategy for preparing PC-derivatives that can be used to produce PC-conjugate vaccines against infection by S. pn. and other PC expressing pathogens. PC is an antigen found on virtually all strains of S. pn. as well as many other bacterial, fungal and parasitic protozoan pathogens. The relevancy of anti-PC antibodies to protection from challenge by various pathogens in humans is demonstrated by the observation that passive immunization of mice with anti-PC specific antibodies purified from humans provide the mice with protection from challenge by S. pn. We are continuing to develop and examine PC-conjugates as potential vaccines against S. pn. infection for use in humans and are developing mouse models for determining whether PC-conjugate vaccines may also provide protection against other pathogens which express PC. In addition, ongoing studies are underway to define the immune response to PC and other phospholipids in humans. These studies should provide further insight into factors which contribute to a protective and beneficial immune response and those which are harmful and detrimental to the host resulting in autoimmune pathologies.
The clonal selection theory and associated corollary (that a single cell expresses a single receptor with a single antigen specificity) has been a dominant tenet in shaping our thinking of the development of the immune system and immune response to antigenic challenge. Based on our earlier observations in PC transgenic mouse models we proposed that this corollary could be compromised and that dual receptor expression or "receptor dilution" was a mechanism by which a host can balance the necessity to avoid self reactivity (which could result in holes in the available repertoire) with the evolutionary pressure to provide protection against specific pathogens. We have recently demonstrated in wild type, nontransgenic C57BL/6 mice that dual receptor expressing B cells are a part of the normal wild type B cell repertoire. We continue to examine the VH and VK genes expressed by this small population of dual isotype expressing B cells in wild type C57BL/6 mice and have noted that the inferred specificities for the expressed VH and VK genes are to both autoreactive antigens as well as antigens expressed on various pathogens. These observations suggest that coexpression may be a general mechanism for shaping this subpopulation of the B cell repertoire. Ongoing experiments should also provide additional evidence for the necessity to conserve the specificities expressed by these dual receptor expressing B cells as well as to delineate the developmental and molecular processes which result in generation and maintenance of this dual receptor expressing B cell population. Continued examination of the contribution these and other components play in shaping the immune repertoire will further expand our understanding of the mechanisms that distinguish between protective, ineffective and detrimental immune responses.
Cyclosporin A-Resistant Costimulation via CD28: The CD28-mediated co-stimulatory signal plays a pivotal role in many immune responses including T cell responses against tumors, virus-infected cells, and transplanted alloantigens. Depending on the nature of primary stimulation, CD28 can initiate multiple intracellular signaling pathways that can be broadly classified into two groups: one is calcium-dependent and sensitive to cyclosporin A (CsA), and the other one is calcium-independent and resistant to CsA. The CsA-resistant pathway has been thought to be responsible for the ineffectiveness of CsA in the treatment of graft-versus-host disease following allogeneic bone marrow transplantation. Our primary objectives are focused on three areas: (1) characterization of the CsA-resistant co-stimulatory pathway; (2) examination of the physiological significance of this pathway; and (3) evaluation of the effect of aging on this pathway.
Role of TGF-b-Receptor II in Resistance to TGF-b-mediated Growth Suppression in A B-cell Lymphoma Cell Line: Transforming growth factor (TGF)-b1 is a member of the TGF-b superfamily that regulates cell growth and differentiation in a variety of cell types. TGF-b inhibits cell proliferation by arresting cells in G1 phase of the cell cycle. Resistance to TGF-b-mediated growth suppression in tumor cells is often associated with the functional loss of TGF-b1 receptors. We are studying a diffuse large B-cell lymphoma cell line, DB, which lacks TGF-b responsiveness with respect to growth suppression. Our goal is to identify the deficit in TGF-b signaling pathway in DB cells. Preliminary data indicate that DB cells lack functional TGF-b receptor II [(TbRII) on the cell surface in contrast to a TGF-b-responsive B-cell lymphoma cell line RL, whereas both cell lines carry comparable levels of receptor I (TbRI)]. Lack of functional TbRII correlates with the lack of TGF-b-induced nuclear translocation of phospho-Smad3 and phospho-Smad2, and lack of nuclear expression of p21Cip1/WAF1 and down regulation of nuclear c-Myc in DB cells. Ectopically-expressed wild type, but not c-terminally- truncated TbRII, renders the DB cell line responsive to TGF-b1-mediated growth suppression, and correlates with up-regulation of nuclear p21Cip1/WAF1 and down-regulation of c-Myc. Analysis of the TbRII gene reveals a truncated message in DB cells. We are currently investigating the nature of modifications in the TbRII gene.

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Laboratory of Molecular Biology and Immunology
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Updated: Saturday October 20, 2012